Single-Cell Analyses of a Novel Mouse Urothelial Carcinoma Model Reveal a Role of Tumor-Associated Macrophages in Response to Anti-PD-1 Therapy

Abstract

Approximately 80% of patients with advanced bladder cancer do not respond to immune checkpoint inhibitor (ICI) immunotherapy. Therefore, there is an urgent unmet need to develop clinically relevant preclinical models so that factors governing immunotherapy responses can be studied in immunocompetent mice. We developed a line of mouse triple knockout (TKO: Trp53, Pten, Rb1) urothelial carcinoma organoids transplanted into immunocompetent mice. These bladder tumors recapitulate the molecular phenotypes and heterogeneous immunotherapy responses observed in human bladder cancers. The TKO organoids were characterized in vivo and in vitro and compared to the widely used MB49 murine bladder cancer model. RNAseq analysis of the TKO tumors demonstrated a basal subtype. The TKO xenografts demonstrated the expression of urothelial markers (CK5, CK7, GATA3, and p63), whereas MB49 subcutaneous xenografts did not express urothelial markers. Anti-PD-1 immunotherapy resulted in a mixed pattern of treatment responses for individual tumors. Eight immune cell types were identified (basophils, B cells, dendritic cells, macrophages, monocytes, neutrophils, NK cells, and T cells) in ICI-treated xenografts. Responder xenografts displayed significantly increased immune cell infiltration (15.3%, 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 (1.0% vs. 0.4%, p = 0.002) and macrophages (8.6% vs. 6.4%, p = 0.0002) in responder xenografts than in non-responder xenografts. In conclusion, we have developed a novel preclinical model that exhibits a mixed pattern of response to anti-PD-1 immunotherapy. The higher percentage of macrophage tumor infiltration in responders suggests a potential role for the innate immune microenvironment in regulating ICI treatment responses.

Preclinical models for bladder cancer therapy research

“…TKO tumours exhibited basal-like features and expressed, in contrast to lesions generated by implantation of MB49 cells, urothelial lineage markers. Treatment of subcutaneous TKO tumours with anti-PD-1 antibodies resulted in a mixed pattern of response reflecting immunotherapy responses observed in the clinics [ 33 ▪ ]. Tsuji et al generated orthotopic BC models using the cell lines MBT-2 and MB49 and female C3H/HeJ or C57BL/6J mice to assess the effects of the novel bacterial minicell-based integrin-targeted oncolytic agent VAX014.…”

The bladder cancer immune micro-environment in the context of response to immune checkpoint inhibition

“…One preclinical study used a conditional knockout BC mouse model which showed a heterogeneous response to treatment with PD-1 inhibition. Responding tumors showed a higher number of intratumoral macrophages ( 86 ).…”