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Additional file 2: Table S1. Log2 fold change and the function of the genes which were significantly up-regulated in the unirradiated tumors treated with NBTXR3+XRT+PLT vs. NBTXR3+XRT+αPD1.

Additional file 1: Figure S1. Log2 fold change in the expression of TIGIT and LAG3 in tumors treated with NBTXR3 + XRT + αPD1. The 344SQR cells (5 × 104) were subcutaneously injected into the right legs of the 129/SvEv syngeneic female mice (n = 4, 8–12 weeks old) on day 0 to establish the “primary” tumor and into the left legs on day 4 to establish the “secondary” tumor. The primary tumors were intratumorally injected with NBTXR3 on day 7, followed by three fractions of 12 Gy radiation on days 8, 9, and 10. αPD1 (200 µg) was intraperitoneally injected into the mice on days 5, 8, 11, and 14. The expression of LAG3 and TIGIT in the irradiated and unirradiated tumors harvested on day 21 were measured by Nanostring. The mice inoculated with both primary and secondary tumors but did not receive any treatment served as control. P < 0.05 was considered statistically significant. **P < 0.01, ***P < 0.001. Figure S2. Individual tumor growth curves in mice receiving treatment as indicated in Fig. 1.Figure S3. Blockade of LAG3 and TIGIT with the combination of NBTXR3 + XRT + αPD1 reduces the number of lung metastases. The mice (n = 5) were treated with various combination therapies, as illustrated in Fig. 1, and were euthanized on day 21. The lungs were harvested and stored in Bouin’s fixative solution for three days, after which metastatic nodules were counted. The number of lung metastases was compared by t-test and were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. Figure S4. Body weight of mice (n = 5) treated with combination therapy of NBTXR3 + XRT + αPD1 + αLAG3 + αTIGIT as shown ing Fig. 1A. Figure S5. Dual blockade of TIGIT and LAG3 promotes proliferation of CD4+ and CD8+ T cells. (A) Representative flow cytometry images of Ki67+CD4+ and Ki67+CD8+ T in primary tumors. (B) Representative flow cytometry images of Ki67+CD4+ and Ki67+CD8+ T cells in secondary tumors. (C) Representative flow cytometry images of Ki67+CD4+ and Ki67+CD8+ T cells in the blood. The mice (n = 5) were treated with various combination therapies, including XRT + αPD1, NBTXR3 + XRT + αPD1, NBTXR3 + XRT + αPD1 + αLAG3, NBTXR3 + XRT + αPD1 + αTIGIT, and NBTXR3 + XRT + PLT as indicated in Fig. 1A and were sacrificed on day 21. The mice which were inoculated with tumors only served as control. Immune cells from primary tumors, secondary tumors, and blood were processed and stained with αCD45-APC-Cy7, αCD3-PE-Cy7, αCD4-alexa 700, αCD8-PercpCy5.5, and αKi67-alexa 647. The cells were run with a Gallios Flow Cytometer (Beckman Coulter) and analyzed with Kaluza software Version 2.1. Figure S6. Flow cytometry gating strategy in Fig. 3. Figure S7. Nanostring cell score of various immune cells treated with different combinations of NBTXR3, XRT, αPD1, αLAG3 and αTIGIT in both primary and secondary tumors. Mice (n = 4) were treated with various combination therapies as described in Fig. 1 and were euthanized 11 d post last fraction of radiation. The RNA from the irradiated and unirradiated tumors was extracted, and the immune-related genes were measured with a nCounter PanCancer Immune Profiling Panel and a nCounter MAX Analysis System. The data were analyzed with the PanCancer Immune Profiling Advanced Analysis Module. The cell scores were compared by t-test and were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant. ***P < 0.001, NS, not significant. Figure S8. Multiple immune-related genes are significantly differentially expressed in the tumors of mice following supplementation of NBTXR3 + XRT + αPD1 with αLAG3, αTIGIT, or both. Statistical significance was assessed via a two-tailed t-test, with a p < 0.05. A Heatmap of genes significantly up- or down-regulated in at least one of the three treatment groups relative to NBTXR3 + XRT + αPD-1 in the primary tumor. B Heatmap of genes significantly up- or down-regulated in at least one of the three treatment groups relative to NBTXR3 + XRT + αPD1 in the secondary tumor. Two hundred and forty-six (246) such genes were identified and are here ranked alphabetically. C Genes identified as significantly upregulated in NBTXR3 + XRT + PLT-treated mice compared to NBTXR3 + XRT + αPD1-treated mice were manually grouped according to their established function. Functional groups were plotted according to the log2 fold change from baseline (NBTXR3 + XRT + αPD1) of their constituent genes, represented in aggregate via violin plot.

Background: while improvements in immunoradiotherapy have significantly improved outcomes for cancer patients, this treatment approach has nevertheless proven ineffective at controlling the majority of malignancies. One of the mechanisms of resistance to immunoradiotherapy is that immune cells may be suppressed via the myriad of different immune checkpoint receptors. Therefore, simultaneous blockade of multiple immune checkpoint receptors may enhance the treatment efficacy of immunoradiotherapy. Methods: we combined NBTXR3-enhanced localized radiation with the simultaneous blockade of three different checkpoint receptors: PD1, LAG3, and TIGIT, and tested the treatment efficacy in an anti-PD1-resistant lung cancer model in mice. 129 Sv/Ev mice were inoculated with fifty thousand alpha PD1-resistant 344SQR cells in the right leg on day 0 to establish primary tumors and with the same number of cells in the left leg on day 4 to establish the secondary tumors. NBTXR3 was intratumorally injected into the primary tumors on day 7, which were irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 mu g), alpha LAG3 (200 mu g), and alpha TIGIT (200 mu g) were given to mice by intraperitoneal injections on days 5, 8, 11, 14, 21, 28, 35, and 42. Results: this nanoparticle-mediated combination therapy is effective at controlling the growth of irradiated and distant unirradiated tumors, enhancing animal survival, and is the only one that led to the destruction of both tumors in approximately 30% of the treated mice. Corresponding with this improved response is robust activation of the immune response, as manifested by increased numbers of immune cells along with a transcriptional signature of both innate and adaptive immunity within the tumor. Furthermore, mice treated with this combinatorial therapy display immunological memory response when rechallenged by the same cancer cells, preventing tumor engraftment. Conclusion: our results strongly attest to the efficacy and validity of combining nanoparticle-enhanced radio-therapy and simultaneous blockade of multiple immune checkpoint receptors and provide a pre-clinical rationale for investigating its translation into human patients., This work was supported by Cancer Center Support (Core) Grant CA016672 to The University of Texas MD Anderson Cancer Center; the Goodwin family research fund; the family of M. Adnan Hamed, and the Orr Family Foundation to MD Anderson Cancer Center's Thoracic Radiation Oncology program; an MD Anderson Knowledge Gap award; Nanobiotix.