Androgen Deprivation Therapy Drives a Distinct Immune Phenotype in Localized Prostate Cancer

Abstract

Purpose: Androgen deprivation therapy (ADT) remains the backbone of prostate cancer treatment. Beyond the suppression of testosterone and tumor cell growth, emerging evidence suggests that ADT also modulates the immune tumor microenvironment. However, a more precise understanding of the timing and intricacies of these immunologic shifts is needed.

Experimental design: In this study, we analyzed 49 primary prostate cancers, comparing those surgically removed either without treatment or following treatment with degarelix at 4, 7, and 14 days before surgery. Utilizing next-generation DNA and RNA sequencing and multiplexed immunofluorescence, we examined alterations in immune phenotypes in the presence or absence of ADT.

Results: Our findings reveal that ADT rapidly transforms the typically bland prostate tumor microenvironment into an inflamed environment within days. Notably, we observed an increase in activated CD8 T cells along with an increase in suppressive regulatory T cells (Treg). We also found an expansion of the myeloid compartment, particularly proinflammatory M1-like tumor-associated macrophages. Intriguingly, discernable changes which have not previously been described also occurred in tumor cells, including upregulation of antigen presentation by MHC classes I and II and, unexpectedly, a decrease in the "do not eat me" signal CD47.

Conclusions: These observations underscore the critical role of timing and disease context in order to optimize the therapeutic efficacy of immune modulators combined with androgen ablation, for which the presurgical neoadjuvant setting may be ideal. Our findings warrant future prospective validation, which is currently underway.

Figure 1.

Schema and somatic molecular alterations. A, Schema depicting treatment and timing of degarelix administration prior to radical prostatectomy. B, Co-mutation plot with tumor mutational profiles in untreated and post-ADT samples.

Figure 2.

Transcriptomic changes following ADT in primary prostate tumors over time show enrichment of immune-related pathways. A, Hierarchical plot of enriched Gene Ontology (GO) processes in degarelix-treated vs untreated patients by log-fold-change ranked gene set enrichment. Image is thresholded to GO sets with enrichment p-value <10e-9. Lines depict relationships between pathways in the GO plot. B, Heatmap of differential expression of immune-related genes by functional group in degarelix treated and untreated specimens. Timing of degarelix administration also depicted with untreated (red) and degarelix treatment either 4 days (green), 7 days (blue), or 14 days (purple) prior to radical prostatectomy. C, Boxplots of normalized T cell gene expression (CD8, CD4), macrophage gene expression (CD68, CD163), immune checkpoint gene expression (CTLA4, PDCD1), antigen presentation gene expression including MHC Class I (B2M) and II (CD74) by treatment group. Statistical comparisons were performed by t-test with Bonferroni multiple-testing correction, such that * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.005. D, Tumor Inflammation Score (TIS) by treatment group. E, Boxplots of CIBERSORT-inferred Total Immune Cell Infiltrate by treatment group. P-values were obtained by t-test, where ** indicates p<0.01.

Figure 3.

ADT increases MHC Class II and decreases CD47+ expression on tumor cells with a concurrent increase in the immune cell infiltrate. A, Representative multiplexed immunofluorescence image for markers of interest including AMACR (red), CD3 (yellow), CD68 (cyan), CTLA-4 (magenta), CD47 (green), CD74 (orange). B, Side-by-side comparison of representative images from untreated (red) versus degarelix-treated patients (blue). C, Boxplots of immunofluorescent cell count frequencies between treatment groups, showing treatment-induced increase in frequency of CD3+ T-cells, CD3+/CTLA4+ T-cells, and AMACR+/CD74+ tumor cells, along with decrease in AMACR+CD47+ tumor cells and CD68+CD74+ macrophages. Count frequencies for each cell type between treatment groups was assessed by Fisher’s exact test with Bonferroni multiple-testing correction, such that * indicates p<0.05 and *** indicates p<0.005

Figure 4.

ADT induces a robust T cell and pro-inflammatory macrophage infiltrate with upregulation of antigen-presenting machinery. A, Composite sample image by CODEX showing marker expression including PSMA (red), pan-cytokeratin (cyan), CD163 (yellow), CD68 (grey), and CD3e (green); cell phenotyping including B-cells, cytotoxic T-cells, endothelial cells, epithelial cells, Tregs, activated cytotoxic T-cells, M1 and M2 macrophages, T helper cells, and tumor cells, with heatmap showing mean marker expression per cell type. Representative images in treated and untreated samples with boxplots shown for B, Overall T-cells (CD3e) and macrophages (CD68+), C, CD8 T-cells (CD8+) and activated CD8 T-cells (GranzymeB+CD8+), D, CD4 T-cells (CD4+), Tregs (CD4+FoxP3+), E, M1-like macrophages (CD68+CD163-CD206-) and M2-like macrophages (CD68+CD163+CD206+) and F, tumor cell expression of MHC-I (PSMA+HLA-A+) and MHC-II (PSMA+HLA-DR+).