Background Immunotherapies such as checkpoint blockade therapy (CBT) can be an effective approach to treat patients with metastatic tumors. In non-small cell lung cancer (NSCLC), four major immune subsets have been described, (a) the CBT-sensitive with T cell infiltration and PD-L1 expression, (b) the immune cell desert that lacks CD8+ T cell infiltration, (c) the non-functional T cell phenotype comprising T cells yet no PD-L1 expression, and (d) the T cell excluded phenotype, characterized by effector T cells surrounding the tumor.1Patients with a non-T cell-infiltrated tumor microenvironment (TME) correlate with a poor response to CBT.2 Data from our lab showed that NSCLC patients can be classified into T cell-infiltrated and non-T cell-infiltrated using a T cell gene signature. Interestingly, we observed that Sox2 upregulation correlates with a lack of T cell infiltration. Using a syngeneic mouse model, we aim to investigate how tumor cell-intrinsic expression of Sox2 mediated immune evasion in NSCLC.
Methods We used a lung adenocarcinoma cell line (KPCt) derived from a KrasG12D/+ and Tp53-/- mouse to overexpress Sox2 (KPS2). Subcutaneous or lung tumors were treated with anti-PD-L1 and anti-CTLA-4 blocking antibodies and analyzed for tumor burden. Tumor infiltration of T cells was evaluated by fluorescence microscopy. To characterize tumor-specific T cell responses, we engineered the KPCt and KPS2 cell lines to express the model antigen SIY. Finally, we performed a bulk RNA-seq analysis of KPCt.SIY and KPS2.SIY cell lines and subcutaneous tumors to determine candidate genes downstream Sox2 that could negatively affect T cell infiltration into the tumor.
Results We found that Sox2 overexpression induces resistance to CBT mediated by T cell exclusion from the tumor core. Analyses of tumor-reactive T cells indicated that T cell priming and differentiation into cytotoxic effector T cells were not affected, yet, cytotoxic T cells failed to infiltrate KPS2 tumors while enriched in the peritumoral regions. Bulk RNA-seq data showed that Sox2 overexpression changed the composition of the extracellular matrix. Furthermore, we observed in KPS2.SIY tumors an increase in endothelial vessel density; however, the size of the vessels was significantly reduced compared to KPCt.SIY tumors.
Conclusions Our results show that tumor cell-intrinsic activation of Sox2 in NSCLC promotes immune evasion and contributes to immunotherapy resistance by inducing changes in the TME. Understanding the molecular and immunological mechanisms mediating T cell exclusion from the lung TME will facilitate the development of novel combination treatment strategies for NSCLC patients.
Acknowledgements This work was supported by the Ludwig Center for Molecular Oncology at MIT and by the SITC-Nektar Therapeutics Equity and Inclusion in Cancer Immunotherapy Fellowship.
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Ethics Approval Animal procedures were approved by the Committee on Animal Care (CAC/IACUC) at MIT, DHHS Animal Welfare Assurance # D16-00078
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