Impacts of Combining PD-L1 inhibitor and Radiotherapy on the Tumour immune microenvironment in a Mouse Model of Esophageal Squamous Cell Carcinoma

Impacts of Combining PD-L1 inhibitor and Radiotherapy on the Tumour immune microenvironment in a Mouse Model of Esophageal Squamous Cell Carcinoma | 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 Impacts of Combining PD-L1 inhibitor and Radiotherapy on the Tumour immune microenvironment in a Mouse Model of Esophageal Squamous Cell Carcinoma Zihao Yin, Qinghua Deng, Ke Zhang, Jing Yue, Yaping Wang, Qingqing Yu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4338719/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background： The combination of radiation with immune checkpoint inhibitors (ICIs) has been demonstrated to display synergistic effects in solid cancers. Nevertheless, the anti-tumor effect of combining radiation with programmed cell death 1 ligand 1 (PD-L1) inhibitor in esophageal squamous cell carcinoma (ESCC) remains unclear. Therefore, the objectives of our study were to evaluate the anti-tumor effects of PD-L1 inhibitors combined with radiotherapy in ESCC mouse model and to depict the immune landscape within the tumor microenvironment (TME). Methods: A syngeneic C57BL/6 subcutaneous xenograft mouse model was applied to evaluate the anti-tumor efficacy of different treatment protocols according to tumor growth curve and survival time. Tumour immune microenvironment was assessed by flow cytometry including CD4 + T cells, CD8 + T cells, regulatory T cells (Tregs), tumor-derived macrophage (TAM), myeloid-derived suppressor cell (MDSC), and the expression of CD8 + T cell activation, exhaustion, and memory state markers. In addition, transcriptomic analysis was used to examine the immune gene expression changes in tumor microenvironment. Results: Radiotherapy combined with anti-PD-L1 inhibitors synergistically enhanced anti-tumor immune response via boosted the infiltration of CD8 + T cells, increased the ratio of CD8 + T cells to Tregs and population of central memory CD8 + T cells (T CM ), enhanced interferon gamma (IFN-γ) secretion by tumor-infiltrating CD8 + T cells, and reduced the accumulation of M2-type TAMs and Tregs in the TME in mouse model. In addition, radioimmunotherapy also induced a better immunophenotype in spleen and tumor draining lymph node (TDLN). Consequently, radioimmunotherapy appeared greater benefit in antitumor effects and mice survival. Moreover, our transcriptomic analysis suggested that radioimmunotherapy promoted the expression of immunostimulation-related regulatory pathways and cytokines that shape the immunoinflammatory tumor microenvironment. Conclusions: Our research indicated that anti-PD-L1 inhibitors combined with RT promotes systemic anti-tumor immunity by improving the immune microenvironment in a mouse model of ESCC. Programmed death ligand 1 radiotherapy tumor microenvironment Esophageal Squamous Cell Carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction ESCC is a common malignant tumor worldwide( 1 ). Due to the inconspicuous early symptoms and the lack of effective early diagnosis methods, ESCC is often diagnosed at advanced stages and patients have a poor prognosis( 2 ). Definitive chemoradiotherapy is the current standard protocol for unresectable locally advanced ESCC. Previous studies have explored optimal chemotherapy protocols and substituted radiation dose fractionation, but local recurrence rates remain high, with 5-year OS rates of only 20%-25%( 3 – 5 ). Immune checkpoint inhibitors have demonstrated as a prospective therapeutic strategy in the treatment of ESCC patients. Several studies, including KEYNOTE-590( 6 ), Escort-1st( 7 )have demonstrated that PD-1/PD-L1 monotherapy improved the survival of advanced ESCC patients, demonstrating reliable efficacy and a favorable safety. However, due to the heterogeneity of tumors and differences in the immune microenvironment, the application of ICI monotherapy is limited by remission rates of only roughly 20%. To overcome this constraint, multiple researches have investigated the protocols of combining immunotherapy with alternate treatment( 8 , 9 ). Radiotherapy (RT) is be involvement in all stages of ESCC. Radiation has been proved to produce significant effects on the immune response of various types of tumors( 10 , 11 ). Briefly, radiation induces the death of immunogenic tumour cells and production of tumour antigens, which function as in situ vaccines to activate systemic anti-tumour immune responses. Additionally, local radiation contributes to tumor remission via promoting the recruitment of CD8 + TILs and secretion of IFN-γ( 12 ). Nevertheless, radiation increases the expression of PD-L1 on the surface of tumor cells, which could then promote immune tolerance through suppressing the activation of lymphocytes, suggesting the potential synergistic effects of anti-PD-1/PD-L1 immunotherapy( 13 , 14 ). Moreover, ICIs can contribute to the activation of effector T cells to eliminate tumor cells and normalization of tumor vasculature, increasing lymphocyte infiltration and sensitivity to radiotherapy( 15 , 16 ). In summary, radiotherapy and immunotherapy complement each other and exert a stronger antitumor immune response. Previous studies have confirmed that radiotherapy in combination with PD-L1 inhibitors can enhance systemic anti-tumor effects in preclinical models of lung( 17 , 18 ), melanoma( 19 ), colorectal cancer( 13 ), breast cancer( 13 ), head and neck squamous cell carcinoma( 20 , 21 ), prostate cancer( 22 , 23 ), and pancreatic cancer( 24 ). But this combination has not been explored in esophageal cancer models. In a phase Ib clinical trial of combing radiotherapy with the PD-1 inhibitor Camrelizumab, involving 19 patients with newly diagnosed locally advanced ESCC who refused or could not tolerate concurrent chemoradiotherapy, the median OS was 16.7 months. This is comparable to the duration of patients receiving concurrent chemoradiotherapy regimens (median OS time of about 18.1–19 months)( 25 ). At present, phase III clinical trials of concurrent chemoradiotherapy combined with anti-PD-L1 therapy in the treatment of patients with ESCC are currently underway( 2 , 26 ). Radiotherapy combined with anti-PD-L1 immunotherapy has emerged as a prospective application in ESCC. However, the synergistic effect and molecular mechanism of radioimmunotherapy in ESCC are not completely understood. Therefore, this research aims to evaluate the synergistic anti-tumor effect in a ESCC preclinical model and define the molecular mechanism through changes of immune cells and cytokines in the immune microenvironment when radiotherapy combined with PD-L1 inhibitors. Materials and methods Preparation of cell lines The murine ESCC cell line mEC25 was purchased from Shenzhen Wenhua Times Technology Co. and cultured in RPMI-1640 medium (11875119, Gibco, CA, USA) with 10% fetal bovine serum (SA211.02, Cellmax, Beijing, China) and maintained in a 5% CO 2 humidified incubator at 37℃. In vivo studies Female C57BL/6 mice (4–6 weeks old) were provided by the Laboratory Animal Center, Zhejiang Academy of Medical Sciences (Hangzhou, China). All mice were maintained and treated depending on the Institutional Animal Care and Use Committee regulations at the Clinical Research Institute. Mice were injected subcutaneously with 5x10 5 mEC25 cells on the right flank. When tumors reached 90–120 mm 3 (Day 1), each mouse was randomly divided into 4 groups: Isotype control; RT (4Gy×3) + isotype; Anti-PD-L1 antibodies; RT + Anti-PD-L1 antibodies. For radiation therapy, tumors were irradiated in 3 fractions for a total dose of 12 Gy. RT was administered via an electron beam on days 1, 4, and 7 from the beginning of therapy. Briefly, mice were anesthetized and placed under a lead shield with a 1 cm 2 hole to limit tumor exposure. Anti-PD-L1(BioXCell, clone 10F.9G2) or isotype control (BioXCell, rat IgG2b) were delivered by intra-peritoneal injection concurrent with radiotherapy. We measured the size of each tumor using calipers and calculated its volume (length×width 2 ×0.5). Survival time of mice was recorded (day 1 until tumor volume reached 1500 mm 3 or animals were executed). As soon as the mice reached a limit point (tumor volume ≥ 1500 mm 3 , pain, significant necrosis), mice were anesthetized and sacrificed by cervical dislocation. Flow cytometry analysis The tumor, spleen and tumor draining lymph nodes (TDLNs) were isolated on days 10 and 14. Tumor tissue were cut into small pieces and incubated at 37°C for 30 minutes (tumor) or 10 minutes (TDLN) in RPMI medium with collagenase type IV and deoxyribonuclease type I, subsequently mechanically separated on frosted slides. Spleens were mechanically separated directly on frosted slides. Single-cell suspension was obtained by filtering the cell suspension through a 70-µm cell strainer (Corning). A lysis buffer (NH4Cl, NaHCO3, EDTA) was used to lyse erythrocytes at room temperature and quenched with RPMI. Fixable Viability Stain 440UV (566332, BD) and α-CD16/32 (553142 BD) were used to label and block the cells. For surface staining, monoclonal antibodies ( supplemental table 1 ) were used to stain the samples for 30 minutes at room temperature. Each analysis consisted of at least three replicates. The data were obtained by using FACSCalibur machines and analyzed using Flowjo software. Intracellular staining To assess the secretion of IFN-γ and granzyme B, the cell suspensions were stimulated with 1 µM phorbol 12- myristate 13- acetate (PMA, Sigma) and ionomycin (Sigma) for 4 hours at 37°C. Then, together with intracellular staining of Foxp3, cells were fixed and permeabilized via FOXP3 Fix/Perm Buffer Set (562574, BD). And then stained with monoclonal antibodies ( supplemental table 1 ) for 30 min on ice depending on the manufacturer’s instructions. Immunohistochemistry (IHC) 4% paraformaldehyde 4% was used to fix tumor tissues and paraffin was used to embed them. Sections were deparaffinized, rehydrated, antigenically repaired and stained overnight at 4°C with the primary antibodies as follows: CD8(D4W2Z, 98941), FOXP3(D608R, 12653), CD206(E6T5J, 24595), CD86(E5W6H, 19589), GZMB(13588-1-AP). Secondary antibodies were incubated with the sections on the second day and reacted with ABC kits (Vector, Burlingame, CA). Stained sections were observed using ECLIPSE E100 light microscope (NIKON) and K-Viewer software. Quantitative analysis of optical density was evaluated by Image J software (National Institutes of Health). The average density of 3 slices per sample was calculated. Homologous transplantation tumour mRNA sequencing and bioinformatic analysis The total RNA was extracted by Trizol Reagent (15596026, Thermo Fisher Scientific Inc., MA, USA). RNA-seq assay was performed using the Illumina Hiseq platform from Wuhan SeqHealth. Musmusculus GRCm38 was the reference genome. Differentially expressed genes (DEGs) were screened using the edgeR package in R software. GO and KEGG 2018 databases were used for gene set enrichment analysis. RNA extraction and quantitative real-time PCR (qRT-PCR) The extraction of total RNA was performed using Trizol Reagent. Reverse transcription was accomplished by using PrimeScript™ RT Master Mix (RR036A, Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). Quantitative RT-PCR (qRT-PCR) was carried out with TB green (RR420A, Takara Biomedical Technology Co., Ltd., Beijing, China). Furthermore, relative expression of each targeted gene including CXCL9 and CXCL10 (primers were listed in Supplemental Table 2 ) was calculated and normalized using 2 −ΔΔCt method. Statistical analysis One-way ANOVA analysis was used to statistical comparisons between three or more groups, followed by Tukey’s multiple comparison test. For mice tumor volume experiments, statistical analysis was performed using mixed-effects model with Tukey’s multiple comparison test. Survival curves for different groups of mice were generated using the Kaplan- Meier method. The the log- rank Mantel- Cox test was used to evaluated the survival curves of mice in each group. Tumor volumes or loads of at least three animals in each group were subjected to t-tests, and the results were expressed as mean ± standard error of the mean (SEM). P < 0.05 was regarded statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). The statistical analyses were performed with GraphPad Prism statistical analysis and graphing software (GraphPad 9). Results Anti-PD-L1 inhibitors combined with radiotherapy synergistically enhanced antitumor effects in esophageal cancer mice model In our previous study, we demonstrated that fractionated radiotherapy increased the expression of PD-L1 in esophageal cancer cell lines (Supplementary Fig. 1) . We firstly examined whether anti-PD-L1 inhibitor combined with radiotherapy could synergistically reinforce anti-tumor efficacy. We subcutaneously injected mEC25 murine cancer cells into syngeneic mice and subjected the mice to different treatment regimens-radiation therapy (RT) alone, anti-PD-L1 alone, and radioimmunotherapy (RT combined with anti-PD-L1) ( Fig. 1 a ) . We found that the radioimmunotherapy demonstrated the strongest inhibition of tumor growth ( Fig. 1 b ) . Radioimmunotherapy significant slowed down tumor growth rate compared with RT ( p = 0.008), anti-PD-L1 ( p = 0.01) and control groups ( p = 0.01). In addition, radioimmunotherapy also improved survival ( Fig. 1 d ) compared to RT ( p = 0.0007), anti-PD-L1 ( p = 0.0248) and control groups ( p = 0.0001). Compared to 33 days for radioimmunotherapy, the median survival for RT was 26 days, for anti-PD-L1 was 28 days and for control group was 24 days. Radioimmunotherapy impacts T cell infiltration To assess alterations of composition in tumor microenvironment after radioimmunotherapy, lymphocytes were collected from the tumor, spleen and tumor draining lymph nodes (TDLN) at Days 10 and 14. The extent of tumor-infiltrating CD8 + T cells (TILs) was associated with suppression of tumor growth. In the tumor, at Day 10, non-significant increase of the proportion of CD8 + TILs in radioimmunotherapy was observed compared with other groups. At Day 14, radioimmunotherapy significantly enhanced the frequency of CD8 + TILs compared to RT and control groups ( p = 0.0049, p = 0.0413 respectively, Fig. 2 a, b). Similarly, in the TDLN, radioimmunotherapy increased the proportion of CD8 + TILs compared to RT and control groups at Day 10(39.8%±0.6% radioimmunotherapy vs 31.2%±3.1% RT p = 0.0472; 30.4%±0.2% control p = 0.0316; Fig. 2 B). In spleen, there were no significant difference among the groups at any time point ( Fig. 2 b ) . Meanwhile, there were no significant differences in the population of CD4 + T cells at any time point in the tumor, spleen and TDLNs (Supplementary Fig. 2a) . Therefore, we further investigated the impact of radioimmunotherapy specifically on the regulatory T (Treg) cell population, which are an immunosuppressive subset of CD4 + T cells. In the tumor, at Day 14, radioimmunotherapy decreased the ratio of infiltrating Tregs compared to those of the other groups (radioimmunotherapy vs RT p < 0.0001; Anti-PD-L1 p = 0.0105; control p < 0.0001, Fig. 2 c, d). The Anti-PD-L1 group also significantly reduced infiltrating Tregs compared with the RT and control groups. Interestingly, in the spleen at Day 10, we found that radiation increased the proportion of Tregs compared to the control group, suggesting that RT can induce the upregulation of Tregs as an immunosuppressive regulator. Notably, radioimmunotherapy reversed this upregulated proportion of infiltrating Tregs (Fig. 2 d). In addition, the proportion of Tregs was markedly reduced in the anti-PD-L1 group compared with RT and control groups (Fig. 2 d). We also investigated the proportion of CD8 + TILs/Foxp3 + Tregs. In tumor, radioimmunotherapy increased CD8TILs + /Treg ratio compared with those of other groups at Day 14, with a same phenomenon seen in the spleen and TDLNs at Day 14( Tumor : 75.4 ± 18.1 radioimmunotherapy vs 14.6 ± 0.4 radiation p = 0.0105; vs 25.7 ± 5.8 Anti-PD-L1 p = 0.0305; vs 20.1 ± 1.7 control p = 0.0175; Spleen : 90.8 ± 3.9 radioimmunotherapy vs 55.1 ± 7.5 radiation p = 0.0170; vs 60.4 ± 7.3 Anti-PD-L1 p = 0.0380; vs 55.3 ± 5.8 control p = 0.0175; TDLNs : 67.1 ± 2.8 radioimmunotherapy vs 39.9 ± 1.9 radiation p = 0.0003; vs 46.5 ± 2.6 Anti-PD-L1 p = 0.0022; vs 53.0 ± 2.7 control p = 0.0206; Fig. 2 e). No significant differences were found at Day 10 in the tumor, spleen and TDLNs. In addition, these results were consistent with the concomitant immunohistochemistry on day 14 ( Fig. 2 f ) . Therefore, these results demonstrated that radioimmunotherapy can upregulate the ratio of CD8 + TILs/Tregs by improving the recruitment of CD8 + TILs and reduced the accumulation of Tregs. Radioimmunotherapy enhances immune memory activation We further investigated whether the T cell activation status was different in different treatment groups. We defined the effector memory phenotype as CD44 + CD62L − , and central memory cells as CD44 + CD62L + . Firstly, we accessed CD8 + TILs populations expressing CD44. CD44 is an activated marker of T cells after antigen stimulation. At Day 10, the percentage of CD44 + CD8 + TILs of radioimmunotherapy and anti-PD-L1 was lower than RT and control groups in the tumor. In the spleen, at Day 10, radioimmunotherapy increased the proportion of CD44 + CD8 + T cells compared to other groups (48.2%±2.2% radioimmunotherapy vs 37.2%±1.9% RT p = 0.031; 36.8%±0.1% Anti-PD-L1 p = 0.0257; 32.1%±3.2% control p = 0.0046; Fig. 3 a). Moreover, radioimmunotherapy enhance the frequency of CD44 + CD8 + TILs compared with RT and control groups at Day 14( p < 0.0001). In the TDLNs, radioimmunotherapy upregulated the percentage of CD44 + CD8 + TILs compared to RT and control groups at Day 10( p = 0.0391; p = 0.0356, respectively). At Day 14, radioimmunotherapy upregulated the percentage of CD44 + CD8 + TILs compared to anti-PD-L1 and control groups ( p = 0.0316; p = 0.0002, respectively; Fig. 3 a). For the infiltrating central memory CD8 + T cells (T CM ), we can see that radioimmunotherapy enhanced the accumulation of CD8 + T CM at Day 14 in the tumor compared to those of other groups ( p < 0.0001; Fig. 3 b, c). And the same trends were also observed in the TDLNs at Days 10 and 14 (Day 10 : 11.1%±0.5% radioimmunotherapy vs 5.8%±0.9% RT p = 0.0011; 6.7%±0.5% Anti PD-L1 p = 0.0035; 6.1%±0.4% control p = 0.0014; Day 14 : 13.8%±0.8% radioimmunotherapy vs 9.8%±0.3% RT p = 0.0087; 3.2%±0.4% Anti PD-L1 p < 0.0001; 2.3%±0.6% control p < 0.0001; Fig. 3 b, c ) . In spleen, compared with Anti-PD-L1 and control groups at Day 10, radioimmunotherapy increased the CD8 + T cm cells infiltration ( 8.0 ± 0.2% radioimmunotherapy vs 4.4%±0.8% Anti-PD-L1 p = 0.0022; 4.1% control p = 0.0012; Fig. 3 b, c ) . At Day 14, compared with RT and control groups, radioimmunotherapy significantly increased the CD8 + T cm cells infiltration ( p = 0.0368; p = 0.0004, respectively). These results indicate that radioimmunotherapy can increase the population of T CM . For infiltrating CD8 + effector memory T cells (T EM ), in the tumor, we observed the same phenomenon consistent with CD44 + CD8 + TILs at Days 10 and 14. In the spleen, no statistical significance were observed. In the TDLNs, the proportion of CD8 + T EM cells were higher in the anti-PD-L1 compared with other groups at Day 14. ( Fig. 3 d ) . Radioimmunotherapy promotes CD8 + T cell effector function Additionally, we assessed the activation status and effector function of the CD8 + TILs based on IFN-γ and Granzyme B (GZMB) expression. At Day 10 in the tumor, an significant increase in the proportion of IFN-γ + CD8 + TILs was observed in radioimmunotherapy compared with the RT and control groups ( p = 0.0047, p = 0.0013, respectively, Fig. 4 a, b). Similarly, we observed the same phenomenon in the spleen at Day 14. Moreover, radioimmunotherapy showed a significantly higher frequency of IFN-γ + CD8 + TILs compared to other groups in the tumor and TDLNs at Day 14 (Tumor : p < 0.0001; TDLN : p = 0.0051; p = 0.0070; p = 0.0017; respectively, Fig. 4 a, b). Meanwhile, similar phenomenon was seen in spleen at Day 10. Compared with the RT at Day 10, radioimmunotherapy also significantly enhanced the accumulation of IFN-γ + CD8 + TILs in the TDLNs ( p = 0.0338, Fig. 4 a, b ) . Concurrently, we evaluated the GZMB secretion of CD8 + T cells. At Day 10 in the tumor and TDLNs, a significantly enhanced proportion of CD8 + TILs expressing GZMB in radioimmunotherapy compared with other groups (Tumor : 31.6%±0.3% radioimmunotherapy vs 11.0%±0.2% RT p < 0.0001; 18.7%±0.1% Anti-PD-L1 p < 0.0001; 12.7%±0.2% control p < 0.0001; TDLN : 12.2%±0.5% radioimmunotherapy vs 7.8%±0.2% RT p = 0.0081; 8.0%±1.1% Anti-PD-L1 p = 0.0002; 6.9%±0.4% control p < 0.0001; Fig. 4 c ) . Meanwhile, we observed the same phenomenon at Day 14 in the tumor by flow cytometry and immunohistochemistry ( Fig. 4 c, d ) . There were no significant differences in the spleen ( Fig. 4 c ) . In summary, radioimmunotherapy can enhance both activation and cytotoxicity of CD8 + TILs. Radioimmunotherapy enhances the expression of immune exhaustion markers In tumor, compared with control group, we found that the group treated with radioimmunotherapy showed a higher amount of PD-1 + CD8 + TILs at Day 14 ( p = 0.0365; Fig. 4 e ) . Similarly, the same phenomenon was observed at Day 10 in the spleen. No differences were observed in the TDLN. In addition, radioimmunotherapy notably enhanced the proportion of PD-1 + CD4 + TILs at Day 10 compared with those of other groups (Supplementary Fig. 2b) . And no differences were revealed in the spleen and TDLNs. Taken together, radioimmunotherapy led to the highest proportion of CD8 + and CD4 + T cells expressing PD-1. Radioimmunotherapy inhibits the recruitment of different immunosuppressive cell populations Aside from Tregs and CD8 + T cells, we also investigated the relative immuno-suppressive populations of tumor-infiltrating mononuclear myeloid-derived suppressor cell (MDSCs) and TAMs. In the tumor, radioimmunotherapy significantly reduced the percentage of M2-TAMs compared to RT or anti-PD-L1 groups at Day 10 ( p = 0.0231, p = 0.0476, respectively). Radioimmunotherapy also reduced the percentage of M2-TAMs compared to those of other groups at Day 14 ( radioimmunotherapy vs RT p = 0.0141; vs Anti-PD-L1 p = 0.0020; vs control p = 0.0314; Fig. 5 a ) . A similar effect was also observed in the spleen at Day 14(radioimmunotherapy vs RT p = 0.0469; Anti-PD-L1 p = 0.0095; control p = 0.0024). In the TDLNs, we found that radioimmunotherapy and anti-PD-L1 groups decreased the percentage of M2 TAMs compared with the RT and control groups at Days 10 and 14 ( Fig. 5 a ) . In addition, we measured the expression of CD206 (M2-TAM marker) and CD86 (M1-TAM marker) in the tumor tissue at Day 14 by immunohistochemistry. We found the radioimmunotherapy group had the highest proportion of M1-TAMs and the lowest proportion of M2-TAMs compared to those of other groups ( Fig. 5 b ) . These results indicated that the radioimmunotherapy impaired the proliferation of M2-TAM. In addition, compared with RT group, radioimmunotherapy and anti-PD-L1 significantly decreased tumor-infiltrating MDSCs in tumor and TDLNs at Day 10 ( Fig. 5 c ). No differences were observed in the spleen. Radioimmunotherapy leads to changes in inflammation and immune profiles To compare the immune responses of radioimmunotherapy, transcriptome analyses were performed. The 20 most significantly enriched signaling pathways including cytokine-cytokine receptor interaction, chemokine signaling pathway and antigen processing and presentation genes are displayed in Fig. 6 a ( Fig. 6 a ). In addition, compared to the RT group, mice treated with radioimmunotherapy had the most profound changes in “regulation of cell-cell adhesion”, “regulation of immune effector process” and “response to IFN-gamma”, suggesting alteration of antitumoral activity ( Fig. 6 b ) . Next, we observed that the gene expression of chemokines involved in the attraction of T cells (eg, CXCL9, CXCL10 and CXCL11) increased in the tumor obtained to the radioimmunotherapy group ( Fig. 6 c ). We subsequently evaluated the expression of CXCL9 and CXCL10 at tumor sites by RT-PCR. Compared with RT, radioimmunotherapy improved the expression of CXCL9 and CXCL10 ( Fig. 6 d ) . These data indicating that radioimmunotherapy can enhance the production of IFN-γ and chemokines induced by IFN-γ. In addition, we found antigen presentation associated genes (MHC-I/II and Tap1/2), immune killing genes (Prf1, Ifn-γ, Gzma, and Gzmb) and innate immunity genes (Irf7, Isg15, Usp18) were upregulated in the radioimmunotherapy group ( Fig. 6 e ) . Taken together, these data demonstrated that the radioimmunotherapy can trigger both innate and adaptive immune response, providing a more immunostimulatory tumor microenvironment. Discussion In our study, we demonstrated that anti-PD-L1 treatment combined with radiotherapy resulted in better tumor control and prolonged survival in mice. Importantly, we demonstrate that radioimmunotherapy stimulated CD8 + T cell infiltration, improved the activity of effector CD8 + TILs, increased the infiltration of CD8 + T CM and reduced the accumulation of M2-TAMs and Tregs, therefore reprogramming an immune-stimulating tumor microenvironment and improving the anti-tumor efficacy in the mEC25 syngeneic mouse model. Similar changes were also seen in the spleen and TDLN. This is the first preclinical research depicting the synergistic antitumor immunity combining PD-L1 inhibitors with radiation in ESCC. Our results shown that radioimmunotherapy can increase the population of CD8 + TILs and the proportion of IFN-γ + CD8 + TILs in tumor microenvironment, indicating that radioimmunotherapy enhanced CD8 + T cell infiltration and activation. Previous observations have demonstrated that IFN-γ secreted by CD8 + TILs accounts for up-regulation of PD-L1 after delivery of fractionated radiotherapy of tumor cells( 14 , 27 ). In our study, we also found that PD-L1 expression is enhanced after fractionated radiotherapy in ESCC lines. In addition, radioimmunotherapy increased the GZMB secretion of CD8 + TILs, indicating that radioimmunotherapy augmented the cytotoxicity of CD8 + TILs. Moreover, our results showed that radioimmunotherapy eliminated regulatory T cells (Tregs). Previous studies have shown that Tregs can effectively inhibit effector T cells and revealed that radiation can recruit immunosuppressive Tregs( 28 ), which limit the immune response to radiotherapy. However, our study proves that radioimmunotherapy enhanced CD8 + TILs/Treg ratio via both boosting infiltration of CD8 + T cells and eradicating Tregs population, thereby improving antitumor immunity. The CD8 + TILs /Treg ratio has been recognized as a predictor of response to immunotherapy( 29 , 30 ). These results are in concordance with the results of Gong et al in a mouse model of lung cancer( 18 ). M2-type macrophages promote tumor immune tolerance, tumor invasion and metastasis( 31 ). Previous studies showed that radiation can recruits M2-type macrophages, thereby limiting radiation-induced adaptive antitumor immunity( 32 ). In addition, PD-L1 on the surface of macrophages binds to PD-1 on T cells, thereby inhibiting the co-stimulation of T cells by macrophage and resulting in T cell incompetence( 33 ). Previous studies have shown that PD-L1 blockade induces IFN-γ secretion of CD8 + TILs, further driving M1 polarization of macrophages( 31 ). Alternatively, Jones et al demonstrated that macrophage exhaustion partially impaired immunosuppression after radiotherapy, but additional anti-PD-L1 therapy was essential to accomplish tumor remission( 34 ). In our study, only radioimmunotherapy significantly reduced the frequency of M2-type TAMs, which may be due to the fact that radiotherapy promotes the expression of PD-L1 on the surface of macrophages, thereby increasing the targeted sites of PD-L1 inhibitors. MDSC can inhibit T cell function and promote immune escape( 18 ). Previous studies demonstrated that radioimmunotherapy eventually results in a decreased amount of MDSC and PD-L1 + MDSCs in irradiated and non-irradiated tumors( 13 , 24 ). In our experiments, both radioimmunotherapy and anti-PD-L1 inhibitor inhibited MDSC recruitment. In conclusion, the addition of PD-L1 inhibitor attenuated the effects on immunosuppressive cell populations that were seen in the radiotherapy. Therefore, radioimmunotherapy reshaped the immune-stimulated microenvironment via elimination of immunosuppressive cells and promoting CD8 + T cells infiltration in TME. T CM cells are characterized by self-renewal and long-term survival in vivo and can be rapidly differentiated into effector T cells after tumor antigens stimulation( 35 ). Several researchers have revealed that combining radiotherapy with ICIs could add the account of CD8 + T CM and inhibit tumor recurrence( 35 , 36 ). We demonstrated that the proportion of infiltrating CD8 + T CM cells were highest in radioimmunotherapy group in TME, spleen and TDLN, which may be helpful to delay tumor recurrence. For the investigation of immune related gene expression, our data showed that CXCL9 and 10 induced by IFN-γ stimulation were enhanced in radioimmunotherapy arm. The elevated CXCL9 and CXCL10 level was associated with recruiting antitumor CD8 + T cells and augmented activation and cytotoxic responses of CD8 + TILs( 37 – 39 ). The expression of interferon-stimulated genes (Irf7, Isg15, Usp18) were up-regulated in the radioimmunotherapy group. Notably, type I IFN stimulates antigen-presenting cells (APCs) to deliver tumor antigens for maintaining CD8 + T cell initiation( 12 , 40 ), while suppressing Treg function to reinforce antitumor efficacy( 41 , 42 ). Overall, radioimmunotherapy reprogrammed a more immunostimulatory tumor microenvironment. T cell exhaustion is inevitable under the continuous stimulation of tumor antigens. In our study, radioimmunotherapy increased the infiltration of PD-1 + CD8 + T cells and PD-1 + CD4 + T cells, which was consistent with the findings of Dudzinski et al in prostate cancer model, indicating that radioimmunotherapy increased tumor antigen presentation and T cell depletion( 22 ). Exhausted T cells (Tex) can be categorized into stem-like exhausted and terminally exhausted populations. Stem-like T cells contribute to the generation of durable anti-tumor immunity( 43 ). Wei et al shown that combing radiotherapy with PD-1 inhibitor reverses the terminal exhaustion into stem-like exhausted PD-1 + CD8 + T cells( 44 ). The expression of other immune checkpoints is on behalf of advanced T cell exhaustion( 45 ). Therefore, it is necessary to further evaluate the status of PD-1 + CD8 + T cells. Our studies reveal the dynamic changes of immune related cell populations in spleen and TDLN. In the spleen, radioimmunotherapy significantly and consistently increased the accumulation of IFN-γ + CD8 + TILs. Additionally, antigen-specific CD8 + T CM and CD8 + TILs/Treg ratio are also greatly increased in the spleen. And radioimmunotherapy also reduced M2-type TAM. In draining lymph nodes, radioimmunotherapy increased the infiltration of IFN-γ + CD8 + T cells and CD8 + TILs/Treg ratio, expanded the number of CD8 + T CM and inhibited the recruitment of M2-type TAM. Some studies have pointed out that the immune microenvironment of TDLN may be a theoretical supplement to TME, and alleviating immunosuppression in TDLN can promote systemic anti-tumor T cell immunity, thereby effectively controlling distant tumor sites. Intact TDLN is essential in the anti-tumor immune response of radioimmunotherapy by promoting CD8 + T cell accumulation as well as M1/M2 macrophage ratio( 46 , 47 ). In conclusion, changes in immunostimulatory activity in the spleen and TDLNs may enhance systemic antitumor effects and increase the incidence of abscopal effects. The 4Gy×3 fractionated radiotherapy scheme was selected in our study. Since most patients received low-dose radiotherapy in clinic, this scheme may be more suitable for clinical patients than the 8Gy×3 fractionated radiotherapy, which has important significance for the design of future clinical trials. In this study, mEC25 mouse syngeneic tumor model was selected, which was derived from the orthotopic esophageal tumor of C57BL/6 mice. This subcutaneous model provides the ability to easily establish tumors in an immunocompetent host and further characterize the tumor-infiltrating lymphocytes and microenvironment, which contribute to investigate the therapeutic effects and related mechanisms in the presence of an intact immune system. However, there are some limitations to our experiment. First, the combination therapy did not completely eliminate the transplanted tumors in the mice. As in the study of Philippou et al( 23 ), the reason may be that the radiation dose of 3×4Gy is not enough to produce CD8 + T cell-dependent anti-tumor response. Therefore, in order to successfully apply radioimmunotherapy to clinical patients with esophageal cancer, treatment combinations such as timing/sequence of treatment and radiation dose/fractionation are required for examining in preclinical models and early clinical trials to define best approach. In addition, the data of this study are limited to a single mouse solid malignant tumor model, and diversified mouse esophageal squamous cell carcinoma models should be used. More importantly, in future studies, we should pay attention to the phenomenon of "abscopal effect" caused by radioimmunotherapy and the influence of different dose sequence combinations on this phenomenon. In addition, the role of TDLNs in the anti-tumor effect of radioimmunotherapy needs to be further clarified. Overall, our study showed that radiotherapy combined with PD-L1 inhibitors is a promising synergistic treatment option for ESCC by improving the immunosuppressive tumor microenvironment. By providing preclinical data, our study provides theoretical support for the design or interpretation of clinical studies of the radioimmunotherapy. Conclusion Based on preliminary observations, our results increase the persuasiveness of radiotherapy combining with PD-L1 inhibitor as a potential synergistic treatment modality for ESCC. Importantly, this study provides a foundation for clinical researches of ESCC comprehensive treatment. Declarations Acknowledgements We thank the staff of Hangzhou Cancer Institution, Affiliated Hangzhou Cancer Hospital, for their technical support. Author contributions DQH, ZHF and ZK conceived and supervised the experiments. the experiments were performed by YZH, YQQ, YJ, WYP, YZH, YQQ and ZHF interpreted the results of the experiments. YZH and ZK completed the manuscript. All authors read and approved the final manuscript. Funding This study was supported by Clinical Research Fund Project of Zhejiang Medical Association (2021ZYC-Z05); Zhejiang Province Health Department Project(2022KY101); "Great Medical Sincerity" cancer prevention and treatment research and academic exchange public welfare program; Hangzhou Agricultural and social Development Research Project(2020ZDSJ0552). Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests Ethics approval We state that animal experiment protocols were approved by the Institutional Animal Care and Use Committee (IACUC 20010652). 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Cancer Commun (Lond). 42: 971-86. doi: 10.1002/cac2.12348 Fransen MF, Schoonderwoerd M, Knopf P, Camps MG, Hawinkels LJ, Kneilling M, van Hall T, Ossendorp F (2018) Tumor-draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy. JCI Insight. 3. doi: 10.1172/jci.insight.124507 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-4338719","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299874980,"identity":"16181f40-015c-4173-b1c3-79c46231ab27","order_by":0,"name":"Zihao Yin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIie3Rv0oDMRzA8ZRAuvx6t+ZQrj5CjhtUKPVV8vOgk9AHcPBXCukrnC8ijr+SocthV6GLUnC+Gx0Uz1nIdXTIBzLkz5cQIkQU/UeyH0hCqDHhW/c9gzSlE5ME+FjUanGe1XzCTaM+yTV+nIHyM0M2fNrsJr59f57nCvxCA+zBCB613V0g8Yk12FSlGrvqSusDXEqS2eNTKAFj0Ul00OCrMQe4JlZyMpAwugd0etlqa1/6qR1MCkLn+8TeamYeTjIPpUC3KxVwVayogqzeroNvSfZN2X26+3y6ITx+0fwmTdfbtgskF/x37febAqbh7SiKoqj3AzqpVZ3f6nuBAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hangzhou Cancer Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zihao","middleName":"","lastName":"Yin","suffix":""},{"id":299874981,"identity":"818b0d3d-bd65-4ec8-80be-01a9929718af","order_by":1,"name":"Qinghua Deng","email":"","orcid":"","institution":"Affiliated Hangzhou Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Deng","suffix":""},{"id":299874982,"identity":"a61fa50e-1ca9-425b-a13f-3469c2ee1a08","order_by":2,"name":"Ke Zhang","email":"","orcid":"","institution":"Affiliated Hangzhou Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Zhang","suffix":""},{"id":299874983,"identity":"0b34e4ad-198f-4bcb-9839-c3fa05aa69a6","order_by":3,"name":"Jing Yue","email":"","orcid":"","institution":"Hangzhou Cancer Institution, Affiliated Hangzhou Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yue","suffix":""},{"id":299874984,"identity":"10aa21e6-f994-47c8-b37c-6049e256bde6","order_by":4,"name":"Yaping Wang","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Wang","suffix":""},{"id":299874985,"identity":"f08ff421-213a-47c4-896d-7aca27f68dd7","order_by":5,"name":"Qingqing Yu","email":"","orcid":"","institution":"Affiliated Hangzhou Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qingqing","middleName":"","lastName":"Yu","suffix":""},{"id":299874986,"identity":"357ef509-6927-4ad2-a7be-7daabc557a91","order_by":6,"name":"Hongfang Zhang","email":"","orcid":"","institution":"Hangzhou Cancer Institution, Affiliated Hangzhou Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongfang","middleName":"","lastName":"Zhang","suffix":""},{"id":299874987,"identity":"ed7b4312-de46-4d7d-9ff0-6b3b7812b7d2","order_by":7,"name":"Rongjun Tang","email":"","orcid":"","institution":"Affiliated Hangzhou Cancer Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rongjun","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2024-04-28 16:25:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4338719/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4338719/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56116125,"identity":"b17ff026-7481-498b-8a76-94b7c1051c04","added_by":"auto","created_at":"2024-05-08 17:53:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":645893,"visible":true,"origin":"","legend":"\u003cp\u003eRadioimmunotherapy controlled tumor growth and improved survival in mice. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic diagram of radiation therapy (RT) and anti-PD-L1 therapy. (\u003cstrong\u003eb\u003c/strong\u003e) Change in tumor volume over time for mice treated with different regimens. (\u003cstrong\u003ec\u003c/strong\u003e) Kaplan Meier survival curves for each treatment regimen. n = 10 in each group except the anti-PD-L1 alone group (n=7). (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.005; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0005; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"OnlineFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4338719/v1/0487994a8bfdd5bc92680518.jpg"},{"id":56115889,"identity":"45d5c07d-7bc1-48c3-8983-22b8269772be","added_by":"auto","created_at":"2024-05-08 17:45:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3048568,"visible":true,"origin":"","legend":"\u003cp\u003eRadioimmunotherapy impacts T cell infiltration. (\u003cstrong\u003ea\u003c/strong\u003e) Representative plots of CD8\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e T cells in tumors at Day 14. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of infiltrating CD8\u003csup\u003e+\u003c/sup\u003eTILs at Days 10 and 14 in tumor, spleen and TDLNs. (\u003cstrong\u003ec\u003c/strong\u003e) Representative plots of CD25\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells in tumors at Day 14. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of CD25\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003eTreg in the tumor, spleen and TDLNs at Days 10 and 14. (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of the ratio of CD8\u003csup\u003e+\u003c/sup\u003eTILs/CD25\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003eTreg at Days 10 and 14 in the tumor, spleen and TDLNs. (\u003cstrong\u003ef\u003c/strong\u003e) Representative immunohistochemistry images of CD8 and FOXP3 expression in tumors from each treatment group at Day 14. The original magnification was 200×(n=3 mice each group). (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.005; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0005; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"OnlineFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4338719/v1/fc8faec3fcdb9b8ddc91d5fd.jpg"},{"id":56115892,"identity":"51c28d6c-1072-4fff-bc4a-e38d789de079","added_by":"auto","created_at":"2024-05-08 17:45:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1506451,"visible":true,"origin":"","legend":"\u003cp\u003eRadioimmunotherapy promotes immune memory activation. (\u003cstrong\u003ea\u003c/strong\u003e) Quantification of infiltrating CD44\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e memory T cells at Days 10 and 14 in the tumor, spleen and TDLNs. (\u003cstrong\u003eb\u003c/strong\u003e) Representative graphs of CD62L and CD44 expression on CD8\u003csup\u003e+\u003c/sup\u003eTILs at Day 14 in the tumor, spleen and TDLNs. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of infiltrating CD8\u003csup\u003e+\u003c/sup\u003eT\u003csub\u003eCM\u003c/sub\u003e(CD62L\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e) cells and (\u003cstrong\u003ed\u003c/strong\u003e) CD8\u003csup\u003e+\u003c/sup\u003eT\u003csub\u003eEM\u003c/sub\u003e(CD62L\u003csup\u003e-\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e) cells at Days 10 and 14 in the tumor, spleen and TDLNs. (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.005; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0005; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"OnlineFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4338719/v1/2e96678f9ea9d98cc13a7eca.jpg"},{"id":56115887,"identity":"cc7d9060-4b63-4a0e-b23a-807e68f80a22","added_by":"auto","created_at":"2024-05-08 17:45:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2491635,"visible":true,"origin":"","legend":"\u003cp\u003eRadioimmunotherapy promotes CD8\u003csup\u003e+\u003c/sup\u003e T cell effector function and increased expression of CD8\u003csup\u003e+\u003c/sup\u003eTILs exhaustion marker. (\u003cstrong\u003ea\u003c/strong\u003e) Representative graphs of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e TILs in tumors, spleen and TDLNS at Day 14. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+ \u003c/sup\u003eTILs in mouse tumors, spleen and TDLNs at Days10 and 14. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of GZMB\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e TILs in mouse tumors spleen and TDLNs at Days 10 and 14. (\u003cstrong\u003ed\u003c/strong\u003e) Representative images of GZMB by immunohistochemistry in tumors at Day 14\u003cstrong\u003e \u003c/strong\u003efrom each group. (\u003cstrong\u003ee\u003c/strong\u003e) Quantification of PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eTILs in the tumor, spleen, and TDLNs at Days 10 and 14. (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.005; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0005; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"OnlineFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4338719/v1/72c76069a0cf6e54a903788f.jpg"},{"id":56115890,"identity":"c2f7f5b5-5e85-4366-a0d7-b8f33f7eb9c2","added_by":"auto","created_at":"2024-05-08 17:45:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2912131,"visible":true,"origin":"","legend":"\u003cp\u003eRadioimmunotherapy influences the recruitment of different immunosuppressive cell populations. (\u003cstrong\u003ea\u003c/strong\u003e) Quantification of M2-TAM in the tumor, spleen and TDLNs at Days 10 and 14. (\u003cstrong\u003eb\u003c/strong\u003e) Representative images of CD206 and CD86 by immunohistochemistry in tumors at Day 14 from each treatment group. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of MDSCs in the tumor, spleen and TDLNs at Days 10 and 14. ( *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.005; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0005; ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"OnlineFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4338719/v1/d7f2d985f6412d564127e612.jpg"},{"id":56115891,"identity":"9774e858-ce85-4a9c-90a8-6bccb1df59ef","added_by":"auto","created_at":"2024-05-08 17:45:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1139536,"visible":true,"origin":"","legend":"\u003cp\u003eRadioimmunotherpy promoted an immunestimulatory antitumor microenvironment. (\u003cstrong\u003ea\u003c/strong\u003e) The enrichment of gene by KEGG pathways between radiation group and radioimmunotherapy groups. (\u003cstrong\u003eb\u003c/strong\u003e) GO analysis of top upregulated pathways in radioimmunotherapy group compared to radiation group. (\u003cstrong\u003ec\u003c/strong\u003e) Heatmap of enriched genes between radiation group and radioimmunotherapy groups in cytokine-cytokine receptor interaction and chemokine signaling pathway. (\u003cstrong\u003ed\u003c/strong\u003e) The mRNA expression of Cxcl9 and 10 genes in tumor mass. (\u003cstrong\u003ee\u003c/strong\u003e) Heatmap showing the expression of immunomodulatory genes in mEC25 tumor samples for radiation and radioimmunotherapy groups, such as regulating antigen processing and presentation genes, immune activation and cytotoxicity of T cells and innate immune response. The intensity of the heatmap represents z-scores. Data was analyzed using RNA-seq 14 days after irradiation (n=3 mice per group). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 with an unpaired two- tailed t- test.\u003c/p\u003e","description":"","filename":"OnlineFigure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4338719/v1/8e82e7b46e675c420d65055e.jpg"},{"id":56413675,"identity":"ce5e756e-0844-463e-a66d-a94265f614e5","added_by":"auto","created_at":"2024-05-13 21:54:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1666300,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4338719/v1/3bcb05a6-d56f-41d6-837a-b9774fe6e87e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impacts of Combining PD-L1 inhibitor and Radiotherapy on the Tumour immune microenvironment in a Mouse Model of Esophageal Squamous Cell Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eESCC is a common malignant tumor worldwide(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Due to the inconspicuous early symptoms and the lack of effective early diagnosis methods, ESCC is often diagnosed at advanced stages and patients have a poor prognosis(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Definitive chemoradiotherapy is the current standard protocol for unresectable locally advanced ESCC. Previous studies have explored optimal chemotherapy protocols and substituted radiation dose fractionation, but local recurrence rates remain high, with 5-year OS rates of only 20%-25%(\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImmune checkpoint inhibitors have demonstrated as a prospective therapeutic strategy in the treatment of ESCC patients. Several studies, including KEYNOTE-590(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), Escort-1st(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)have demonstrated that PD-1/PD-L1 monotherapy improved the survival of advanced ESCC patients, demonstrating reliable efficacy and a favorable safety. However, due to the heterogeneity of tumors and differences in the immune microenvironment, the application of ICI monotherapy is limited by remission rates of only roughly 20%. To overcome this constraint, multiple researches have investigated the protocols of combining immunotherapy with alternate treatment(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRadiotherapy (RT) is be involvement in all stages of ESCC. Radiation has been proved to produce significant effects on the immune response of various types of tumors(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Briefly, radiation induces the death of immunogenic tumour cells and production of tumour antigens, which function as in situ vaccines to activate systemic anti-tumour immune responses.\u003c/p\u003e \u003cp\u003eAdditionally, local radiation contributes to tumor remission via promoting the recruitment of CD8\u003csup\u003e+\u003c/sup\u003eTILs and secretion of IFN-γ(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Nevertheless, radiation increases the expression of PD-L1 on the surface of tumor cells, which could then promote immune tolerance through suppressing the activation of lymphocytes, suggesting the potential synergistic effects of anti-PD-1/PD-L1 immunotherapy(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Moreover, ICIs can contribute to the activation of effector T cells to eliminate tumor cells and normalization of tumor vasculature, increasing lymphocyte infiltration and sensitivity to radiotherapy(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In summary, radiotherapy and immunotherapy complement each other and exert a stronger antitumor immune response. Previous studies have confirmed that radiotherapy in combination with PD-L1 inhibitors can enhance systemic anti-tumor effects in preclinical models of lung(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), melanoma(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), colorectal cancer(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), breast cancer(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), head and neck squamous cell carcinoma(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), prostate cancer(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), and pancreatic cancer(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). But this combination has not been explored in esophageal cancer models.\u003c/p\u003e \u003cp\u003eIn a phase Ib clinical trial of combing radiotherapy with the PD-1 inhibitor Camrelizumab, involving 19 patients with newly diagnosed locally advanced ESCC who refused or could not tolerate concurrent chemoradiotherapy, the median OS was 16.7 months. This is comparable to the duration of patients receiving concurrent chemoradiotherapy regimens (median OS time of about 18.1\u0026ndash;19 months)(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). At present, phase III clinical trials of concurrent chemoradiotherapy combined with anti-PD-L1 therapy in the treatment of patients with ESCC are currently underway(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRadiotherapy combined with anti-PD-L1 immunotherapy has emerged as a prospective application in ESCC. However, the synergistic effect and molecular mechanism of radioimmunotherapy in ESCC are not completely understood. Therefore, this research aims to evaluate the synergistic anti-tumor effect in a ESCC preclinical model and define the molecular mechanism through changes of immune cells and cytokines in the immune microenvironment when radiotherapy combined with PD-L1 inhibitors.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of cell lines\u003c/h2\u003e \u003cp\u003eThe murine ESCC cell line mEC25 was purchased from Shenzhen Wenhua Times Technology Co. and cultured in RPMI-1640 medium (11875119, Gibco, CA, USA) with 10% fetal bovine serum (SA211.02, Cellmax, Beijing, China) and maintained in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator at 37℃.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vivo studies\u003c/h3\u003e\n\u003cp\u003eFemale C57BL/6 mice (4\u0026ndash;6 weeks old) were provided by the Laboratory Animal Center, Zhejiang Academy of Medical Sciences (Hangzhou, China). All mice were maintained and treated depending on the Institutional Animal Care and Use Committee regulations at the Clinical Research Institute.\u003c/p\u003e \u003cp\u003eMice were injected subcutaneously with 5x10\u003csup\u003e5\u003c/sup\u003e mEC25 cells on the right flank. When tumors reached 90\u0026ndash;120 mm\u003csup\u003e3\u003c/sup\u003e (Day 1), each mouse was randomly divided into 4 groups: Isotype control; RT (4Gy\u0026times;3)\u0026thinsp;+\u0026thinsp;isotype; Anti-PD-L1 antibodies; RT\u0026thinsp;+\u0026thinsp;Anti-PD-L1 antibodies. For radiation therapy, tumors were irradiated in 3 fractions for a total dose of 12 Gy. RT was administered via an electron beam on days 1, 4, and 7 from the beginning of therapy. Briefly, mice were anesthetized and placed under a lead shield with a 1 cm\u003csup\u003e2\u003c/sup\u003e hole to limit tumor exposure. Anti-PD-L1(BioXCell, clone 10F.9G2) or isotype control (BioXCell, rat IgG2b) were delivered by intra-peritoneal injection concurrent with radiotherapy. We measured the size of each tumor using calipers and calculated its volume (length\u0026times;width\u003csup\u003e2\u003c/sup\u003e\u0026times;0.5). Survival time of mice was recorded (day 1 until tumor volume reached 1500 mm\u003csup\u003e3\u003c/sup\u003e or animals were executed). As soon as the mice reached a limit point (tumor volume\u0026thinsp;\u0026ge;\u0026thinsp;1500 mm\u003csup\u003e3\u003c/sup\u003e, pain, significant necrosis), mice were anesthetized and sacrificed by cervical dislocation.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analysis\u003c/h3\u003e\n\u003cp\u003eThe tumor, spleen and tumor draining lymph nodes (TDLNs) were isolated on days 10 and 14. Tumor tissue were cut into small pieces and incubated at 37\u0026deg;C for 30 minutes (tumor) or 10 minutes (TDLN) in RPMI medium with collagenase type IV and deoxyribonuclease type I, subsequently mechanically separated on frosted slides. Spleens were mechanically separated directly on frosted slides. Single-cell suspension was obtained by filtering the cell suspension through a 70-\u0026micro;m cell strainer (Corning). A lysis buffer (NH4Cl, NaHCO3, EDTA) was used to lyse erythrocytes at room temperature and quenched with RPMI. Fixable Viability Stain 440UV (566332, BD) and α-CD16/32 (553142 BD) were used to label and block the cells.\u003c/p\u003e \u003cp\u003eFor surface staining, monoclonal antibodies (\u003cb\u003esupplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/b\u003e) were used to stain the samples for 30 minutes at room temperature. Each analysis consisted of at least three replicates. The data were obtained by using FACSCalibur machines and analyzed using Flowjo software.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular staining\u003c/h2\u003e \u003cp\u003eTo assess the secretion of IFN-γ and granzyme B, the cell suspensions were stimulated with 1 \u0026micro;M phorbol 12- myristate 13- acetate (PMA, Sigma) and ionomycin (Sigma) for 4 hours at 37\u0026deg;C. Then, together with intracellular staining of Foxp3, cells were fixed and permeabilized via FOXP3 Fix/Perm Buffer Set (562574, BD). And then stained with monoclonal antibodies (\u003cb\u003esupplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/b\u003e) for 30 min on ice depending on the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003e4% paraformaldehyde 4% was used to fix tumor tissues and paraffin was used to embed them. Sections were deparaffinized, rehydrated, antigenically repaired and stained overnight at 4\u0026deg;C with the primary antibodies as follows: CD8(D4W2Z, 98941), FOXP3(D608R, 12653), CD206(E6T5J, 24595), CD86(E5W6H, 19589), GZMB(13588-1-AP). Secondary antibodies were incubated with the sections on the second day and reacted with ABC kits (Vector, Burlingame, CA). Stained sections were observed using ECLIPSE E100 light microscope (NIKON) and K-Viewer software. Quantitative analysis of optical density was evaluated by Image J software (National Institutes of Health). The average density of 3 slices per sample was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHomologous transplantation tumour mRNA sequencing and bioinformatic analysis\u003c/h2\u003e \u003cp\u003eThe total RNA was extracted by Trizol Reagent (15596026, Thermo Fisher Scientific Inc., MA, USA). RNA-seq assay was performed using the Illumina Hiseq platform from Wuhan SeqHealth. Musmusculus GRCm38 was the reference genome. Differentially expressed genes (DEGs) were screened using the edgeR package in R software. GO and KEGG 2018 databases were used for gene set enrichment analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eThe extraction of total RNA was performed using Trizol Reagent. Reverse transcription was accomplished by using PrimeScript\u0026trade; RT Master Mix (RR036A, Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). Quantitative RT-PCR (qRT-PCR) was carried out with TB green (RR420A, Takara Biomedical Technology Co., Ltd., Beijing, China). Furthermore, relative expression of each targeted gene including CXCL9 and CXCL10 (primers were listed in \u003cb\u003eSupplemental Table\u0026nbsp;2\u003c/b\u003e) was calculated and normalized using 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eOne-way ANOVA analysis was used to statistical comparisons between three or more groups, followed by Tukey\u0026rsquo;s multiple comparison test. For mice tumor volume experiments, statistical analysis was performed using mixed-effects model with Tukey\u0026rsquo;s multiple comparison test. Survival curves for different groups of mice were generated using the Kaplan- Meier method. The the log- rank Mantel- Cox test was used to evaluated the survival curves of mice in each group. Tumor volumes or loads of at least three animals in each group were subjected to t-tests, and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded statistically significant (*, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The statistical analyses were performed with GraphPad Prism statistical analysis and graphing software (GraphPad 9).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnti-PD-L1 inhibitors combined with radiotherapy synergistically enhanced antitumor effects in esophageal cancer mice model\u003c/h2\u003e \u003cp\u003eIn our previous study, we demonstrated that fractionated radiotherapy increased the expression of PD-L1 in esophageal cancer cell lines \u003cb\u003e(Supplementary Fig.\u0026nbsp;1)\u003c/b\u003e. We firstly examined whether anti-PD-L1 inhibitor combined with radiotherapy could synergistically reinforce anti-tumor efficacy. We subcutaneously injected mEC25 murine cancer cells into syngeneic mice and subjected the mice to different treatment regimens-radiation therapy (RT) alone, anti-PD-L1 alone, and radioimmunotherapy (RT combined with anti-PD-L1) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. We found that the radioimmunotherapy demonstrated the strongest inhibition of tumor growth \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Radioimmunotherapy significant slowed down tumor growth rate compared with RT (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008), anti-PD-L1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) and control groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01). In addition, radioimmunotherapy also improved survival \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e compared to RT (\u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.0007), anti-PD-L1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0248) and control groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001). Compared to 33 days for radioimmunotherapy, the median survival for RT was 26 days, for anti-PD-L1 was 28 days and for control group was 24 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRadioimmunotherapy impacts T cell infiltration\u003c/h2\u003e \u003cp\u003eTo assess alterations of composition in tumor microenvironment after radioimmunotherapy, lymphocytes were collected from the tumor, spleen and tumor draining lymph nodes (TDLN) at Days 10 and 14. The extent of tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells (TILs) was associated with suppression of tumor growth. In the tumor, at Day 10, non-significant increase of the proportion of CD8\u003csup\u003e+\u003c/sup\u003e TILs in radioimmunotherapy was observed compared with other groups. At Day 14, radioimmunotherapy significantly enhanced the frequency of CD8\u003csup\u003e+\u003c/sup\u003e TILs compared to RT and control groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0049, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0413 respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Similarly, in the TDLN, radioimmunotherapy increased the proportion of CD8\u003csup\u003e+\u003c/sup\u003eTILs compared to RT and control groups at Day 10(39.8%\u0026plusmn;0.6% radioimmunotherapy vs 31.2%\u0026plusmn;3.1% RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0472; 30.4%\u0026plusmn;0.2% control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0316; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In spleen, there were no significant difference among the groups at any time point \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMeanwhile, there were no significant differences in the population of CD4\u003csup\u003e+\u003c/sup\u003e T cells at any time point in the tumor, spleen and TDLNs \u003cb\u003e(Supplementary Fig.\u0026nbsp;2a)\u003c/b\u003e. Therefore, we further investigated the impact of radioimmunotherapy specifically on the regulatory T (Treg) cell population, which are an immunosuppressive subset of CD4\u003csup\u003e+\u003c/sup\u003e T cells. In the tumor, at Day 14, radioimmunotherapy decreased the ratio of infiltrating Tregs compared to those of the other groups (radioimmunotherapy vs RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0105; control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). The Anti-PD-L1 group also significantly reduced infiltrating Tregs compared with the RT and control groups. Interestingly, in the spleen at Day 10, we found that radiation increased the proportion of Tregs compared to the control group, suggesting that RT can induce the upregulation of Tregs as an immunosuppressive regulator. Notably, radioimmunotherapy reversed this upregulated proportion of infiltrating Tregs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In addition, the proportion of Tregs was markedly reduced in the anti-PD-L1 group compared with RT and control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eWe also investigated the proportion of CD8\u003csup\u003e+\u003c/sup\u003eTILs/Foxp3\u003csup\u003e+\u003c/sup\u003eTregs. In tumor, radioimmunotherapy increased CD8TILs\u003csup\u003e+\u003c/sup\u003e/Treg ratio compared with those of other groups at Day 14, with a same phenomenon seen in the spleen and TDLNs at Day 14(\u003cb\u003eTumor\u003c/b\u003e: 75.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18.1 radioimmunotherapy vs 14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 radiation \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0105; vs 25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0305; vs 20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0175; \u003cb\u003eSpleen\u003c/b\u003e: 90.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 radioimmunotherapy vs 55.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5 radiation \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0170; vs 60.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3 Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0380; vs 55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0175; \u003cb\u003eTDLNs\u003c/b\u003e: 67.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 radioimmunotherapy vs 39.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 radiation \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003; vs 46.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0022; vs 53.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0206; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). No significant differences were found at Day 10 in the tumor, spleen and TDLNs. In addition, these results were consistent with the concomitant immunohistochemistry on day 14\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e. Therefore, these results demonstrated that radioimmunotherapy can upregulate the ratio of CD8\u003csup\u003e+\u003c/sup\u003eTILs/Tregs by improving the recruitment of CD8\u003csup\u003e+\u003c/sup\u003e TILs and reduced the accumulation of Tregs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRadioimmunotherapy enhances immune memory activation\u003c/h2\u003e \u003cp\u003eWe further investigated whether the T cell activation status was different in different treatment groups. We defined the effector memory phenotype as CD44\u003csup\u003e+\u003c/sup\u003e CD62L\u003csup\u003e\u0026minus;\u003c/sup\u003e, and central memory cells as CD44\u003csup\u003e+\u003c/sup\u003e CD62L\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFirstly, we accessed CD8\u003csup\u003e+\u003c/sup\u003e TILs populations expressing CD44. CD44 is an activated marker of T cells after antigen stimulation. At Day 10, the percentage of CD44\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e TILs of radioimmunotherapy and anti-PD-L1 was lower than RT and control groups in the tumor. In the spleen, at Day 10, radioimmunotherapy increased the proportion of CD44\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells compared to other groups (48.2%\u0026plusmn;2.2% radioimmunotherapy vs 37.2%\u0026plusmn;1.9% RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031; 36.8%\u0026plusmn;0.1% Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0257; 32.1%\u0026plusmn;3.2% control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0046; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Moreover, radioimmunotherapy enhance the frequency of CD44\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e TILs compared with RT and control groups at Day 14(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In the TDLNs, radioimmunotherapy upregulated the percentage of CD44\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e TILs compared to RT and control groups at Day 10(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0391; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0356, respectively). At Day 14, radioimmunotherapy upregulated the percentage of CD44\u003csup\u003e+\u003c/sup\u003eCD8\u0026thinsp;+\u0026thinsp;TILs compared to anti-PD-L1 and control groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0316; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eFor the infiltrating central memory CD8\u003csup\u003e+\u003c/sup\u003eT cells (T\u003csub\u003eCM\u003c/sub\u003e), we can see that radioimmunotherapy enhanced the accumulation of CD8\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eCM\u003c/sub\u003e at Day 14 in the tumor compared to those of other groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). And the same trends were also observed in the TDLNs at Days 10 and 14 \u003cb\u003e(Day 10\u003c/b\u003e: 11.1%\u0026plusmn;0.5% radioimmunotherapy vs 5.8%\u0026plusmn;0.9% RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0011; 6.7%\u0026plusmn;0.5% Anti PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0035; 6.1%\u0026plusmn;0.4% control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0014; \u003cb\u003eDay 14\u003c/b\u003e: 13.8%\u0026plusmn;0.8% radioimmunotherapy vs 9.8%\u0026plusmn;0.3% RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0087; 3.2%\u0026plusmn;0.4% Anti PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 2.3%\u0026plusmn;0.6% control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c\u003cb\u003e)\u003c/b\u003e. In spleen, compared with Anti-PD-L1 and control groups at Day 10, radioimmunotherapy increased the CD8\u003csup\u003e+\u003c/sup\u003eT\u003csub\u003ecm\u003c/sub\u003e cells infiltration \u003cb\u003e(\u003c/b\u003e8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% radioimmunotherapy vs 4.4%\u0026plusmn;0.8% Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0022; 4.1% control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0012; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c\u003cb\u003e)\u003c/b\u003e. At Day 14, compared with RT and control groups, radioimmunotherapy significantly increased the CD8\u003csup\u003e+\u003c/sup\u003eT\u003csub\u003ecm\u003c/sub\u003e cells infiltration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0368; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004, respectively). These results indicate that radioimmunotherapy can increase the population of T\u003csub\u003eCM\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFor infiltrating CD8\u003csup\u003e+\u003c/sup\u003e effector memory T cells (T\u003csub\u003eEM\u003c/sub\u003e), in the tumor, we observed the same phenomenon consistent with CD44\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eTILs at Days 10 and 14. In the spleen, no statistical significance were observed. In the TDLNs, the proportion of CD8\u003csup\u003e+\u003c/sup\u003eT\u003csub\u003eEM\u003c/sub\u003e cells were higher in the anti-PD-L1 compared with other groups at Day 14. \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 \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRadioimmunotherapy promotes CD8\u003csup\u003e+\u003c/sup\u003e T cell effector function\u003c/h2\u003e \u003cp\u003eAdditionally, we assessed the activation status and effector function of the CD8\u003csup\u003e+\u003c/sup\u003e TILs based on IFN-γ and Granzyme B (GZMB) expression. At Day 10 in the tumor, an significant increase in the proportion of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eTILs was observed in radioimmunotherapy compared with the RT and control groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0047, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0013, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Similarly, we observed the same phenomenon in the spleen at Day 14. Moreover, radioimmunotherapy showed a significantly higher frequency of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eTILs compared to other groups in the tumor and TDLNs at Day 14 \u003cb\u003e(Tumor\u003c/b\u003e: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003eTDLN\u003c/b\u003e: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0051; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0070; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0017; respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Meanwhile, similar phenomenon was seen in spleen at Day 10. Compared with the RT at Day 10, radioimmunotherapy also significantly enhanced the accumulation of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e TILs in the TDLNs \u003cb\u003e(\u003c/b\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0338, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eConcurrently, we evaluated the GZMB secretion of CD8\u003csup\u003e+\u003c/sup\u003e T cells. At Day 10 in the tumor and TDLNs, a significantly enhanced proportion of CD8\u003csup\u003e+\u003c/sup\u003e TILs expressing GZMB in radioimmunotherapy compared with other groups \u003cb\u003e(Tumor\u003c/b\u003e: 31.6%\u0026plusmn;0.3% radioimmunotherapy vs 11.0%\u0026plusmn;0.2% RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 18.7%\u0026plusmn;0.1% Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 12.7%\u0026plusmn;0.2% control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003eTDLN\u003c/b\u003e: 12.2%\u0026plusmn;0.5% radioimmunotherapy vs 7.8%\u0026plusmn;0.2% RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0081; 8.0%\u0026plusmn;1.1% Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002; 6.9%\u0026plusmn;0.4% control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Meanwhile, we observed the same phenomenon at Day 14 in the tumor by flow cytometry and immunohistochemistry \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d\u003cb\u003e)\u003c/b\u003e. There were no significant differences in the spleen \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. In summary, radioimmunotherapy can enhance both activation and cytotoxicity of CD8\u003csup\u003e+\u003c/sup\u003e TILs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRadioimmunotherapy enhances the expression of immune exhaustion markers\u003c/h2\u003e \u003cp\u003eIn tumor, compared with control group, we found that the group treated with radioimmunotherapy showed a higher amount of PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eTILs at Day 14 \u003cb\u003e(\u003c/b\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0365; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Similarly, the same phenomenon was observed at Day 10 in the spleen. No differences were observed in the TDLN. In addition, radioimmunotherapy notably enhanced the proportion of PD-1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e TILs at Day 10 compared with those of other groups \u003cb\u003e(Supplementary Fig.\u0026nbsp;2b)\u003c/b\u003e. And no differences were revealed in the spleen and TDLNs. Taken together, radioimmunotherapy led to the highest proportion of CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells expressing PD-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRadioimmunotherapy inhibits the recruitment of different immunosuppressive cell populations\u003c/h2\u003e \u003cp\u003eAside from Tregs and CD8\u003csup\u003e+\u003c/sup\u003eT cells, we also investigated the relative immuno-suppressive populations of tumor-infiltrating mononuclear myeloid-derived suppressor cell (MDSCs) and TAMs. In the tumor, radioimmunotherapy significantly reduced the percentage of M2-TAMs compared to RT or anti-PD-L1 groups at Day 10 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0231, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0476, respectively). Radioimmunotherapy also reduced the percentage of M2-TAMs compared to those of other groups at Day 14 \u003cb\u003e(\u003c/b\u003eradioimmunotherapy vs RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0141; vs Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0020; vs control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0314; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. A similar effect was also observed in the spleen at Day 14(radioimmunotherapy vs RT \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0469; Anti-PD-L1 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0095; control \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0024). In the TDLNs, we found that radioimmunotherapy and anti-PD-L1 groups decreased the percentage of M2 TAMs compared with the RT and control groups at Days 10 and 14 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. In addition, we measured the expression of CD206 (M2-TAM marker) and CD86 (M1-TAM marker) in the tumor tissue at Day 14 by immunohistochemistry. We found the radioimmunotherapy group had the highest proportion of M1-TAMs and the lowest proportion of M2-TAMs compared to those of other groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. These results indicated that the radioimmunotherapy impaired the proliferation of M2-TAM.\u003c/p\u003e \u003cp\u003eIn addition, compared with RT group, radioimmunotherapy and anti-PD-L1 significantly decreased tumor-infiltrating MDSCs in tumor and TDLNs at Day 10 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e No differences were observed in the spleen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRadioimmunotherapy leads to changes in inflammation and immune profiles\u003c/h2\u003e \u003cp\u003eTo compare the immune responses of radioimmunotherapy, transcriptome analyses were performed. The 20 most significantly enriched signaling pathways including cytokine-cytokine receptor interaction, chemokine signaling pathway and antigen processing and presentation genes are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e In addition, compared to the RT group, mice treated with radioimmunotherapy had the most profound changes in \u0026ldquo;regulation of cell-cell adhesion\u0026rdquo;, \u0026ldquo;regulation of immune effector process\u0026rdquo; and \u0026ldquo;response to IFN-gamma\u0026rdquo;, suggesting alteration of antitumoral activity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Next, we observed that the gene expression of chemokines involved in the attraction of T cells (eg, CXCL9, CXCL10 and CXCL11) increased in the tumor obtained to the radioimmunotherapy group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e We subsequently evaluated the expression of CXCL9 and CXCL10 at tumor sites by RT-PCR. Compared with RT, radioimmunotherapy improved the expression of CXCL9 and CXCL10\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. These data indicating that radioimmunotherapy can enhance the production of IFN-γ and chemokines induced by IFN-γ. In addition, we found antigen presentation associated genes (MHC-I/II and Tap1/2), immune killing genes (Prf1, Ifn-γ, Gzma, and Gzmb) and innate immunity genes (Irf7, Isg15, Usp18) were upregulated in the radioimmunotherapy group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Taken together, these data demonstrated that the radioimmunotherapy can trigger both innate and adaptive immune response, providing a more immunostimulatory tumor microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our study, we demonstrated that anti-PD-L1 treatment combined with radiotherapy resulted in better tumor control and prolonged survival in mice. Importantly, we demonstrate that radioimmunotherapy stimulated CD8\u003csup\u003e+\u003c/sup\u003eT cell infiltration, improved the activity of effector CD8\u003csup\u003e+\u003c/sup\u003e TILs, increased the infiltration of CD8\u003csup\u003e+\u003c/sup\u003eT\u003csub\u003eCM\u003c/sub\u003e and reduced the accumulation of M2-TAMs and Tregs, therefore reprogramming an immune-stimulating tumor microenvironment and improving the anti-tumor efficacy in the mEC25 syngeneic mouse model. Similar changes were also seen in the spleen and TDLN. This is the first preclinical research depicting the synergistic antitumor immunity combining PD-L1 inhibitors with radiation in ESCC.\u003c/p\u003e \u003cp\u003eOur results shown that radioimmunotherapy can increase the population of CD8\u003csup\u003e+\u003c/sup\u003e TILs and the proportion of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eTILs in tumor microenvironment, indicating that radioimmunotherapy enhanced CD8\u003csup\u003e+\u003c/sup\u003eT cell infiltration and activation. Previous observations have demonstrated that IFN-γ secreted by CD8\u003csup\u003e+\u003c/sup\u003e TILs accounts for up-regulation of PD-L1 after delivery of fractionated radiotherapy of tumor cells(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In our study, we also found that PD-L1 expression is enhanced after fractionated radiotherapy in ESCC lines. In addition, radioimmunotherapy increased the GZMB secretion of CD8\u003csup\u003e+\u003c/sup\u003e TILs, indicating that radioimmunotherapy augmented the cytotoxicity of CD8\u003csup\u003e+\u003c/sup\u003e TILs.\u003c/p\u003e \u003cp\u003eMoreover, our results showed that radioimmunotherapy eliminated regulatory T cells (Tregs). Previous studies have shown that Tregs can effectively inhibit effector T cells and revealed that radiation can recruit immunosuppressive Tregs(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), which limit the immune response to radiotherapy. However, our study proves that radioimmunotherapy enhanced CD8\u003csup\u003e+\u003c/sup\u003eTILs/Treg ratio via both boosting infiltration of CD8\u003csup\u003e+\u003c/sup\u003eT cells and eradicating Tregs population, thereby improving antitumor immunity. The CD8\u003csup\u003e+\u003c/sup\u003eTILs /Treg ratio has been recognized as a predictor of response to immunotherapy(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). These results are in concordance with the results of Gong et al in a mouse model of lung cancer(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eM2-type macrophages promote tumor immune tolerance, tumor invasion and metastasis(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Previous studies showed that radiation can recruits M2-type macrophages, thereby limiting radiation-induced adaptive antitumor immunity(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In addition, PD-L1 on the surface of macrophages binds to PD-1 on T cells, thereby inhibiting the co-stimulation of T cells by macrophage and resulting in T cell incompetence(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Previous studies have shown that PD-L1 blockade induces IFN-γ secretion of CD8\u003csup\u003e+\u003c/sup\u003eTILs, further driving M1 polarization of macrophages(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Alternatively, Jones et al demonstrated that macrophage exhaustion partially impaired immunosuppression after radiotherapy, but additional anti-PD-L1 therapy was essential to accomplish tumor remission(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In our study, only radioimmunotherapy significantly reduced the frequency of M2-type TAMs, which may be due to the fact that radiotherapy promotes the expression of PD-L1 on the surface of macrophages, thereby increasing the targeted sites of PD-L1 inhibitors.\u003c/p\u003e \u003cp\u003eMDSC can inhibit T cell function and promote immune escape(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Previous studies demonstrated that radioimmunotherapy eventually results in a decreased amount of MDSC and PD-L1\u003csup\u003e+\u003c/sup\u003eMDSCs in irradiated and non-irradiated tumors(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In our experiments, both radioimmunotherapy and anti-PD-L1 inhibitor inhibited MDSC recruitment. In conclusion, the addition of PD-L1 inhibitor attenuated the effects on immunosuppressive cell populations that were seen in the radiotherapy. Therefore, radioimmunotherapy reshaped the immune-stimulated microenvironment via elimination of immunosuppressive cells and promoting CD8\u003csup\u003e+\u003c/sup\u003eT cells infiltration in TME.\u003c/p\u003e \u003cp\u003eT\u003csub\u003eCM\u003c/sub\u003e cells are characterized by self-renewal and long-term survival in vivo and can be rapidly differentiated into effector T cells after tumor antigens stimulation(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Several researchers have revealed that combining radiotherapy with ICIs could add the account of CD8\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eCM\u003c/sub\u003e and inhibit tumor recurrence(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). We demonstrated that the proportion of infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eCM\u003c/sub\u003e cells were highest in radioimmunotherapy group in TME, spleen and TDLN, which may be helpful to delay tumor recurrence.\u003c/p\u003e \u003cp\u003eFor the investigation of immune related gene expression, our data showed that CXCL9 and 10 induced by IFN-γ stimulation were enhanced in radioimmunotherapy arm. The elevated CXCL9 and CXCL10 level was associated with recruiting antitumor CD8\u003csup\u003e+\u003c/sup\u003eT cells and augmented activation and cytotoxic responses of CD8\u003csup\u003e+\u003c/sup\u003eTILs(\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The expression of interferon-stimulated genes (Irf7, Isg15, Usp18) were up-regulated in the radioimmunotherapy group. Notably, type I IFN stimulates antigen-presenting cells (APCs) to deliver tumor antigens for maintaining CD8\u003csup\u003e+\u003c/sup\u003eT cell initiation(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), while suppressing Treg function to reinforce antitumor efficacy(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Overall, radioimmunotherapy reprogrammed a more immunostimulatory tumor microenvironment.\u003c/p\u003e \u003cp\u003eT cell exhaustion is inevitable under the continuous stimulation of tumor antigens. In our study, radioimmunotherapy increased the infiltration of PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells and PD-1\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells, which was consistent with the findings of Dudzinski et al in prostate cancer model, indicating that radioimmunotherapy increased tumor antigen presentation and T cell depletion(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Exhausted T cells (Tex) can be categorized into stem-like exhausted and terminally exhausted populations. Stem-like T cells contribute to the generation of durable anti-tumor immunity(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Wei et al shown that combing radiotherapy with PD-1 inhibitor reverses the terminal exhaustion into stem-like exhausted PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). The expression of other immune checkpoints is on behalf of advanced T cell exhaustion(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Therefore, it is necessary to further evaluate the status of PD-1\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells.\u003c/p\u003e \u003cp\u003eOur studies reveal the dynamic changes of immune related cell populations in spleen and TDLN. In the spleen, radioimmunotherapy significantly and consistently increased the accumulation of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eTILs. Additionally, antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T\u003csub\u003eCM\u003c/sub\u003e and CD8\u003csup\u003e+\u003c/sup\u003eTILs/Treg ratio are also greatly increased in the spleen. And radioimmunotherapy also reduced M2-type TAM.\u003c/p\u003e \u003cp\u003eIn draining lymph nodes, radioimmunotherapy increased the infiltration of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells and CD8\u003csup\u003e+\u003c/sup\u003eTILs/Treg ratio, expanded the number of CD8\u003csup\u003e+\u003c/sup\u003eT\u003csub\u003eCM\u003c/sub\u003e and inhibited the recruitment of M2-type TAM. Some studies have pointed out that the immune microenvironment of TDLN may be a theoretical supplement to TME, and alleviating immunosuppression in TDLN can promote systemic anti-tumor T cell immunity, thereby effectively controlling distant tumor sites. Intact TDLN is essential in the anti-tumor immune response of radioimmunotherapy by promoting CD8\u003csup\u003e+\u003c/sup\u003eT cell accumulation as well as M1/M2 macrophage ratio(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In conclusion, changes in immunostimulatory activity in the spleen and TDLNs may enhance systemic antitumor effects and increase the incidence of abscopal effects.\u003c/p\u003e \u003cp\u003eThe 4Gy\u0026times;3 fractionated radiotherapy scheme was selected in our study. Since most patients received low-dose radiotherapy in clinic, this scheme may be more suitable for clinical patients than the 8Gy\u0026times;3 fractionated radiotherapy, which has important significance for the design of future clinical trials.\u003c/p\u003e \u003cp\u003eIn this study, mEC25 mouse syngeneic tumor model was selected, which was derived from the orthotopic esophageal tumor of C57BL/6 mice. This subcutaneous model provides the ability to easily establish tumors in an immunocompetent host and further characterize the tumor-infiltrating lymphocytes and microenvironment, which contribute to investigate the therapeutic effects and related mechanisms in the presence of an intact immune system.\u003c/p\u003e \u003cp\u003eHowever, there are some limitations to our experiment. First, the combination therapy did not completely eliminate the transplanted tumors in the mice. As in the study of Philippou et al(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), the reason may be that the radiation dose of 3\u0026times;4Gy is not enough to produce CD8\u003csup\u003e+\u003c/sup\u003e T cell-dependent anti-tumor response. Therefore, in order to successfully apply radioimmunotherapy to clinical patients with esophageal cancer, treatment combinations such as timing/sequence of treatment and radiation dose/fractionation are required for examining in preclinical models and early clinical trials to define best approach. In addition, the data of this study are limited to a single mouse solid malignant tumor model, and diversified mouse esophageal squamous cell carcinoma models should be used. More importantly, in future studies, we should pay attention to the phenomenon of \"abscopal effect\" caused by radioimmunotherapy and the influence of different dose sequence combinations on this phenomenon. In addition, the role of TDLNs in the anti-tumor effect of radioimmunotherapy needs to be further clarified.\u003c/p\u003e \u003cp\u003eOverall, our study showed that radiotherapy combined with PD-L1 inhibitors is a promising synergistic treatment option for ESCC by improving the immunosuppressive tumor microenvironment. By providing preclinical data, our study provides theoretical support for the design or interpretation of clinical studies of the radioimmunotherapy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on preliminary observations, our results increase the persuasiveness of radiotherapy combining with PD-L1 inhibitor as a potential synergistic treatment modality for ESCC. Importantly, this study provides a foundation for clinical researches of ESCC comprehensive treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the staff of Hangzhou Cancer Institution, Affiliated Hangzhou Cancer Hospital, for their technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDQH, ZHF and ZK conceived and supervised the experiments. the experiments were performed by YZH, YQQ, YJ, WYP, YZH, YQQ and ZHF interpreted the results of the experiments. YZH and ZK completed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Clinical Research Fund Project of Zhejiang Medical Association (2021ZYC-Z05); Zhejiang Province Health Department Project(2022KY101); \"Great Medical Sincerity\" cancer prevention and treatment research and academic exchange public welfare program; Hangzhou Agricultural and social Development Research Project(2020ZDSJ0552).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe state that animal experiment protocols were approved by the Institutional Animal Care and Use Committee (IACUC 20010652).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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JCI Insight. 3. doi: 10.1172/jci.insight.124507\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Programmed death ligand 1, radiotherapy, tumor microenvironment, Esophageal Squamous Cell Carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-4338719/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4338719/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground：\u003c/strong\u003eThe combination of radiation with immune checkpoint inhibitors (ICIs) has been demonstrated to display synergistic effects in solid cancers. Nevertheless, the anti-tumor effect of combining radiation with programmed cell death 1 ligand 1 (PD-L1) inhibitor in esophageal squamous cell carcinoma (ESCC) remains unclear. Therefore, the objectives of our study were to evaluate the anti-tumor effects of PD-L1 inhibitors combined with radiotherapy in ESCC mouse model and to depict the immune landscape within the tumor microenvironment (TME).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA syngeneic C57BL/6 subcutaneous xenograft mouse model was applied to evaluate the anti-tumor efficacy of different treatment protocols according to tumor growth curve and survival time. Tumour immune microenvironment was assessed by flow cytometry including CD4\u003csup\u003e+\u003c/sup\u003eT cells, CD8\u003csup\u003e+\u003c/sup\u003eT cells, regulatory T cells (Tregs), tumor-derived macrophage (TAM), myeloid-derived suppressor cell (MDSC), and the expression of CD8\u003csup\u003e+\u003c/sup\u003eT cell activation, exhaustion, and memory state markers. In addition, transcriptomic analysis was used to examine the immune gene expression changes in tumor microenvironment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eRadiotherapy combined with anti-PD-L1 inhibitors synergistically enhanced anti-tumor immune response via boosted the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells, increased the ratio of CD8\u003csup\u003e+\u003c/sup\u003e T cells to Tregs and population of central memory CD8\u003csup\u003e+\u003c/sup\u003e T cells (T\u003csub\u003eCM\u003c/sub\u003e),\u003cstrong\u003e \u003c/strong\u003eenhanced interferon gamma (IFN-γ) secretion by tumor-infiltrating CD8\u003csup\u003e+ \u003c/sup\u003eT cells, and reduced the accumulation of M2-type TAMs and Tregs in the TME in mouse model. In addition, radioimmunotherapy also induced a better immunophenotype in spleen and tumor draining lymph node (TDLN). Consequently, radioimmunotherapy appeared greater benefit in antitumor effects and mice survival. Moreover, our transcriptomic analysis suggested that radioimmunotherapy promoted the expression of immunostimulation-related regulatory pathways and cytokines that shape the immunoinflammatory tumor microenvironment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur research indicated that anti-PD-L1 inhibitors combined with RT promotes systemic anti-tumor immunity by improving the immune microenvironment in a mouse model of ESCC.\u003c/p\u003e","manuscriptTitle":"Impacts of Combining PD-L1 inhibitor and Radiotherapy on the Tumour immune microenvironment in a Mouse Model of Esophageal Squamous Cell Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 17:45:11","doi":"10.21203/rs.3.rs-4338719/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bff764a2-3851-431a-8b3f-4bd2c5061b60","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T02:44:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-08 17:45:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4338719","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4338719","identity":"rs-4338719","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}