Abstract 6857: Evolution of myeloid-mediated mechanisms of immunotherapy resistance at single-cell resolution with prostate cancer progression

Abstract Background: Patients with metastatic castration-resistant prostate cancer (mCRPC) are largely refractory to immune checkpoint inhibitors (ICIs). This resistance is, in part, attributed to the presence of immunosuppressive myeloid cells in tumors. However, attempts to target these cells broadly, such as CSF1R antagonism, have thus far failed clinically primarily due to their heterogeneous nature. Thus, we hypothesized that a more comprehensive understanding of immunosuppressive myeloid subsets and their molecular mechanisms at the single-cell level is critical to enhance the effectiveness of immunotherapy. Methods: We performed single-cell profiling of patient biopsies with either localized, metastatic hormone-sensitive prostate cancer, or mCRPC. We then reverse translated these findings in a syngeneic mouse model of CRPC, where we performed multi-omic single-cell assessment and conducted studies to assess tumor efficacy and gain mechanistic insights. Results: We identified a specific subset of tumor-associated macrophages with elevated hypoxia signatures, including higher SPP1 transcript levels (SPP1hi-TAMs). These macrophages become significantly more abundant as the disease advances, expressing elevated immunosuppressive molecular programs. Notably, CSF1R transcripts were significantly reduced in SPP1hi-TAMs, suggesting their potential role in the clinical ineffectiveness of CSF1R blockade. In a syngeneic mouse CRPC model, we identified an analogous macrophage subset through single-cell profiling and flow cytometry. These macrophages possess the ability to suppress T cell activity in co-culture. Consistent with these findings, adoptive transfer of Spp1hi-TAMs into CRPC promoted ICI resistance and worsened survival in vivo. Importantly, these macrophages proved resistant to anti-CSF1R treatment, underscoring the need to target their immunosuppressive signals as a potential therapeutic approach. Pathway analysis revealed enrichment of gene signatures associated with adenosine signaling in SPP1hi-TAMs both in patients and in mice, implicating this pathway as their immunosuppressive mechanism. Indeed, blocking adenosine signaling using antibodies or pharmacologic inhibitors resulted in a significant reduction in T cell suppression mediated by Spp1hi-TAMs in culture. Furthermore, pharmacologic blockade of adenosine receptors in vivo significantly decreased tumor growth and enhanced the responsiveness of tumor cells to ICI therapies. Conclusions: Our studies demonstrate that SPP1hi-TAMs become prevalent as the disease progresses. They are resistant to CSF1R blockade and play a key role in mediating immunotherapeutic resistance in vivo by activating adenosine signaling pathways. These findings highlight the potential of the associated signals as promising targets for therapeutic intervention. Citation Format: Aram Lyu, Zenghua Fan, Diamond Luong, Ali Setayesh, Alec Starzinski, Laura Valderrábano, Eliezer Van Allen, Lawrence Fong. Evolution of myeloid-mediated mechanisms of immunotherapy resistance at single-cell resolution with prostate cancer progression abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6857.