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Abstract
Background Stereotactic Radiotherapy is used to manage tumor burden/progression in metastatic lesions of cancer patients, often presenting with synchronous liver metastases (LM). LM correlates with poor immunotherapy response. Our work in an immunocompetent dual-tumor LM murine model demonstrated LM’s induction of systemic tumor-specific immune tolerance.1 Radiotherapy (RT) can stimulate or suppress immune responses in tumor-bearing hosts, but its specific immunomodulatory effects in the context of LM remain unclear. We studied how RT targeted at the more immunogenic subcutaneous (SQ) tumor affects the synchronous LM model in the context of checkpoint inhibitor (CPI) therapies.
Methods C57BL/6 mice were orthotopically injected with MC38 cells both subcutaneously and intrahepatically (LM) to mimic synchronous liver and a distant tissue tumor growth seen in stage IV cancer patients, confirmed by IVIS imaging. The SQ tumor alone were targeted with an ablative dose of 1x14 Gy, facilitated by the SARRP image-guided small animal stereotactic irradiator. The irradiated SQ and non-irradiated LM tumor growth rates were measured via bioluminescence IVIS imaging in the context of RT alone and the combination of RT and CPI therapies (both anti-PD-1 monotherapy and anti-PD-1 plus anti-CTLA-4). Innate and adaptive immune compartments in the tumor microenvironment at both sites were analyzed by multi-color spectral FACS analysis with tumor-specific CD8 tetramer staining.
Results Without CPI treatment, RT targeting the SQ site controlled the local tumor growth but resulted in 3 out of 9 mice with faster tumor growth at the non-irradiated LM site compared to no RT controls. Similar results were obtained when done in lymphocyte-deficient NRG mice. Adding anti-PD-1 to SQ RT did not significantly improve LM response nor result in any abscopal effects (figure 1). Remarkably, combining anti-PD-1 plus anti-CTLA-4 treatment with SQ RT significantly improved responses at both RT and non-RT abscopal sites, and achieved ~40% complete response (figure 2). Interestingly, FACS analysis of SQ RT treated mice showed reduced ICOS+ and Ki67+ tumor-specific CD8 cells at the abscopal liver tumor and higher expression of PD-L1 on MDSCs at the SQ site compared to no RT control (figure 3). The triple combination group, in contrast to control and RT plus anti-PD-1 groups, resulted in increased ICOS+ tumor-specific CD8 T cells at both sites (figure 4).
Conclusions Our data suggest that SQ RT may adversely impact systemic antitumor immunity via innate immune cells in the presence of LM to promote distal, non-irradiated, liver tumor growth. Combining RT with dual check-point inhibitors may overcome this immunosuppressive effect.
Acknowledgements Research support provided by UCSF Department of Radiation Oncology, UCSF Liver Center, Parker Institute for Cancer Immunotherapy, and U.S. Congressionally Directed Medical Research Program – Melanoma Research Program.
Reference
Lee JC, Mehdizadeh S, Smith J, et al. Regulatory T cell control of systemic immunity and immunotherapy response in liver metastasis. Science Immunology. 2020;5(52):eaba0759.
Ethics Approval The animal study was approved by UCSF IACUC, approval number AN202115.
SQ and liver tumor growth with SQ RT alone or in combination with anti-PD-1. A. SQ RT control local tumor growth but even promote the distal liver tumor growth (3/4, data not shown) compared to no RT control. B-C. Adding anti-PD-1 to SQ RT did not significantly improve LM response nor result in any abscopal effects
SQ and liver tumor growth with triple SQ RT plus anti-PD-1 and anti-CTLA-4 therapy. Combining anti-PD-1 plus anti-CTLA-4 treatment with SQ RT significantly improved responses at both RT and non-RT abscopal sites, and achieved ~40% complete response
FACS analysis of tumor microenvironment from SQ tumor and liver tumor sites. SQ RT treated mice showed reduced ICOS+ and Ki67+ tumor-specific CD8 cells at the abscopal liver tumor and higher expression of PD-L1 on MDSCs at the SQ site compared to no RT control
FACS analysis of tumor-specific CD8 T cells from SQ tumor and liver tumor sites. The triple combination group (SQ RT+anti-PD-1+anti-CTLA-4), in contrast to control and RT plus anti-PD-1 groups, resulted in increased ICOS+ tumor-specific CD8 T cells at both sites
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