The TROP2 targeting antibody-drug conjugate IMMU-132 enhances the efficacy of radiation therapy for lung cancer

The TROP2 targeting antibody-drug conjugate IMMU-132 enhances the efficacy of radiation therapy for lung cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The TROP2 targeting antibody-drug conjugate IMMU-132 enhances the efficacy of radiation therapy for lung cancer Vaishali Kapoor, Harendra Shah, Sai Prem, Ron Bose, Abhay Singh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7593735/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Studies suggest that the human trophoblast cell-surface antigen (TROP2) is highly expressed in most lung cancers and is associated with poor prognosis. Currently, there are no TROP2-directed ADCs approved for treating lung cancer patients. IMMU-132 ADC (Sacituzumab govitecan) is a TROP-2-directed ADC recently approved for metastatic triple-negative breast cancer and urothelial cancer. However, its role in non-small cell carcinoma (NSCLC) has not been explored. Here, we examined the impact of IMMU-132 alone and in combination with radiation on NSCLC cells in vitro and in vivo . We found cell surface expression of TROP-2 on NSCLC cell lines, internalization of IMMU-132, induction of cell cycle arrest at the G2/M phase, and promotion of programmed cell death (apoptosis) following irradiation. Furthermore, IMMU-132 enhanced radiosensitivity by decreasing clonogenic survival through increased DNA double-strand break formation (as indicated by the γH2AX level), modulating DNA damage repair, inhibiting survival pathways, and inducing PARP-mediated apoptosis. In vivo , the combination of IMMU-132 and radiation therapy increased tumor control and improved overall survival in mice bearing H441 and H460 cell xenografts, as well as in the syngeneic LLC tumor. Tumor radio-sensitization with IMMU-132 promotes the inhibition of prosurvival signaling (PI3K/AKT/mTOR/, MEK/ERK, p38MAPK/JNK) together with induced apoptosis by increasing PARP, cleaved caspase-3 and reducing anti-apoptotic proteins (BCL-xL, BCL-2, XIAP) as well as modulating DNA damage repair (ATM, ATR, RAD51, p53, DNA-PK). Together, our data suggests that the targeted delivery of IMMU-132 radiosensitizer at sub-therapeutic doses could broaden the therapeutic window of radiation therapy in lung cancer and may decrease the possibility of side effects. Hence, the combination of IMMU-132 and radiation therapy could be a promising therapeutic strategy for NSCLC. Health sciences/Oncology/Cancer/Cancer therapy/Targeted therapies Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer Health sciences/Oncology/Cancer/Cancer therapy/Radiotherapy TROP2 Radiation Lung cancer Radiation sensitization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Lung cancer is the leading cause of death worldwide 1 , 2 . Non-small cell lung cancer (NSCLC) is the predominant form of lung cancer, accounting for nearly 85% of all lung cancer cases 3 . Despite advances in treatment strategies, the five-year survival rate for lung cancer patients is still very low (23%) 1,2 . Chemotherapy combined with radiotherapy, followed by immunotherapy, is the standard of care for patients with locally advanced NSCLC 4 – 6 . Radiation-induced tumor death is mainly attributed to DNA double-strand breaks through the formation of chromosomal aberrations, cell cycle arrest, and apoptosis 7 . Various signaling pathways, including MAPK and PI3K/AKT/mTOR, that regulate tumor cell growth, survival, and proliferation, play a crucial role in developing radioresistance in NSCLC 8 – 13 . The application of radiation-sensitizing chemotherapy in cancer treatment has displayed encouraging potential, but harmful side effects impede the administration of chemotherapy at elevated dosages. Antibody-drug conjugates (ADCs) are an emerging class of therapeutics that combine a highly specific monoclonal antibody with a potent cytotoxic payload linked via a chemical linker 14 . The payloads typically consist of either antimicrotubule agents or DNA-damaging compounds, which induce cell-cycle arrest and promote apoptosis in target cells 14 , 15 . ADC technology relies on using a linker that remains stable in circulation to minimize off-target effects and ensure selective delivery of the cytotoxic payload to target cells 14 , 15 . Fam-trastuzumab deruxtecan-nxki (Enhertu) was approved in August 2022 for adults with unresectable or metastatic HER2-mutant NSCLC who have received prior systemic therapy 16 . Trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein consisting of an extracellular domain, a transmembrane region, and a short cytoplasmic tail that can undergo phosphorylation 17 , 18 . The transmembrane domain is a calcium signal transducer, influencing cell cycle-related signaling pathways. TROP2 interacts with proteins such as insulin-like growth factor 1, claudin-1 and − 7, cyclin D1, and protein kinase C (PKC), triggering downstream effects that promote cell proliferation and apoptosis 19 , 20 . The cytoplasmic tail regulates protein-protein interactions and contains a PKC phosphorylation site that modulates calcium signaling. TROP2 is weakly expressed or not expressed in normal tissue but is overexpressed in several types of cancer, including lung cancer 21 , 22 . TROP2 expression has been observed in up to 64% of adenocarcinoma and up to 75% of squamous cell carcinoma NSCLC, making it a promising target for cancer therapy 23 , 24 . Sacituzumab govitecan-hziy (IMMU-132) is an anti-TROP2 ADC that consists of a humanized anti-TROP2 monoclonal antibody linked to the topoisomerase I inhibitor SN-38 by a hydrolyzable pH-sensitive linker with a high drug-to-antibody ratio (DAR) of 7.6 18 . IMMU-132 has been approved for the treatment of triple-negative metastatic breast cancer and metastatic urothelial cancer 18 , 25 . SN-38 is the active metabolite of irinotecan and mediates the upregulation of early proapoptotic proteins, such as p53, resulting in caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, and inhibition of NF-κB and AKT signaling in cancer 26 – 29 . Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), activating ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR), and DNA-dependent protein kinase (DNA-Pk) 30 . Additionally, IR activates several cellular processes, including apoptosis, cell cycle arrest, and DNA repair in cancer cells, which promotes radioresistance 31 , 32 . Thus, targeting these prosurvival pathways with a subtherapeutic dose of IMMU-132 could have great potential for sensitizing lung cancer cells to radiation therapy. Therefore, we hypothesized that sub-therapeutic doses of IMMU-132 would enhance the therapeutic index of radiotherapy (XRT) in lung cancer. In this study, we demonstrated that NSCLC cell lines exhibit surface expression of TROP2, facilitating efficient binding and internalization of the TROP2-targeting ADC IMMU-132. Upon internalization, IMMU-132 induced G2/M phase cell cycle arrest, a phase known to confer heightened radiosensitivity 33 . Treatment with IMMU-132 enhanced the radiosensitivity of lung tumor cells by reducing clonogenic survival and impairing DNA damage response pathways. Additionally, IMMU-132 attenuated irradiation-induced activation of key prosurvival signaling pathways, including PI3K/AKT, MEK1/2, ERK1/2, mTOR, and SAPK/JNK. It also promoted apoptosis through the upregulation of proapoptotic BIM, increased cleavage of caspase-3 and PARP, and downregulation of anti-apoptotic BCL-xL. In vivo , administering a subtherapeutic dose of IMMU-132 in nude mice bearing H441 and H460 tumor xenografts significantly improved tumor control and survival when combined with radiation therapy. These findings suggest that IMMU-132 may serve as an effective radiosensitizer, offering a targeted strategy to enhance the therapeutic efficacy of radiation in lung cancer. Materials and methods Cell Lines and Irradiation Human NSCLC cell lines H441 (ATCC Cat# HTB-174, RRID: CVCL_1561), H460 (ATCC Cat# HTB-177, RRID:CVCL_0459), and A549 (ATCC Cat# CCL-185, RRID:CVCL_0023), and the murine NSCLC cell line Lewis lung carcinoma (LLC1/LL2) (ATCC Cat#CRL-1642, RRID:CVCL_4358) were obtained from the American Type Culture Collection. H441 and H460 cells were cultured in RPMI-1640 medium (Gibco), while A549 and LLC cells were cultured in DMEM/F12 (Gibco), each medium supplemented with 10% FBS and 1% penicillin and streptomycin (Gibco), at 37°C in a humidified incubator with 5% CO 2 . Mycoplasma testing was performed every three months using the MycoAlert™ PLUS mycoplasma detection kit (Lonza) and was consistently negative. All experiments were performed within 10 passages of thawing; short tandem repeat profiling was performed for human cell lines before use. IMMU-132 (Sacituzumab govitecan) was provided by Dr. Ron Bose (Washington University in St. Louis). Irradiation was delivered using an RS 2000 X-ray irradiator (Rad Source, USA) operating at 160 kV and 25 mA, at a dose of 0.0682Gy/s ( in vitro) and 0.0167Gy/s ( in vivo) . For animal irradiation, mice were anesthetized with 2% isoflurane, and the body was shielded with lead to expose only the hindlimb tumors to radiation. Cell surface expression of TROP2 by Flow Cytometry Cells were incubated with Live/Dead fixable violet dead cell stain (Invitrogen, Cat# L34964) for 30 minutes, washed with flow buffer (PBS containing 2% FBS and 0.1% sodium azide), and stained with PE anti-human TACSTD2 (BioLegend, Cat# 363803, RRID: AB_2572021) for 30 minutes at 4°C. Samples were acquired on a MACSQuant Analyzer flow cytometer (RRID: SCR_020268) and analyzed with FlowJo software (BD Biosciences). Cell surface expression of TROP2 by Immunofluorescence Cells were seeded on coverslip in 12-well plates, fixed in 4% paraformaldehyde for 20 minutes, washed with PBS, and blocked with 1% BSA in PBS for 1 hour at room temperature. Cells were incubated with anti-TROP2 antibody (Abcam, Cat# ab214488, RRID: AB_2811182) overnight at 4°C, followed by Alexa Fluor 488-conjugated Goat anti-Rabbit IgG (H + L) secondary antibody (Thermo Scientific, Cat# 11034, RRID: AB_2576217)) for 1 hour at room temperature in the dark. Nuclei were stained with nuclear blue live-ready probes reagent (Invitrogen, USA, Cat# R37605) for 15 minutes, and slides were mounted with ProLong Glass Antifade Mountant (Invitrogen, Cat#P36980). Images were acquired on LSM 510 confocal microscope (Carl Zeiss), and fluorescence intensity was quantified with ImageJ. Cell surface binding of IMMU-132 by Flow Cytometry H441, H460, A549, and LLC cells were treated with 5 Gy radiation, and 24 hours later, stained with viability dye as above. Cells were incubated with IMMU-132 (500nM or 1000nM) for 1 hour at 4°C, washed, and stained with PE-conjugated anti-human IgG secondary antibody (BioLegend, Cat# 410708, RRID: AB_2565786) for 30 minutes at 4°C. Data acquisition and analysis were performed as described above. IMMU-132 internalization by Immunofluorescence Cancer cells (1x 10 6 ) were seeded in four-chamber slides (Cat# PEZGS0416) overnight. IMMU-132 was labeled with Alexa Flour 488 using ZIP protein labeling kit (Thermo Scientific, USA, Cat# Z11233) per the manufacturer instructions. Cells were incubated with labeled IMMU-132 (30 µg/ml) for 2 or 24 hours at 37°C, washed with acidic buffer (0.5% acetic acid and 0.5M NaCl) for 30 seconds to remove surface-bound ADC, and stained with nuclear blue live-ready probes reagent as above. After fixation with 2% paraformaldehyde for 10 minutes, slides were mounted and imaged on an LSM 510 confocal microscope (Carl Zeiss). Green fluorescence intensity was quantified with ImageJ. Cell cytotoxicity assay Cell viability following IMMU-132 treatment was assessed using the CellTiter-Glo® Luminescent cell viability assay (Promega, Cat# G7571). H441, H460, A549, and LLC cells were seeded at 5000 cells/well in 96-well plates and treated the following day with IMMU-132 (120nm, 30nM, 7.5nM, 1.875nM, and 0.4685nM) for 48 h. Plates were equilibrated to room temperature for 30 minutes before adding an equal volume of CellTiter-Glow reagent. After orbital shaking for 2 min to induce lysis, plates were incubated further for 10 minutes at room temperature and luminescence measured on a SpectraMax i3 Plate reader (Molecular Devices). Viability was normalized to untreated controls, and data were expressed as mean ± SD from three replicates. Colony formation assay Radiation-sensitizing effects of IMMU-132 were evaluated by colony formation assay (CFA) as previously described 34 . Cells (2000–10000/well) were seeded in 6-well plates, treated the next day with IMMU-132 for 24 hours, and irradiated with 0, 1, 3, 5, or 7 Gy. Plates were incubated for 7–9 days and stained with 0.5% crystal violet. Colonies containing ≥ 50 cells were counted using a StemiDV4 Stereo Microscope (Zeiss). Survival fraction was calculated as (colonies at given dose relative to colonies of untreated) x 100. Combination effects were analyzed using Combenefit software. Cell cycle and DNA damage analysis by flow cytometry H441, H460, A549, and LLC (3x10 5 ) cells were seeded in 6-well plates and treated next day with IMMU-132 (1 nM or 5 nM) for 24 h, followed by 2 Gy irradiation. Cells were harvested at 1 h and 24 h post-irradiation. The cells were harvested, fixed with 70% ice-cold ethanol, incubated overnight at 4°C, washed, and then incubated with FITC-conjugated γ-H2AX antibody for 1 h at 4°C. After washing, cells were resuspended in flow buffer containing PI (5µg/ml; Cat# 51-66211E) and RNase (100µg/ml) for 10 minutes. Samples were analyzed in a MACSQuant flow cytometer (Miltenyi Biotec), and data processed with FlowJo software as previously described 35 . Annexin-V/PI analysis by flow cytometry Apoptosis was assessed using the BD Pharmingen FITC Annexin-V/PI Detection Kit (BD Biosciences Cat# 556547, RRID: AB_2869082). Cells (1x10 5 ) were seeded in 12-well plates, treated the next day with IMMU-132 (1nM and 5nM) for 24 h, then irradiated (2 Gy or 5 Gy). After 24 h, cells were harvested, washed with ice-cold PBS, and resuspended in 1x binding buffer. Annexin-V and PI were added at the recommended concentrations and incubated for 15 minutes at room temperature in the dark. Samples were analyzed by flow cytometry and analysis were performed as previously described 36 . Immunofluorescence analysis of γH2AX expression Cells (2 × 10 4 ) were seeded on coverslips in 24-well plates and treated with (1nM or 5nM) for 24 h before 2 Gy irradiation. At 1 h post-irradiation, cells were washed, fixed in 4% formaldehyde for 20 min, permeabilized in 0.5% Triton X-100 for 15 min, and blocked in 5% BSA for 30 min at 37 o C. Cells were incubated with γH2AX (Ser139) antibody (Clone JBW301, Millipore, Sigma, Cat# 05-636, RRID: AB_309864) for 1 h at 37°C, followed by Alexa Fluor 488-conjugated secondary antibody for 45 min at 37°C. Coverslips were mounted with a ProLong Diamond Antifade mounting medium containing DAPI (Thermo Scientific, Cat#P36962). Foci were visualized using an LSM 510 confocal microscope (Carl Zeiss) and counted by ImageJ software. In vivo tumor xenograft studies All animal experiments were approved by the Washington University Animal Studies Committee and conducted in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. Athymic nude mice (6-week-old; Charles River Labs) received subcutaneous injections of H441 (3x10 6 cells) or H460 (2x10 6 cells) into the right hind limb. Upon palpation of tumors, mice were randomized into four groups (6–8 per group): vehicle (PBS) ± XRT, or IMMU-132 (25 mg/kg) 37 ± XRT. The treatment regimens are detailed in Supplementary Fig. 5A-B. Briefly, H441 xenograft received two doses of IMMU-132 plus six 2 Gy fractions (2 cycles); H460 received two doses of IMMU-132 plus ten 3 Gy fractions (2 cycles). Tumor dimensions (length, width, and height) were assessed using a digital caliper. Tumor volume was calculated by length x width x height. Body weights were monitored for toxicity. Mice were euthanized once tumors reached ~ 2000mm 3 , and tumors were harvested. For syngeneic studies, LLC cells (5x10 5 ) were injected subcutaneously into 6-week-old C57BL/6 mice with treatment groups and schedule as in Supplementary Fig. 5E . Western Blot Protein lysates were prepared from H441 and H460 tumor tissues harvested from xenograft studies, as well as from H441 and H460 cells. Cells were seeded at a density of 0.3 X 10 6 cells per well in 6-well plates and treated with IMMU-132 (1nM or 5nM) for 24 hours prior irradiation (2 Gy). At 48 h post-irradiation, cells were washed with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo Scientific, Cat#78444). Protein concentration was determined, and 30 µg of protein per sample was resolved on 4–20% SDS polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin (BSA) in TBST (Tris Buffered Saline (G-Biosciences, Cat#R029) containing 0.1% Tween (Sigma, Cat#P9416), for 1 hour at room temperature. Blots were incubated overnight at 4°C with primary antibodies (listed in Supplementary Table 1), followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Immunoreactive bands were detected using a chemiluminescence kit (Perkin Elmer, Cat# NEL103001EA) and imaged with a ChemiDoc™ MP Imaging System (Bio-Rad, Cat#12003154). Statistical analysis Statistical analyses were performed using the student’s t-test and one-way or two-way analysis of variance (ANOVA). All analyses were performed using GraphPad Prism Software (RRID: SCR_002798), and statistical significance is indicated in each graph where appropriate. Results Surface expression of TROP2 on NSCLC cells We performed this study on three human (H441, H460, and A549) and a murine (LLC) NSCLC cell line. Given that IMMU-132 is an antibody-drug conjugate (ADC) targeting the TROP2 antigen, we first assessed cell surface TROP2 expression in all four cell lines (H441, H460, A549, and LLC) with and without radiation exposure using flow cytometry and immunofluorescence staining, as well as total TROP2 levels by Western blot. Among the cell lines tested, H441 demonstrated the highest surface TROP2 expression. Radiation exposure led to a significant increase in surface TROP2 in H441 cells, with the median fluorescence intensity (MFI) rising from 10,644.5 ± 504.2 (sham) to 16,079 ± 294.2 (XRT), corresponding to a 1.5-fold increase (p = 0.01) ( Fig. 1 A ) . Similarly, total TROP2 protein levels were elevated in irradiated H441 cells compared to sham-treated controls ( Fig. 1 B ) . In H460 cells, surface TROP2 expression also increased post-radiation, with an MFI of 36 ± 5.6 in sham versus 66.5 ± 7.8 in the XRT group, reflecting more than a 1.5-fold change (p = 0.03) (Fig. 1 C ) . Consistently, western blot analysis revealed an increased total TROP2 protein in irradiated H460 cells ( Fig. 1 D ) . In A549 and LLC cells, flow cytometry showed a significant increase (A549 p = 0.02; LLC p = 0.0002) in MFI of surface TROP2 expression following irradiation compared to sham-treated cells (Supplementary Figs. 1A and 1C) . This upregulation was corroborated by immunoblot analyses, which also demonstrated elevated total TROP2 protein levels in the XRT groups (Supplementary Figs. 1B and 1D), respectively. The flow cytometry data were supported by immunofluorescence analysis, which demonstrated a significant increase in surface TROP2 expression following radiation in H441 (p = 0.0001) ( Figs. 1 E and F) , H460 (p = 0.0001) ( Figs. 1 G and H) , A549 (p = 0.0001) (Supplementary Figs. 1E and F) , and LLC cells (p = 0.0001) (Supplementary Figs. 1G and H) compared to their respective sham-treated controls. IMMU-132 binds to the surface of cancer cells and undergoes endocytosis Given the confirmed cell surface expression of TROP2 in NSCLC cell lines and the TROP2 specificity of IMMU-132, we next assessed the surface binding and internalization of IMMU-132 in these cells. Flow cytometry analysis demonstrated robust binding of IMMU-132 to H441, H460, A549, and LLC cells. Specifically, H441 and H460 cells showed 88.3% and 83.7% binding in the sham group, which increased to 94.6% and 96.7%, respectively, following radiation ( Fig. 1 I and 1 K ) . Similarly, A549 and LLC cells exhibited 87.5% and 41.6% binding in the sham group, increasing to 90.1% and 47.5%, respectively, in the XRT group (Supplementary Fig. 1I and 1K). The median fluorescence intensity (MFI) of IMMU-132 surface binding correlated with TROP2 expression, with H441 cells showing the highest MFI, followed by H460, A549, and LLC cells. Comparative analysis between sham and irradiated cells revealed significantly elevated MFI in the XRT group for H441 (4333 ± 168.3 vs. 4877.5 ± 20.5; p < 0.05) ( Fig. 1 J ) and H460 (549 ± 33.2 vs. 1214.5; p < 0.001) cells ( Fig. 1 L ) . Similarly, A549 (609 ± 15.6 vs. 694 ± 33.0; p < 0.05) and LLC (284 ± 43.8 vs. 346 ± 29.7; p < 0.0001) cells showed significantly higher MFI in the XRT group compared to sham (Supplementary Fig. 1J and 1L) . To evaluate internalization, we monitored uptake of fluorescently labeled IMMU-132 over time. H441 and H460 cells displayed time-dependent internalization (Fig. 1 M and 1 O), with significantly increased mean green fluorescence intensity (GFI) at 24 h compared to 2 h (p < 0.0001) ( Figs. 1 N and 1 P ) . A similar trend was observed in A549 and LLC cells, with internalization evident at 2 hours and reaching a maximum at 24 hours (Supplementary Figs. 1M–1P) . These findings indicate efficient, time-dependent internalization of IMMU-132 across all cell lines. IMMU-132 reduces cell viability and enhances the efficacy of radiation in vitro The cytotoxic effect of IMMU-132 on H441, H460, A549, and LLC cells was evaluated by assessing cell viability after treatment with a range of concentrations (0.4685 nM to 120 nM). A dose-dependent decrease in cell viability was observed across all cell lines, with calculated IC₅₀ values of 11.43 nM (H441; Fig. 2 A), 12.41 nM (H460; Fig. 2 F), 10.33 nM (A549; Supplementary Fig. 2A ), and 23.37 nM (LLC; Supplementary Fig. 2F ). To investigate the radiosensitizing potential of IMMU-132, clonogenic survival assays were performed using increasing concentrations of IMMU-132 (0–1 nM) in combination with graded doses of radiation (0–5 Gy). Across all four cell lines, the combination of IMMU-132 and radiation significantly reduced clonogenic survival compared to either treatment alone, with a clear dose-dependent decrease in colony formation of H441 cells ( Fig. 2 B ) , H460 cells ( Fig. 2 G ) , A549 cells (Supplementary Fig. 2B) and LLC cells (Supplementary Fig. 2G) . Synergy analysis revealed additive and synergistic effects between IMMU-132 and radiation in H441 ( Fig. 2 C ) , H460 ( Fig. 2 H ) , and A549 cells ( Supplementary Fig. 2C) , as indicated by synergy maps. Whereas LLC cells exhibited only additive effects (Supplementary Fig. 2H) . Effective concentration (EC₅₀) values for IMMU-132 in the combination setting were determined as 0.388 nM of H441 ( Fig. 2 D ) , 3.91 nM of H460 ( Fig. 2 I ) , 0.811 nM of A549 Supplementary Fig. 2D, and 2 .57 nM of LLC (Supplementary Fig. 2I) . Similarly, the EC₅₀ values for radiation in the combination setting were 0.94 Gy (H441; Fig. 2 E), 1.61 Gy (H460; Fig. 2 J), 3.4 Gy (A549; Supplementary Fig. 2E ), and 6.02 Gy (LLC; Supplementary Fig. 2J ). Notably, the combination of IMMU-132 and radiation significantly reduced the surviving fraction in all cell lines compared to IMMU-132 and radiation alone. These findings demonstrate that IMMU-132 enhances radiosensitivity in NSCLC models (H441, H460, and A549), with evidence of synergistic interactions when combined with ionizing radiation. Combination treatment alters cell cycle distribution Cell cycle analysis was performed using flow cytometry after treatment with 1 and 5 nM IMMU-132 and 2 Gy XRT. Figures 3 A and 3 B show the histograms of G0, G1, S, and G2/M cell cycle phases in H441 and H460 cells, respectively. Cell cycle analysis revealed an increased percentage of H441 cells arrested in the G2 phase following treatment with IMMU-132 (1 nM, 63.7 ± 6.36 and 5 nM, 77.2 ± 0.71) as compared to untreated (39.35 ± 1.77); and IMMU-132 + XRT (1 nM, 67.55 ± 0.64 and 5 nM, 69.45 ± 1.77) as compared to and XRT alone (55.8 ± 1.13) ( Fig. 3 C ) . Similarly, we found an increased percentage of H460 cells arrested in the G2 phase treated with IMMU-132 (1 nM, 40.75 ± 3.18 and 5 nM, 49.4 ± 0.85) as compared to untreated (32.4 ± 2.33); and IMMU-132 + XRT (1nM, 44.3 ± 0.71 and 5 nM, 54.35 ± 3.04) as compared to XRT alone (36.25 ± 1.34) ( Fig. 3 D ) . Moreover, A549 and LLC cells were also arrested in the G2 phase following treatment with IMMU-132 and XRT as shown in Supplementary Fig. 3A and 3B , respectively. The percentage of A549 cells in the G2 phase was higher in IMMU-132 (5 nM, 41.85 ± 1.91) and IMMU-132 + XRT (5 nM, 41.25 ± 0.35) as compared to untreated (25 ± 0.424) and XRT alone (27.5 ± 0.78) (Supplementary Fig. 3C) . The percentage of LLC cells arrested at G2 was higher in IMMU-132 (5 nM, 49.7 ± 2.55) and IMMU-132 + XRT (5 nM, 48.65 ± 6.71) as compared to untreated (30.6 ± 5.51) and XRT alone (40.19 ± 1.99) (Supplementary Fig. 3D). At the molecular level, we investigated the total and phosphorylated protein levels of AKT, extracellular signal-regulated kinase (ERK1/2), mitogen-activated protein kinase (MEK1/2) and p53, which regulate cell cycle progression survival. We found reduced phosphorylation of AKT, MEK1/2, ERK1/2, and overexpression of p53 in H441 ( Fig. 3 E ) and H460 ( Fig. 3 F ) cells treated with IMMU-132 + 2Gy as compared to IMMU-132 alone. AKT, ERK, and MEK phosphorylation are associated with cell cycle progression and cell survival 38 – 40 . p53 acts as a tumor suppressor protein and phosphorylation of p53 plays an important role in cell cycle arrest and apoptosis 41 . Hence, reduced phosphorylation of AKT, ERK, and MEK regulatory proteins in combined treatment confirm cell cycle arrest, and induction of phosphorylated p53 signifies acceleration of apoptosis. IMMU-132 Combined with Radiation Enhances DNA Damage and Inhibits DNA Repair γH2AX phosphorylation is a marker for DNA damage 42 43 . We evaluated γH2AX expression in all NSCLC cells by immunofluorescence and flow cytometry. Cells revealed γH2AX foci (green fluorescence) following staining with FITC-conjugated γH2AX in H441 ( Fig. 4 A ) , H460 ( Fig. 4 C). Quantification of mean number of γH2AX per nucleus in H441 demonstrated a significant increase (p < 0.0001) with IMMU-132 treatment (1 nM, 21.31 ± 1.79 and 5 nM, 24.38 ± 1.71) compared to untreated controls (8.52 ± 0.92). There was a further increase in combination with XRT compared to IMMU-132 alone (1 nM IMMU-132 + XRT, 27.57 ± 2.62 vs 1nM IMMU-132, 21.31 ± 1.79; p < 0.0001 and 5 nM IMMU-132 + XRT, 28.66 ± 0.94 vs. 5nM IMMU-132, 24.38 ± 1.71; p < 0.0001) ( Fig. 4 B ). Similar data was obtained in H460 cells, where the mean number of γH2AX per nucleus significantly (p < 0.0001) increased with IMMU-132 treatment (1 nM, 3.32 ± 1 and 5 nM, 8.22 ± 3.35) compared to untreated controls (1.2 ± 0.53). XRT further increased the foci compared to ADC alone (1 nM IMMU-132 + XRT, 12.59 ± 2.5 vs 1nM IMMU-132, 3.32 ± 1; p < 0.0001 and 5 nM IMMU-132 + XRT, 14.47 ± 2.46 vs. 5nM IMMU-132, 8.22 ± 3.35; p < 0.0001) ( Fig. 4 D ). This was further confirmed by flow cytometry analysis ( Figs. 4 E-H ) . Figures 4 E and G show the dot plots of γH2AX and propidium iodide positive cells for H441 and H460 cells, respectively. The percentage of γH2AX positive cells were significantly higher in H441 treated with XRT (19.1 ± 1.84) vs sham (8.11 ± 0.19), p < 0.02; 1 nM IMMU-132 (24.35 ± 1.63) vs. 0 nM (8.11 ± 0.191), p < 0.003; 1 nM IMMU-132 + XRT (34.3 ± 0.99) vs. 1 nM IMMU-132 (24.35 ± 1.63), p < 0.04; 5 nM (39.3 ± 3.67) vs 1nM IMMU-132 (24.35 ± 1.62), p < 0.0001; 1nM + XRT (34.3 ± 0.99) vs 0nM XRT (19.1 ± 1.84), p < 0.004; 5nM + XRT (43.8 ± 1.98) vs. 1nM + XRT (34.3 ± .099), p < 0.04; ( Fig. 4 F ). Similarly, the percentage of γH2AX positive H460 cells were also significantly high in XRT (13.2 ± 2.4) vs sham (2.98 ± 0.67), p < 0.04; 1nM IMMU-132 (13.05 ± 3.61) vs. 0nM (2.98 ± 0.67), p < 0.04; 1nM + XRT (23.75 ± 0.1) vs. 1nM IMMU-132 (13.05 ± 3.61), p < 0.03; 1nM + XRT (23.75 ± 0.1) vs 0nM XRT (13.2 ± 2.4), p < 0.001; ( Fig. 4 H ) . To elucidate the mechanism underlying the enhanced cytotoxicity of IMMU-132 combined with radiation, we examined key mediators of the DNA damage response by western blot analysis in H441 and H460 lung cancer cells ( Figs. 4 I and 4 J ) . Combination treatment (IMMU-132 + XRT) markedly increased phosphorylation of ATM and ATR compared to IMMU-132 alone, radiation alone, or untreated controls, indicating robust activation of DNA damage signaling pathways. This effect correlated with a pronounced accumulation of phosphorylated γH2AX, a surrogate marker of persistent DNA double-strand breaks. We also assessed the expression of DNA-PK, a critical regulator of DNA repair. DNA-PK phosphorylation levels were substantially reduced following IMMU-132 + XRT treatment, suggesting impaired DNA repair capacity ( Figs. 4 I and 4 J ). IMMU-132 Enhances Radiation-Induced Apoptosis in NSCLC Cells To assess the apoptotic response to IMMU-132 combined with radiation, H441, H460, A549, and LLC cells were treated with either 1 nM or 5 nM IMMU-132 for 24 hours, followed by irradiation with 2 Gy. Additionally, A549 and LLC cells were exposed to 5 Gy in combination with 1 nM or 5 nM IMMU-132 to evaluate dose-dependent effects. Apoptosis was measured by Annexin V/propidium iodide (PI) staining 24 hours post-irradiation. Flow cytometric dot plots revealed distinct early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic populations in H441 ( Fig. 5 A ) , H460 ( Fig. 5 D ) , A549 (Supplementary Fig. 5A) , and LLC (Supplementary Fig. 5D) cells. In H441 cells, early apoptosis was significantly increased following irradiation alone (7.58 ± 0.81%) compared to sham (4.41 ± 0.51%, p < 0.03). This effect was enhanced by IMMU-132, with further increases observed in the 1 nM + XRT group (10.12 ± 2.23% vs. 1 nM sham: 5.21 ± 1.24%, p < 0.006) and a similar trend at 5 nM + XRT (10.04 ± 0.79% vs. 5 nM sham: 7.6 ± 0.89%, p < 0.09; Fig. 5 B). A comparable pattern was seen in H460 cells, where early apoptosis significantly increased in all irradiated groups, including XRT alone (18.8 ± 2.69%) vs. sham (8.06 ± 1.31%, p < 0.03), 1 nM + XRT (24.5 ± 2.69%) vs. 1 nM sham (12.6 ± 0.28%, p < 0.02), and 5 nM + XRT (43.25 ± 3.32%) vs. 5 nM sham (32.6 ± 1.13%, p < 0.03). A dose-dependent increase was evident, with significantly higher apoptosis in 5 nM vs. 1 nM groups both at baseline and with radiation (p < 0.001 and p < 0.002, respectively; Fig. 5 E). Late apoptosis also increased significantly in H441 cells upon combination treatment. Compared to sham-treated controls, late apoptosis was higher in the XRT group (14.45 ± 3.47% vs. 5.2 ± 1.13%, p < 0.006), and further enhanced with IMMU-132: 1 nM + XRT (22.8 ± 2.68%) vs. 1 nM sham (14.41 ± 2.25%, p < 0.01); 5 nM + XRT (30 ± 1.69%) vs. 5 nM sham (24.3 ± 1.69%, p < 0.04; Fig. 5 C). A similar pattern was observed in H460 cells, where late apoptosis significantly increased in all irradiated groups, including 0 nM + XRT (16.3 ± 0.71%) vs. 0 nM sham (7.52 ± 0.1%, p < 0.002), 1 nM + XRT (24.1 ± 2.55%) vs. 1 nM sham (9.2 ± 0.11%, p < 0.0001), and 5 nM + XRT (40.85 ± 0.91%) vs. 5 nM sham (24.8 ± 0.28%, p < 0.0001; Fig. 5 F). Treatment with low-dose IMMU-132 (1 nM) and 2 Gy radiation did not significantly increase early or late apoptotic cell populations in A549 or LLC cells (Supplementary Fig. 5) . However, increasing the IMMU-132 concentration to 5 nM and the radiation dose to 5 Gy led to a significant increase in both early and late apoptosis. In A549 cells, 5 nM IMMU-132 + 5 Gy XRT significantly elevated early apoptotic cells compared to controls and lower-dose treatments (e.g., vs. 0 nM + 5 Gy, p < 0.003; vs. 5 nM alone, p < 0.03; Supplementary Fig. 5B ). Similarly, late apoptotic cells increased significantly in the same group (vs. 5 nM sham, p < 0.0001; vs. 5 nM + 2 Gy, p < 0.0001; Supplementary Fig. 5C ). In LLC cells, a similar dose-dependent increase in apoptosis was observed. Early apoptotic populations were significantly higher in the 5 nM + 5 Gy group compared to all other conditions (e.g., vs. 5 nM alone, p < 0.0001; vs. 1 nM + 5 Gy, p < 0.0008; Supplementary Fig. 5E ). Late apoptotic populations were also markedly elevated with combination treatment (e.g., 5 nM + 5 Gy vs. 5 nM alone, p < 0.0001; vs. 0 nM + 5 Gy, p < 0.0001; Supplementary Fig. 5F ). To elucidate the mechanisms underlying the enhancement of radiation-induced apoptosis by IMMU-132 treatment, we evaluated apoptosis regulators caspase-3, PARP, X-chromosome-linked inhibitor of apoptosis protein (XIAP), and Bcl-2-like protein 11 (Bim) (Figs. 5 G and H) . We observed an increase in the cleavage of caspase-3 and PARP, as well as an increase in the expression of the pro-apoptotic protein Bim. XIAP levels were substantially reduced following IMMU-132 + XRT treatment, suggesting pro-apoptotic signaling (Figs. 5 G and H) . These findings demonstrate that IMMU-132 potentiates the effects of radiation by exacerbating DNA damage, suppressing repair pathways, and promoting apoptosis. IMMU-132 enhances the efficacy of radiation therapy in vivo We evaluated the efficacy of IMMU-132 in combination with radiation therapy in nude mice bearing H441 and H460 tumors ( Fig. 6 ) . Mice were implanted subcutaneously with H441 and H460 cells into the hind limb. The treatment schema for IMMU-132 and XRT for H441 and H460 is shown in Supplementary Fig. 5A and 5B , respectively. IMMU-132 in combination with XRT significantly delayed H441 tumor growth as compared to IMMU-132 alone (p < 0.001) or XRT alone (p < 0.0001) ( Fig. 6 A ). A similar delay in tumor growth was also observed in the H460 model ( Fig. 6 D ) . Treatment-related side effects were not observed, as indicated by stable body weights of the mice ( Supplementary Fig. 5C and 5D) . Analysis of the survival data revealed that the median survival of untreated H441 tumor-bearing mice was 35 days, those treated with XRT had 67 and IMMU-132 treated mice had 72 days. All mice were alive at the end of the study when treated with the combination of IMMU-132 and XRT ( Fig. 6 B ) . The probability of survival at the endpoint was 0% for the untreated and XRT alone groups, 28.5% for the IMMU-132 group, and 100% for the IMMU-132 + XRT group ( Fig. 6 B ) . For the H460 tumor-bearing mice, the probability of survival at the endpoint was 0% for the untreated and IMMU-132 alone groups, 80% for the XRT group, and 100% for the IMMU-132 + XRT group ( Fig. 6 E ) . We also evaluated the efficacy of IMMU-132 in combination with XRT in immunocompetent mice (C57BL/6) bearing LLC tumors. The treatment scheme is shown in Supplementary Fig. 5E. IMMU-132 combined with XRT significantly reduced tumor growth as compared to XRT and IMMU-132 alone (Supplementary Fig. 5F) . Moreover, increased survival was observed in the IMMU-132 + XRT group compared to the other groups (Supplementary Fig. 5G) . The body weights of the mice were stable among all the groups (Supplementary Fig. 5H) . Pathway analysis was performed on resected tumors to understand the molecular mechanisms underlying the IMMU-132-induced radiosensitivity in vivo . We found reduced protein levels of DNA-PK, RAD51, and anti-apoptotic proteins such as p-BCL-2, BCL-XL, p-BAD, and XIAP, as well as high levels of apoptotic proteins, including phospho-p38, c-caspase-3, and c-PARP, in both H441 and H460 tumors treated with IMMU-132 plus XRT compared to IMMU-132, XRT alone, and untreated controls ( Fig. 6 C and 6 F ) . The PI3K/AKT and RAS/RAF/MEK/ERK pathways have been shown to induce cell survival responses to radiation. We found reduced phosphorylation of AKT, MEK1/2, ERK1/2, mTOR, SAPK/JNK, and downregulation of PIK3C2B (Anti-Phosphoinositide-3-kinase, class 2, β polypeptide) and cyclic adenosine monophosphate-responsive element-binding protein (CREB) transcription factor in IMMU-132 + XRT groups as compared to XRT alone, IMMU-132, and the sham group ( Fig. 6 C and 6 F ) . Furthermore, the ratio of phospho-protein to total protein was lower in IMMU-132 + XRT compared to other groups. Moreover, phosphatase and tensin homolog (PTEN), which inhibits PI3K/AKT signaling, was found to be upregulated in H441 and H460 cells treated with the IMMU-132 + XRT group compared to the IMMU-132, XRT alone, and untreated groups. Discussion IMMU-132 (also called sacituzumab govitecan) is approved for the treatment of triple-negative metastatic breast cancer and metastatic urothelial cancer 18 , 25 . In this study, we demonstrated that IMMU-132 enhances the radiosensitivity of NSCLC both in vitro and in vivo . These findings provide compelling evidence for the therapeutic potential of combining IMMU-132 with RT, particularly in TROP2-expressing NSCLC. We observed TROP2 surface expressions in H441, H460, A549, and LLC. Among these, H441 showed high expression, while the remaining three cell lines displayed low expression of TROP2. Based on TROP2 expression, we selected a high TROP2-expressing H441 cell line and low TROP2-expressing cell lines (H460, A549, and LLC) to study the radiosensitization capability of IMMU-132 following radiation therapy. We evaluated radiation-induced upregulation of TROP2 expression and found that XRT indeed induced TROP2 in NSCLC. This observation is consistent with prior studies reporting TROP2 induction under cellular stress conditions. The enhanced TROP2 expression correlated with increased IMMU-132 binding and internalization, suggesting that radiation improves ADC uptake by increasing antigen expression, a mechanism we have previously reported for other antigens 44 – 47 . We found a dose-dependent cytotoxicity effect of IMMU-132 on NSCLC cell lines, which correlated with the antigen expression. Studies have shown that SN-38, the cytotoxic component of IMMU-132, is a radiation-sensitizing anticancer agent 48 , 49 , 50 . However, the non-targeted nature of the free drug results in inadvertent side effects, limiting its dose. Thus, we postulated that the targeted delivery of SN-38 via IMMU-132 may reduce side effects and sensitize lung cancer cells to radiation therapy, especially at sub-therapeutic doses. Our results confirmed that IMMU-132 acts as a radiosensitizer across all NSCLC cells tested, as evidenced by reduced colony formation, synergistic cytotoxicity in clonogenic assays with XRT, and increased DNA damage. Mechanistically, IMMU-132 + XRT treatment resulted in G2/M phase arrest, which is known to be the most radiosensitive phase of the cell cycle, thereby contributing to the radiosensitizing effect. SN-38 arrests cell cycle in the G2/M phase, contributing the therapeutic effect 51 . We observed increased markers of DNA damage (γH2AX) and apoptosis (annexin V/PI, cleaved PARP, caspase-3) in cells treated with IMMU-132 plus radiation, indicating that the combination amplifies radiation-induced cytotoxicity by disrupting DNA repair and activating apoptotic cascades. Mechanistically, this effect was associated with a reduction in DNA repair mediators RAD51 and DNA-PKcs, which are essential for homologous recombination and non-homologous end joining, respectively 52 , 53 . Concurrently, we detected heightened activation of ATM and ATR kinases, upstream regulators of DNA damage response that phosphorylate downstream substrates such as p53, thereby enforcing G2/M arrest and promoting apoptotic signaling 54 , 55 . The inability of NSCLC cells to sustain RAD51- and DNA-PKcs–mediated repair in the context of IMMU-132 plus radiation underscores a critical vulnerability in the DNA repair machinery that facilitates apoptotic progression. In parallel, we identified suppression of major survival signaling networks, including PI3K/AKT/mTOR and MEK/ERK, which are typically induced following radiation 56 , 57 to promote DNA repair, cell survival, and proliferation. Notably, the dual suppression of PI3K/AKT and MEK/ERK signaling by IMMU-132 in combination with XRT is likely to overcome the compensatory crosstalk between these pathways, a known mechanism of resistance in KRAS-mutated NSCLC cell lines such as H460 and A549 58 . Downregulation of anti-apoptotic proteins (p-BCL2, XIAP, BCL-XL, and p-BAD) further tips the balance toward apoptosis, reinforcing the mechanistic link between impaired repair capacity and diminished survival signaling. Together, these molecular alterations delineate a coordinated mechanism whereby IMMU-132 sensitizes NSCLC cells to radiation: by simultaneously disabling DNA repair pathways and suppressing compensatory pro-survival signaling, thereby committing cells to apoptosis. In vivo , the combination of ADC + XRT significantly reduced tumor growth and improved survival in both xenograft (H441, H460) and syngeneic (LLC) mouse models, supporting the translational potential of this approach. The differential responses to IMMU-132 monotherapy among all cell lines reflected their TROP2 expression levels, reaffirming the importance of TROP2 as a predictive biomarker for ADC efficacy. Importantly, treatment was well tolerated, with no significant weight loss in mice, indicating an acceptable therapeutic index. Mechanistically, ERK1/2 activation following radiation is known to induce the G2/M DNA damage checkpoint via ATR and to facilitate NHEJ-mediated repair through DNA-PK 59–62 . ERK1/2 also promotes survival by activating CREB, upregulating anti-apoptotic proteins (Bcl-xL), and suppressing pro-apoptotic proteins (Bim, Bad, caspase-9) 68–71 . Pharmacologic inhibition of ERK1/2 or upstream EGFR/MEK signaling reverses these effects, inducing Bim expression and enhancing apoptosis in NSCLC 73 . Consistent with this, our data suggest that IMMU-132 inhibits MEK/ERK signaling in the context of radiation, thereby radiosensitizing NSCLC cells by diminishing DNA repair capacity, reducing the expression of anti-apoptotic proteins, and preventing phosphorylation-mediated inactivation of pro-apoptotic mediators. Similarly, PI3K/AKT/mTOR signaling—frequently activated after radiation—plays a central role in survival and radioresistance. PTEN loss leads to aberrant Akt activation, which phosphorylates downstream effectors such as mTOR and Bad to drive growth and inhibit apoptosis 74 . Stress-activated MAPK family members, including JNK, ERK, and p38, further contribute to survival by phosphorylating and inactivating Bad 7677 . Notably, combined inhibition of MEK and mTOR has been shown to induce DNA damage, prolong G2/M arrest, and sensitize NSCLC xenografts to radiotherapy 78 . Our results align with these observations, demonstrating that IMMU-132 suppresses both PI3K/AKT/mTOR and MEK/ERK signaling, thereby abrogating redundant pro-survival pathways. Taken together, our findings highlight the promise of IMMU-132 as a potent radiosensitizer in NSCLC through TROP2-mediated targeted delivery of SN-38. The combination with radiation therapy not only enhances DNA damage and apoptosis but also abrogates key survival and DNA repair pathways, particularly in TROP2-high tumors. These results provide a strong rationale for clinical evaluation of IMMU-132 in combination with radiotherapy for NSCLC, potentially offering an effective strategy to overcome radioresistance and improve patient outcomes. Declarations Conflicts of interest: Ron Bose reports a research grant from Puma Biotechnology and Consulting for Genentech. All other authors have no conflict. Author contributions: AKS and VK developed the hypothesis, designed the studies, analyzed the data, wrote the manuscript, provided funding, and supervised the project. HS and SP designed the studies, performed experiments, analyzed data, and wrote the manuscript. RB offered helpful advice and reviewed the manuscript. All authors reviewed the manuscript and contributed to discussions. Acknowledgments: This study was supported by startup funds, the Elsa U Pardee Foundation grant, and in part by the Center for Drug Discovery, Washington University in Saint Louis, to VK. We thank Alison Clay for her technical support with the studies and Amanda Klaas for the tail vein injections in mice. References Siegel RL, Miller KD, Fuchs HE, Jemal A (2022) Cancer statistics, 2022. CA Cancer J Clin 72:7–33. https://doi.org:10.3322/caac.21708 Sung H et al (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209–249. https://doi.org:10.3322/caac.21660 Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA (2008) Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 83:584–594. https://doi.org:10.4065/83.5.584 Antonia SJ et al (2018) Overall Survival with Durvalumab after Chemoradiotherapy in Stage III NSCLC. New Engl J Med 379:2342–2350. https://doi.org:10.1056/NEJMoa1809697 Raben D et al (2018) Overall Survival with Durvalumab versus Placebo after Chemoradiotherapy in Stage III NSCLC. Int J Radiat Oncol 102:1607–1608. https://doi.org:DOI 10.1016/j.ijrobp.2018.08.057 Spigel DR et al (2021) Five-year survival outcomes with durvalumab after chemoradiotherapy in unresectable stage III NSCLC: An update from the PACIFIC trial. J Clin Oncol 39. https://doi.org:10.1200/JCO.2021.39.15_suppl.8511 Huang R-X, Zhou P-K (2020) DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer. Signal Transduct Target therapy 5:60 Marampon F, Ciccarelli C, Zani BM (2019) Biological rationale for targeting MEK/ERK pathways in anti-cancer therapy and to potentiate tumour responses to radiation. Int J Mol Sci 20:2530 Lin SH et al (2014) A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer. J Thorac Oncol 9:965–973. https://doi.org:10.1097/jto.0000000000000199 Chen Y et al (2019) mTORC1 inhibitor RAD001 (everolimus) enhances non-small cell lung cancer cell radiosensitivity in vitro via suppressing epithelial–mesenchymal transition. Acta Pharmacol Sin 40:1085–1094 Schuurbiers OC et al (2009) The PI3-K/AKT-pathway and radiation resistance mechanisms in non-small cell lung cancer. J Thorac Oncol 4:761–767 Shannon AM et al (2009) The mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) enhances the radiation responsiveness of lung and colorectal tumor xenografts. Clin Cancer Res 15:6619–6629 Brognard J, Clark AS, Ni Y, Dennis PA (2001) Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res 61:3986–3997 Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J (2020) Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res 18:3–19. https://doi.org:10.1158/1541-7786.MCR-19-0582 Chau CH, Steeg PS, Figg WD (2019) Antibody-drug conjugates for cancer. Lancet 394:793–804. https://doi.org:10.1016/S0140-6736(19)31774-X Mehta GU et al (2024) FDA approval summary: fam-trastuzumab deruxtecan-nxki for unresectable or metastatic non-small cell lung cancer with activating HER2 mutations. Oncologist 29:667–671. https://doi.org:10.1093/oncolo/oyae151 Qiu S et al (2023) Targeting Trop-2 in cancer: Recent research progress and clinical application. Biochim Biophys Acta Rev Cancer 1878:188902. https://doi.org:10.1016/j.bbcan.2023.188902 Belluomini L et al (2023) Antibody-drug conjugates (ADCs) targeting TROP-2 in lung cancer. Expert Opin Biol Ther 23:1077–1087. https://doi.org:10.1080/14712598.2023.2198087 Cubas R, Zhang S, Li M, Chen C, Yao Q (2010) Trop2 expression contributes to tumor pathogenesis by activating the ERK MAPK pathway. Mol Cancer 9:1–13 Guerra E et al (2013) The Trop-2 signalling network in cancer growth. Oncogene 32:1594–1600 Heist RS et al (2017) Therapy of Advanced Non-Small-Cell Lung Cancer With an SN-38-Anti-Trop-2 Drug Conjugate, Sacituzumab Govitecan. J Clin Oncol 35:2790–2797. https://doi.org:10.1200/jco.2016.72.1894 Wang J et al (2011) Loss of Trop2 promotes carcinogenesis and features of epithelial to mesenchymal transition in squamous cell carcinoma. Mol Cancer Res 9:1686–1695 Pak MG, Shin DH, Lee CH, Lee MK (2012) Significance of EpCAM and TROP2 expression in non-small cell lung cancer. World J Surg Oncol 10:53. https://doi.org:10.1186/1477-7819-10-53 Li Z, Jiang X, Zhang W (2016) TROP2 overexpression promotes proliferation and invasion of lung adenocarcinoma cells. Biochem Biophys Res Commun 470:197–204. https://doi.org/10.1016/j.bbrc.2016.01.032 . https://doi.org: Shastry M, Jacob S, Rugo HS, Hamilton E (2022) Antibody-drug conjugates targeting TROP-2: Clinical development in metastatic breast cancer. Breast 66:169–177. https://doi.org:10.1016/j.breast.2022.10.007 Whitacre CM, Zborowska E, Willson JK, Berger NA (1999) Detection of poly (ADP-ribose) polymerase cleavage in response to treatment with topoisomerase I inhibitors: a potential surrogate end point to assess treatment effectiveness. Clin Cancer Res 5:665–672 Lagadec P et al (2008) Pharmacological targeting of NF-κB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells. Br J Cancer 98:335–344 Liu Y et al (2009) Inhibition of Akt signaling by SN-38 induces apoptosis in cervical cancer. Cancer Lett 274:47–53 Cusack JC Jr et al (2001) Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: implications for systemic nuclear factor-κB inhibition. Cancer Res 61:3535–3540 Wei L et al (2022) Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells. Biomed Environ Sci 35:419–436. https://doi.org:10.3967/bes2022.058 Gewirtz DA (2000) Growth arrest and cell death in the breast tumor cell in response to ionizing radiation and chemotherapeutic agents which induce DNA damage. Breast Cancer Res Treat 62:223–235. https://doi.org:10.1023/a:1006414422919 Ouellette MM, Yan Y (2019) Radiation-activated prosurvival signaling pathways in cancer cells. Precision Radiation Oncol 3:111–120. https://doi.org/10.1002/pro6.1076 . https://doi.org: O'Connell MJ, Cimprich KA (2005) G2 damage checkpoints: what is the turn-on? J Cell Sci 118:1–6. https://doi.org:10.1242/jcs.01626 Lewis CD et al (2021) Targeting a Radiosensitizing Antibody-Drug Conjugate to a Radiation-Inducible Antigen. Clin Cancer Res 27:3224–3233. https://doi.org:10.1158/1078-0432.CCR-20-1725 de Feraudy S et al (2007) Pol η is required for DNA replication during nucleotide deprivation by hydroxyurea. Oncogene 26:5713–5721. https://doi.org:10.1038/sj.onc.1210385 Singh AK et al (2023) Blocking the functional domain of TIP1 by antibodies sensitizes cancer to radiation therapy. Biomed Pharmacother 166:115341. https://doi.org/10.1016/j.biopha.2023.115341 . https://doi.org: Cardillo TM et al (2020) Predictive biomarkers for sacituzumab govitecan efficacy in Trop-2-expressing triple-negative breast cancer. Oncotarget 11:3849–3862. https://doi.org:10.18632/oncotarget.27766 Steelman LS et al (2011) Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy-implications for cancer and aging. Aging 3:192–222. https://doi.org:10.18632/aging.100296 He Y et al (2021) Targeting PI3K/Akt signal transduction for cancer therapy. Signal Transduct Target Therapy 6:425. https://doi.org:10.1038/s41392-021-00828-5 McCubrey JA et al (2007) Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim et Biophys Acta (BBA) - Mol Cell Res 1773:1263–1284. https://doi.org/10.1016/j.bbamcr.2006.10.001 . https://doi.org: Chen J (2016) The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. Cold Spring Harb Perspect Med 6, a026104 https://doi.org:10.1101/cshperspect.a026104 Mah LJ, El-Osta A, Karagiannis TC (2010) γH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 24:679–686. https://doi.org:10.1038/leu.2010.6 Sharma A, Singh K, Almasan A (2012) Histone H2AX phosphorylation: a marker for DNA damage. Methods Mol Biol 920:613–626. https://doi.org:10.1007/978-1-61779-998-3_40 Dadey DYA et al (2017) Antibody Targeting GRP78 Enhances the Efficacy of Radiation Therapy in Human Glioblastoma and Non-Small Cell Lung Cancer Cell Lines and Tumor Models. Clin Cancer Res 23:2556–2564. https://doi.org:10.1158/1078-0432.CCR-16-1935 Lewis CD et al (2021) Targeting a Radiosensitizing Antibody–Drug Conjugate to a Radiation-Inducible Antigen. Clin Cancer Res 27:3224–3233. https://doi.org:10.1158/1078-0432.Ccr-20-1725 Singh AK et al (2023) Blocking the functional domain of TIP1 by antibodies sensitizes cancer to radiation therapy. Biomed Pharmacother 166:115341. https://doi.org:10.1016/j.biopha.2023.115341 Singh AK et al (2024) Development of a [89Zr]Zr-labeled Human Antibody using a Novel Phage-displayed Human scFv Library. Clin Cancer Res 30:1293–1306. https://doi.org:10.1158/1078-0432.CCR-23-3647 Sasai K et al (1998) Effects of SN-38 (an active metabolite of CPT-11) on responses of human and rodent cells to irradiation. Int J Radiation Oncology*Biology*Physics 42:785–788. https://doi.org/10.1016/S0360-3016(98)00326-5 . https://doi.org: Tamura K et al (1997) Enhancement of Tumor Radio-response by Irinotecan in Human Lung Tumor Xenografts. Jpn J Cancer Res 88:218–223. https://doi.org/10.1111/j.1349-7006.1997.tb00369.x . https://doi.org: OKUNO T et al (2018) SN-38 Acts as a Radiosensitizer for Colorectal Cancer by Inhibiting the Radiation-induced Up-regulation of HIF-1α. Anticancer Res 38:3323–3331. https://doi.org:10.21873/anticanres.12598 Sun X, Wu L, Du L, Xu W, Han M (2024) Targeting the organelle for radiosensitization in cancer radiotherapy. Asian J Pharm Sci 19:100903. https://doi.org:https://doi.org/10.1016/j.ajps.2024.100903 Raleigh DR, Haas-Kogan DA (2013) Molecular targets and mechanisms of radiosensitization using DNA damage response pathways. Future Oncol 9:219–233. https://doi.org:10.2217/fon.12.185 Schürmann L, Schumacher L, Roquette K, Brozovic A, Fritz G (2021) Inhibition of the DSB repair protein RAD51 potentiates the cytotoxic efficacy of doxorubicin via promoting apoptosis-related death pathways. Cancer Lett 520:361–373. https://doi.org:https://doi.org/10.1016/j.canlet.2021.08.006 Fedak EA, Adler FR, Abegglen LM, Schiffman JD (2021) ATM and ATR Activation Through Crosstalk Between DNA Damage Response Pathways. Bull Math Biol 83:38. https://doi.org:10.1007/s11538-021-00868-6 Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB (2007) p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11:175–189. https://doi.org:10.1016/j.ccr.2006.11.024 Hong E-H et al (2010) Ionizing radiation induces cellular senescence of articular chondrocytes via negative regulation of SIRT1 by p38 kinase. J Biol Chem 285:1283–1295 Valerie K (2007) Radiation?induced cell signaling: inside?out and outside?in. Mol Cancer Ther 6:789–801 Hu H, Tan LY, Xie S, He XX, Luo J, Wang F (2020) Anlotinib Exerts Anti-Cancer Effects on KRAS-Mutated Lung Cancer Cell Through Suppressing the MEK/ERK Pathway. Dove Press 2020:12 3579–3587. https://doi.org:https://doi.org/10.2147/CMAR.S243660 Abbott DW (1999) Mitogen?activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation. J Biol Chem 274:2732–2742 Yan Y, Irradiation?induced (2007) G2/M checkpoint response requires ERK1/2 activation. Oncogene 26:4689–4698 Wei F (2013) Inhibition of ERK activation enhances the repair of double?stranded breaks via non?homologous end joining by increasing DNA?PKcs activation. Biochim Biophys Acta 1833:90–100 Cohen?Armon M (2007) PARP?1 activation in the ERK signaling pathway. Trends Pharmacol Sci (Regular ed) 28:556–560 Vivanco I et al (2010) The phosphatase and tensin homolog regulates epidermal growth factor receptor (EGFR) inhibitor response by targeting EGFR for degradation. Proc Natl Acad Sci U S A 107:6459–6464. https://doi.org:10.1073/pnas.0911188107 Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2:489–501. https://doi.org:10.1038/nrc839 Weston CR, Davis RJ (2007) The JNK signal transduction pathway. Curr Opin Cell Biol 19:142–149. https://doi.org:10.1016/j.ceb.2007.02.001 Yu C et al (2004) JNK suppresses apoptosis via phosphorylation of the proapoptotic Bcl-2 family protein BAD. Mol Cell 13:329–340. https://doi.org:10.1016/s1097-2765(04)00028-0 KIM SY, JEONG E-H, KIM LEET-G, H.-R., KIM CH (2021) The Combination of Trametinib, a MEK Inhibitor, and Temsirolimus, an mTOR Inhibitor, Radiosensitizes Lung Cancer Cells. Anticancer Res 41:2885–2894. https://doi.org:10.21873/anticanres.15070 Meng J et al (2010) Apoptosis Induction by MEK Inhibition in Human Lung Cancer Cells Is Mediated by Bim. PLoS ONE 5:e13026. https://doi.org:10.1371/journal.pone.0013026 Additional Declarations Yes there is potential Competing Interest. Ron Bose reports a research grant from Puma Biotechnology and Consulting for Genentech. All other authors have no conflict. Supplementary Files SupplementaryFigureLegends.docx Supplementary Figure legends supplementaryfigurefinal.pdf Supplementary figures Cite Share Download PDF Status: Under Review Version 1 posted Unknown event 01 Oct, 2025 Editorial decision: Reject before peer review 25 Sep, 2025 Editor assigned by journal 15 Sep, 2025 Submission checks completed at journal 11 Sep, 2025 First submitted to journal 11 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7593735","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":515359744,"identity":"24f14aa5-4f3c-4eed-b2a5-56e3e81b2a64","order_by":0,"name":"Vaishali Kapoor","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYDACHgglByYfMDAwNhCrxZgBpDqBFC2JDURrMThz+NmDDxX30jfcSH/+IIHBRnbDAUJazraZG844U5y74UaOIdCWNGOCWiT7GcykedsScrfdyAE57HAiEVrYv4G0pJvdSH8I1PKfsBZ+3h6wLQlmNxJADjtAhBaeM+VAvyQY7j/zxnBGgkGy8UxCWth40rcBQyxBXrI9/cGHDxV2sn2EtIB0IbENCCtH1zIKRsEoGAWjAAsAABchRmsMDoPKAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7581-1487","institution":"Washington University in St. Louis School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Vaishali","middleName":"","lastName":"Kapoor","suffix":""},{"id":515359745,"identity":"287195ee-f138-4d0b-8e6e-bc59ac4baa75","order_by":1,"name":"Harendra Shah","email":"","orcid":"","institution":"Washington University in St. Louis School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Harendra","middleName":"","lastName":"Shah","suffix":""},{"id":515359746,"identity":"856e13d9-cb48-4bfb-a983-4823329e4aeb","order_by":2,"name":"Sai Prem","email":"","orcid":"","institution":"Washington University in St. Louis School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sai","middleName":"","lastName":"Prem","suffix":""},{"id":515359747,"identity":"f35478b9-475a-447d-bfdb-940681716b9b","order_by":3,"name":"Ron Bose","email":"","orcid":"https://orcid.org/0000-0003-1977-8894","institution":"Washington University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ron","middleName":"","lastName":"Bose","suffix":""},{"id":515359748,"identity":"cedc9e64-854b-4f41-a1fd-3334b0e4b08a","order_by":4,"name":"Abhay Singh","email":"","orcid":"","institution":"MilliporeSigma","correspondingAuthor":false,"prefix":"","firstName":"Abhay","middleName":"","lastName":"Singh","suffix":""}],"badges":[],"createdAt":"2025-09-11 16:20:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7593735/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7593735/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91962674,"identity":"dc4e2454-6d88-4aaf-9dfa-af7b77b46b92","added_by":"auto","created_at":"2025-09-23 07:58:01","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158865,"visible":true,"origin":"","legend":"","description":"","filename":"20250911Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/424cf5a593f7e8e2462093b2.docx"},{"id":91962677,"identity":"75b1fdfa-cc85-4e7c-bf56-37672b14fa18","added_by":"auto","created_at":"2025-09-23 07:58:01","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1375032,"visible":true,"origin":"","legend":"","description":"","filename":"Fig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/c9046ba34a868b66f7dc0385.tif"},{"id":91965297,"identity":"1fc3b2f8-235a-4123-ab66-7bde799df873","added_by":"auto","created_at":"2025-09-23 08:22:01","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2233228,"visible":true,"origin":"","legend":"","description":"","filename":"Fig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/c598b49a6d8b18de9ced1866.tif"},{"id":91961020,"identity":"c6ec2c9c-e73c-4c0e-b798-f20a1163bf5a","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":415996,"visible":true,"origin":"","legend":"","description":"","filename":"Fig3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/3211a41968ed11e5e02b9cb1.tif"},{"id":91961019,"identity":"1f303ba7-2c6e-4dff-8dcf-615438e73f2a","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1318660,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/dedf122110aef1d9af95e356.tif"},{"id":91961025,"identity":"a498c403-9e14-4e4a-ab27-2d0ccc1be69a","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":700144,"visible":true,"origin":"","legend":"","description":"","filename":"Fig5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/347cb9373e7442e5d58f794d.tif"},{"id":91963425,"identity":"0b42634e-03b2-49e4-87ce-ec2f56221675","added_by":"auto","created_at":"2025-09-23 08:06:01","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":700200,"visible":true,"origin":"","legend":"","description":"","filename":"Fig6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/492055067aaa8226487726a5.tif"},{"id":91961028,"identity":"3c1581e6-c216-4a28-8cf8-9837678bd424","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"json","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7326,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2570920.json","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/d2f16f835474fe5de21ea45e.json"},{"id":91961030,"identity":"6e050dae-18bc-4a9c-acd9-1a5b8be0d647","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16855,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/ae4338893e797035877b2e29.docx"},{"id":91963427,"identity":"24748c0e-2342-4465-941a-04bd134c7f1d","added_by":"auto","created_at":"2025-09-23 08:06:01","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1447934,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigurefinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/bcc486006632a9ed877f3b7f.pdf"},{"id":91961040,"identity":"a6e59fc7-74eb-493b-920c-4df0740f6c8d","added_by":"auto","created_at":"2025-09-23 07:50:02","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172491,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25709200enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/4f2000c4e6fe5167c7f913f3.xml"},{"id":91962684,"identity":"bb65c71c-47f0-47af-a489-1848ce104baa","added_by":"auto","created_at":"2025-09-23 07:58:01","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1375032,"visible":true,"origin":"","legend":"","description":"","filename":"Fig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/d4d114ed26662e14d9fad7dd.tif"},{"id":91962683,"identity":"84bb555d-b918-4b6f-80f5-aaf1e24259c8","added_by":"auto","created_at":"2025-09-23 07:58:01","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2233228,"visible":true,"origin":"","legend":"","description":"","filename":"Fig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/e509d61d3819d079353ba479.tif"},{"id":91961031,"identity":"13148820-6eff-42c7-81eb-ac920426203e","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":415996,"visible":true,"origin":"","legend":"","description":"","filename":"Fig3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/caa24446331c954fd3a29dc6.tif"},{"id":91961038,"identity":"fe8c796e-4a23-49ef-9005-66bff2571a96","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1318660,"visible":true,"origin":"","legend":"","description":"","filename":"Fig4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/4fc3f40f90d25424e845672f.tif"},{"id":91961034,"identity":"d12adce8-a0be-4738-b184-8771b2d4c600","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":700144,"visible":true,"origin":"","legend":"","description":"","filename":"Fig5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/8e122646e855adb53f6e6c3f.tif"},{"id":91963426,"identity":"ee4bf22b-7f53-45e6-8ed8-15d41b53b60f","added_by":"auto","created_at":"2025-09-23 08:06:01","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":700200,"visible":true,"origin":"","legend":"","description":"","filename":"Fig6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/8fa432590179cc9952969633.tif"},{"id":91961032,"identity":"ae507f57-37fb-42db-983b-53f008bada14","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167279,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25709200structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/665f6561782ae4e1c72008ac.xml"},{"id":91961035,"identity":"564bfed1-4859-4cc8-92d5-47ff0c304e49","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185724,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/e58994342a2ee8fd6183ecee.html"},{"id":91961014,"identity":"0608cfff-6453-43dd-873a-aa166c4059d6","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":183134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell surface binding and internalization of IMMU-132 in NSCLC cells. A and C \u003c/strong\u003eBar graphs showing fold-change in median fluorescent intensity (MFI) of cell surface expression of TROP2 in H441 (A) and H460 (C) cells following 5Gy XRT by flow cytometry. MFI was determined by analyzing the data in FlowJo software. \u003cstrong\u003eB and D\u003c/strong\u003e Western blots from whole cell lysates showing the expression of TROP2 in H441 (B) and H460 cells (D). Numbers below the blots represent the normalized protein expression. \u003cstrong\u003eE and G\u003c/strong\u003e Immunofluorescence images of surface TROP2 on H441 (E) and H460 (G) cells following XRT (5 Gy). The nucleus was stained with DAPI (blue) and TROP2 with an Alexa Fluor 488-labeled anti-TROP2 antibody (green). Images were captured using a Carl Zeiss confocal microscope at 63x magnification. Scale bar represents 20 µM. \u003cstrong\u003eF and H \u003c/strong\u003eGraphs showing mean green fluorescence intensity (GFI) of surface TROP2 on H441 (F) and H460 (H). Each dot represents data obtained from measuring the intensities on 50 cells. \u003cstrong\u003eI and K \u003c/strong\u003eOverlay histograms showing the binding of IMMU-132 on H441 (I) and H460 (K) cells following radiation. Cells were incubated with IMMU-132 (500nM) for 1h at room temperature followed by PE-conjugated anti-human secondary antibody and acquired by flow cytometry. Black, blue and red histograms represent Isotype control, Sham-irradiated cells, and cells irradiated with 5Gy, respectively. \u003cstrong\u003eThe J and L\u003c/strong\u003e Bar diagram representing the median fluorescent intensity (MFI) of IMMU-132 binding on H441 (J) and H460 (L) cells. \u003cstrong\u003eM and O\u003c/strong\u003e Time dependent internalization (2 \u0026amp; 24 h) of Alexa Fluor 488-labelled IMMU-132 (green) in H441 (M) and H460 (O) cells. Nucleus was stained with NucBlue (blue). Images were taken in Carl Zeiss confocal microscope at 63X magnification. Scale bar represents 20 µM. \u003cstrong\u003eN and P\u003c/strong\u003e Mean green fluorescence intensity (GFI) of internalized IMMU-132 in H441 (N) and H460 (P) following 2 and 24h incubation. *P\u0026lt;0.05, **p\u0026lt;0.001, and ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/7e0665a322a2782679844771.png"},{"id":91961017,"identity":"70749290-9736-41d9-b715-24ec311ce26d","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose-dependent cytotoxicity and radiosensitization of NSCLC cells by IMMU-132. (A and F) \u003c/strong\u003eH441 (A) and H460 (F) cells were treated with various doses (120nm, 30nM, 7.5nM, 1.875nM, and 0.4685nM) of IMMU-132 for 48 h, followed by Cell Titer Glo Luminescent cell viability assay. Cell viability was normalized to untreated cells (control) and represented as mean with SD from three treatments. Data was fitted using a non-linear fit equation, log(inhibitor) vs. normalized response, in GraphPad Prism software. \u003cstrong\u003e(B and G) \u003c/strong\u003eClonogenic assay of H441 (B) and H460 (G) cells treated with IMMU-132 and radiation. Cells were seeded in 6-well plates in triplicate, followed by treatment with 0, 0.2, 0.4, 0.6, 0.8, and 1 nM IMMU-132, and then exposed to 0, 1, 3, and 5 Gy. Plates were stained with crystal violet, and colonies comprising 50 cells or more were counted using a microscope. Photos of the plates show clonogenic survival at 7 days following exposure to IMMU-132 and/or XRT. \u003cstrong\u003e(C and H) \u003c/strong\u003eSynergy plots demonstrate additive (green) or synergistic (blue) effects for the combination of IMMU-132 with XRT in H441 (C) and H460 (H) cells. \u003cstrong\u003e(D and I)\u003c/strong\u003e H441 (D) and H460 (I) cells showing effective dose (EC50) of IMMU-132. \u003cstrong\u003e(E and J)\u003c/strong\u003e H441(E) and H460 (J) showing effective dose (EC50) of radiation therapy. Synergy plot and EC50 were estimated by Combenefit software (Cancer Research UK Cambridge Institute).\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/a31523f08c4bf089066d4e3a.png"},{"id":91961016,"identity":"3305f116-895b-4d1a-adb0-d8e95d61a12b","added_by":"auto","created_at":"2025-09-23 07:50:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":99559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIMMU-132 blocks NSCLC cells in the G2/M phase of the cell cycle.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH441 and H460 cells were treated with 1 and 5nM of IMMU-132 24 h before XRT (2Gy), and the cell cycle profile was analyzed after 1h of irradiation (2Gy). Cell cycle analysis was performed using PI staining and analyzed by flow cytometry. Flow cytometry analysis reveals the gating pattern of the G0, G1, S, and G2/M phases in H441 (A) and H460 cells (B). Bar diagram showing percentage of H441 cells (C) and H460 (D) in G0, G1, S, and G2/M phase. H441 cells (E) and H460 cells (F) were incubated with 0, 1nM, and 5nM of IMMU-132 24h before irradiation (2Gy) and harvested 48h post-irradiation. After electrophoresis, proteins were detected using the indicated antibodies. Band intensities were quantified by densitometry with Image Lab software (Bio-Rad) and normalized to GAPDH expression. The corresponding intensities are listed below for each band.\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/444959c0a5acbc88cbd1308c.png"},{"id":91964928,"identity":"7e5c376b-2552-42f7-b702-b11f9f108b53","added_by":"auto","created_at":"2025-09-23 08:14:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIMMU-132 increases DNA damage and inhibits DNA repair in irradiated NSCLC cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH441 and H460 cells were incubated with 1nM and 5nM of IMMU-132 24 h before XRT (2Gy), and cells were analyzed after 24h following radiation (2Gy). Representative images showing immunofluorescence staining of nuclear γH2AX foci in H441 (A) and H460 (C) cells. Images were taken in Carl Zeiss confocal microscope at 63X magnification. Scale bar represents 20 µM. Mean number of γH2AX foci per nucleus in H441 (B) and H460 (D) obtained by counting 50 cells. Flow cytometry profiles showing γH2AX levels and DNA content of H441 cells (E) and H460 cells (G). The quantification data represent the percentage of γH2AX-positive H441 (F) and H460 (H). H441 (I) and H460 (J) cells were incubated with 0, 1nM, and 5nM of IMMU-132 24h before irradiation (2Gy) and harvested 48h post-irradiation. After electrophoresis, proteins were detected using the indicated antibodies. Band intensities were quantified by densitometry with the Image Lab software (Bio-Rad) and normalized to GAPDH expression. The corresponding intensities are listed below for each band. Representatives immunoblot of GAPDH are shown in the figure. *P\u0026lt;0.05, **p\u0026lt;0.001, ***p=0.0004, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/46995b1a9f100c8d134fcec4.png"},{"id":91984795,"identity":"018b6584-4b4a-476d-9e7c-20566ab8d978","added_by":"auto","created_at":"2025-09-23 11:44:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA combination of IMMU-132 and radiation enhances apoptosis in NSCLC Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH441 and H460 cells were treated with 1nM and 5nM of IMMU-132 24 h before XRT (2Gy), and apoptotic cells were analyzed after 24h following radiation (2Gy) using annexin V/PI-staining. Flow cytometry analysis showing dot plots of annexin V/PI-stained H441 cells (A) and H460 cells (D). Bar diagrams display the percentage of early apoptotic (annexin V-positive and PI-negative) cells in H441 cells (B) and H460 cells (E), and late apoptotic (double-positive) cells in H441 cells (C) and H460 cells (F). H441 (G) and H460 (H) cells were incubated with 0, 1 nM, and 5 nM of IMMU-132 24 hours before irradiation (2 Gy) and harvested 48 hours post-irradiation. After electrophoresis, proteins were detected using the indicated antibodies. Band intensities were quantified by densitometry with Image Lab software (Bio-Rad) and normalized to GAPDH expression. The corresponding intensities are listed below for each band. A representative immunoblot of GAPDH is shown in the figure. *P\u0026lt;0.05, **p\u0026lt;0.001, ***p≥0.0001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/95e7dc9fbdc123e6ab17a427.png"},{"id":91963421,"identity":"97547ec4-1230-4811-a8f7-4b15f54a5251","added_by":"auto","created_at":"2025-09-23 08:06:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":132167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficacy of IMMU-132 in nude mice bearing human lung carcinoma xenografts. \u003c/strong\u003e(A) Tumor growth curve of H441 (A) and H460 (D). Survival curve of H441 xenograft (B) and H460 (E) for untreated, IMMU-132 alone, XRT, and IMMU-132+XRT treated animals (n=6- 8 mice). Survival curves were plotted at the endpoint of tumor progression, defined as \u0026gt;2000 mm³. H441 (C) and H460 (F) tumor tissue isolated from all the groups of mice with endpoint tumor size. Protein isolated from the tumor tissue was subjected to electrophoresis. After electrophoresis, the protein of interest was detected using the appropriate antibodies. Band intensities were quantified by densitometry with Image Lab (Bio-Rad) and normalized to GAPDH expression. The corresponding intensities are listed below for each band. A representative immunoblot of GAPDH is shown in the figure.\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/01aa732a3af0cfe35fbc7b48.png"},{"id":92190844,"identity":"cfc979b5-b32f-4102-bdfa-88b4aa503c68","added_by":"auto","created_at":"2025-09-25 15:06:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2956516,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/a29c914e-c412-4cd0-af70-3a096d723130.pdf"},{"id":91963420,"identity":"2c67dd34-f213-4658-a654-d1a43bdf4242","added_by":"auto","created_at":"2025-09-23 08:06:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16855,"visible":true,"origin":"","legend":"Supplementary Figure legends","description":"","filename":"SupplementaryFigureLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/fe7c135b3d541325b869cd13.docx"},{"id":91962682,"identity":"24b00f1c-117f-49e3-9f17-d112a143ae0a","added_by":"auto","created_at":"2025-09-23 07:58:01","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1447934,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"supplementaryfigurefinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7593735/v1/65a323e2f613e35ffefd6087.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nRon Bose reports a research grant from Puma Biotechnology and Consulting for Genentech. All other authors have no conflict.","formattedTitle":"The TROP2 targeting antibody-drug conjugate IMMU-132 enhances the efficacy of radiation therapy for lung cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is the leading cause of death worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Non-small cell lung cancer (NSCLC) is the predominant form of lung cancer, accounting for nearly 85% of all lung cancer cases\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Despite advances in treatment strategies, the five-year survival rate for lung cancer patients is still very low (23%)\u003csup\u003e1,2\u003c/sup\u003e. Chemotherapy combined with radiotherapy, followed by immunotherapy, is the standard of care for patients with locally advanced NSCLC\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Radiation-induced tumor death is mainly attributed to DNA double-strand breaks through the formation of chromosomal aberrations, cell cycle arrest, and apoptosis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Various signaling pathways, including MAPK and PI3K/AKT/mTOR, that regulate tumor cell growth, survival, and proliferation, play a crucial role in developing radioresistance in NSCLC\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The application of radiation-sensitizing chemotherapy in cancer treatment has displayed encouraging potential, but harmful side effects impede the administration of chemotherapy at elevated dosages.\u003c/p\u003e\u003cp\u003eAntibody-drug conjugates (ADCs) are an emerging class of therapeutics that combine a highly specific monoclonal antibody with a potent cytotoxic payload linked via a chemical linker\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The payloads typically consist of either antimicrotubule agents or DNA-damaging compounds, which induce cell-cycle arrest and promote apoptosis in target cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. ADC technology relies on using a linker that remains stable in circulation to minimize off-target effects and ensure selective delivery of the cytotoxic payload to target cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Fam-trastuzumab deruxtecan-nxki (Enhertu) was approved in August 2022 for adults with unresectable or metastatic HER2-mutant NSCLC who have received prior systemic therapy\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTrophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein consisting of an extracellular domain, a transmembrane region, and a short cytoplasmic tail that can undergo phosphorylation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The transmembrane domain is a calcium signal transducer, influencing cell cycle-related signaling pathways. TROP2 interacts with proteins such as insulin-like growth factor 1, claudin-1 and \u0026minus;\u0026thinsp;7, cyclin D1, and protein kinase C (PKC), triggering downstream effects that promote cell proliferation and apoptosis\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The cytoplasmic tail regulates protein-protein interactions and contains a PKC phosphorylation site that modulates calcium signaling. TROP2 is weakly expressed or not expressed in normal tissue but is overexpressed in several types of cancer, including lung cancer\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. TROP2 expression has been observed in up to 64% of adenocarcinoma and up to 75% of squamous cell carcinoma NSCLC, making it a promising target for cancer therapy\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSacituzumab govitecan-hziy (IMMU-132) is an anti-TROP2 ADC that consists of a humanized anti-TROP2 monoclonal antibody linked to the topoisomerase I inhibitor SN-38 by a hydrolyzable pH-sensitive linker with a high drug-to-antibody ratio (DAR) of 7.6\u003csup\u003e18\u003c/sup\u003e. IMMU-132 has been approved for the treatment of triple-negative metastatic breast cancer and metastatic urothelial cancer\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. SN-38 is the active metabolite of irinotecan and mediates the upregulation of early proapoptotic proteins, such as p53, resulting in caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, and inhibition of NF-κB and AKT signaling in cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), activating ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR), and DNA-dependent protein kinase (DNA-Pk)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Additionally, IR activates several cellular processes, including apoptosis, cell cycle arrest, and DNA repair in cancer cells, which promotes radioresistance\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thus, targeting these prosurvival pathways with a subtherapeutic dose of IMMU-132 could have great potential for sensitizing lung cancer cells to radiation therapy. Therefore, we hypothesized that sub-therapeutic doses of IMMU-132 would enhance the therapeutic index of radiotherapy (XRT) in lung cancer.\u003c/p\u003e\u003cp\u003eIn this study, we demonstrated that NSCLC cell lines exhibit surface expression of TROP2, facilitating efficient binding and internalization of the TROP2-targeting ADC IMMU-132. Upon internalization, IMMU-132 induced G2/M phase cell cycle arrest, a phase known to confer heightened radiosensitivity\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Treatment with IMMU-132 enhanced the radiosensitivity of lung tumor cells by reducing clonogenic survival and impairing DNA damage response pathways. Additionally, IMMU-132 attenuated irradiation-induced activation of key prosurvival signaling pathways, including PI3K/AKT, MEK1/2, ERK1/2, mTOR, and SAPK/JNK. It also promoted apoptosis through the upregulation of proapoptotic BIM, increased cleavage of caspase-3 and PARP, and downregulation of anti-apoptotic BCL-xL. \u003cem\u003eIn vivo\u003c/em\u003e, administering a subtherapeutic dose of IMMU-132 in nude mice bearing H441 and H460 tumor xenografts significantly improved tumor control and survival when combined with radiation therapy. These findings suggest that IMMU-132 may serve as an effective radiosensitizer, offering a targeted strategy to enhance the therapeutic efficacy of radiation in lung cancer.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell Lines and Irradiation\u003c/h2\u003e\u003cp\u003eHuman NSCLC cell lines H441 (ATCC Cat# HTB-174, RRID: CVCL_1561), H460 (ATCC Cat# HTB-177, RRID:CVCL_0459), and A549 (ATCC Cat# CCL-185, RRID:CVCL_0023), and the murine NSCLC cell line Lewis lung carcinoma (LLC1/LL2) (ATCC Cat#CRL-1642, RRID:CVCL_4358) were obtained from the American Type Culture Collection. H441 and H460 cells were cultured in RPMI-1640 medium (Gibco), while A549 and LLC cells were cultured in DMEM/F12 (Gibco), each medium supplemented with 10% FBS and 1% penicillin and streptomycin (Gibco), at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Mycoplasma testing was performed every three months using the MycoAlert\u0026trade; PLUS mycoplasma detection kit (Lonza) and was consistently negative. All experiments were performed within 10 passages of thawing; short tandem repeat profiling was performed for human cell lines before use. IMMU-132 (Sacituzumab govitecan) was provided by Dr. Ron Bose (Washington University in St. Louis).\u003c/p\u003e\u003cp\u003eIrradiation was delivered using an RS 2000 X-ray irradiator (Rad Source, USA) operating at 160 kV and 25 mA, at a dose of 0.0682Gy/s (\u003cem\u003ein vitro)\u003c/em\u003e and 0.0167Gy/s (\u003cem\u003ein vivo)\u003c/em\u003e. For animal irradiation, mice were anesthetized with 2% isoflurane, and the body was shielded with lead to expose only the hindlimb tumors to radiation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell surface expression of TROP2 by Flow Cytometry\u003c/h3\u003e\n\u003cp\u003eCells were incubated with Live/Dead fixable violet dead cell stain (Invitrogen, Cat# L34964) for 30 minutes, washed with flow buffer (PBS containing 2% FBS and 0.1% sodium azide), and stained with PE anti-human TACSTD2 (BioLegend, Cat# 363803, RRID: AB_2572021) for 30 minutes at 4\u0026deg;C. Samples were acquired on a MACSQuant Analyzer flow cytometer (RRID: SCR_020268) and analyzed with FlowJo software (BD Biosciences).\u003c/p\u003e\n\u003ch3\u003eCell surface expression of TROP2 by Immunofluorescence\u003c/h3\u003e\n\u003cp\u003eCells were seeded on coverslip in 12-well plates, fixed in 4% paraformaldehyde for 20 minutes, washed with PBS, and blocked with 1% BSA in PBS for 1 hour at room temperature. Cells were incubated with anti-TROP2 antibody (Abcam, Cat# ab214488, RRID: AB_2811182) overnight at 4\u0026deg;C, followed by Alexa Fluor 488-conjugated Goat anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibody (Thermo Scientific, Cat# 11034, RRID: AB_2576217)) for 1 hour at room temperature in the dark. Nuclei were stained with nuclear blue live-ready probes reagent (Invitrogen, USA, Cat# R37605) for 15 minutes, and slides were mounted with ProLong Glass Antifade Mountant (Invitrogen, Cat#P36980). Images were acquired on LSM 510 confocal microscope (Carl Zeiss), and fluorescence intensity was quantified with ImageJ.\u003c/p\u003e\n\u003ch3\u003eCell surface binding of IMMU-132 by Flow Cytometry\u003c/h3\u003e\n\u003cp\u003eH441, H460, A549, and LLC cells were treated with 5 Gy radiation, and 24 hours later, stained with viability dye as above. Cells were incubated with IMMU-132 (500nM or 1000nM) for 1 hour at 4\u0026deg;C, washed, and stained with PE-conjugated anti-human IgG secondary antibody (BioLegend, Cat# 410708, RRID: AB_2565786) for 30 minutes at 4\u0026deg;C. Data acquisition and analysis were performed as described above.\u003c/p\u003e\n\u003ch3\u003eIMMU-132 internalization by Immunofluorescence\u003c/h3\u003e\n\u003cp\u003eCancer cells (1x 10\u003csup\u003e6\u003c/sup\u003e) were seeded in four-chamber slides (Cat# PEZGS0416) overnight. IMMU-132 was labeled with Alexa Flour 488 using ZIP protein labeling kit (Thermo Scientific, USA, Cat# Z11233) per the manufacturer instructions. Cells were incubated with labeled IMMU-132 (30 \u0026micro;g/ml) for 2 or 24 hours at 37\u0026deg;C, washed with acidic buffer (0.5% acetic acid and 0.5M NaCl) for 30 seconds to remove surface-bound ADC, and stained with nuclear blue live-ready probes reagent as above. After fixation with 2% paraformaldehyde for 10 minutes, slides were mounted and imaged on an LSM 510 confocal microscope (Carl Zeiss). Green fluorescence intensity was quantified with ImageJ.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell cytotoxicity assay\u003c/h2\u003e\u003cp\u003eCell viability following IMMU-132 treatment was assessed using the CellTiter-Glo\u0026reg; Luminescent cell viability assay (Promega, Cat# G7571). H441, H460, A549, and LLC cells were seeded at 5000 cells/well in 96-well plates and treated the following day with IMMU-132 (120nm, 30nM, 7.5nM, 1.875nM, and 0.4685nM) for 48 h. Plates were equilibrated to room temperature for 30 minutes before adding an equal volume of CellTiter-Glow reagent. After orbital shaking for 2 min to induce lysis, plates were incubated further for 10 minutes at room temperature and luminescence measured on a SpectraMax i3 Plate reader (Molecular Devices). Viability was normalized to untreated controls, and data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three replicates.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eRadiation-sensitizing effects of IMMU-132 were evaluated by colony formation assay (CFA) as previously described\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Cells (2000\u0026ndash;10000/well) were seeded in 6-well plates, treated the next day with IMMU-132 for 24 hours, and irradiated with 0, 1, 3, 5, or 7 Gy. Plates were incubated for 7\u0026ndash;9 days and stained with 0.5% crystal violet. Colonies containing\u0026thinsp;\u0026ge;\u0026thinsp;50 cells were counted using a StemiDV4 Stereo Microscope (Zeiss). Survival fraction was calculated as (colonies at given dose relative to colonies of untreated) x 100. Combination effects were analyzed using Combenefit software.\u003c/p\u003e\n\u003ch3\u003eCell cycle and DNA damage analysis by flow cytometry\u003c/h3\u003e\n\u003cp\u003eH441, H460, A549, and LLC (3x10\u003csup\u003e5\u003c/sup\u003e) cells were seeded in 6-well plates and treated next day with IMMU-132 (1 nM or 5 nM) for 24 h, followed by 2 Gy irradiation. Cells were harvested at 1 h and 24 h post-irradiation. The cells were harvested, fixed with 70% ice-cold ethanol, incubated overnight at 4\u0026deg;C, washed, and then incubated with FITC-conjugated γ-H2AX antibody for 1 h at 4\u0026deg;C. After washing, cells were resuspended in flow buffer containing PI (5\u0026micro;g/ml; Cat# 51-66211E) and RNase (100\u0026micro;g/ml) for 10 minutes. Samples were analyzed in a MACSQuant flow cytometer (Miltenyi Biotec), and data processed with FlowJo software as previously described\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAnnexin-V/PI analysis by flow cytometry\u003c/h2\u003e\u003cp\u003eApoptosis was assessed using the BD Pharmingen FITC Annexin-V/PI Detection Kit (BD Biosciences Cat# 556547, RRID: AB_2869082). Cells (1x10\u003csup\u003e5\u003c/sup\u003e) were seeded in 12-well plates, treated the next day with IMMU-132 (1nM and 5nM) for 24 h, then irradiated (2 Gy or 5 Gy). After 24 h, cells were harvested, washed with ice-cold PBS, and resuspended in 1x binding buffer. Annexin-V and PI were added at the recommended concentrations and incubated for 15 minutes at room temperature in the dark. Samples were analyzed by flow cytometry and analysis were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence analysis of γH2AX expression\u003c/h2\u003e\u003cp\u003eCells (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were seeded on coverslips in 24-well plates and treated with (1nM or 5nM) for 24 h before 2 Gy irradiation. At 1 h post-irradiation, cells were washed, fixed in 4% formaldehyde for 20 min, permeabilized in 0.5% Triton X-100 for 15 min, and blocked in 5% BSA for 30 min at 37\u003csup\u003eo\u003c/sup\u003eC. Cells were incubated with γH2AX (Ser139) antibody (Clone JBW301, Millipore, Sigma, Cat# 05-636, RRID: AB_309864) for 1 h at 37\u0026deg;C, followed by Alexa Fluor 488-conjugated secondary antibody for 45 min at 37\u0026deg;C. Coverslips were mounted with a ProLong Diamond Antifade mounting medium containing DAPI (Thermo Scientific, Cat#P36962). Foci were visualized using an LSM 510 confocal microscope (Carl Zeiss) and counted by ImageJ software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003etumor xenograft studies\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All animal experiments were approved by the Washington University Animal Studies Committee and conducted in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. Athymic nude mice (6-week-old; Charles River Labs) received subcutaneous injections of H441 (3x10\u003csup\u003e6\u003c/sup\u003e cells) or H460 (2x10\u003csup\u003e6\u003c/sup\u003e cells) into the right hind limb. Upon palpation of tumors, mice were randomized into four groups (6\u0026ndash;8 per group): vehicle (PBS)\u0026thinsp;\u0026plusmn;\u0026thinsp;XRT, or IMMU-132 (25 mg/kg)\u003csup\u003e37\u003c/sup\u003e \u0026plusmn; XRT. The treatment regimens are detailed in Supplementary Fig.\u0026nbsp;5A-B. Briefly, H441 xenograft received two doses of IMMU-132 plus six 2 Gy fractions (2 cycles); H460 received two doses of IMMU-132 plus ten 3 Gy fractions (2 cycles). Tumor dimensions (length, width, and height) were assessed using a digital caliper. Tumor volume was calculated by length x width x height. Body weights were monitored for toxicity. Mice were euthanized once tumors reached\u0026thinsp;~\u0026thinsp;2000mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and tumors were harvested.\u003c/p\u003e\u003cp\u003eFor syngeneic studies, LLC cells (5x10\u003csup\u003e5\u003c/sup\u003e) were injected subcutaneously into 6-week-old C57BL/6 mice with treatment groups and schedule as in \u003cb\u003eSupplementary Fig.\u0026nbsp;5E\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eWestern Blot\u003c/h2\u003e\u003cp\u003eProtein lysates were prepared from H441 and H460 tumor tissues harvested from xenograft studies, as well as from H441 and H460 cells. Cells were seeded at a density of 0.3 X 10\u003csup\u003e6\u003c/sup\u003e cells per well in 6-well plates and treated with IMMU-132 (1nM or 5nM) for 24 hours prior irradiation (2 Gy). At 48 h post-irradiation, cells were washed with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo Scientific, Cat#78444). Protein concentration was determined, and 30 \u0026micro;g of protein per sample was resolved on 4\u0026ndash;20% SDS polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin (BSA) in TBST (Tris Buffered Saline (G-Biosciences, Cat#R029) containing 0.1% Tween (Sigma, Cat#P9416), for 1 hour at room temperature. Blots were incubated overnight at 4\u0026deg;C with primary antibodies (listed in Supplementary Table\u0026nbsp;1), followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Immunoreactive bands were detected using a chemiluminescence kit (Perkin Elmer, Cat# NEL103001EA) and imaged with a ChemiDoc\u0026trade; MP Imaging System (Bio-Rad, Cat#12003154).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using the student\u0026rsquo;s t-test and one-way or two-way analysis of variance (ANOVA). All analyses were performed using GraphPad Prism Software (RRID: SCR_002798), and statistical significance is indicated in each graph where appropriate.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSurface expression of TROP2 on NSCLC cells\u003c/h2\u003e\u003cp\u003eWe performed this study on three human (H441, H460, and A549) and a murine (LLC) NSCLC cell line. Given that IMMU-132 is an antibody-drug conjugate (ADC) targeting the TROP2 antigen, we first assessed cell surface TROP2 expression in all four cell lines (H441, H460, A549, and LLC) with and without radiation exposure using flow cytometry and immunofluorescence staining, as well as total TROP2 levels by Western blot.\u003c/p\u003e\u003cp\u003eAmong the cell lines tested, H441 demonstrated the highest surface TROP2 expression. Radiation exposure led to a significant increase in surface TROP2 in H441 cells, with the median fluorescence intensity (MFI) rising from 10,644.5\u0026thinsp;\u0026plusmn;\u0026thinsp;504.2 (sham) to 16,079\u0026thinsp;\u0026plusmn;\u0026thinsp;294.2 (XRT), corresponding to a 1.5-fold increase (p\u0026thinsp;=\u0026thinsp;0.01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Similarly, total TROP2 protein levels were elevated in irradiated H441 cells compared to sham-treated controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. In H460 cells, surface TROP2 expression also increased post-radiation, with an MFI of 36\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6 in sham versus 66.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8 in the XRT group, reflecting more than a 1.5-fold change (p\u0026thinsp;=\u0026thinsp;0.03) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Consistently, western blot analysis revealed an increased total TROP2 protein in irradiated H460 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn A549 and LLC cells, flow cytometry showed a significant increase (A549 p\u0026thinsp;=\u0026thinsp;0.02; LLC p\u0026thinsp;=\u0026thinsp;0.0002) in MFI of surface TROP2 expression following irradiation compared to sham-treated cells \u003cb\u003e(Supplementary Figs.\u0026nbsp;1A and 1C)\u003c/b\u003e. This upregulation was corroborated by immunoblot analyses, which also demonstrated elevated total TROP2 protein levels in the XRT groups \u003cb\u003e(Supplementary Figs.\u0026nbsp;1B and 1D), respectively.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe flow cytometry data were supported by immunofluorescence analysis, which demonstrated a significant increase in surface TROP2 expression following radiation in H441 (p\u0026thinsp;=\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE \u003cb\u003eand F)\u003c/b\u003e, H460 (p\u0026thinsp;=\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG \u003cb\u003eand H)\u003c/b\u003e, A549 (p\u0026thinsp;=\u0026thinsp;0.0001) \u003cb\u003e(Supplementary Figs.\u0026nbsp;1E and F)\u003c/b\u003e, and LLC cells (p\u0026thinsp;=\u0026thinsp;0.0001) \u003cb\u003e(Supplementary Figs.\u0026nbsp;1G and H)\u003c/b\u003e compared to their respective sham-treated controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eIMMU-132 binds to the surface of cancer cells and undergoes endocytosis\u003c/h2\u003e\u003cp\u003eGiven the confirmed cell surface expression of TROP2 in NSCLC cell lines and the TROP2 specificity of IMMU-132, we next assessed the surface binding and internalization of IMMU-132 in these cells. Flow cytometry analysis demonstrated robust binding of IMMU-132 to H441, H460, A549, and LLC cells. Specifically, H441 and H460 cells showed 88.3% and 83.7% binding in the sham group, which increased to 94.6% and 96.7%, respectively, following radiation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e. Similarly, A549 and LLC cells exhibited 87.5% and 41.6% binding in the sham group, increasing to 90.1% and 47.5%, respectively, in the XRT group \u003cb\u003e(Supplementary Fig.\u0026nbsp;1I and 1K).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe median fluorescence intensity (MFI) of IMMU-132 surface binding correlated with TROP2 expression, with H441 cells showing the highest MFI, followed by H460, A549, and LLC cells. Comparative analysis between sham and irradiated cells revealed significantly elevated MFI in the XRT group for H441 (4333\u0026thinsp;\u0026plusmn;\u0026thinsp;168.3 vs. 4877.5\u0026thinsp;\u0026plusmn;\u0026thinsp;20.5; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e and H460 (549\u0026thinsp;\u0026plusmn;\u0026thinsp;33.2 vs. 1214.5; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e. Similarly, A549 (609\u0026thinsp;\u0026plusmn;\u0026thinsp;15.6 vs. 694\u0026thinsp;\u0026plusmn;\u0026thinsp;33.0; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and LLC (284\u0026thinsp;\u0026plusmn;\u0026thinsp;43.8 vs. 346\u0026thinsp;\u0026plusmn;\u0026thinsp;29.7; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) cells showed significantly higher MFI in the XRT group compared to sham \u003cb\u003e(Supplementary Fig.\u0026nbsp;1J and 1L)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo evaluate internalization, we monitored uptake of fluorescently labeled IMMU-132 over time. H441 and H460 cells displayed time-dependent internalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO), with significantly increased mean green fluorescence intensity (GFI) at 24 h compared to 2 h (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP\u003cb\u003e)\u003c/b\u003e. A similar trend was observed in A549 and LLC cells, with internalization evident at 2 hours and reaching a maximum at 24 hours \u003cb\u003e(Supplementary Figs.\u0026nbsp;1M\u0026ndash;1P)\u003c/b\u003e. These findings indicate efficient, time-dependent internalization of IMMU-132 across all cell lines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIMMU-132 reduces cell viability and enhances the efficacy of radiation\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cytotoxic effect of IMMU-132 on H441, H460, A549, and LLC cells was evaluated by assessing cell viability after treatment with a range of concentrations (0.4685 nM to 120 nM). A dose-dependent decrease in cell viability was observed across all cell lines, with calculated IC₅₀ values of 11.43 nM (H441; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), 12.41 nM (H460; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), 10.33 nM (A549; \u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e), and 23.37 nM (LLC; \u003cb\u003eSupplementary Fig.\u0026nbsp;2F\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the radiosensitizing potential of IMMU-132, clonogenic survival assays were performed using increasing concentrations of IMMU-132 (0\u0026ndash;1 nM) in combination with graded doses of radiation (0\u0026ndash;5 Gy). Across all four cell lines, the combination of IMMU-132 and radiation significantly reduced clonogenic survival compared to either treatment alone, with a clear dose-dependent decrease in colony formation of H441 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, H460 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e, A549 cells \u003cb\u003e(Supplementary Fig.\u0026nbsp;2B)\u003c/b\u003e and LLC cells \u003cb\u003e(Supplementary Fig.\u0026nbsp;2G)\u003c/b\u003e. Synergy analysis revealed additive and synergistic effects between IMMU-132 and radiation in H441 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, H460 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e, and A549 cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;2C)\u003c/b\u003e, as indicated by synergy maps. Whereas LLC cells exhibited only additive effects \u003cb\u003e(Supplementary Fig.\u0026nbsp;2H)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eEffective concentration (EC₅₀) values for IMMU-132 in the combination setting were determined as 0.388 nM of H441 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e, 3.91 nM of H460 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e, 0.811 nM of A549 \u003cb\u003eSupplementary Fig.\u0026nbsp;2D, and 2\u003c/b\u003e.57 nM of LLC \u003cb\u003e(Supplementary Fig.\u0026nbsp;2I)\u003c/b\u003e. Similarly, the EC₅₀ values for radiation in the combination setting were 0.94 Gy (H441; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), 1.61 Gy (H460; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ), 3.4 Gy (A549; \u003cb\u003eSupplementary Fig.\u0026nbsp;2E\u003c/b\u003e), and 6.02 Gy (LLC; \u003cb\u003eSupplementary Fig.\u0026nbsp;2J\u003c/b\u003e). Notably, the combination of IMMU-132 and radiation significantly reduced the surviving fraction in all cell lines compared to IMMU-132 and radiation alone. These findings demonstrate that IMMU-132 enhances radiosensitivity in NSCLC models (H441, H460, and A549), with evidence of synergistic interactions when combined with ionizing radiation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCombination treatment alters cell cycle distribution\u003c/h2\u003e\u003cp\u003eCell cycle analysis was performed using flow cytometry after treatment with 1 and 5 nM IMMU-132 and 2 Gy XRT. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB show the histograms of G0, G1, S, and G2/M cell cycle phases in H441 and H460 cells, respectively. Cell cycle analysis revealed an increased percentage of H441 cells arrested in the G2 phase following treatment with IMMU-132 (1 nM, 63.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.36 and 5 nM, 77.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71) as compared to untreated (39.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77); and IMMU-132\u0026thinsp;+\u0026thinsp;XRT (1 nM, 67.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 and 5 nM, 69.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77) as compared to and XRT alone (55.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Similarly, we found an increased percentage of H460 cells arrested in the G2 phase treated with IMMU-132 (1 nM, 40.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18 and 5 nM, 49.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85) as compared to untreated (32.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33); and IMMU-132\u0026thinsp;+\u0026thinsp;XRT (1nM, 44.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 and 5 nM, 54.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04) as compared to XRT alone (36.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, A549 and LLC cells were also arrested in the G2 phase following treatment with IMMU-132 and XRT as shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;3A and 3B\u003c/b\u003e, respectively. The percentage of A549 cells in the G2 phase was higher in IMMU-132 (5 nM, 41.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91) and IMMU-132\u0026thinsp;+\u0026thinsp;XRT (5 nM, 41.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35) as compared to untreated (25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.424) and XRT alone (27.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78) \u003cb\u003e(Supplementary Fig.\u0026nbsp;3C)\u003c/b\u003e. The percentage of LLC cells arrested at G2 was higher in IMMU-132 (5 nM, 49.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55) and IMMU-132\u0026thinsp;+\u0026thinsp;XRT (5 nM, 48.65\u0026thinsp;\u0026plusmn;\u0026thinsp;6.71) as compared to untreated (30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51) and XRT alone (40.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99) \u003cb\u003e(Supplementary Fig.\u0026nbsp;3D).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the molecular level, we investigated the total and phosphorylated protein levels of AKT, extracellular signal-regulated kinase (ERK1/2), mitogen-activated protein kinase (MEK1/2) and p53, which regulate cell cycle progression survival. We found reduced phosphorylation of AKT, MEK1/2, ERK1/2, and overexpression of p53 in H441 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e and H460 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e cells treated with IMMU-132\u0026thinsp;+\u0026thinsp;2Gy as compared to IMMU-132 alone. AKT, ERK, and MEK phosphorylation are associated with cell cycle progression and cell survival\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. p53 acts as a tumor suppressor protein and phosphorylation of p53 plays an important role in cell cycle arrest and apoptosis\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Hence, reduced phosphorylation of AKT, ERK, and MEK regulatory proteins in combined treatment confirm cell cycle arrest, and induction of phosphorylated p53 signifies acceleration of apoptosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIMMU-132 Combined with Radiation Enhances DNA Damage and Inhibits DNA Repair\u003c/b\u003e γH2AX phosphorylation is a marker for DNA damage \u003csup\u003e42 43\u003c/sup\u003e. We evaluated γH2AX expression in all NSCLC cells by immunofluorescence and flow cytometry. Cells revealed γH2AX foci (green fluorescence) following staining with FITC-conjugated γH2AX in H441 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, H460 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Quantification of mean number of γH2AX per nucleus in H441 demonstrated a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with IMMU-132 treatment (1 nM, 21.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 and 5 nM, 24.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71) compared to untreated controls (8.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92). There was a further increase in combination with XRT compared to IMMU-132 alone (1 nM IMMU-132\u0026thinsp;+\u0026thinsp;XRT, 27.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62 vs 1nM IMMU-132, 21.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and 5 nM IMMU-132\u0026thinsp;+\u0026thinsp;XRT, 28.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 vs. 5nM IMMU-132, 24.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e Similar data was obtained in H460 cells, where the mean number of γH2AX per nucleus significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) increased with IMMU-132 treatment (1 nM, 3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1 and 5 nM, 8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35) compared to untreated controls (1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53). XRT further increased the foci compared to ADC alone (1 nM IMMU-132\u0026thinsp;+\u0026thinsp;XRT, 12.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 vs 1nM IMMU-132, 3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and 5 nM IMMU-132\u0026thinsp;+\u0026thinsp;XRT, 14.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46 vs. 5nM IMMU-132, 8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis was further confirmed by flow cytometry analysis \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H\u003cb\u003e)\u003c/b\u003e. Figures\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE \u003cb\u003eand G\u003c/b\u003e show the dot plots of γH2AX and propidium iodide positive cells for H441 and H460 cells, respectively. The percentage of γH2AX positive cells were significantly higher in H441 treated with XRT (19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84) vs sham (8.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19), p\u0026thinsp;\u0026lt;\u0026thinsp;0.02; 1 nM IMMU-132 (24.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63) vs. 0 nM (8.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.191), p\u0026thinsp;\u0026lt;\u0026thinsp;0.003; 1 nM IMMU-132\u0026thinsp;+\u0026thinsp;XRT (34.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99) vs. 1 nM IMMU-132 (24.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63), p\u0026thinsp;\u0026lt;\u0026thinsp;0.04; 5 nM (39.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67) vs 1nM IMMU-132 (24.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62), p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 1nM\u0026thinsp;+\u0026thinsp;XRT (34.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99) vs 0nM XRT (19.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84), p\u0026thinsp;\u0026lt;\u0026thinsp;0.004; 5nM\u0026thinsp;+\u0026thinsp;XRT (43.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98) vs. 1nM\u0026thinsp;+\u0026thinsp;XRT (34.3\u0026thinsp;\u0026plusmn;\u0026thinsp;.099), p\u0026thinsp;\u0026lt;\u0026thinsp;0.04; \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u003cb\u003e).\u003c/b\u003e Similarly, the percentage of γH2AX positive H460 cells were also significantly high in XRT (13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4) vs sham (2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67), p\u0026thinsp;\u0026lt;\u0026thinsp;0.04; 1nM IMMU-132 (13.05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61) vs. 0nM (2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67), p\u0026thinsp;\u0026lt;\u0026thinsp;0.04; 1nM\u0026thinsp;+\u0026thinsp;XRT (23.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1) vs. 1nM IMMU-132 (13.05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61), p\u0026thinsp;\u0026lt;\u0026thinsp;0.03; 1nM\u0026thinsp;+\u0026thinsp;XRT (23.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1) vs 0nM XRT (13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4), p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo elucidate the mechanism underlying the enhanced cytotoxicity of IMMU-132 combined with radiation, we examined key mediators of the DNA damage response by western blot analysis in H441 and H460 lung cancer cells \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. Combination treatment (IMMU-132\u0026thinsp;+\u0026thinsp;XRT) markedly increased phosphorylation of ATM and ATR compared to IMMU-132 alone, radiation alone, or untreated controls, indicating robust activation of DNA damage signaling pathways. This effect correlated with a pronounced accumulation of phosphorylated γH2AX, a surrogate marker of persistent DNA double-strand breaks. We also assessed the expression of DNA-PK, a critical regulator of DNA repair. DNA-PK phosphorylation levels were substantially reduced following IMMU-132\u0026thinsp;+\u0026thinsp;XRT treatment, suggesting impaired DNA repair capacity \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eIMMU-132 Enhances Radiation-Induced Apoptosis in NSCLC Cells\u003c/h2\u003e\u003cp\u003eTo assess the apoptotic response to IMMU-132 combined with radiation, H441, H460, A549, and LLC cells were treated with either 1 nM or 5 nM IMMU-132 for 24 hours, followed by irradiation with 2 Gy. Additionally, A549 and LLC cells were exposed to 5 Gy in combination with 1 nM or 5 nM IMMU-132 to evaluate dose-dependent effects. Apoptosis was measured by Annexin V/propidium iodide (PI) staining 24 hours post-irradiation. Flow cytometric dot plots revealed distinct early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic populations in H441 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, H460 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e, A549 \u003cb\u003e(Supplementary Fig.\u0026nbsp;5A)\u003c/b\u003e, and LLC \u003cb\u003e(Supplementary Fig.\u0026nbsp;5D)\u003c/b\u003e cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn H441 cells, early apoptosis was significantly increased following irradiation alone (7.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81%) compared to sham (4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.03). This effect was enhanced by IMMU-132, with further increases observed in the 1 nM\u0026thinsp;+\u0026thinsp;XRT group (10.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23% vs. 1 nM sham: 5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.006) and a similar trend at 5 nM\u0026thinsp;+\u0026thinsp;XRT (10.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79% vs. 5 nM sham: 7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.09; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). A comparable pattern was seen in H460 cells, where early apoptosis significantly increased in all irradiated groups, including XRT alone (18.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69%) vs. sham (8.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.03), 1 nM\u0026thinsp;+\u0026thinsp;XRT (24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69%) vs. 1 nM sham (12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.02), and 5 nM\u0026thinsp;+\u0026thinsp;XRT (43.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32%) vs. 5 nM sham (32.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.03). A dose-dependent increase was evident, with significantly higher apoptosis in 5 nM vs. 1 nM groups both at baseline and with radiation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.002, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eLate apoptosis also increased significantly in H441 cells upon combination treatment. Compared to sham-treated controls, late apoptosis was higher in the XRT group (14.45\u0026thinsp;\u0026plusmn;\u0026thinsp;3.47% vs. 5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.006), and further enhanced with IMMU-132: 1 nM\u0026thinsp;+\u0026thinsp;XRT (22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68%) vs. 1 nM sham (14.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); 5 nM\u0026thinsp;+\u0026thinsp;XRT (30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69%) vs. 5 nM sham (24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.04; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). A similar pattern was observed in H460 cells, where late apoptosis significantly increased in all irradiated groups, including 0 nM\u0026thinsp;+\u0026thinsp;XRT (16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71%) vs. 0 nM sham (7.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.002), 1 nM\u0026thinsp;+\u0026thinsp;XRT (24.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55%) vs. 1 nM sham (9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and 5 nM\u0026thinsp;+\u0026thinsp;XRT (40.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91%) vs. 5 nM sham (24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eTreatment with low-dose IMMU-132 (1 nM) and 2 Gy radiation did not significantly increase early or late apoptotic cell populations in A549 or LLC cells \u003cb\u003e(Supplementary Fig.\u0026nbsp;5)\u003c/b\u003e. However, increasing the IMMU-132 concentration to 5 nM and the radiation dose to 5 Gy led to a significant increase in both early and late apoptosis. In A549 cells, 5 nM IMMU-132\u0026thinsp;+\u0026thinsp;5 Gy XRT significantly elevated early apoptotic cells compared to controls and lower-dose treatments (e.g., vs. 0 nM\u0026thinsp;+\u0026thinsp;5 Gy, p\u0026thinsp;\u0026lt;\u0026thinsp;0.003; vs. 5 nM alone, p\u0026thinsp;\u0026lt;\u0026thinsp;0.03; \u003cb\u003eSupplementary Fig.\u0026nbsp;5B\u003c/b\u003e). Similarly, late apoptotic cells increased significantly in the same group (vs. 5 nM sham, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; vs. 5 nM\u0026thinsp;+\u0026thinsp;2 Gy, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003eSupplementary Fig.\u0026nbsp;5C\u003c/b\u003e). In LLC cells, a similar dose-dependent increase in apoptosis was observed. Early apoptotic populations were significantly higher in the 5 nM\u0026thinsp;+\u0026thinsp;5 Gy group compared to all other conditions (e.g., vs. 5 nM alone, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; vs. 1 nM\u0026thinsp;+\u0026thinsp;5 Gy, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0008; \u003cb\u003eSupplementary Fig.\u0026nbsp;5E\u003c/b\u003e). Late apoptotic populations were also markedly elevated with combination treatment (e.g., 5 nM\u0026thinsp;+\u0026thinsp;5 Gy vs. 5 nM alone, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; vs. 0 nM\u0026thinsp;+\u0026thinsp;5 Gy, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003eSupplementary Fig.\u0026nbsp;5F\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo elucidate the mechanisms underlying the enhancement of radiation-induced apoptosis by IMMU-132 treatment, we evaluated apoptosis regulators caspase-3, PARP, X-chromosome-linked inhibitor of apoptosis protein (XIAP), and Bcl-2-like protein 11 (Bim) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG \u003cb\u003eand H)\u003c/b\u003e. We observed an increase in the cleavage of caspase-3 and PARP, as well as an increase in the expression of the pro-apoptotic protein Bim. XIAP levels were substantially reduced following IMMU-132\u0026thinsp;+\u0026thinsp;XRT treatment, suggesting pro-apoptotic signaling (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG \u003cb\u003eand H)\u003c/b\u003e. These findings demonstrate that IMMU-132 potentiates the effects of radiation by exacerbating DNA damage, suppressing repair pathways, and promoting apoptosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIMMU-132 enhances the efficacy of radiation therapy\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe evaluated the efficacy of IMMU-132 in combination with radiation therapy in nude mice bearing H441 and H460 tumors \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Mice were implanted subcutaneously with H441 and H460 cells into the hind limb. The treatment schema for IMMU-132 and XRT for H441 and H460 is shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;5A and 5B\u003c/b\u003e, respectively. IMMU-132 in combination with XRT significantly delayed H441 tumor growth as compared to IMMU-132 alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) or XRT alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e A similar delay in tumor growth was also observed in the H460 model \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Treatment-related side effects were not observed, as indicated by stable body weights of the mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;5C and 5D)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of the survival data revealed that the median survival of untreated H441 tumor-bearing mice was 35 days, those treated with XRT had 67 and IMMU-132 treated mice had 72 days. All mice were alive at the end of the study when treated with the combination of IMMU-132 and XRT \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The probability of survival at the endpoint was 0% for the untreated and XRT alone groups, 28.5% for the IMMU-132 group, and 100% for the IMMU-132\u0026thinsp;+\u0026thinsp;XRT group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. For the H460 tumor-bearing mice, the probability of survival at the endpoint was 0% for the untreated and IMMU-132 alone groups, 80% for the XRT group, and 100% for the IMMU-132\u0026thinsp;+\u0026thinsp;XRT group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWe also evaluated the efficacy of IMMU-132 in combination with XRT in immunocompetent mice (C57BL/6) bearing LLC tumors. The treatment scheme is shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;5E.\u003c/b\u003e IMMU-132 combined with XRT significantly reduced tumor growth as compared to XRT and IMMU-132 alone \u003cb\u003e(Supplementary Fig.\u0026nbsp;5F)\u003c/b\u003e. Moreover, increased survival was observed in the IMMU-132\u0026thinsp;+\u0026thinsp;XRT group compared to the other groups \u003cb\u003e(Supplementary Fig.\u0026nbsp;5G)\u003c/b\u003e. The body weights of the mice were stable among all the groups \u003cb\u003e(Supplementary Fig.\u0026nbsp;5H)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003ePathway analysis was performed on resected tumors to understand the molecular mechanisms underlying the IMMU-132-induced radiosensitivity \u003cem\u003ein vivo\u003c/em\u003e. We found reduced protein levels of DNA-PK, RAD51, and anti-apoptotic proteins such as p-BCL-2, BCL-XL, p-BAD, and XIAP, as well as high levels of apoptotic proteins, including phospho-p38, c-caspase-3, and c-PARP, in both H441 and H460 tumors treated with IMMU-132 plus XRT compared to IMMU-132, XRT alone, and untreated controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. The PI3K/AKT and RAS/RAF/MEK/ERK pathways have been shown to induce cell survival responses to radiation. We found reduced phosphorylation of AKT, MEK1/2, ERK1/2, mTOR, SAPK/JNK, and downregulation of PIK3C2B (Anti-Phosphoinositide-3-kinase, class 2, β polypeptide) and cyclic adenosine monophosphate-responsive element-binding protein (CREB) transcription factor in IMMU-132\u0026thinsp;+\u0026thinsp;XRT groups as compared to XRT alone, IMMU-132, and the sham group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Furthermore, the ratio of phospho-protein to total protein was lower in IMMU-132\u0026thinsp;+\u0026thinsp;XRT compared to other groups. Moreover, phosphatase and tensin homolog (PTEN), which inhibits PI3K/AKT signaling, was found to be upregulated in H441 and H460 cells treated with the IMMU-132\u0026thinsp;+\u0026thinsp;XRT group compared to the IMMU-132, XRT alone, and untreated groups.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIMMU-132 (also called sacituzumab govitecan) is approved for the treatment of triple-negative metastatic breast cancer and metastatic urothelial cancer\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In this study, we demonstrated that IMMU-132 enhances the radiosensitivity of NSCLC both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. These findings provide compelling evidence for the therapeutic potential of combining IMMU-132 with RT, particularly in TROP2-expressing NSCLC.\u003c/p\u003e\u003cp\u003eWe observed TROP2 surface expressions in H441, H460, A549, and LLC. Among these, H441 showed high expression, while the remaining three cell lines displayed low expression of TROP2. Based on TROP2 expression, we selected a high TROP2-expressing H441 cell line and low TROP2-expressing cell lines (H460, A549, and LLC) to study the radiosensitization capability of IMMU-132 following radiation therapy. We evaluated radiation-induced upregulation of TROP2 expression and found that XRT indeed induced TROP2 in NSCLC. This observation is consistent with prior studies reporting TROP2 induction under cellular stress conditions. The enhanced TROP2 expression correlated with increased IMMU-132 binding and internalization, suggesting that radiation improves ADC uptake by increasing antigen expression, a mechanism we have previously reported for other antigens\u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe found a dose-dependent cytotoxicity effect of IMMU-132 on NSCLC cell lines, which correlated with the antigen expression. Studies have shown that SN-38, the cytotoxic component of IMMU-132, is a radiation-sensitizing anticancer agent\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, the non-targeted nature of the free drug results in inadvertent side effects, limiting its dose. Thus, we postulated that the targeted delivery of SN-38 via IMMU-132 may reduce side effects and sensitize lung cancer cells to radiation therapy, especially at sub-therapeutic doses. Our results confirmed that IMMU-132 acts as a radiosensitizer across all NSCLC cells tested, as evidenced by reduced colony formation, synergistic cytotoxicity in clonogenic assays with XRT, and increased DNA damage. Mechanistically, IMMU-132\u0026thinsp;+\u0026thinsp;XRT treatment resulted in G2/M phase arrest, which is known to be the most radiosensitive phase of the cell cycle, thereby contributing to the radiosensitizing effect. SN-38 arrests cell cycle in the G2/M phase, contributing the therapeutic effect\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe observed increased markers of DNA damage (γH2AX) and apoptosis (annexin V/PI, cleaved PARP, caspase-3) in cells treated with IMMU-132 plus radiation, indicating that the combination amplifies radiation-induced cytotoxicity by disrupting DNA repair and activating apoptotic cascades. Mechanistically, this effect was associated with a reduction in DNA repair mediators RAD51 and DNA-PKcs, which are essential for homologous recombination and non-homologous end joining, respectively\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Concurrently, we detected heightened activation of ATM and ATR kinases, upstream regulators of DNA damage response that phosphorylate downstream substrates such as p53, thereby enforcing G2/M arrest and promoting apoptotic signaling\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The inability of NSCLC cells to sustain RAD51- and DNA-PKcs\u0026ndash;mediated repair in the context of IMMU-132 plus radiation underscores a critical vulnerability in the DNA repair machinery that facilitates apoptotic progression.\u003c/p\u003e\u003cp\u003eIn parallel, we identified suppression of major survival signaling networks, including PI3K/AKT/mTOR and MEK/ERK, which are typically induced following radiation\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e to promote DNA repair, cell survival, and proliferation. Notably, the dual suppression of PI3K/AKT and MEK/ERK signaling by IMMU-132 in combination with XRT is likely to overcome the compensatory crosstalk between these pathways, a known mechanism of resistance in KRAS-mutated NSCLC cell lines such as H460 and A549\u003csup\u003e58\u003c/sup\u003e. Downregulation of anti-apoptotic proteins (p-BCL2, XIAP, BCL-XL, and p-BAD) further tips the balance toward apoptosis, reinforcing the mechanistic link between impaired repair capacity and diminished survival signaling. Together, these molecular alterations delineate a coordinated mechanism whereby IMMU-132 sensitizes NSCLC cells to radiation: by simultaneously disabling DNA repair pathways and suppressing compensatory pro-survival signaling, thereby committing cells to apoptosis.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e, the combination of ADC\u0026thinsp;+\u0026thinsp;XRT significantly reduced tumor growth and improved survival in both xenograft (H441, H460) and syngeneic (LLC) mouse models, supporting the translational potential of this approach. The differential responses to IMMU-132 monotherapy among all cell lines reflected their TROP2 expression levels, reaffirming the importance of TROP2 as a predictive biomarker for ADC efficacy. Importantly, treatment was well tolerated, with no significant weight loss in mice, indicating an acceptable therapeutic index.\u003c/p\u003e\u003cp\u003eMechanistically, ERK1/2 activation following radiation is known to induce the G2/M DNA damage checkpoint via ATR and to facilitate NHEJ-mediated repair through DNA-PK\u003csup\u003e59\u0026ndash;62\u003c/sup\u003e. ERK1/2 also promotes survival by activating CREB, upregulating anti-apoptotic proteins (Bcl-xL), and suppressing pro-apoptotic proteins (Bim, Bad, caspase-9)\u003csup\u003e68\u0026ndash;71\u003c/sup\u003e. Pharmacologic inhibition of ERK1/2 or upstream EGFR/MEK signaling reverses these effects, inducing Bim expression and enhancing apoptosis in NSCLC\u003csup\u003e73\u003c/sup\u003e. Consistent with this, our data suggest that IMMU-132 inhibits MEK/ERK signaling in the context of radiation, thereby radiosensitizing NSCLC cells by diminishing DNA repair capacity, reducing the expression of anti-apoptotic proteins, and preventing phosphorylation-mediated inactivation of pro-apoptotic mediators.\u003c/p\u003e\u003cp\u003eSimilarly, PI3K/AKT/mTOR signaling\u0026mdash;frequently activated after radiation\u0026mdash;plays a central role in survival and radioresistance. PTEN loss leads to aberrant Akt activation, which phosphorylates downstream effectors such as mTOR and Bad to drive growth and inhibit apoptosis\u003csup\u003e74\u003c/sup\u003e. Stress-activated MAPK family members, including JNK, ERK, and p38, further contribute to survival by phosphorylating and inactivating Bad\u003csup\u003e7677\u003c/sup\u003e. Notably, combined inhibition of MEK and mTOR has been shown to induce DNA damage, prolong G2/M arrest, and sensitize NSCLC xenografts to radiotherapy\u003csup\u003e78\u003c/sup\u003e. Our results align with these observations, demonstrating that IMMU-132 suppresses both PI3K/AKT/mTOR and MEK/ERK signaling, thereby abrogating redundant pro-survival pathways.\u003c/p\u003e\u003cp\u003eTaken together, our findings highlight the promise of IMMU-132 as a potent radiosensitizer in NSCLC through TROP2-mediated targeted delivery of SN-38. The combination with radiation therapy not only enhances DNA damage and apoptosis but also abrogates key survival and DNA repair pathways, particularly in TROP2-high tumors. These results provide a strong rationale for clinical evaluation of IMMU-132 in combination with radiotherapy for NSCLC, potentially offering an effective strategy to overcome radioresistance and improve patient outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of interest:\u003c/h2\u003e\u003cp\u003eRon Bose reports a research grant from Puma Biotechnology and Consulting for Genentech. All other authors have no conflict.\u003c/p\u003e\u003ch2\u003eAuthor contributions:\u003c/h2\u003e\u003cp\u003eAKS and VK developed the hypothesis, designed the studies, analyzed the data, wrote the manuscript, provided funding, and supervised the project. HS and SP designed the studies, performed experiments, analyzed data, and wrote the manuscript. RB offered helpful advice and reviewed the manuscript. All authors reviewed the manuscript and contributed to discussions.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\u003cp\u003eThis study was supported by startup funds, the Elsa U Pardee Foundation grant, and in part by the Center for Drug Discovery, Washington University in Saint Louis, to VK. We thank Alison Clay for her technical support with the studies and Amanda Klaas for the tail vein injections in mice.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A (2022) Cancer statistics, 2022. CA Cancer J Clin 72:7\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3322/caac.21708\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3322/caac.21708\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSung H et al (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209\u0026ndash;249. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3322/caac.21660\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3322/caac.21660\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMolina JR, Yang P, Cassivi SD, Schild SE, Adjei AA (2008) Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 83:584\u0026ndash;594. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.4065/83.5.584\u003c/span\u003e\u003cspan address=\"https://doi.org:10.4065/83.5.584\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAntonia SJ et al (2018) Overall Survival with Durvalumab after Chemoradiotherapy in Stage III NSCLC. New Engl J Med 379:2342\u0026ndash;2350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1056/NEJMoa1809697\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1056/NEJMoa1809697\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaben D et al (2018) Overall Survival with Durvalumab versus Placebo after Chemoradiotherapy in Stage III NSCLC. Int J Radiat Oncol 102:1607\u0026ndash;1608. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:DOI\u003c/span\u003e\u003cspan address=\"https://doi.org:DOI\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijrobp.2018.08.057\u003c/span\u003e\u003cspan address=\"10.1016/j.ijrobp.2018.08.057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpigel DR et al (2021) Five-year survival outcomes with durvalumab after chemoradiotherapy in unresectable stage III NSCLC: An update from the PACIFIC trial. J Clin Oncol 39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1200/JCO.2021.39.15_suppl.8511\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1200/JCO.2021.39.15_suppl.8511\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang R-X, Zhou P-K (2020) DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer. Signal Transduct Target therapy 5:60\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarampon F, Ciccarelli C, Zani BM (2019) Biological rationale for targeting MEK/ERK pathways in anti-cancer therapy and to potentiate tumour responses to radiation. Int J Mol Sci 20:2530\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin SH et al (2014) A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer. J Thorac Oncol 9:965\u0026ndash;973. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1097/jto.0000000000000199\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1097/jto.0000000000000199\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen Y et al (2019) mTORC1 inhibitor RAD001 (everolimus) enhances non-small cell lung cancer cell radiosensitivity in vitro via suppressing epithelial\u0026ndash;mesenchymal transition. Acta Pharmacol Sin 40:1085\u0026ndash;1094\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchuurbiers OC et al (2009) The PI3-K/AKT-pathway and radiation resistance mechanisms in non-small cell lung cancer. J Thorac Oncol 4:761\u0026ndash;767\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShannon AM et al (2009) The mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) enhances the radiation responsiveness of lung and colorectal tumor xenografts. Clin Cancer Res 15:6619\u0026ndash;6629\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrognard J, Clark AS, Ni Y, Dennis PA (2001) Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res 61:3986\u0026ndash;3997\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J (2020) Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res 18:3\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1158/1541-7786.MCR-19-0582\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1158/1541-7786.MCR-19-0582\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChau CH, Steeg PS, Figg WD (2019) Antibody-drug conjugates for cancer. Lancet 394:793\u0026ndash;804. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/S0140-6736(19)31774-X\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/S0140-6736(19)31774-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMehta GU et al (2024) FDA approval summary: fam-trastuzumab deruxtecan-nxki for unresectable or metastatic non-small cell lung cancer with activating HER2 mutations. Oncologist 29:667\u0026ndash;671. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1093/oncolo/oyae151\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1093/oncolo/oyae151\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQiu S et al (2023) Targeting Trop-2 in cancer: Recent research progress and clinical application. Biochim Biophys Acta Rev Cancer 1878:188902. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.bbcan.2023.188902\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.bbcan.2023.188902\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBelluomini L et al (2023) Antibody-drug conjugates (ADCs) targeting TROP-2 in lung cancer. Expert Opin Biol Ther 23:1077\u0026ndash;1087. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1080/14712598.2023.2198087\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1080/14712598.2023.2198087\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCubas R, Zhang S, Li M, Chen C, Yao Q (2010) Trop2 expression contributes to tumor pathogenesis by activating the ERK MAPK pathway. Mol Cancer 9:1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuerra E et al (2013) The Trop-2 signalling network in cancer growth. Oncogene 32:1594\u0026ndash;1600\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeist RS et al (2017) Therapy of Advanced Non-Small-Cell Lung Cancer With an SN-38-Anti-Trop-2 Drug Conjugate, Sacituzumab Govitecan. J Clin Oncol 35:2790\u0026ndash;2797. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1200/jco.2016.72.1894\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1200/jco.2016.72.1894\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang J et al (2011) Loss of Trop2 promotes carcinogenesis and features of epithelial to mesenchymal transition in squamous cell carcinoma. Mol Cancer Res 9:1686\u0026ndash;1695\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePak MG, Shin DH, Lee CH, Lee MK (2012) Significance of EpCAM and TROP2 expression in non-small cell lung cancer. World J Surg Oncol 10:53. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1186/1477-7819-10-53\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1186/1477-7819-10-53\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Z, Jiang X, Zhang W (2016) TROP2 overexpression promotes proliferation and invasion of lung adenocarcinoma cells. Biochem Biophys Res Commun 470:197\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2016.01.032\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2016.01.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org:\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShastry M, Jacob S, Rugo HS, Hamilton E (2022) Antibody-drug conjugates targeting TROP-2: Clinical development in metastatic breast cancer. Breast 66:169\u0026ndash;177. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.breast.2022.10.007\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.breast.2022.10.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWhitacre CM, Zborowska E, Willson JK, Berger NA (1999) Detection of poly (ADP-ribose) polymerase cleavage in response to treatment with topoisomerase I inhibitors: a potential surrogate end point to assess treatment effectiveness. Clin Cancer Res 5:665\u0026ndash;672\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLagadec P et al (2008) Pharmacological targeting of NF-κB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells. Br J Cancer 98:335\u0026ndash;344\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu Y et al (2009) Inhibition of Akt signaling by SN-38 induces apoptosis in cervical cancer. Cancer Lett 274:47\u0026ndash;53\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCusack JC Jr et al (2001) Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: implications for systemic nuclear factor-κB inhibition. Cancer Res 61:3535\u0026ndash;3540\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei L et al (2022) Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells. Biomed Environ Sci 35:419\u0026ndash;436. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3967/bes2022.058\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3967/bes2022.058\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGewirtz DA (2000) Growth arrest and cell death in the breast tumor cell in response to ionizing radiation and chemotherapeutic agents which induce DNA damage. Breast Cancer Res Treat 62:223\u0026ndash;235. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1023/a:1006414422919\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1023/a:1006414422919\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOuellette MM, Yan Y (2019) Radiation-activated prosurvival signaling pathways in cancer cells. Precision Radiation Oncol 3:111\u0026ndash;120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pro6.1076\u003c/span\u003e\u003cspan address=\"10.1002/pro6.1076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org:\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eO'Connell MJ, Cimprich KA (2005) G2 damage checkpoints: what is the turn-on? J Cell Sci 118:1\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1242/jcs.01626\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1242/jcs.01626\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLewis CD et al (2021) Targeting a Radiosensitizing Antibody-Drug Conjugate to a Radiation-Inducible Antigen. Clin Cancer Res 27:3224\u0026ndash;3233. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1158/1078-0432.CCR-20-1725\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1158/1078-0432.CCR-20-1725\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Feraudy S et al (2007) Pol η is required for DNA replication during nucleotide deprivation by hydroxyurea. Oncogene 26:5713\u0026ndash;5721. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/sj.onc.1210385\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/sj.onc.1210385\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh AK et al (2023) Blocking the functional domain of TIP1 by antibodies sensitizes cancer to radiation therapy. Biomed Pharmacother 166:115341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2023.115341\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2023.115341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org:\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCardillo TM et al (2020) Predictive biomarkers for sacituzumab govitecan efficacy in Trop-2-expressing triple-negative breast cancer. Oncotarget 11:3849\u0026ndash;3862. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.18632/oncotarget.27766\u003c/span\u003e\u003cspan address=\"https://doi.org:10.18632/oncotarget.27766\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSteelman LS et al (2011) Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy-implications for cancer and aging. Aging 3:192\u0026ndash;222. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.18632/aging.100296\u003c/span\u003e\u003cspan address=\"https://doi.org:10.18632/aging.100296\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe Y et al (2021) Targeting PI3K/Akt signal transduction for cancer therapy. Signal Transduct Target Therapy 6:425. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/s41392-021-00828-5\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/s41392-021-00828-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcCubrey JA et al (2007) Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim et Biophys Acta (BBA) - Mol Cell Res 1773:1263\u0026ndash;1284. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbamcr.2006.10.001\u003c/span\u003e\u003cspan address=\"10.1016/j.bbamcr.2006.10.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org:\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen J (2016) The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. \u003cem\u003eCold Spring Harb Perspect Med\u003c/em\u003e 6, a026104 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1101/cshperspect.a026104\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1101/cshperspect.a026104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMah LJ, El-Osta A, Karagiannis TC (2010) γH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 24:679\u0026ndash;686. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/leu.2010.6\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/leu.2010.6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharma A, Singh K, Almasan A (2012) Histone H2AX phosphorylation: a marker for DNA damage. Methods Mol Biol 920:613\u0026ndash;626. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/978-1-61779-998-3_40\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/978-1-61779-998-3_40\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDadey DYA et al (2017) Antibody Targeting GRP78 Enhances the Efficacy of Radiation Therapy in Human Glioblastoma and Non-Small Cell Lung Cancer Cell Lines and Tumor Models. Clin Cancer Res 23:2556\u0026ndash;2564. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1158/1078-0432.CCR-16-1935\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1158/1078-0432.CCR-16-1935\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLewis CD et al (2021) Targeting a Radiosensitizing Antibody\u0026ndash;Drug Conjugate to a Radiation-Inducible Antigen. Clin Cancer Res 27:3224\u0026ndash;3233. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1158/1078-0432.Ccr-20-1725\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1158/1078-0432.Ccr-20-1725\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh AK et al (2023) Blocking the functional domain of TIP1 by antibodies sensitizes cancer to radiation therapy. Biomed Pharmacother 166:115341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.biopha.2023.115341\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.biopha.2023.115341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh AK et al (2024) Development of a [89Zr]Zr-labeled Human Antibody using a Novel Phage-displayed Human scFv Library. Clin Cancer Res 30:1293\u0026ndash;1306. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1158/1078-0432.CCR-23-3647\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1158/1078-0432.CCR-23-3647\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSasai K et al (1998) Effects of SN-38 (an active metabolite of CPT-11) on responses of human and rodent cells to irradiation. Int J Radiation Oncology*Biology*Physics 42:785\u0026ndash;788. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0360-3016(98)00326-5\u003c/span\u003e\u003cspan address=\"10.1016/S0360-3016(98)00326-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org:\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTamura K et al (1997) Enhancement of Tumor Radio-response by Irinotecan in Human Lung Tumor Xenografts. Jpn J Cancer Res 88:218\u0026ndash;223. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1349-7006.1997.tb00369.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1349-7006.1997.tb00369.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org:\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOKUNO T et al (2018) SN-38 Acts as a Radiosensitizer for Colorectal Cancer by Inhibiting the Radiation-induced Up-regulation of HIF-1α. Anticancer Res 38:3323\u0026ndash;3331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.21873/anticanres.12598\u003c/span\u003e\u003cspan address=\"https://doi.org:10.21873/anticanres.12598\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun X, Wu L, Du L, Xu W, Han M (2024) Targeting the organelle for radiosensitization in cancer radiotherapy. Asian J Pharm Sci 19:100903. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:https://doi.org/10.1016/j.ajps.2024.100903\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.ajps.2024.100903\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaleigh DR, Haas-Kogan DA (2013) Molecular targets and mechanisms of radiosensitization using DNA damage response pathways. Future Oncol 9:219\u0026ndash;233. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.2217/fon.12.185\u003c/span\u003e\u003cspan address=\"https://doi.org:10.2217/fon.12.185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSch\u0026uuml;rmann L, Schumacher L, Roquette K, Brozovic A, Fritz G (2021) Inhibition of the DSB repair protein RAD51 potentiates the cytotoxic efficacy of doxorubicin via promoting apoptosis-related death pathways. Cancer Lett 520:361\u0026ndash;373. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:https://doi.org/10.1016/j.canlet.2021.08.006\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.canlet.2021.08.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFedak EA, Adler FR, Abegglen LM, Schiffman JD (2021) ATM and ATR Activation Through Crosstalk Between DNA Damage Response Pathways. Bull Math Biol 83:38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s11538-021-00868-6\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s11538-021-00868-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReinhardt HC, Aslanian AS, Lees JA, Yaffe MB (2007) p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11:175\u0026ndash;189. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.ccr.2006.11.024\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.ccr.2006.11.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHong E-H et al (2010) Ionizing radiation induces cellular senescence of articular chondrocytes via negative regulation of SIRT1 by p38 kinase. J Biol Chem 285:1283\u0026ndash;1295\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eValerie K (2007) Radiation?induced cell signaling: inside?out and outside?in. Mol Cancer Ther 6:789\u0026ndash;801\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu H, Tan LY, Xie S, He XX, Luo J, Wang F (2020) Anlotinib Exerts Anti-Cancer Effects on KRAS-Mutated Lung Cancer Cell Through Suppressing the MEK/ERK Pathway. Dove Press 2020:12 3579\u0026ndash;3587. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:https://doi.org/10.2147/CMAR.S243660\u003c/span\u003e\u003cspan address=\"https://doi.org:10.2147/CMAR.S243660\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbbott DW (1999) Mitogen?activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation. J Biol Chem 274:2732\u0026ndash;2742\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYan Y, Irradiation?induced (2007) G2/M checkpoint response requires ERK1/2 activation. Oncogene 26:4689\u0026ndash;4698\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei F (2013) Inhibition of ERK activation enhances the repair of double?stranded breaks via non?homologous end joining by increasing DNA?PKcs activation. Biochim Biophys Acta 1833:90\u0026ndash;100\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCohen?Armon M (2007) PARP?1 activation in the ERK signaling pathway. Trends Pharmacol Sci (Regular ed) 28:556\u0026ndash;560\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVivanco I et al (2010) The phosphatase and tensin homolog regulates epidermal growth factor receptor (EGFR) inhibitor response by targeting EGFR for degradation. Proc Natl Acad Sci U S A 107:6459\u0026ndash;6464. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1073/pnas.0911188107\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1073/pnas.0911188107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVivanco I, Sawyers CL (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2:489\u0026ndash;501. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/nrc839\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/nrc839\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeston CR, Davis RJ (2007) The JNK signal transduction pathway. Curr Opin Cell Biol 19:142\u0026ndash;149. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.ceb.2007.02.001\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.ceb.2007.02.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu C et al (2004) JNK suppresses apoptosis via phosphorylation of the proapoptotic Bcl-2 family protein BAD. Mol Cell 13:329\u0026ndash;340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/s1097-2765(04)00028-0\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/s1097-2765(04)00028-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKIM SY, JEONG E-H, KIM LEET-G, H.-R., KIM CH (2021) The Combination of Trametinib, a MEK Inhibitor, and Temsirolimus, an mTOR Inhibitor, Radiosensitizes Lung Cancer Cells. Anticancer Res 41:2885\u0026ndash;2894. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.21873/anticanres.15070\u003c/span\u003e\u003cspan address=\"https://doi.org:10.21873/anticanres.15070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng J et al (2010) Apoptosis Induction by MEK Inhibition in Human Lung Cancer Cells Is Mediated by Bim. PLoS ONE 5:e13026. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1371/journal.pone.0013026\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1371/journal.pone.0013026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TROP2, Radiation, Lung cancer, Radiation sensitization","lastPublishedDoi":"10.21203/rs.3.rs-7593735/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7593735/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStudies suggest that the human trophoblast cell-surface antigen (TROP2) is highly expressed in most lung cancers and is associated with poor prognosis. Currently, there are no TROP2-directed ADCs approved for treating lung cancer patients. IMMU-132 ADC (Sacituzumab govitecan) is a TROP-2-directed ADC recently approved for metastatic triple-negative breast cancer and urothelial cancer. However, its role in non-small cell carcinoma (NSCLC) has not been explored. Here, we examined the impact of IMMU-132 alone and in combination with radiation on NSCLC cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eWe found cell surface expression of TROP-2 on NSCLC cell lines, internalization of IMMU-132, induction of cell cycle arrest at the G2/M phase, and promotion of programmed cell death (apoptosis) following irradiation. Furthermore, IMMU-132 enhanced radiosensitivity by decreasing clonogenic survival through increased DNA double-strand break formation (as indicated by the γH2AX level), modulating DNA damage repair, inhibiting survival pathways, and inducing PARP-mediated apoptosis. \u003cem\u003eIn vivo\u003c/em\u003e, the combination of IMMU-132 and radiation therapy increased tumor control and improved overall survival in mice bearing H441 and H460 cell xenografts, as well as in the syngeneic LLC tumor. Tumor radio-sensitization with IMMU-132 promotes the inhibition of prosurvival signaling (PI3K/AKT/mTOR/, MEK/ERK, p38MAPK/JNK) together with induced apoptosis by increasing PARP, cleaved caspase-3 and reducing anti-apoptotic proteins (BCL-xL, BCL-2, XIAP) as well as modulating DNA damage repair (ATM, ATR, RAD51, p53, DNA-PK).\u003c/p\u003e\u003cp\u003eTogether, our data suggests that the targeted delivery of IMMU-132 radiosensitizer at sub-therapeutic doses could broaden the therapeutic window of radiation therapy in lung cancer and may decrease the possibility of side effects. Hence, the combination of IMMU-132 and radiation therapy could be a promising therapeutic strategy for NSCLC.\u003c/p\u003e","manuscriptTitle":"The TROP2 targeting antibody-drug conjugate IMMU-132 enhances the efficacy of radiation therapy for lung cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 07:49:56","doi":"10.21203/rs.3.rs-7593735/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"transferred","content":"Cell Death \u0026 Disease","date":"2025-10-01T16:36:50+00:00","index":"","fulltext":""},{"type":"decision","content":"Reject before peer review","date":"2025-09-25T14:56:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-15T13:36:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T21:13:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Nature Communications","date":"2025-09-11T16:19:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fc6e3acd-7a8b-4bb7-a87f-655f73363830","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54742983,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Targeted therapies"},{"id":54742984,"name":"Biological sciences/Cancer/Lung cancer/Non-small-cell lung cancer"},{"id":54742985,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Radiotherapy"}],"tags":[],"updatedAt":"2025-10-22T09:20:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 07:49:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7593735","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7593735","identity":"rs-7593735","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}