Dissecting Mechanisms of Sarcoma Response to Immunotherapy and Radiotherapy in Primary Tumor Mouse Models

Abstract

In recent years, immunotherapy has surfaced as an innovative and promising strategy for treating cancer. It was named “Breakthrough of the Year” by Science in 2013 due to the impressive clinical success achieved by immune checkpoint inhibitors (ICI) (Ribas and Wolchok 2018). ICIs are especially effective in non-small cell lung carcinoma (NSCLC) and melanoma patients with an objective response rate of around 45% (Darvin et al. 2018). However, in most solid tumors, only about 15-30% of patients respond to ICI treatments (Das and Johnson 2019). Most patients with locally advanced or metastatic cancer either fail to respond to ICI or relapse shortly after the initial response (Y. Yang 2015; Chiriva-Internati and Bot 2015; Das and Johnson 2019). A clinical trial in patients with metastatic soft tissue or bone sarcoma showed that approximately 17.5% of patients respond to pembrolizumab monotherapy, which blocks programmed cell death protein 1 (PD-1), supporting previous postulations that sarcoma might respond poorly to immunotherapy due to low immunogenicity compared to other tumors, such as melanoma and renal cell carcinoma (Keung et al. 2020; Kawaguchi et al. 2005; Raj, Miller, and Triozzi 2018; S. Wang et al. 2019). Tumors with high mutational burden and abundant T cell infiltration tend to respond favorably to ICI and are commonly referred to as “hot tumors” (Bonaventura et al. 2019; Maleki Vareki 2018; S. Wang et al. 2019). On the contrary, “cold tumors” are more likely to have low mutational load and lower immune infiltrates. These cold tumors tend to respond poorly to immunotherapy. Many studies have evaluated therapeutic approaches to turn cold tumors into hot ones. However, the majority of preclinical studies investigating the mechanisms behind immunotherapy resistance are based on models of transplanted tumors, where the tumor does not coevolve with the host's immune system. Therapeutic approaches that elicit impressive survival benefits in these models often do not translate into improved outcomes for patients (Günther, Däbritz, and Wirthgen 2019; Luke et al. 2018). Using a genetically engineered mouse model of autochthonous soft tissue sarcoma with high mutational load (p53/MCA model), we have shown that ICI alone or in combination with radiation therapy fails to cure the autochthonous soft tissue sarcoma. However, the combination of ICI and radiation therapy cures all transplant tumors generated from the same sarcoma model and injected into syngeneic mice (Lee et al. 2019). These results indicate that the coevolution of the immune system and tumor cells in the autochthonous sarcoma model might drive the tumor microenvironment to become more immune tolerant and less effective at eliminating malignant cells. Therefore, we plan to address transplant tumor models’ limitations by using carcinogen-induced and genetically engineered mouse models (GEMMs) of cancer in which the tumor gradually develops over several weeks under surveillance by an intact immune system (Huang et al. 2017; Lee et al. 2019). We will utilize an autochthonous model of soft tissue sarcoma (p53-MCA model) induced by CRISPR/Cas9-mediated loss of p53 and exposure to the carcinogen 3-methylcholanthrene (MCA). According to the Cancer Research and Surveillance, Epidemiology, and End-Results (SEER) database linked to U.S. Census data, approximately 50% of all cancer patients received radiation therapy during the course of their treatments (Moding, Kastan, and Kirsch 2013). The utility of radiation therapy for treating local diseases within the radiation field was rapidly established. There have been reports of abscopal responses where non-irradiated secondary tumors decrease in size after irradiation of a primary tumor (Dewan et al. 2009). Since radiation therapy can stimulate an anti-tumor immune response, multiple clinical trials are currently testing the efficacy of a combination of ICB and radiotherapy (Kang, Demaria, and Formenti 2016). My thesis work seeks to explore a combination of immunotherapy and radiotherapy that might turn a “cold sarcoma” into a “hot sarcoma” that would have an enhanced response to treatments. It is also my goal to study the underlying mechanisms through which combination therapy mediates its treatment effects. Using p53/MCA sarcoma model, we showed that toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to radiation therapy (RT) in mouse sarcomas. Bulk RNA-seq, single-cell RNA-seq, and mass cytometry were used to assess the immune response to RT + CpG in sarcomas. Results from these experiments showed increased intratumoral infiltration of CD8 T cells after treatment with 20 Gy and CpG. These CD8 T cells also express markers associated with activation, such as Ki-67, Granzyme B, and IFN-γ. Myeloid cells also exhibited an upregulation in mRNA associated with antigen presentation pathways in response to RT + CpG. Additionally, TCR clonality analysis suggests clonal T-cell dominance is increased in sarcomas treated with RT + CpG. Genetic or immunodepletion of CD8+ T cells in vivo abolishes the treatment effect of the combination therapy. Taken together, these results suggest that the combination therapy delays sarcoma growth in a CD8 T cell-dependent manner.