Tumor-Infiltrating Myeloid Cells Confer de novo Resistance to PD-L1 Blockade through EMT-stromal and Tgf-beta Dependent Mechanisms

Most bladder cancers are poorly responsive to immune checkpoint blockade (ICB) of PD-L1. Thus, there is a need to define mechanisms of de novo resistance including contributions from tumor infiltrating immune cells. In this study, we used single-cell transcriptional profiling to map and define infiltrating myeloid cells in 10 human bladder tumors. Human data sets were qualitatively compared with myeloid data sets from the carcinogen (BBN) induced mouse model of bladder cancer which we have demonstrated to be poorly responsive to PD-L1 blockade. We previously established a signature of acquired ICB resistance which we apply here to new human and murine tumor data sets that have not received prior ICB (or systemic treatment). In doing so, we reveal conservation in EMT-stromal core genes and TGF beta signaling between human and mouse myeloid cells consistent with signatures of de novo ICB resistance. Untreated BBN tumors were highly infiltrated with M0-M2 macrophages, low in T-NK infiltration and modeled a patient subpopulation with poor survival outcome. Concordantly, the combined targeting of TGFb + PD-L1 reverted immune cell exclusion and resulted in increased survival and delayed BBN mouse tumor progression. These data constitute the stromal and myeloid cell populations as providing a coordinate mechanism de novo resistance to PD-L1 blockade in a TGF-beta dependent manner. Overall design: In this study, 10 human primary bladder cancer samples were obtained from patients who underwent either trans urethral resection bladder tumor (TURBT) or cystectomy under approval of the Mount Sinai School of Medicine IRB and written consent from all subjects. Resected tumor sample were examined by a genitourinary pathologist who confirmed the pathological staging and tumor content. Tumor tissues were then resected and dissociated to the single cell level in a solution of RPMI-10% FBS (Gibco) + 1% type 1 collagenase (Gibco). The dissociate is subject to mechanical dissociation through a 16G needle and syringe (5-10 passes) and then a 21.5G needle (5-10 passes). Single cell dissociates were then sorted into CD45 positive/negative cells using a BD Aria after cell staining using a BV510-CD45 antibody (Biolegend) and DAPI stain. Single cell suspensions of all sorted samples were then resuspended in BSA (0.2 % in PBS) at a concentration of 2x10^6 cells/ml followed by barcoding with a 10x Chromium Controller (10x Genomics). Sequencing was done on a Nextseq 500 platform.