548 Evolution of myeloid-mediated mechanisms of immunotherapy resistance at single-cell resolution with human prostate cancer progression

Background

Patients with metastatic castration-resistant prostate cancer (mCRPC) are refractory to immune checkpoint inhibitors (ICIs).^{1 2} Although prostate cancer is highly populated by multiple immunosuppressive myeloid cells especially after androgen deprivation therapy (ADT),³ efforts to therapeutically target myeloid cells broadly have thus far failed clinically, potentially due to myeloid cell heterogeneity and complexity.^{4 5} Understanding the specific myeloid populations relevant in prostate cancer and the molecular mechanisms mediating immunosuppression is needed to improve the efficacy of immunotherapy in this disease.

Methods

We performed multi-omic single-cell RNA sequencing (scRNA-seq) on biopsies from prostate cancer patients with either ADT-naïve localized prostate cancer, metastatic hormone-sensitive prostate cancer on ADT, or mCRPC progressing on ADT. We also performed scRNA-seq on immune and non-immune cells isolated from a syngeneic mouse model of CRPC. Functional significance of specific myeloid populations was assessed with immune assays in vitro and tumor efficacy in vivo with this syngeneic model.

Results

We identified a specific population of tumor-associated macrophages expressing elevated SPP1 transcripts (SPP1^hi-TAMs) significantly enriched in mCRPC relative to earlier states of prostate cancer. This myeloid population expresses elevated levels of immunosuppressive molecular programs.^6–8 Notably, CSF1R transcript levels were drastically diminished in SPP1^hi-TAMs relative to other macrophages, implicating this population as a potential mechanism underlying the clinical ineffectiveness of CSF1R blockade. We then utilized a syngeneic mouse model of CRPC to investigate the specific mechanisms by which SPP1^hi-TAMs mediate resistance to ICIs. Using scRNA-seq, we identified an analogous macrophage state and demonstrated their ability to suppress T-cell proliferation and activation in vitro. Administration of an anti-CSF1R antibody failed to ablate Spp1^hi-TAMs in CRPC, pointing to their immunosuppressive signals as potential immunotherapeutic targets. Adoptive transfer of Spp1^hi-TAMs into tumors promoted resistance to ICIs and worsened survival in vivo. Pathway analyses revealed enrichment of gene signatures associated with the adenosine pathway in SPP1^hi-TAMs both in patients and in mice.^{9 10} Analyses of The Cancer Genome Atlas (TCGA) database from prostate cancer patients as well as our own patient scRNA-seq dataset indicated that enrichment of the adenosine pathway correlates with SPP1^hi-TAM abundance and CD8⁺ T-cell exhaustion in tumors. Finally, pharmacologic inhibition of A2AR abrogated Spp1^hi-TAM-mediated immunosuppression on activated T cells in vitro.

Conclusions

Our studies demonstrate that as prostate cancer progresses, the abundance of SPP1^hi-TAMs increases. Furthermore, this myeloid population can mediate resistance to ICIs in mCRPC by activating adenosine signaling, supporting the relevance of therapeutically targeting of this pathway in this disease.

References

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