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Background Radiotherapy is a powerful and widely used technique for the treatment of solid tumors. Beyond its ability to destroy tumor cells, it has been demonstrated that radiotherapy can stimulate the anti-tumor immune response. Unfortunately, this effect is mainly restricted to the irradiated lesion, as tumor control outside the treated field (called the ‘abscopal effect’) is rarely obtained. In addition, many pro-tumoral factors prevent this anti-tumor immune response from being sustained and efficient. We previously reported that radiotherapy-activated NBTXR3 produced a significant CD8-dependent abscopal effect in immunocompetent mice bearing CT26.WT tumors, while radiotherapy failed to generate such a response. Methods To identify the mechanisms that may explain this response, we evaluated the capacity of radiotherapy-activated NBTXR3 to modulate the immunogenicity of tumor cells by analysis of immunogenic cell death biomarkers and immunopeptidome sequencing. In vivo, we analyzed treated tumors for CD4+, CD8 + and CD68 + cell infiltrates by immunohistochemistry and digital pathology and sequenced the T cell receptor (TCR) repertoire in both treated and untreated distant tumors. Results We showed that NBTXR3 activated by radiotherapy both increased immunogenic cell death biomarkers and modulated the immunopeptidome profile of CT26.WT cells. Immunohistochemistry analysis of treated tumors revealed a significant increase in CD4+, CD8 + and CD68 + cell infiltrates for NBTXR3 activated by radiotherapy group, compared to radiotherapy. We also measured significant modifications in TCR repertoire diversity in the radiotherapy-activated NBTXR3 group, both in treated and distant untreated tumors, compared to radiotherapy alone. Conclusions These results indicate that radiotherapy-activated NBTXR3 can act as an effective immunomodulator, modifying tumor cell immunogenicity and impacting the lymphocyte population. Graphical Abstract

Since many decades, radiotherapy (RT) is worldwide used and crucial therapeutic approach for the treatment of cancers. Nowadays, about 50% of cancer patients will receive radiotherapy during their treatment. Nevertheless, the use of RT is limited by the damage induced by ionizing radiation on healthy tissues. To overcome this constraint, we have developed high electron density of functionalized hafnium oxide nanoparticles (NBTXR3), allowing a high probability of interaction with incoming ionizing radiation, increasing energy dose deposit within cells. Many preclinical data have shown that RT-activated NBTXR3 (NBTXR3+RT) increase cancer cell destruction as well as better control treated tumor growth, compared to RT alone. In clinic, the benefit of NBTXR3+RT compared to RT alone has been demonstrated for patients with locally advanced Soft Tissue Sarcoma, in the randomized controlled phase II/III Act.in.Sarc study (NCT02379845). We also demonstrated in preclinical settings that, beyond the destruction of tumor cells, NBTXR3+RT was also able to induce an antitumor immune response capable to produce a significant abscopal effect, linked to the increase of CD8 infiltrates in tumors. It has been recently reported that NBTXR3+RT can also restore anti-PD1 efficacy in an anti-PD1 resistant mouse model. At the cancer cell level, we reported that NBTXR3+RT could modulate the immunopeptidome of cancer cells, induce the activation of the cGAS-STING pathway and increase the expression of ICD biomarkers. All these data indicate that NBTXR3+RT has the ability to transform the tumor into an in situ vaccine more efficiently than RT alone and could have important implications for the use of NBTXR3+RT in combination with immunotherapy. Nevertheless, we currently have relatively few elements on the early cellular events that could trigger or explain the above reported performances of NBTXR3+RT, compared to RT alone. To address this question, we have carried out a series of in vitro studies (cytometry, microscopy) on different cancer models (human and murine). We have thus identified that the vast majority of NBTXR3 nanoparticles are located in lysosomes after endocytosis by tumor cells. Once activated by RT, we detected lysosomal membrane permeabilization (LMP) only in cells treated with NBTXR3+RT, but not in cells only treated by RT. Following LMP, we measured a significant increase of in lipid peroxidation. These first elements allow a better understanding of the early biological consequences induced by NBTXR3+RT, compared to RT alone. Citation Format: Jordan Da Silva, Célia Bienassis, Sebastien Paris. Early biological impacts of radiotherapy-activated NBTXR3 nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2414.

Introduction: Immunoradiotherapy combining radiotherapy and immune checkpoint blockade, has achieved impressive treatment outcomes. We have previously demonstrated that photon (XRT) and proton (PRT) localized radiotherapy in combination with immune checkpoint blockade and NBTXR3, a radioenhancing nanoparticle, could induce a systemic antitumor immune response. However, no studies have compared the treatment efficacy of PRT and XRT immunoradiotherapy. Here, we compared the capabilities of PRT and XRT when combined with NBTXR3 and PD1 blockade for inducing an antitumor immune response. Methods: 344SQR cells were inoculated in the right and left hindlimbs of 129Sv/Ev mice on day 0 and day 4 to establish primary and secondary tumors, respectively. Mice were divided into five treatment groups: 1) untreated control; 2) XRT+αPD1; 3) NBTXR3+XRT+αPD1; 4) PRT+αPD1; 5) NBTXR3+PRT+αPD1. The primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7, followed by two fractions of 12 Gy XRT or PRT on days 8 and 9 (total dose of 24 Gy). Two hundred μg of αPD1 was given to the mice on days 7, 10, 14, 21, 28, 35, and 42 through intraperitoneal injection. We assayed whole tumor RNAs with a Nanostring pan-cancer immune panel to profile the tumor immune microenvironment changes. In addition, immune cells extracted from the tumors were analyzed with single-cell RNA sequencing (scRNAseq). The survivor mice treated with the NBTXR3+PRT+αPD1 were rechallenged with 344SQR and 344SQP cells. Results: PRT+ αPD1 resulted in significantly better control of the primary tumors than XRT+ αPD1. No significant difference was observed in the volume of the secondary tumors treated with XRT+ αPD1 and the control. In contrast, PRT+ αPD1 resulted in a pronounced abscopal effect. Adding NBTXR3 to XRT+ αPD1 and PRT+ αPD1 improved the control of both the primary and secondary tumors. In addition, NBTXR3+PRT+αPD1 achieved significantly slower growth of the two tumors and more prolonged survival than NBTXR3+XRT+αPD1. Remarkably, NBTXR3+PRT+αPD1 eradicated both primary and secondary tumors in 40% of the treated mice, while no mice were cured by NBTXR3+XRT+αPD1. Nanostring analysis and scRNAseq revealed better activation and infiltration of antitumoral immune cells to the two tumors when treated with PRT than XRT. The survivor mice treated with NBTXR3+PRT+αPD1 effectively rejected tumor growth after rechallenge with 344SQR and 344SQP cells. Conclusions: When combined with NBTXR3, PRT with αPD1 exhibited significantly better treatment efficacy than XRT with αPD1. The superior efficacy of NBTXR3+PRT+αPD1 is accompanied by a more robust immune activation and immune cell infiltration in irradiated and unirradiated tumors. Citation Format: Yun Hu, Sébastien Paris, Narayan Sahoo, Qi Wang, Qianxia Wang, Hampartsoum B. Barsoumian, Jordan Da Silva, Célia Bienassis, Ailing Huang, Nahum Puebla-Osorio, Saumil Gandhi, Quynh-Nhu Nguyen, Jing Wang, Maria A. Cortez, James W. Welsh. NBTXR3 radio-enhancing nanoparticle achieves a more robust antitumor immune response when combined with proton radiotherapy than photon radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2415.

Introduction: Checkpoint inhibition (CPI) has been a game-changer for cancer therapy, but most cancers do not respond. Part of the problem is that cancers employ multiple checkpoints; thus, targeting multiple checkpoint receptors simultaneously may yield increased benefit. Previously, we found that radiotherapy (XRT) in concert with a radiation-enhancing nanoparticle (NBTXR3) and PD1 blockade significantly improved tumor control both at the irradiative site and in remote, unirradiated tumors in a mouse model of anti-PD1 (αPD1)-resistant lung cancer (344SQR). Here, we tested whether blockade of TIGIT, LAG3, and PD1 in combination with NBTXR3-enhanced XRT could improve antitumor immune responses in our 344SQR tumor model. Methods: Fifty thousand 344SQR cells were inoculated in the right and left hindlimbs of 129Sv/Ev mice on day 0 and day 4 to establish primary and secondary tumors, respectively. Mice were divided into six treatment groups: 1) untreated; 2) XRT+αPD1; 3) NBTXR3+XRT+αPD1; 4) NBTXR3+XRT+αPD1+αLAG3; 5) NBTXR3+XRT+αPD1+αTIGIT; and 6) NBTXR3+XRT+αPD1+αLAG3 +αTIGIT. Primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7 and received 3 fractions of 12 Gy X-ray radiation on days 8, 9, and 10. Immune checkpoint inhibitors, composed of αPD1 (200 μg), αLAG3 (200 μg), and αTIGIT (200 μg), were intraperitoneally administered to the mice on days 5, 8, 11, 14. The expression of 770 immune-related genes were measured via NanoString from the RNA extracted from the primary and secondary tumors on day 21. Results: CPIs+NBTXR3-enhanced radiation significantly promoted the upregulation of mRNA transcripts involved in innate immunity, the humoral response, B cell function, dendritic cell (DC) function, and antigen processing within primary, irradiated tumors relative to untreated controls. No additional increase in immune gene activity in the irradiated tumors was observed when LAG3, TIGIT, or both were blocked in addition to PD1. However, within the non-irradiated tumors, triple blockade of PD1, LAG3, and TIGIT in concert with NBTXR3+XRT produced elevations in multiple immune-related pathways that were significantly higher than those produced by other treatment combinations. These pathways included both adaptive and innate immunity; B, T, and natural killer (NK) cell, and DC function; and antigen processing. Conclusions: Simultaneous inhibition of LAG3 and TIGIT in tandem with PD1 blockade and NBTXR3-enhanced radiotherapy promotes immune activation at the irradiated site. In addition, abscopal immune responses are improved with blockade of LAG3 and TIGIT. These results suggest that blockade of multiple immune checkpoints in parallel with NBTXR3-mediated radiotherapy may be effective in metastatic cancers. [Y.H. and B.G. contributed equally to this work.] Citation Format: Yun Hu, Genevieve Bertolet, Sebastien Paris, Hampartsoum Barsoumian, Jordan DA SILVA, Saumil Gandhi, Quynh-Nhu Nguyen, Maria A. Cortez, James W. Welsh. Nanoparticle-enhanced radiotherapy combined with triple blockade of PD1, LAG3, and TIGIT enhances anti-tumor immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5516.

BackgroundWhen exposed to radiotherapy (RT), NBTXR3 nanoparticles increase radiation dose deposition from within the cancer cells. NBTXR3 is intended for a single intratumor injection. Results from a phase II/III clinical trial in patients with locally advanced Soft Tissue Sarcoma demonstrated significant superiority and clinical benefits of NBTXR3 activated by RT compared to RT alone, and was well tolerated. NBTXR3 is currently being evaluated in several other tumors including head and neck, liver, and pancreatic cancer as a single agent or in combination with anti-PD1. Moreover, preclinical studies have demonstrated that NBTXR3 can produce a significant abscopal effect, whereas RT alone cannot. Here, we explored the impact of NBTXR3 activated by RT on CD8+ infiltrates and TcR repertoire diversity change, and the effect on the immunopeptidome of cancer cells.MethodsCT26 (murine colorectal cancer cells) were subcutaneously injected in BALB/c mice in one flank. Then, tumors were intratumorally injected with NBTXR3 (or vehicle) and irradiated 24 hours later with 4Gy per fraction for 3 consecutive days. Tumors were collected 3 days after the last RT fraction and immune cell infiltrates were measured using immunohistochemistry (IHC) and digital pathology. For TcR repertoire sequencing, the same workflow was followed, except cells were injected in both flanks. Only right tumors received treatment, while left tumors remained untreated. For immunopeptidome analysis, in vitro cells were irradiated by 4Gy. After one day, cells were collected for isolation and sequencing of MHC I-loaded peptides.ResultsIHC analyses showed a significant increase of CD8+ T cell infiltrates in tumors of mice treated with NBTXR3+RT, while RT alone had no significant effect. In addition, NBTXR3+RT treatment was able to increase TcR repertoire diversity, both in treated and untreated tumors, compared to RT alone. Finally, analysis of immunopeptidome showed that NBTXR3+RT changed the profile of MHC-I-loaded peptides.ConclusionsOur in vivo data indicate that NBTXR3+RT can modulate the microenvironment of treated tumors, leading to enhanced CD8+ T cell infiltration as well as modification of the TcR repertoire, both in treated and distant untreated tumors. These NBTXR3+RT-induced responses may be related to changes in the immunopeptidome of cancer cells triggered by this treatment.

International audience; BACKGROUND: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.METHODS: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.RESULTS: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.CONCLUSIONS: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSAbinding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.