400 Background: Cells of the tumor microenvironment (TME) have been implicated in prostate cancer (PCa) progression. However, the specific TME cell subtypes and cell–cell interactions driving disease progression in patients remains unclear. Traditional tumor profiling techniques lack the resolution needed to assess complex single-cell and spatial biomarkers. Imaging Mass Cytometry (IMC) can identify a much wider range of cancer, stromal and immune cell subtypes in patient tumors than previously possible using spatial proteomics. We profiled the tumors of a large prospective biopsy cohort with longitudinal clinical outcomes using two custom IMC assays to reveal potentially targetable TME cells driving localized PCa. Methods: Spatial single-cell expression profiling was performed on primary PCa tumor biopsies using two IMC assays (“Tumor Panel” enriched for PCa tumor markers and “TME Panel” enriched for stromal and immune cell markers). Cell subtypes were defined for both assays independently by clustering, then integrated using affine registration of paired images. Reference cell subtypes were defined using Tumor Panel, and TME cell subtypes were refined using co-registered TME Panel data. Per-sample “cell fraction” of each cell subtype was defined as cell count of that subtype divided by total cell count. Biochemical progression-free survival (bPFS) and cancer-specific survival (CSS) were prespecified clinical endpoints. Survival analyses stratified by cell fraction tertiles were performed using a Cox proportional hazards model. Cell–cell interactions were defined as colocalization using knn graphs, and significance was assessed using a permutation test (α=0.05). Results: Protein co-expression patterns in >6.4 million cells comprising 604 biopsy samples obtained from 393 patients were measured. Initial single-cell analysis of Tumor Panel data revealed 16 cell subtypes including luminal PCa cells, basal epithelial cells, lymphocytes, and MHC-II+ immune cells. Patients with tumors enriched for MHC-II+ immune cells demonstrated impaired bPFS ( P =0.001) and CSS ( P =0.002). Multivariable analysis including GS revealed MHC-II+ immune cell enrichment, Canary risk score, and Prolaris genomic risk score to be all independently prognostic of bPFS and CSS ( P <0.01). TME Panel showed the MHC-II+ cells to co-express CD68 and CD163, consistent with M2 macrophages. Spatial interaction testing revealed significant interaction between these cells and CD4+ FOXP3+ putative Tregs (P<0.05), suggesting they may cooperate to promote PCa progression. Conclusions: We identified a single-cell biomarker independently associated with poor prognosis in localized PCa. Validation in an independent cohort is needed. Our findings support new therapeutic strategies targeting TME immune cells and highlight the utility of spatial tumor profiling for discovering next-generation biomarkers.
Chen et al. (Sun,) studied this question.