Introduction: Approximately 80% of patients with advanced bladder cancer do not respond to immune checkpoint inhibitor (ICI) immunotherapy. The molecular mechanism of ICI treatment response remains unknown largely due to the lack of clinically relevant immunocompetent mouse models.
Methods: We developed a syngeneic triple knockout (TKO: Trp53/Pten/Rb1) bladder cancer model in immunocompetent C57 BL/6J mice. Mouse urothelial cells (Trp53 f/f, Pten f/f, Rb1f/f) were transfected ex vivo with Ad5CMVCre (Adenovirus Cre recombinase driven by a CMV promoter). The TKO urothelial cells were subcutaneously injected into immunocompetent C57 BL/6J for tumor formation. The tumors were treated with an anti-PD1 antibody (BioXcell, RMP1-14, 200 µg, intraperitoneally twice weekly) (n=9) or a control IgG (n=9). Single cell analyses were performed for tumor xenografts from control IgG (n=2), responders (30% less than the median of control tumors) (n=2) and non-responders (n=2) using PTPRC (also named CD45) expression as an immune cell marker. Seurat package was used for data analysis including dimension reduction, cell clustering, and identification of cell markers. SingleR package was utilized to identify the cell types. Immunohistochemistry (IHC) of CD8 and F4/80 was performed to identify T cells and macrophages respectively.
Results: Anti-PD-1 immunotherapy resulted in a mixed pattern of treatment responses in individual tumors. Eight clusters of immune cells were identified (basophils, B cells, dendritic cells, macrophages, monocytes, neutrophils, NK cell, and T cells) in ICI treated xenografts. Responder xenografts displayed significantly increased immune cell infiltration (15.2%, 742 immune cells/4861 total cells) compared to the non-responder tumors (10.1%, 452 immune cells/4459 total cells, Fisher Exact Test p<0.0001). Specifically, there were more T cells (46/4861 vs 18/4459, p=0.002), and macrophages (420/4861 vs 287/4459, p=0.002) in responder xenografts than in non-responder xenografts. Compared to control IgG tumors, a near complete response tumor exhibited an immune-inflamed phenotype with significant infiltration of T cells and macrophages, whereas non-responder tumors showed no significant change of T cells and macrophages infiltration.
Conclusions: The higher percentage of T cells and macrophages tumor infiltration in responders suggests a potential role of innate immune microenvironment for the ICI treatment response. Modulating tumor associated macrophage may overcome the resistance to ICI immunotherapy.
Source of Funding: This research work was supported in part by NIH Grants, K08CA252161(Li, Q), P30CA016056 (NCI Cancer Center Core Support Grant), the Roswell Park Alliance Foundation and the Friends of Urology Foundation.
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