Unraveling myeloid cell-mediated mechanisms of resistance to immune checkpoint blockade in bladder cancer

Abstract Background: Only 15–25% of bladder cancer (BC) patients respond to PD-(L)1 immune checkpoint blockade (ICB) therapy. We previously used pre-treatment tumor to derive an ICB response gene signature that was enriched in adaptive immune genes and an ICB resistance signature enriched in innate immune and inflammatory genes. Methods: We performed single-cell RNA sequencing (scRNAseq) on 27 bladder tumors and 3 normal-adjacent tissue specimens to resolve these signatures. We profiled tumor macrophages (MΦs) underlying the resistance signature with flow cytometry (FC). To recapitulate them, we used healthy donor blood monocytes differentiated with M-CSF, skewed with predicted ligands, and characterized by FC, RTqPCR, and bulk RNAseq. To examine for peripheral biomarkers, we conducted scRNAseq on paired blood and urine and performed O-Link and ELISA on plasma. Results: Our ICB response and resistance signatures were enriched in distinct MΦ subsets: immunostimulatory (is)MΦs and pro-tumorigenic (pt)MΦs, respectively. ptMΦs upregulated SPP1, TREM1, and CLEC5Aand pro-inflammatory and hypoxic programs whereas isMΦs upregulated antigen presentation and complement machinery. ptMΦs were enriched in tumor versus normal tissue. When we tested ptMΦ predicted drivers, IL-1β induced expression of Clec5a and Trem1 protein and transcription of SPP1, TREM1, and CLEC5A. In the first-ever BC urine scRNAseq, we discovered urine contains ptMΦs. Corresponding inflammatory cytokines (IL-1β, IL-6, IL-8) were elevated in advanced patient plasma compared to early stage and healthy donors. In conclusion, we identified pro-inflammatory SPP1+CLEC5A+TREM1+MΦs that may underlie ICB resistance, be targeted via IL-1β and monitored via plasma and urine. Supported by grants from NIH (F30 CA275269-01, R01 CA249175-01).