Dissecting the immune infiltrate induced by ADT and PD-1 blockade in men with metastatic hormone-sensitive prostate cancer using single-cell RNA-sequencing.

Abstract

e17548 Background: Overall survival of men with de novo metastatic, hormone-sensitive prostate cancer (mHSPC) is improved by treatment intensification with docetaxel and hormone therapy compared to androgen deprivation therapy (ADT) alone. However, castration-resistant prostate cancer (CRPC) invariably develops. We and others have shown that ADT induces a robust and complex immune infiltrate in localized prostate cancer, in both animal models and humans. We are conducting a clinical trial (NCT03951831) to test the hypothesis that the ADT-induced immune infiltrate can be further augmented with chemotherapy and an anti-PD-1 inhibitor in men with mHSPC. Here, we present single-cell RNA-sequencing analysis of paired metastatic tumor biopsies at baseline and on-treatment. Methods: In our phase 2, open-label, single-center trial, eligible patients with mHSPC are treated with phased administration of ADT, cemiplimab (anti-PD-1 inhibitor), and docetaxel. All patients undergo baseline metastatic tumor biopsies as well as on-treatment biopsies after either ADT alone or ADT in combination with cemiplimab. Biopsies are dissociated by gentleMACS protocol, and single cells are sequenced using the 10X Chromium platform. Cells are Quality-control filtered to those with over 1000 Unique Molecular Identifiers and less than 25% mitochondrial RNA, then clustered on global gene expression and on inferred protein activity by unsupervised Louvain algorithm with silhouette score selection of optimal clustering resolution. Cell types are assigned by the singleR algorithm and tumor cell identity is verified by inference of Copy Number Variations (InferCNV). Differential gene expression across sub-clusters is performed, and baselines will be compared to their paired on-treatment sample (N = 3). Results: Preliminary results showed a significant increase in the normalized fold-change of CD8+ T cells in on-treatment samples relative to baseline samples (P < 0.001). There was also a dramatic increase in other immune cell subtypes with treatment. In addition, we found a shift in transcriptional state of tumor cells and that tumor cells expressing KLK2/KLK3 significantly dissipate with treatment (normalized fold-change 0.56, P < 0.001). Conclusions: Taken together these show the feasibility of SS-RNA-seq on PC metastatic lesions and provide initial support for the hypothesis that ADT induces complex immunological changes in the tumor microenvironment (TME) in metastatic prostate tumors.