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Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non–small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells—such as M2 tumor-associated macrophages (TAM)—thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti–PD-1–resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFβ levels. The addition of anti–PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

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Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non–small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells—such as M2 tumor-associated macrophages (TAM)—thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti–PD-1–resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFβ levels. The addition of anti–PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

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.