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BACKGROUND: Multiple synergistic combination approaches with cancer drugs are developed to overcome primary resistance to immunotherapy; however, the mechanistic rationale to combine chemoradiotherapy (CRT) with immune checkpoint inhibitors remains elusive. METHODS: This study described the immunological landscape of tumor microenvironment (TME) exposed to CRT. Tumor samples from patients with rectal cancer (n=43) treated with neoadjuvant CRT or radiotherapy were analyzed by nanostring and immunohistochemistry. Studies in mice were performed using three syngeneic tumors (TC1, CT26 and MC38). Tumor-bearing mice were treated either with platinum-based CRT, radiotherapy or chemotherapy. Anti-CTLA-4 and/or anti-Programmed Cell Death Receptor-1 (PD-1) therapy was used in combination with CRT. The therapy-exposed TME was screened by RNA sequencing and flow cytometry and tumor-infiltrating T lymphocyte functionality was evaluated by interferon (IFN)-γ ELIspot and intracellular cytokine staining. RESULTS: Front-to-front comparison analysis revealed the synergistic effect of CRT to establish a highly inflamed and Th1-polarized immune signature in the TME of patients and mice. In both settings, CRT-exposed TMEs were highly enriched in newly-infiltrated tumor-specific CD8+ T cells as well as tissue resident memory CD103+CD8+ T cells. In mice, CD8 T cells were involved in the antitumor response mediated by CRT and were primed by CRT-activated CD103+ dendritic cells. In the three tumor models, we showed that concurrent combination of CRT with a dual CTLA-4 and PD-1 blockade was required to achieve an optimal antitumor effect and to establish a broad and long-lasting protective antitumor T cell immunity. CONCLUSIONS: Our results highlight the ability of CRT to stimulate strong antitumor T-cell-mediated immunity and tissue resident memory T activation in TME, to foster immune checkpoint inhibitors action. These findings have implications in clinic for the design clinical trials combining chemoradiation with immunotherapy.

Additional file 3: Supplementary Table S1. List of monoclonal antibodies used for flow cytometry.

Additional file 2: Supplementary Figure S2. Kaplan-Meier overall survival (OS) curves in patients according to TERT specific T-cell responses and immunosuppressive cells levels before and after CRT. OS according toTERT-specific responses levels (A), MDSC levels (B), andTreg levels (C).

Additional file 1: Supplementary Figure S1. Gating strategy for flow cytometry analyses. The figure shows the gating strategy to Treg (A) and MDSC (B) populations. Frequencies of Treg cells were observed in CD4 T-cell population. Expression of CD127, FoxP3, and CTLA4 were analyzed on Treg (A). MDSC populations were analyzed after exclusion of lineage (CD3, CD56, CD19)(B).

Figure S1. Gating strategy for lymphoid cells identification and quantification in tumor tissue. Figure S2. Gating strategy for myeloid and tumor cells identification, quantification and phenotype (PD-L1 expression) in tumor tissue. Figure S3. Gating strategy for lymphoid cells functionality quantification in tumor tissue. Figure S4. Efficacy evaluation of immunotherapy (anti-PD-L1 and/or anti-TIGIT) and RT (3x8Gy) in B16-F10 model. Growth of irradiated tumors in mice treated with IgG + 0Gy (black), IgG + 3x8Gy (red), anti-TIGIT + anti-PD-L1 (blue) and with anti-TIGIT + anti-PD-L1 + 3x8Gy (purple). Mean ± SEM. X axes express the number of days since the beginning of RT. Y axes express the tumor volume (mm3). Experimental groups contained at 5 mice per group. Non-parametric Mann-Whitney test was used. Figure S5. Immunomonitoring of Treg and CD8+ T cells and their KI67 and PD-1 status after radiotherapy. Ten days after the injection of CT26 colon murine cancer, mice were assigned in 4 groups: control (at day 7), 1 × 16.4Gy (red), 3x8Gy (blue), 18x2Gy (purple) (a). Seven, 14 and 30 days after the beginning of RT, flow cytometry monitoring (FCM) was performed on dissociated tumors. CD8+ T cells (a) and Treg T cells (b) were analyzed according to their status for KI67 and PD-1 labelling. All data are shown with box and whiskers with min to max values obtained from 8 independent samples per point (duplicate, n = 8 per condition). In the second part of a) and b), representative cytometry analysis was highlighted for each condition at day 7 and day 14 after treatment. *p

Céline Mirjolet,1 Julien Boudon,2 Alexis Loiseau,2 Sandy Chevrier,1 Romain Boidot,1 Alexandra Oudot,3 Bertrand Collin,3 Etienne Martin,1 Pattayil Alias Joy,4 Nadine Millot,2 Gilles Créhange1 1Department of Radiation Oncology, Center Georges-François Leclerc, Dijon, France; 2Laboratoire Interdisciplinaire Carnot de Bourgogne, Dijon, France; 3Preclinical Imaging Platform, Nuclear Medicine Department, Center Georges-François Leclerc, Dijon, France; 4CSIR-National Chemical Laboratory (CSIR-NCL), Pune, India Abstract: Around 40% of high-risk prostate cancer patients who undergo radiotherapy (RT) will experience biochemical failure. Chemotherapy, such as docetaxel (DTX), can enhance the efficacy of RT. Multidrug resistance mechanisms often limit drug efficacy by decreasing intracellular concentrations of drugs in tumor cells. It is, therefore, of interest to develop nanocarriers of DTX to maintain the drug inside cancer cells and thus improve treatment efficacy. The purpose of this study was to investigate the use of titanate nanotubes (TiONts) to develop a TiONts-DTX nanocarrier and to evaluate its radiosensitizing in vivo efficacy in a prostate cancer model. In vitro cytotoxic activity of TiONts-DTX was evaluated using an MTS assay. The biodistribution of TiONts-DTX was analyzed in vivo by single-photon emission computed tomography. The benefit of TiONts-DTX associated with RT was evaluated in vivo. Eight groups with seven mice in each were used to evaluate the efficacy of the nanohybrid combined with RT: control with buffer IT injection ± RT, free DXL ± RT, TiONts ± RT and TiONts-DXL ± RT. Mouse behavior, health status and tumor volume were monitored twice a week until the tumor volume reached a maximum of 2,000 mm3. More than 70% of nanohybrids were localized inside the tumor 96 h after administration. Tumor growth was significantly slowed by TiONts-DTX associated with RT, compared with free DTX in the same conditions (P=0.013). These results suggest that TiONts-DTX improved RT efficacy and might enhance local control in high-risk localized prostate cancer. Keywords: prostate cancer, docetaxel nanocarrier, titanate nanotubes, radiosensitivity, nanoparticle