Background PD-(L)1 immune checkpoint blockade (ICB) therapy yields objective responses in 15–25% of patients with bladder cancer (BC), suggesting tumor-associated resistance mechanisms undermine their efficacy.1–6 We previously used pre-treatment tumor to derive an ICB response gene signature enriched in adaptive immune genes and an ICB resistance signature enriched in innate immune and inflammatory genes.7
Methods Single-cell RNA sequencing (scRNAseq) was performed on 27 BC tumors and 3 normal-adjacent tissue specimens to resolve cells underlying these signatures. We profiled tumor macrophages (MΦs) underlying the resistance signature with flow cytometry (FC). To recapitulate them, we used healthy donor (HD) blood monocytes differentiated into MΦs with M-CSF and skewed with bioinformatically predicted ligands, characterizing them with FC, RTqPCR, and bulk RNAseq. To discover peripheral biomarkers, we conducted scRNAseq, O-Link, and ELISAs on matched blood. To evaluate the impact of these MΦs on T cells, we employed functional assays on HD T cells, adding cytokines these MΦs produce and patient plasma. Finally, we performed Visium spatial transcriptomics to assess the intratumoral organization of these cells.
Results From our scRNAseq, we discovered the adaptive immune response and pro-tumorigenic inflammation signatures were enriched in distinct MΦ subsets: immunostimulatory (is)MΦs and pro-tumorigenic (pt)MΦs, respectively. ptMΦs upregulated CLEC5A, TREM1, and SPP1; expressed pro-inflammatory and hypoxic programs; and downregulated antigen presentation machinery whereas isMΦs upregulated antigen presentation and complement machinery. ptMΦs were enriched in tumor versus normal-adjacent tissue, as confirmed by FC on additional patients. When we tested predicted drivers of ptMΦs, IL-1β most highly induced Clec5a, Trem1, Spp1 and IL-6 expression in MΦs. We found corresponding cytokines, IL-1β, IL-6, and macrophage-colony stimulating factor (CSF-1), elevated in patient plasma. Our assays revealed IL-6 suppresses naïve CD8+ T cells’ capacity to differentiate into IFNγ-producing effector cells. scRNAseq of patient blood shows IL-6 acts through suppressor of cytokine signaling 3 (SOCS3). Culturing HD T cells in patient plasma induced SOCS3. Finally, spatial transcriptomics revealed IL-6 gradients across tumors that associate with ptMΦs and SOCS3-expressing T cells.
Conclusions We found that intratumoral Clec5a+Trem1+Spp1+ ptMΦs underlie our ICB resistance signature in BC. These ptMΦs produce IL-6 and are recapitulated by culturing HD monocytes with M-CSF and IL-1β. IL-6 is elevated in patient plasma and limits effector CD8+ T cell differentiation via SOCS3. Correspondingly, IL-6-producing ptMΦs and SOCS3-expressing CD8+ T cells are spatially associated within tumors. Overall, we demonstrate a novel axis associated with ICB resistance in BC whereby distinct pro-inflammatory MΦs suppress CD8+ T cells.
Acknowledgements We are very thankful to the Mount Sinai Genomics and Human Immune Monitoring Core (HIMC) Cores for their assistance with sequencing. This research is supported by the NIH NCI R01 Grant 1R01CA249175–01. M.A.T. is supported by the National Institute of Health (NIH) National Cancer Institute (NCI) F30 grant CA275269–01 as well as the Medical Scientist Training Program Training Grant T32GM007280 from the NIH National Institute of General Medical Sciences.
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Ethics Approval The study was approved by Mount Sinai Institution’s Ethics Board, approval number 10–1180. Participants gave informed consent before taking part in the study.
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