Heterogenous activation of multiple suppressive pathways by scRNAseq may underscore resistance to PD-1 therapy in metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is a deadly disease that, until recently, was thought to be resistant to checkpoint blockade. A recent clinical trial revealed that the PD-1 inhibitor, pembrolizumab, when given in combination with the androgen-deprivation therapy (ADT) drug enzalutamide, achieved an unprecedented 18% complete biochemical response rate in mCRPC patients. Though promising, ~80% of these patients failed to respond and the mechanisms of resistance remain unclear. The immunologic landscape of mCRPC is largely unknown. We hypothesize that mechanisms of resistance to anti-PD-1 therapy may reflect the heterogeneity of immune suppression in this disease. Revealing this heterogeneity is difficult to achieve because access to the metastatic disease is limited to core-needle biopsies of largely bone and lymph node disease. Therefore, to reveal pathways of resistance, we developed a protocol for isolating leukocytes and performing single-cell RNAseq from fresh core-needle biopsies taken from enzalutamide-resistant metastatic lesions prior to and after pembrolizumab treatment. The abundance of tumor-infiltrating leukocytes was on average less than 1% of total cells independent of biopsy location. Using our single-cell RNA sequencing data, we generated a leukocyte gene signature of enzalutamide mCRPC and used this to retrospectively deconvolute bulk RNA sequencing data from a cohort of 16 paired biopsies from enzalutamide-naive and -resistant patients. Computational approaches revealed the diverse leukocyte heterogeneity of mCRPC and multiple suppressive pathways beyond PD-1 within the tumor microenvironment. Importantly, the suppressive pathways were shared between metastatic biopsy locations (i.e., bone vs lymph node). To validate these targets in combination with ADT treatment, we developed orthotopic mouse models of mCRPC in immunocompetent mice. These strategies will identify new opportunities for clinical intervention to enhance antitumor responses in this patient population. This abstract is also being presented as Poster A04. Citation Format: Chaojie Wang, Julie Graff, Zheng Xia, Amy Moran. Heterogenous activation of multiple suppressive pathways by scRNAseq may underscore resistance to PD-1 therapy in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR10.