Radio-sensitization of mutated KRAS G12C non-small cell lung cancer with KRAS G12C tyrosine kinase inhibitor and Carbon ions irradiation

Radio-sensitization of mutated KRAS G12C non-small cell lung cancer with KRAS G12C tyrosine kinase inhibitor and Carbon ions irradiation | 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 Research Article Radio-sensitization of mutated KRAS G12C non-small cell lung cancer with KRAS G12C tyrosine kinase inhibitor and Carbon ions irradiation Mathieu Césaire, Kilian Lecrosnier, Juliette Montanari, Mateusz Sitarz, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9469593/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Non-small cell lung cancer (NSCLC) with KRAS G12C mutation is associated with poor prognosis and resistance to standard therapies. Sotorasib, a KRAS G12C tyrosine kinase inhibitor, has shown clinical efficacy, yet its interaction with different irradiation modalities remains unclear. This study aimed to evaluate the potential radiosensitizing effect of Sotorasib in such mutated cells exposed to X-rays or carbon-ion (C-ions) irradiation. Two human NSCLC cell lines (H358, KRAS G12C ; and A549, KRAS G12S ) were treated with Sotorasib alone or in combination with X-rays or C-ions irradiation (1–8 Gy). Cell viability, clonogenic survival, and sphere-forming ability were assessed. Cytokines secretion and mRNA expression of cancer stem cells and epithelial-mesenchymal transition (EMT) -associated markers were analyzed 24 h post-treatments. Sotorasib alone specifically reduced the clonogenic capacity of H358 cells to ~ 30% while no effect was observed with A549 cells. Interestingly, Sotorasib combined with X-ray irradiation decreased clonogenic survival compared with either treatment alone, without evidence of synergy. In contrast, a synergistic effect was observed with C-ions in H358 cells (β = −0.36, 95% CI [− 0.52 to − 0.20], p < 0.001; enhancement ratio = 4.21 at SF2). C-ions irradiation alone markedly reduced sphere formation in both cell lines, whereas sotorasib showed no additional impact. GM-CSF expression was downregulated with sotorasib alone while IP-10 was upregulated when H358 cells were irradiated with sotorasib, suggesting an immune-specific modulatory response. A specific radiosensitization of KRAS G12C –mutated NSCLC cells was observed when associating Sotorasib with C-ions. This represents the first evidence of enhanced cytotoxicity from combining KRAS G12C inhibition with high-LET radiation. These findings support further in vivo and translational studies exploring C-ions therapy as a promising strategy for KRAS G12C –mutated NSCLC. Non-small cell lung cancer KRAS inhibitor Carbon ions irradiation cancer stem cell Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Non-small cell lung cancer (NSCLC) is the most common lung cancer (80% of lung cancers). Locally advanced stage III NSCLC represent approximately 30% of NSCLC with a significant number of inoperable cancers. The prognosis of these locally advanced inoperable cancers remains poor with a 5-year survival of less than 50% [ 1 ]. The KRAS (Kirsten rat sarcoma viral oncogene homologue) proto-oncogene encodes a small GTPase protein that transmits growth and differentiation signals from the cell surface to the nucleus by cycling between an active GTP-bound state and an inactive GDP-bound state. Mutated forms of KRAS remains permanently active by staying bound to GTP and continuously send signals that promote uncontrolled cell growth. KRAS mutated inoperable stage III NSLC have been reported to be more resistant to chemoradiotherapy than non KRAS mutated [ 2 ]. Furthermore, data obtained from resected NSCLC found that KRAS G12C mutation was associated with worst survival [ 3 ]. Recently, tyrosine kinase inhibitors (TKI) targeting the KRAS G12C mutation have been reported to promote good responses in metastatic KRAS G12C mutated NSCLC [ 4 – 6 ]. G12C is a single point mutation with a Glycine-to-Cysteine substitution at codon 12 of the exon 2. This substitution favors the activated state of KRAS, resulting in a predominantly GTP-bound KRAS oncoprotein, amplifying signaling pathways that lead to oncogenesis. AMG-510, also called Sotorasib, is a KRAS G12C TKI that inhibits specifically and irreversibly KRAS G12C mutation by trapping the mutated protein in the inactive GDP-bound state [ 7 ]. Treatment with Sotorasib resulted in a response rate of approximately 40% in patients with KRAS G12C mutated NSCLC in a phase II clinical trial [ 5 ]. KRAS G12C inhibitors in association with chemoradiotherapy may represent a good strategy to improve response and survival in KRAS G12C mutated inoperable stage III NSCLC patients, indeed, KRAS mutated A549 cells were radiosentized by inhibiting mutated KRAS G12S expression with siRNA [ 8 ]. KRAS mutations contribute also to the establishment of an immunosuppressive tumor microenvironment by limiting effective T-cell infiltration and promoting the recruitment of suppressive immune populations [ 9 ]. Combining X-rays irradiation and KRAS pathways inhibitors such as MEK inhibitors [ 10 ] or KRAS inhibitors like Adagrasib [ 11 ] can reverse this immune suppressive effect of KRAS mutations and promote a pro inflammatory, anti-tumor phenotype. Another radioresistance mechanism of tumors may be the presence of cancer stem cells (CSCs). CSCs are a subpopulation of dedifferentiated cancer cells with self-renewal capabilities and increased resistance to DNA damage, apoptosis, and oxidative stress [ 12 ]. CSCs are identified by specific stemness markers overexpressed in lung cancer such as CD166 [ 13 ] in adenocarcinoma lung cells. X-rays irradiation can up-regulate CSCs markers in lung cancer cells [ 14 – 16 ] leading to radioresistant phenotype selection. CSCs are mainly in hypoxic niches that could also explain this radioresistance effect [ 17 , 18 ]. Cancer cells can acquire stem-like properties through epithelial–mesenchymal transition (EMT) [ 19 ]. EMT confers mesenchymal characteristics to epithelial cancer cells, including loss of polarity, downregulation of epithelial markers such as E-cadherin, upregulation of Nestin [ 20 ] and Vimetin [ 21 ], acquisition of enhanced migratory and invasive capacities [ 22 ]. EMT facilitates metastasis and tumor recurrence by enabling mesenchymal CSCs to disseminate to distant sites, where they can revert to an epithelial phenotype and initiate metastatic growth [ 23 ]. Vimentin, a key mesenchymal marker, is frequently upregulated in NSCLC, particularly in response to X-ray irradiation [ 14 , 24 ]. Nestin is also upregulated in NSCLC [ 20 , 25 ] and has been implicated in the regulation of redox homeostasis [ 26 ], a poorer tumor differentiation and an increased cancer cell proliferation [ 13 ]. Carbon-ions (C-ions) radiotherapy represents a promising modality in the management of radioresistant tumors due to its unique physical and biological properties. Owing to their high linear energy transfer (LET), C-ions deposit dense ionizations in the tissue at the end of the ion range (Bragg peak). This dense ionization produces clustered and complex DNA lesions that are difficult to repair, enhancing cell-killing efficacy compared with sparsely ionizing photons. The resulting increase in relative biological effectiveness (RBE), generally ranges from 1.5 to 3 for CSC depending on tissue type, dose, and LET distribution [ 27 , 28 ]. In conventional X-rays irradiation, indirect DNA damage through reactive oxygen species generated by water radiolysis is predominant, whereas high-LET radiation primarily induces direct DNA damage. Unlike X-ray irradiation, the cytotoxicity of C-ions is largely preserved under hypoxia due to their reduced dependence on oxygen conditions [ 29 ]. C-ions irradiation have been reported to be more effective on lung cancer cells in hypoxic niches [ 30 , 31 ] and might be more effective on CSC. The combination of high RBE, low oxygen enhancement ratio, and the induction of complex, irreparable DNA damage provides a strong rationale for the use of C-ions radiotherapy in hypoxic niches enriched with CSCs, such as in NSCLC [ 32 ]. Japanese clinical series have demonstrated excellent local control (80–93% at 2 years) and favorable toxicity profiles for use of C-ions radiotherapy in NSCLC [ 33 , 34 ]. To investigate how treatment efficiency could be increased by the combination of irradiation and KRAS G12C inhibitor in lung cancers, we conducted an in vitro study to determine the efficacy of combining the KRAS inhibitor Sotorasib in association with different qualities of irradiation (X-rays and C-ions irradiations) in a KRAS G12C mutated lung cancer cell line, target of Sotorasib and in a KRAS G12S mutated lung cancer cell line, used as a control cells. A specific and robust effect was observed when combining Sotorasib and high LET radiation, reducing the clonogenic survival and proliferation of KRAS G12C mutated cells, and inducing an antitumoral immune response. 2 Material and Methods 2.1 Cell culture The human lung adenocarcinoma cell line A549 (Ref CCL-185) and the human bronchioalveolar carcinoma cell line H358 (Ref NCI-H358), were purchased from ATCC (Manassas, VA, USA). A549 cell line harboring KRAS G12S mutation was isolated from the lung tissue of a 58-year-old male with lung cancer. H358 cell line harboring KRAS G12C heterozygous mutation and P53 homozygous deletion was isolated from the bronchiole of a male patient with bronchioalveolar carcinoma. All cell lines were cultured in RPMI medium (Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal calf serum (Merck KGaA, Darmstadt, Germany), 2 mM glutamine (Merck KGaA, Darmstadt, Germany), and 1% antibiotics (penicillin-streptomycin, Merck KGaA, Darmstadt, Germany), at 37°C in a humidified atmosphere with 5% CO 2 . 2.2 KRAS G12C inhibitors Sotorasib (AMG-510) was purchased from MedChemExpress (New Jersey, USA) and used in vitro at 10 nM according to a dose response experiment. Sotorasib in DMSO at 10 mM was diluted to its final concentration with culture medium, and finally added to the cells 24 h before irradiation and left in the cell culture medium for 24 h. Negative control samples were treated with the same DMSO dilutions. 2.3 Irradiations X-rays irradiation was performed using a Pxi XradSmart 225cX irradiator with a tube tension of 225 kV and an intensity of 10 mA, corresponding to a dose rate of 2 Gy/min. For gene expression and cytokine secretion analysis, H358 cells were exposed to X-rays in a Xstrahl-CIX2 cabinet (Xstrahl, United Kingdom), with an energy of 195 kV and a current of 12.5 mA. C-ions irradiations were performed at GANIL (Caen, France), using the IRABAT beam line, with a native 12 C beam of 95 MeV/A in the plateau region before the Bragg peak. A 16.9-mm-thick PMMA was inserted between the exit of the beam and sample holder in order to reach a LET of 73 keV/µm (2 Gy = 1.71 × 10 7 particles/cm 2 ), as previously described [ 35 ]. 2.4 Western blot Following the irradiation and treatment (48 h after irradiation), the cells were washed with PBS, and the cell pellet was lysed in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA USA), supplemented with 1X protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA USA), as previously described [ 36 ]. The protein samples were then analyzed by western-blotting (see detailed protocol in Sup data 1 ) using tubuline, ERK and phospho-ERK antibodies. 2.5 Clonogenic assay A549 and H358 cells were irradiated in T25 cm 2 flasks at cell confluency. A sham control was included to evaluate the plating efficiency. At 18–24 h after irradiation, the cells were harvested and re-plated at low density. Medium including Sotorasib or DMSO was changed every 2 to 3 days. Colonies were stained with crystal violet solution (0.3% w/v crystal violet in 20% v/v ethanol). Only colonies that comprised over 50 cells were counted by eye under a stereomicroscope. Clonogenic survival of H358 and A549 cells after irradiation was expressed as a function of dose and was normalized with non-irradiated samples. Surviving fractions of X-rays-irradiated samples were plotted using a linear quadratic and a linear model was used for those after C-ions irradiated samples. Delayed plating was used to avoid confounding effects of Sotorasib on colony formation. Immediate plating under continued drug-induced cell-cycle arrest could artificially reduce clonogenic growth independently of radiation-induced reproductive death. Allowing a recovery period before plating ensures that colony formation reflects true clonogenic survival following the combined treatment. 2.6 Proliferation assay Cell proliferation was evaluated with the CellTrace® Far Red Cell Proliferation Kit (ThermoFisher Scientific, Waltham, MA USA) as previously described [ 37 ]. Experiments with X-rays and C-ions were analyzed with a Gallios flow cytometer (Beckman Coulter, Villepinte, France) at the ISOCELL platform (US PLATON, Caen, France). The data were acquired using Gallios software (Beckman Coulter). Data were analyzed with FCS Express 6+ (DeNovo®), and proliferation index were calculated by the software using the non-divided control as reference. Control and irradiated samples were compared with the corresponding Sotorasib-treated samples at the same irradiation dose to evaluate the radiosensitizing effect, defined as the enhancement factor. 2.7 Tumorspheres assay A549 and H358 cells were plated 24 hours after irradiation at 5,000 cells/mL in 96 wells treated to prevent cell adhesion (Corning Costar Ultra-Low Attachment Multiple Well Plate). The culture medium was DMEM F12 (Sigma- Aldrich, D6421), supplemented with 0.5 µL/mL of βFGF and EGF, 10 µL/mL of PS, glutamine, and 3.6% hydrocortisone and finally 20µL/mL of insulin. The plates were supplemented every 2 or 3 days with DMEM medium at 5 µL/mL of EGF and βFGF. The number of spheres formed (from a cell that successfully proliferated without adhering) was counted after 10 days of culture (spheres with a diameter of more than 50 µm). 2.8 RTq PCR Total RNA extraction from control and treated cells, cDNA synthesis and real time qPCR were performed as previously described [ 38 ]. Sequences of primers used to amplify human Nestin, Vimentin and CD166 transcripts are provided in sup table 1 . 2.9 Cytokines quantification The quantification of secreted cytokine in the supernatant of control and treated H358 cells was performed as previously described [ 39 ]. 2.10 Statistics The statistical analysis for cell proliferation, CSC markers, cytokines, and tumorsphere assays was performed using the statistical software Graph Prism with different statistic tests depending on normal distribution (two-way Anova test, Mann-Whitney U + FDR). Data were considered to be significantly different when p < 0.05. Cell survival fractions data were analyzed using linear mixed models. Cell counts were first normalized to the initial number of seeded cells. For each experimental condition, the surviving fraction was calculated by normalizing to the corresponding 0 Gy control within each experiment. The surviving fraction was log-transformed prior to analysis. The models included treatment, radiation type, and their interaction term as fixed effect, with experiment included as a random intercept to account for between-experiment variability. The interaction term was used to assess departure from additivity between treatment and irradiation. Estimated coefficients are reported with their 95% confidence intervals (95% CI). The surviving fraction at 2 Gy (SF2) was analyzed using a two-way ANOVA with treatment and radiation quality as fixed factors. Enhancement ratios (ER) were calculated at 2 Gy (SF2) by comparing survival fractions between treated and untreated conditions and are reported as descriptive measures of radiosensitization. The dose required to achieve 10% survival (D10) was derived from fitted survival curves. 3 Results 3.1 Sotorasib reduced clonogenic survival in H358 cells In order to select an appropriate concentration of Sotorasib for all experiments, we tested the effects of different concentration of Sotorasib on ERK phosphorylation, a downstream signalization pathway of KRAS by western blot assays. We observed a specific reduction of phospho-ERK, in H358 cells at a concentration of 10 nM ( sup data 1 ). Using this Sotorasib concentration, clonogenic survival in H358 cells was about 30% (p < 0,001) (Fig. 1 A, left ) with a characteristic reduction of clone size. As expected, Sotorasib had no effect on clonogenic behavior in A549 cells (Fig. 1 A, right) . 3.2 Sotorasib enhances the radiosensitivity of H358 cells to carbon ions but not to X-rays Sotorasib (10 nM) reduced the survival fraction of H358 cells with all irradiation doses compared to irradiated cells without drug ( Fig. 1 B, left) , whereas no effect was observed on A549 cells ( Fig. 1 B, right) . In H358 cells, the surviving fraction at 2 Gy (SF2) was significantly reduced when Sotorasib was combined with C-ions irradiation compared with C-ions alone (p < 0.005), with a corresponding enhancement ratio (ER) of 4.21. Statistical analysis confirmed a synergistic interaction between Sotorasib and C-ions (β = −0.36, 95% CI [− 0.52; −0.20], p < 0.001). In contrast, no evidence of a synergistic interaction was observed with X-ray irradiation (β = 0.04, 95% CI [− 0.02; 0.10], p = 0.23), and no significant reduction in SF2 was detected, with a corresponding ER of 1.02 (Table 1 ). Consistently, the dose required to achieve 10% survival (D10) was reduced by factors of 1.09 and 2.4 when Sotorasib was combined with X-rays and carbon ions, respectively. Table 1 Calculated parameters of H358 and A549 cells survival after irradiation with X-rays and C-ions with and without Sotorasib Conditions H358 A589 SF2 a (%) D10 b (Gy) ER (SF2) c ER (D10) d SF2 a (%) D10 b (Gy) ER (SF2) c ER (D10) d X-rays 51 5,8 / / 60 7,2 X-rays + Sotorasib 50 5,3 1,02 1,09 88 7,1 0,68 1,01 C-ions 21,5 3,6 / / 12,7 2,2 C-ions + Sotorasib 5,1* 1,5 4,21 2,4 15,5 2,8 0,82 0,79 a: SF2: surviving fraction (%) at 2 Gy. b: D10: dose required to achieve 10% surviving fraction. c: ER (SF2): enhancement ratio calculated as SF2 (with Sotorasib) / SF2 (without Sotorasib). d: ER (D10): enhancement ratio calculated as D10 (with Sotorasib) / D10 (without Sotorasib). Statistical significance was assessed for SF2 values only. Enhancement ratios (ER) are descriptive and were not statistically tested. * p < 0.05 compared with corresponding irradiation condition without Sotorasib. 3.3 Sotorasib decreased cell proliferation in combination with C-ions irradiation Sotorasib alone reduced the proliferation index of H358 cells by approximately 35% (p < 0.05; Fig. 2 ), while no significant effect was observed in A549 cells. X-ray irradiation alone did not alter the cellular index in H358 cells; however, its combination with Sotorasib further decreased the index by 41–44% at 1, 2, and 4 Gy compared with irradiation alone (p < 0.05). Carbon-ion irradiation alone reduced the cellular index of H358 cells by 26.5%, 64%, and 83% at 1, 2, and 4 Gy, respectively (p < 0.05). When combined with Sotorasib, C-ion irradiation at 1 and 2 Gy produced greater decreases of 67% and 82%, (p < 0.05) with the corresponding enhancement factors of 1.55 at 1 Gy and 1.50 at 2 Gy (p < 0.05). In contrast, the reduced proliferation index observed in C-ions-irradiated A549 cells is not further modulated in the presence of Sotorasib. 3.4 Sotorasib did not impact tumor-spheres formation and CSC markers expression Sphere formation was reduced by X-ray at 1 Gy in H358 and 2 Gy in A549 (Fig. 3 top ). C-ions irradiations reduced the formation of spheres in both A549 and H358 cell lines (Fig. 3 bottom ). The addition of Sotorasib alone or with irradiations did not alter any sphere formation in H358 and A549 cells. This observation was confirmed with the quantification of selected CSC and EMT mRNA markers in H358 cells. Sotorasib alone, X-rays, C-ion and combinations did not affect CD166, Vimentin or Nestin expression in H358 cells ( sup data 2 ). 3.5 Sotorasib induced pro-inflammatory and immune anti-tumor phenotype Secretions of pro- and anti-inflammatory cytokines were measured in H358 cells after exposure to Sotorasib combined with irradiations ( Fig. 4 ). Sotorasib alone showed a trend toward increased IP-10 secretion, a pro-inflammatory cytokine, at 24 hours in H358 cells ( Fig. 4 , A) with a significant upregulation of IP-10 secretion 24 hours after X-ray irradiation at 2 and 4 Gy (p < 0.05), a similar upward trend at 8 Gy and across all doses of carbon-ion irradiation (2–8 Gy). The secretion of GM-CSF, involved in the establishment of an immunosuppressive microenvironment, is reduced by Sotorasib alone ( Fig. 4 , B). C-ions irradiation at 8 Gy significantly upregulated GM-CSF secretion in H358 cells, 24 h post-irradiation (p < 0.05). This effect was reversed when combined with Sotorasib, which led to a significant downregulation of GM-CSF at 8 Gy (p < 0.05). A similar decreasing trend in GM-CSF secretion was observed at all C-ions doses. In contrast, Sotorasib did not affect GM-CSF secretion when combined with X-rays irradiation in H358 cells. In contrast, Sotorasib—either alone or combined with X-rays or C-ions irradiation—did not modify IL-6 secretion 24 hours after irradiation ( Fig. 4 , C). 4 Discussion In our study, Sotorasib in combination with X-ray irradiation reduced clonogenic survival compared with either treatment alone; however, no evidence of a radiosensitizing or synergistic interaction was observed in H358 cells.H358 cells harbor KRAS G12C heterozygote mutation. Consistently, Adagrasib, another KRAS G12C inhibitor, has been shown to enhance sensitivity to X-ray in KRAS G12C homozygous mutant cells (CT26, a murine colorectal carcinoma line) but not in heterozygous mutant cells (LL2, a murine Lewis lung carcinoma line), both in vitro and in mouse models [ 11 ]. In contrast, Sotorasib exhibited a synergistic radio-sensitizing effect in H358 cells when combined with C-ions irradiation. To our knowledge, this is the first report of such an effect in human KRAS G12C –mutated cell lines. While C-ions irradiation alone reduced tumor sphere formation, neither Sotorasib nor X-ray irradiation produced a similar effect. Tumor spheres are cell aggregates that grow under non-adherent conditions, as a surrogate indicator of CSC [ 40 ] and radioresistance [ 16 ]. Sotorasib did not affect the formation of non-adherent spheres, suggesting the persistence of a subpopulation of cells that is not responsive to KRAS G12C inhibition. This observation is consistent with previously reported intrinsic and acquired resistance mechanisms to Sotorasib in NSCLC [ 5 ], including adaptive pathway reactivation and cellular heterogeneity. Such resistant cells may exhibit stem-like properties and contribute to tumorsphere formation, which is commonly used as a surrogate for self-renewal capacity. In this context, the persistence of sphere-forming cells may reflect a reservoir of drug-tolerant or resistant cells that could underlie tumor relapse. The effect of high LET C-ions on tumor spheres suggests that C-ions may target a cancer cell population distinct from that affected by Sotorasib. Nestin, an EMT marker, is overexpressed in NSCLC cells following X-ray irradiation and has been associated with increased resistance to oxidative stress [ 41 ]. Vimentin, another EMT marker, is commonly overexpressed in various lung cancers [ 24 ], while CD166 is frequently expressed in lung adenocarcinomas and is considered a potential CSC marker [ 13 ]. In our study, the expression of these EMT and CSC markers showed high variability (data not shown) and was not significantly changed after treatment with Sotorasib, X-rays, or C-ions irradiation. Further investigations are required to determine which CSC subpopulations might be specifically targeted by C-ions irradiation of H358 cells. Sotorasib has been reported to promote a pro-inflammatory tumor microenvironment in mouse models [ 7 ] and KRAS G12C inhibitors such as Adagrasib, when combined with X-ray irradiation, have been shown to elicit anti-tumor immune responses in vivo [ 11 ]. In our study, Sotorasib induced an overexpression of IP-10 and decreased the expression of GM-CSF. GM-CSF can contribute to tumor progression [ 42 ] while IP-10 expression has been shown to sensitize tumors to immune checkpoint inhibitors by promoting CD8⁺ T-cell recruitment [ 43 ]. Our findings suggest that Sotorasib combined with either X-ray or C-ion irradiation can affect cytokine secretion so that the immune microenvironment is modulated to promote the development of a pro-inflammatory, anti-tumor immune contexture. Further investigations on CSCs, EMT markers, and cytokines are still needed in order to elucidate mechanisms underlying the efficacy of combining irradiation with Sotorasib in NSCLC. 5 Conclusion In this study, we first demonstrated the specific radio-sensitizing effect of combining high LET C-ions irradiation with Sotorasib in a KRAS G12C -mutated NSCLC cell line. This effect was observed despite the heterozygous, rather than homozygous, KRAS mutation in H358 cells. These findings support further investigation of the combination of C-ions and KRAS inhibitors in NSCLC, as they may target distinct cancer cell populations and thereby improve therapeutic responses. Additional in vitro and in vivo studies are warranted to elucidate the underlying mechanisms prior to clinical translation. Declarations 6 Acknowledgements The authors want to thank Clément Rouichi, from IRIG-LCBM laboratory and Marilyne Guillamin from the Isocell Cytometry Platform (Service Unit PLATON, University of Caen Normandie) for their technical support. Finally, the authors want to thank the GANIL and CIMAP-CIRIL facilities staff for C-ions calibration and dosimetry. 7 Funding This work was supported by a Grant of “Ligue contre le Cancer – comité de Seine Maritime”, 2022-2023, « Traitement par inhibiteurs de KRAS en association avec l’irradiation X vs ions carbone dans le cancer bronchique non à petite cellule (CBNPC) avec mutation KRAS », by a Grant of Electricité de France 2023-2025, funding from committee « Protection de l’Homme et de l’Environnement (CPPHE) », under the Life Sciences group of the four-way national agreement CEA-EDF-IRSN-Framatome, “Rôle de KRAS dans la modulation de la radiorésistance et du statut inflammatoire dans le cancer bronchique non à petites cellules” by the LABEX PRIMES (grant number ANR-11-LABX-0063), and by GRAL, a program from the Chemistry Biology Health (CBH) Graduate School of University Grenoble Alpes (grant number ANR-17-EURE-0003). Irradiations with C-ions were performed at GANIL (Caen, France) using beam times obtained under experiment number P1317-H (KRAS Inhibitors treatment in association with X vs hadrons Radiations in non-small cell lung cancer” of the iPAC 2022 call, CIMAP-CIRIL. 8 Conflicts of interest none 9 Ethics declaration : not applicable 10 Authors’ contribution: Mathieu Césaire: Formal analysis, Methodology, Writing – original draft, Writing – review and editing. Kilian Lecrosnier: Formal analysis, Methodology. Juliette Montanari: Formal analysis, Methodology. Mateusz Sitarz: Formal analysis, Methodology, Writing – original draft. Sahra Messaoudi: Methodology, Writing – original draft. Elisabeth Chartier-Garcia: Formal analysis, Methodology. Isabelle Testard: Formal analysis, Methodology, Writing – original draft. Guénaëlle Levallet: Writing – review and editing. Serge Candéias: Formal analysis, Methodology, Writing – original draft, Writing – review and editing. 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09:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9469593/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9469593/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108231446,"identity":"3a5486d2-aac0-43d7-a1fb-39498eb81334","added_by":"auto","created_at":"2026-04-30 17:40:04","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":771222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSotorasib radiosensitized H358 cells in combination with Carbon ions irradiation but not with X-rays irradiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003esurvival fraction of H358 cells (\u003cstrong\u003eleft\u003c/strong\u003e) and A549 cells (\u003cstrong\u003eright\u003c/strong\u003e) after treatment with 10 nM sotorasib.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003e survival fraction (%) of H358 (\u003cstrong\u003eleft in orange\u003c/strong\u003e) A549 (\u003cstrong\u003eright in green\u003c/strong\u003e) cells after irradiation with X-rays (\u003cstrong\u003etop\u003c/strong\u003e) or carbon ions (\u003cstrong\u003ebottom\u003c/strong\u003e) with 10 nM Sotorasib (\u003cstrong\u003edotted line\u003c/strong\u003e) or without sotorasib (\u003cstrong\u003esolid line\u003c/strong\u003e). A red dotted line was added in case of H358, representing the effect of sotorasib without normalization on the non-irradiated control (100 %). Cell survival was represented as mean +/- SD of 3 independent experiments performed in triplicate for X-rays and C-ions. Survival curves were fitted according to the linear quadratic model (X-rays), or fitted and extrapolated according to a linear model (carbon ions).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9469593/v1/63857b871e69cea77a670fc5.jpeg"},{"id":108491657,"identity":"711db7ff-6c23-4a4f-a809-94591c4bda8c","added_by":"auto","created_at":"2026-05-05 09:55:04","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":375295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSotorasib decreased cell proliferation in combination with Carbon ions irradiation\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCell proliferation index of H358 cells (\u003cstrong\u003eleft part\u003c/strong\u003e) and A549 cells (\u003cstrong\u003eright part\u003c/strong\u003e) following irradiation with X-rays (\u003cstrong\u003etop part\u003c/strong\u003e) or C-ions irradiation (\u003cstrong\u003ebottom part\u003c/strong\u003e) with DMSO as control (in orange) or KRASi (in blue). Proliferation indexes (%) are shown as mean +/- SD normalised on control without irradiation either KRASi with 3 independent experiments performed in triplicate. *p\u0026lt;0,05 et ***p\u0026lt;0,001 with a non-parametric two-way ANOVA test.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9469593/v1/f2c597d951fa3be9e37b0cd2.jpeg"},{"id":108231450,"identity":"b2da0490-f691-4d9f-884f-b0451ba6d8b0","added_by":"auto","created_at":"2026-04-30 17:40:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":517203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth of tumor spheres (\u0026gt; 50 μm) from H358 and A549 cells in non-adherent conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor sphere formation of H358 (\u003cstrong\u003eleft part\u003c/strong\u003e) and A549 cells (\u003cstrong\u003eright part\u003c/strong\u003e) following treatment with X-rays (\u003cstrong\u003etop part\u003c/strong\u003e) or C-ions irradiation (\u003cstrong\u003ebottom part\u003c/strong\u003e) with DMSO as control (in orange) or KRASi (in blue). *p\u0026lt;0.05 and ***p\u0026lt;0.001 with a non-parametric two-way ANOVA test, ns = non-significant; black bar = dose-sample comparison; blue bar = KRASi-sample comparison; orange bar = DMSO-sample comparison.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9469593/v1/f22d5b59101d69dc44ba1c26.jpeg"},{"id":108231448,"identity":"03e200ca-47a3-44f4-af45-3a890ec08b51","added_by":"auto","created_at":"2026-04-30 17:40:04","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":479205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative expression of IP10, GM-CSF and IL-6 cytokines at 24h after treatment with Sotorasib and X-rays (left) or C-ions irradiation (right) in H358 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative expressions of IP-10 (A), GM-CSF (B) and IL-6 (C) were expressed as a percentage of non-irradiated controls without Sotorasib. * p\u0026lt;0.05 ; ** p\u0026lt;0.001 with a non-parametric two-way ANOVA test.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9469593/v1/c6854033007d233674caffd5.jpeg"},{"id":108494439,"identity":"b8ace9de-9793-48ab-bc91-4bc6a4609baf","added_by":"auto","created_at":"2026-05-05 10:05:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2474035,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9469593/v1/ce4b516f-72f9-4432-a79f-bf9da6a53caf.pdf"},{"id":108231445,"identity":"1cc4ab59-a149-4f71-986f-6bbf41b17114","added_by":"auto","created_at":"2026-04-30 17:40:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":827996,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9469593/v1/f2ddcbd324710d52db87c47d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Radio-sensitization of mutated KRAS G12C non-small cell lung cancer with KRAS G12C tyrosine kinase inhibitor and Carbon ions irradiation","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eNon-small cell lung cancer (NSCLC) is the most common lung cancer (80% of lung cancers). Locally advanced stage III NSCLC represent approximately 30% of NSCLC with a significant number of inoperable cancers. The prognosis of these locally advanced inoperable cancers remains poor with a 5-year survival of less than 50% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eKRAS\u003c/em\u003e (Kirsten rat sarcoma viral oncogene homologue) proto-oncogene encodes a small GTPase protein that transmits growth and differentiation signals from the cell surface to the nucleus by cycling between an active GTP-bound state and an inactive GDP-bound state. Mutated forms of KRAS remains permanently active by staying bound to GTP and continuously send signals that promote uncontrolled cell growth. KRAS mutated inoperable stage III NSLC have been reported to be more resistant to chemoradiotherapy than non KRAS mutated [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Furthermore, data obtained from resected NSCLC found that \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e mutation was associated with worst survival [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Recently, tyrosine kinase inhibitors (TKI) targeting the KRAS\u003csup\u003eG12C\u003c/sup\u003e mutation have been reported to promote good responses in metastatic KRAS\u003csup\u003eG12C\u003c/sup\u003e mutated NSCLC [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. G12C is a single point mutation with a Glycine-to-Cysteine substitution at codon 12 of the exon 2. This substitution favors the activated state of KRAS, resulting in a predominantly GTP-bound KRAS oncoprotein, amplifying signaling pathways that lead to oncogenesis. AMG-510, also called Sotorasib, is a KRAS\u003csup\u003eG12C\u003c/sup\u003e TKI that inhibits specifically and irreversibly \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e mutation by trapping the mutated protein in the inactive GDP-bound state [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Treatment with Sotorasib resulted in a response rate of approximately 40% in patients with KRAS\u003csup\u003eG12C\u003c/sup\u003e mutated NSCLC in a phase II clinical trial [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. KRAS\u003csup\u003eG12C\u003c/sup\u003e inhibitors in association with chemoradiotherapy may represent a good strategy to improve response and survival in \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e mutated inoperable stage III NSCLC patients, indeed, \u003cem\u003eKRAS\u003c/em\u003e mutated A549 cells were radiosentized by inhibiting mutated \u003cem\u003eKRAS\u003c/em\u003e \u003csup\u003e\u003cem\u003eG12S\u003c/em\u003e\u003c/sup\u003e expression with siRNA [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eKRAS\u003c/em\u003e mutations contribute also to the establishment of an immunosuppressive tumor microenvironment by limiting effective T-cell infiltration and promoting the recruitment of suppressive immune populations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Combining X-rays irradiation and KRAS pathways inhibitors such as MEK inhibitors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] or KRAS inhibitors like Adagrasib [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] can reverse this immune suppressive effect of KRAS mutations and promote a pro inflammatory, anti-tumor phenotype.\u003c/p\u003e \u003cp\u003eAnother radioresistance mechanism of tumors may be the presence of cancer stem cells (CSCs). CSCs are a subpopulation of dedifferentiated cancer cells with self-renewal capabilities and increased resistance to DNA damage, apoptosis, and oxidative stress [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. CSCs are identified by specific stemness markers overexpressed in lung cancer such as CD166 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] in adenocarcinoma lung cells. X-rays irradiation can up-regulate CSCs markers in lung cancer cells [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] leading to radioresistant phenotype selection. CSCs are mainly in hypoxic niches that could also explain this radioresistance effect [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Cancer cells can acquire stem-like properties through epithelial\u0026ndash;mesenchymal transition (EMT) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. EMT confers mesenchymal characteristics to epithelial cancer cells, including loss of polarity, downregulation of epithelial markers such as E-cadherin, upregulation of Nestin [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and Vimetin [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], acquisition of enhanced migratory and invasive capacities [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. EMT facilitates metastasis and tumor recurrence by enabling mesenchymal CSCs to disseminate to distant sites, where they can revert to an epithelial phenotype and initiate metastatic growth [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Vimentin, a key mesenchymal marker, is frequently upregulated in NSCLC, particularly in response to X-ray irradiation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Nestin is also upregulated in NSCLC [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and has been implicated in the regulation of redox homeostasis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], a poorer tumor differentiation and an increased cancer cell proliferation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCarbon-ions (C-ions) radiotherapy represents a promising modality in the management of radioresistant tumors due to its unique physical and biological properties. Owing to their high linear energy transfer (LET), C-ions deposit dense ionizations in the tissue at the end of the ion range (Bragg peak). This dense ionization produces clustered and complex DNA lesions that are difficult to repair, enhancing cell-killing efficacy compared with sparsely ionizing photons. The resulting increase in relative biological effectiveness (RBE), generally ranges from 1.5 to 3 for CSC depending on tissue type, dose, and LET distribution [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In conventional X-rays irradiation, indirect DNA damage through reactive oxygen species generated by water radiolysis is predominant, whereas high-LET radiation primarily induces direct DNA damage. Unlike X-ray irradiation, the cytotoxicity of C-ions is largely preserved under hypoxia due to their reduced dependence on oxygen conditions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. C-ions irradiation have been reported to be more effective on lung cancer cells in hypoxic niches [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and might be more effective on CSC. The combination of high RBE, low oxygen enhancement ratio, and the induction of complex, irreparable DNA damage provides a strong rationale for the use of C-ions radiotherapy in hypoxic niches enriched with CSCs, such as in NSCLC [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Japanese clinical series have demonstrated excellent local control (80\u0026ndash;93% at 2 years) and favorable toxicity profiles for use of C-ions radiotherapy in NSCLC [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo investigate how treatment efficiency could be increased by the combination of irradiation and KRAS\u003csup\u003eG12C\u003c/sup\u003e inhibitor in lung cancers, we conducted an \u003cem\u003ein vitro\u003c/em\u003e study to determine the efficacy of combining the \u003cem\u003eKRAS\u003c/em\u003e inhibitor Sotorasib in association with different qualities of irradiation (X-rays and C-ions irradiations) in a \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e mutated lung cancer cell line, target of Sotorasib and in a \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12S\u003c/em\u003e\u003c/sup\u003e mutated lung cancer cell line, used as a control cells. A specific and robust effect was observed when combining Sotorasib and high LET radiation, reducing the clonogenic survival and proliferation of KRAS\u003csup\u003eG12C\u003c/sup\u003e mutated cells, and inducing an antitumoral immune response.\u003c/p\u003e"},{"header":"2 Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture\u003c/h2\u003e \u003cp\u003eThe human lung adenocarcinoma cell line A549 (Ref CCL-185) and the human bronchioalveolar carcinoma cell line H358 (Ref NCI-H358), were purchased from ATCC (Manassas, VA, USA). A549 cell line harboring \u003cem\u003eKRAS G12S\u003c/em\u003e mutation was isolated from the lung tissue of a 58-year-old male with lung cancer. H358 cell line harboring \u003cem\u003eKRAS G12C\u003c/em\u003e heterozygous mutation and \u003cem\u003eP53\u003c/em\u003e homozygous deletion was isolated from the bronchiole of a male patient with bronchioalveolar carcinoma. All cell lines were cultured in RPMI medium (Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal calf serum (Merck KGaA, Darmstadt, Germany), 2 mM glutamine (Merck KGaA, Darmstadt, Germany), and 1% antibiotics (penicillin-streptomycin, Merck KGaA, Darmstadt, Germany), at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 KRAS G12C inhibitors\u003c/h2\u003e \u003cp\u003eSotorasib (AMG-510) was purchased from MedChemExpress (New Jersey, USA) and used \u003cem\u003ein vitro\u003c/em\u003e at 10 nM according to a dose response experiment. Sotorasib in DMSO at 10 mM was diluted to its final concentration with culture medium, and finally added to the cells 24 h before irradiation and left in the cell culture medium for 24 h. Negative control samples were treated with the same DMSO dilutions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Irradiations\u003c/h2\u003e \u003cp\u003eX-rays irradiation was performed using a Pxi XradSmart 225cX irradiator with a tube tension of 225 kV and an intensity of 10 mA, corresponding to a dose rate of 2 Gy/min. For gene expression and cytokine secretion analysis, H358 cells were exposed to X-rays in a Xstrahl-CIX2 cabinet (Xstrahl, United Kingdom), with an energy of 195 kV and a current of 12.5 mA.\u003c/p\u003e \u003cp\u003eC-ions irradiations were performed at GANIL (Caen, France), using the IRABAT beam line, with a native \u003csup\u003e12\u003c/sup\u003eC beam of 95 MeV/A in the plateau region before the Bragg peak. A 16.9-mm-thick PMMA was inserted between the exit of the beam and sample holder in order to reach a LET of 73 keV/\u0026micro;m (2 Gy\u0026thinsp;=\u0026thinsp;1.71 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e particles/cm\u003csup\u003e2\u003c/sup\u003e), as previously described [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western blot\u003c/h2\u003e \u003cp\u003eFollowing the irradiation and treatment (48 h after irradiation), the cells were washed with PBS, and the cell pellet was lysed in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA USA), supplemented with 1X protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA USA), as previously described [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The protein samples were then analyzed by western-blotting (see detailed protocol in \u003cb\u003eSup data 1\u003c/b\u003e) using tubuline, ERK and phospho-ERK antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Clonogenic assay\u003c/h2\u003e \u003cp\u003eA549 and H358 cells were irradiated in T25 cm\u003csup\u003e2\u003c/sup\u003e flasks at cell confluency. A sham control was included to evaluate the plating efficiency. At 18\u0026ndash;24 h after irradiation, the cells were harvested and re-plated at low density. Medium including Sotorasib or DMSO was changed every 2 to 3 days. Colonies were stained with crystal violet solution (0.3% w/v crystal violet in 20% v/v ethanol). Only colonies that comprised over 50 cells were counted by eye under a stereomicroscope. Clonogenic survival of H358 and A549 cells after irradiation was expressed as a function of dose and was normalized with non-irradiated samples. Surviving fractions of X-rays-irradiated samples were plotted using a linear quadratic and a linear model was used for those after C-ions irradiated samples. Delayed plating was used to avoid confounding effects of Sotorasib on colony formation. Immediate plating under continued drug-induced cell-cycle arrest could artificially reduce clonogenic growth independently of radiation-induced reproductive death. Allowing a recovery period before plating ensures that colony formation reflects true clonogenic survival following the combined treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation was evaluated with the CellTrace\u0026reg; Far Red Cell Proliferation Kit (ThermoFisher Scientific, Waltham, MA USA) as previously described [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Experiments with X-rays and C-ions were analyzed with a Gallios flow cytometer (Beckman Coulter, Villepinte, France) at the ISOCELL platform (US PLATON, Caen, France). The data were acquired using Gallios software (Beckman Coulter). Data were analyzed with FCS Express 6+ (DeNovo\u0026reg;), and proliferation index were calculated by the software using the non-divided control as reference. Control and irradiated samples were compared with the corresponding Sotorasib-treated samples at the same irradiation dose to evaluate the radiosensitizing effect, defined as the enhancement factor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Tumorspheres assay\u003c/h2\u003e \u003cp\u003eA549 and H358 cells were plated 24 hours after irradiation at 5,000 cells/mL in 96 wells treated to prevent cell adhesion (Corning Costar Ultra-Low Attachment Multiple Well Plate). The culture medium was DMEM F12 (Sigma- Aldrich, D6421), supplemented with 0.5 \u0026micro;L/mL of βFGF and EGF, 10 \u0026micro;L/mL of PS, glutamine, and 3.6% hydrocortisone and finally 20\u0026micro;L/mL of insulin. The plates were supplemented every 2 or 3 days with DMEM medium at 5 \u0026micro;L/mL of EGF and βFGF. The number of spheres formed (from a cell that successfully proliferated without adhering) was counted after 10 days of culture (spheres with a diameter of more than 50 \u0026micro;m).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 RTq PCR\u003c/h2\u003e \u003cp\u003eTotal RNA extraction from control and treated cells, cDNA synthesis and real time qPCR were performed as previously described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Sequences of primers used to amplify human Nestin, Vimentin and CD166 transcripts are provided in \u003cb\u003esup table 1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cytokines quantification\u003c/h2\u003e \u003cp\u003eThe quantification of secreted cytokine in the supernatant of control and treated H358 cells was performed as previously described [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistics\u003c/h2\u003e \u003cp\u003eThe statistical analysis for cell proliferation, CSC markers, cytokines, and tumorsphere assays was performed using the statistical software Graph Prism with different statistic tests depending on normal distribution (two-way Anova test, Mann-Whitney U\u0026thinsp;+\u0026thinsp;FDR). Data were considered to be significantly different when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eCell survival fractions data were analyzed using linear mixed models. Cell counts were first normalized to the initial number of seeded cells. For each experimental condition, the surviving fraction was calculated by normalizing to the corresponding 0 Gy control within each experiment. The surviving fraction was log-transformed prior to analysis.\u003c/p\u003e \u003cp\u003eThe models included treatment, radiation type, and their interaction term as fixed effect, with experiment included as a random intercept to account for between-experiment variability. The interaction term was used to assess departure from additivity between treatment and irradiation. Estimated coefficients are reported with their 95% confidence intervals (95% CI). The surviving fraction at 2 Gy (SF2) was analyzed using a two-way ANOVA with treatment and radiation quality as fixed factors. Enhancement ratios (ER) were calculated at 2 Gy (SF2) by comparing survival fractions between treated and untreated conditions and are reported as descriptive measures of radiosensitization. The dose required to achieve 10% survival (D10) was derived from fitted survival curves.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sotorasib reduced clonogenic survival in H358 cells\u003c/h2\u003e \u003cp\u003eIn order to select an appropriate concentration of Sotorasib for all experiments, we tested the effects of different concentration of Sotorasib on ERK phosphorylation, a downstream signalization pathway of KRAS by western blot assays. We observed a specific reduction of phospho-ERK, in H358 cells at a concentration of 10 nM (\u003cb\u003esup data 1\u003c/b\u003e). Using this Sotorasib concentration, clonogenic survival in H358 cells was about 30% (p\u0026thinsp;\u0026lt;\u0026thinsp;0,001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cb\u003eleft\u003c/b\u003e) with a characteristic reduction of clone size. As expected, Sotorasib had no effect on clonogenic behavior in A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cb\u003eright)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Sotorasib enhances the radiosensitivity of H358 cells to carbon ions but not to X-rays\u003c/h2\u003e \u003cp\u003eSotorasib (10 nM) reduced the survival fraction of H358 cells with all irradiation doses compared to irradiated cells without drug \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cb\u003eleft)\u003c/b\u003e, whereas no effect was observed on A549 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cb\u003eright)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn H358 cells, the surviving fraction at 2 Gy (SF2) was significantly reduced when Sotorasib was combined with C-ions irradiation compared with C-ions alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), with a corresponding enhancement ratio (ER) of 4.21. Statistical analysis confirmed a synergistic interaction between Sotorasib and C-ions (β = \u0026minus;0.36, 95% CI [\u0026minus;\u0026thinsp;0.52; \u0026minus;0.20], p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eIn contrast, no evidence of a synergistic interaction was observed with X-ray irradiation (β\u0026thinsp;=\u0026thinsp;0.04, 95% CI [\u0026minus;\u0026thinsp;0.02; 0.10], p\u0026thinsp;=\u0026thinsp;0.23), and no significant reduction in SF2 was detected, with a corresponding ER of 1.02 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Consistently, the dose required to achieve 10% survival (D10) was reduced by factors of 1.09 and 2.4 when Sotorasib was combined with X-rays and carbon ions, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCalculated parameters of H358 and A549 cells survival after irradiation with X-rays and C-ions with and without Sotorasib\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eH358\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eA589\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSF2\u003csup\u003ea\u003c/sup\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD10\u003csup\u003eb\u003c/sup\u003e (Gy)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eER (SF2) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eER (D10) \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSF2\u003csup\u003ea\u003c/sup\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD10\u003csup\u003eb\u003c/sup\u003e (Gy)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eER (SF2) \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eER (D10) \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eX-rays\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eX-rays\u0026thinsp;+\u0026thinsp;Sotorasib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0,68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1,01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC-ions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC-ions\u0026thinsp;+\u0026thinsp;Sotorasib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4,21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0,82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0,79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003ea: SF2: surviving fraction (%) at 2 Gy.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eb: D10: dose required to achieve 10% surviving fraction.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003ec: ER (SF2): enhancement ratio calculated as SF2 (with Sotorasib) / SF2 (without Sotorasib).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003ed: ER (D10): enhancement ratio calculated as D10 (with Sotorasib) / D10 (without Sotorasib).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eStatistical significance was assessed for SF2 values only. Enhancement ratios (ER) are descriptive and were not statistically tested. * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared with corresponding irradiation condition without Sotorasib.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Sotorasib decreased cell proliferation in combination with C-ions irradiation\u003c/h2\u003e \u003cp\u003eSotorasib alone reduced the proliferation index of H358 cells by approximately 35% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), while no significant effect was observed in A549 cells. X-ray irradiation alone did not alter the cellular index in H358 cells; however, its combination with Sotorasib further decreased the index by 41\u0026ndash;44% at 1, 2, and 4 Gy compared with irradiation alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Carbon-ion irradiation alone reduced the cellular index of H358 cells by 26.5%, 64%, and 83% at 1, 2, and 4 Gy, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When combined with Sotorasib, C-ion irradiation at 1 and 2 Gy produced greater decreases of 67% and 82%, (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with the corresponding enhancement factors of 1.55 at 1 Gy and 1.50 at 2 Gy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the reduced proliferation index observed in C-ions-irradiated A549 cells is not further modulated in the presence of Sotorasib.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Sotorasib did not impact tumor-spheres formation and CSC markers expression\u003c/h2\u003e \u003cp\u003eSphere formation was reduced by X-ray at 1 Gy in H358 and 2 Gy in A549 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003etop\u003c/b\u003e). C-ions irradiations reduced the formation of spheres in both A549 and H358 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003ebottom\u003c/b\u003e). The addition of Sotorasib alone or with irradiations did not alter any sphere formation in H358 and A549 cells. This observation was confirmed with the quantification of selected CSC and EMT mRNA markers in H358 cells. Sotorasib alone, X-rays, C-ion and combinations did not affect CD166, Vimentin or Nestin expression in H358 cells (\u003cb\u003esup data 2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Sotorasib induced pro-inflammatory and immune anti-tumor phenotype\u003c/h2\u003e \u003cp\u003eSecretions of pro- and anti-inflammatory cytokines were measured in H358 cells after exposure to Sotorasib combined with irradiations \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSotorasib alone showed a trend toward increased IP-10 secretion, a pro-inflammatory cytokine, at 24 hours in H358 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, A) with a significant upregulation of IP-10 secretion 24 hours after X-ray irradiation at 2 and 4 Gy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), a similar upward trend at 8 Gy and across all doses of carbon-ion irradiation (2\u0026ndash;8 Gy).\u003c/p\u003e \u003cp\u003eThe secretion of GM-CSF, involved in the establishment of an immunosuppressive microenvironment, is reduced by Sotorasib alone \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, B). C-ions irradiation at 8 Gy significantly upregulated GM-CSF secretion in H358 cells, 24 h post-irradiation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This effect was reversed when combined with Sotorasib, which led to a significant downregulation of GM-CSF at 8 Gy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A similar decreasing trend in GM-CSF secretion was observed at all C-ions doses. In contrast, Sotorasib did not affect GM-CSF secretion when combined with X-rays irradiation in H358 cells.\u003c/p\u003e \u003cp\u003eIn contrast, Sotorasib\u0026mdash;either alone or combined with X-rays or C-ions irradiation\u0026mdash;did not modify IL-6 secretion 24 hours after irradiation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, C).\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn our study, Sotorasib in combination with X-ray irradiation reduced clonogenic survival compared with either treatment alone; however, no evidence of a radiosensitizing or synergistic interaction was observed in H358 cells.H358 cells harbor \u003cem\u003eKRAS\u003c/em\u003e \u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e heterozygote mutation. Consistently, Adagrasib, another KRAS\u003csup\u003eG12C\u003c/sup\u003e inhibitor, has been shown to enhance sensitivity to X-ray in \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e homozygous mutant cells (CT26, a murine colorectal carcinoma line) but not in heterozygous mutant cells (LL2, a murine Lewis lung carcinoma line), both in vitro and in mouse models [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, Sotorasib exhibited a synergistic radio-sensitizing effect in H358 cells when combined with C-ions irradiation. To our knowledge, this is the first report of such an effect in human \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e\u0026ndash;mutated cell lines. While C-ions irradiation alone reduced tumor sphere formation, neither Sotorasib nor X-ray irradiation produced a similar effect. Tumor spheres are cell aggregates that grow under non-adherent conditions, as a surrogate indicator of CSC [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and radioresistance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Sotorasib did not affect the formation of non-adherent spheres, suggesting the persistence of a subpopulation of cells that is not responsive to KRAS G12C inhibition. This observation is consistent with previously reported intrinsic and acquired resistance mechanisms to Sotorasib in NSCLC [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], including adaptive pathway reactivation and cellular heterogeneity. Such resistant cells may exhibit stem-like properties and contribute to tumorsphere formation, which is commonly used as a surrogate for self-renewal capacity. In this context, the persistence of sphere-forming cells may reflect a reservoir of drug-tolerant or resistant cells that could underlie tumor relapse. The effect of high LET C-ions on tumor spheres suggests that C-ions may target a cancer cell population distinct from that affected by Sotorasib.\u003c/p\u003e \u003cp\u003eNestin, an EMT marker, is overexpressed in NSCLC cells following X-ray irradiation and has been associated with increased resistance to oxidative stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Vimentin, another EMT marker, is commonly overexpressed in various lung cancers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], while CD166 is frequently expressed in lung adenocarcinomas and is considered a potential CSC marker [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our study, the expression of these EMT and CSC markers showed high variability (data not shown) and was not significantly changed after treatment with Sotorasib, X-rays, or C-ions irradiation. Further investigations are required to determine which CSC subpopulations might be specifically targeted by C-ions irradiation of H358 cells.\u003c/p\u003e \u003cp\u003eSotorasib has been reported to promote a pro-inflammatory tumor microenvironment in mouse models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e inhibitors such as Adagrasib, when combined with X-ray irradiation, have been shown to elicit anti-tumor immune responses in vivo [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In our study, Sotorasib induced an overexpression of IP-10 and decreased the expression of GM-CSF. GM-CSF can contribute to tumor progression [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] while IP-10 expression has been shown to sensitize tumors to immune checkpoint inhibitors by promoting CD8⁺ T-cell recruitment [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our findings suggest that Sotorasib combined with either X-ray or C-ion irradiation can affect cytokine secretion so that the immune microenvironment is modulated to promote the development of a pro-inflammatory, anti-tumor immune contexture.\u003c/p\u003e \u003cp\u003eFurther investigations on CSCs, EMT markers, and cytokines are still needed in order to elucidate mechanisms underlying the efficacy of combining irradiation with Sotorasib in NSCLC.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn this study, we first demonstrated the specific radio-sensitizing effect of combining high LET C-ions irradiation with Sotorasib in a \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e-mutated NSCLC cell line. This effect was observed despite the heterozygous, rather than homozygous, KRAS mutation in H358 cells. These findings support further investigation of the combination of C-ions and KRAS inhibitors in NSCLC, as they may target distinct cancer cell populations and thereby improve therapeutic responses. Additional in vitro and in vivo studies are warranted to elucidate the underlying mechanisms prior to clinical translation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6 Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors want to thank Cl\u0026eacute;ment Rouichi, from IRIG-LCBM laboratory and Marilyne Guillamin from the Isocell Cytometry Platform (Service Unit PLATON, University of Caen Normandie) for their technical support. Finally, the authors want to thank the GANIL and CIMAP-CIRIL facilities staff for C-ions calibration and dosimetry.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7 Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a Grant of \u0026ldquo;Ligue contre le Cancer \u0026ndash; comit\u0026eacute; de Seine Maritime\u0026rdquo;, 2022-2023, \u0026laquo; Traitement par inhibiteurs de KRAS en association avec l\u0026rsquo;irradiation X vs ions carbone dans le cancer bronchique non \u0026agrave; petite cellule (CBNPC) avec mutation KRAS \u0026raquo;, by a Grant of Electricit\u0026eacute; de France 2023-2025, funding from committee \u0026laquo; Protection de l\u0026rsquo;Homme et de l\u0026rsquo;Environnement (CPPHE) \u0026raquo;, under the Life Sciences group of the four-way national agreement CEA-EDF-IRSN-Framatome, \u0026ldquo;R\u0026ocirc;le de KRAS dans la modulation de la radior\u0026eacute;sistance et du statut inflammatoire dans le cancer bronchique non \u0026agrave; petites cellules\u0026rdquo; by the LABEX PRIMES (grant number ANR-11-LABX-0063), and by GRAL, a program from the Chemistry Biology Health (CBH) Graduate School of University Grenoble Alpes (grant number ANR-17-EURE-0003). Irradiations with C-ions were performed at GANIL (Caen, France) using beam times obtained under experiment number P1317-H (KRAS Inhibitors treatment in association with X vs hadrons Radiations in non-small cell lung cancer\u0026rdquo; of the iPAC 2022 call, CIMAP-CIRIL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8 Conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9 Ethics declaration\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10 Authors\u0026rsquo; contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMathieu C\u0026eacute;saire: Formal analysis, Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing. Kilian Lecrosnier: Formal analysis, Methodology. Juliette Montanari: Formal analysis, Methodology. Mateusz Sitarz: Formal analysis, Methodology, Writing \u0026ndash; original draft. Sahra Messaoudi: Methodology, Writing \u0026ndash; original draft. Elisabeth Chartier-Garcia: Formal analysis, Methodology. Isabelle Testard: Formal analysis, Methodology, Writing \u0026ndash; original draft. Gu\u0026eacute;na\u0026euml;lle Levallet: Writing \u0026ndash; review and editing. Serge Cand\u0026eacute;ias: Formal analysis, Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing. Fran\u0026ccedil;ois Chevalier: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAntonia SJ, Villegas A, Daniel D, Vicente D, Murakami S, Hui R, Kurata T, Chiappori A, Lee KH, de Wit M, Cho BC, Bourhaba M, Quantin X, Tokito T, Mekhail T, Planchard D, Kim Y-C, Karapetis CS, Hiret S, Ostoros G, Kubota K, Gray JE, Paz-Ares L, de Castro Carpe\u0026ntilde;o J, Faivre-Finn C, Reck M, Vansteenkiste J, Spigel DR, Wadsworth C, Melillo G, Taboada M, Dennis PA, \u0026Ouml;zg\u0026uuml;roğlu M, PACIFIC Investigators (2018) Overall Survival with Durvalumab after Chemoradiotherapy in Stage III NSCLC. 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Cancer Cell 40:136\u0026ndash;152e12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ccell.2021.12.009\u003c/span\u003e\u003cspan address=\"10.1016/j.ccell.2021.12.009\" 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":false,"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":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Non-small cell lung cancer, KRAS inhibitor; Carbon ions irradiation, cancer stem cell","lastPublishedDoi":"10.21203/rs.3.rs-9469593/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9469593/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNon-small cell lung cancer (NSCLC) with \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e mutation is associated with poor prognosis and resistance to standard therapies. Sotorasib, a \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003eG12C\u003c/sup\u003e tyrosine kinase inhibitor, has shown clinical efficacy, yet its interaction with different irradiation modalities remains unclear. This study aimed to evaluate the potential radiosensitizing effect of Sotorasib in such mutated cells exposed to X-rays or carbon-ion (C-ions) irradiation.\u003c/p\u003e \u003cp\u003eTwo human NSCLC cell lines (H358, \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003eG12C\u003c/sup\u003e; and A549, \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003eG12S\u003c/sup\u003e) were treated with Sotorasib alone or in combination with X-rays or C-ions irradiation (1\u0026ndash;8 Gy). Cell viability, clonogenic survival, and sphere-forming ability were assessed. Cytokines secretion and mRNA expression of cancer stem cells and epithelial-mesenchymal transition (EMT) -associated markers were analyzed 24 h post-treatments.\u003c/p\u003e \u003cp\u003eSotorasib alone specifically reduced the clonogenic capacity of H358 cells to ~\u0026thinsp;30% while no effect was observed with A549 cells. Interestingly, Sotorasib combined with X-ray irradiation decreased clonogenic survival compared with either treatment alone, without evidence of synergy. In contrast, a synergistic effect was observed with C-ions in H358 cells (β = \u0026minus;0.36, 95% CI [\u0026minus;\u0026thinsp;0.52 to \u0026minus;\u0026thinsp;0.20], p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; enhancement ratio\u0026thinsp;=\u0026thinsp;4.21 at SF2). C-ions irradiation alone markedly reduced sphere formation in both cell lines, whereas sotorasib showed no additional impact. GM-CSF expression was downregulated with sotorasib alone while IP-10 was upregulated when H358 cells were irradiated with sotorasib, suggesting an immune-specific modulatory response.\u003c/p\u003e \u003cp\u003eA specific radiosensitization of \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003eG12C\u003c/sup\u003e \u0026ndash;mutated NSCLC cells was observed when associating Sotorasib with C-ions. This represents the first evidence of enhanced cytotoxicity from combining \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e inhibition with high-LET radiation. These findings support further \u003cem\u003ein vivo\u003c/em\u003e and translational studies exploring C-ions therapy as a promising strategy for \u003cem\u003eKRAS\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12C\u003c/em\u003e\u003c/sup\u003e\u0026ndash;mutated NSCLC.\u003c/p\u003e","manuscriptTitle":"Radio-sensitization of mutated KRAS G12C non-small cell lung cancer with KRAS G12C tyrosine kinase inhibitor and Carbon ions irradiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 17:39:59","doi":"10.21203/rs.3.rs-9469593/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-05T14:59:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T08:02:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:34:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-02T16:02:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27032012229215021733477521551716973710","date":"2026-04-24T15:15:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243000946120790498817463272691599164979","date":"2026-04-24T07:58:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117353069332848108204683829289612853633","date":"2026-04-24T07:50:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T07:07:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-20T11:08:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-20T11:07:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2026-04-20T08:55:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5dd4eca1-e72c-43e3-a16f-2b108b4cf4fb","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-05T14:59:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T08:02:48+00:00","index":49,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:34:08+00:00","index":48,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-02T16:02:28+00:00","index":47,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T13:08:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 17:39:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9469593","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9469593","identity":"rs-9469593","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}