&lt;em&gt;Ex Vivo&lt;/em&gt; Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Xu, Dongbo; Wang, Li; Wieczorek, Kyle; Wang, Yanqing; Zhang, Xiaojing; Goodrich, David W.; Li, Qiang

doi:10.3791/63855

Cited by 1 publication

(4 citation statements)

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“…The TKO tumors revealed positive protein expression of urothelial lineage markers (CK5, CK14, CK7, GATA3, and p63) and negative expression of Uroplakin 3, CK20. The expression pattern of urothelial markers in intravesical tumors is identical to subcutaneous (published in [22]) and orthotopic tumors (Supplemental Figure S3B). In contrast, the MB49 tumors expressed no urothelial markers.…”

Section: Tko Is a Basal-like Bladder Cancer Modelmentioning

confidence: 56%

“…The use of autochthonous mouse models is time-consuming, laborious, and expensive. To provide a faster and more tractable model, we employed an ex vivo organoid culture system to delete the Trp53, Pten, and Rb1 tumor suppressors from normal mouse urothelium [22]. The genetic inactivation of these three tumor suppressors is relevant because P53/RB1/PTEN pathways are commonly altered in human MIBC [39][40][41].…”

Section: Ex Vivo Inactivation Of Trp53/rb1/pten Results In High-grade...mentioning

confidence: 99%

“…Rapid and efficient ex vivo gene editing in organoids has accelerated the pace of generating clinically relevant experimental models [14]. We have used a rapid ex vivo approach by employing the Cre expressing adenovirus to delete clinically relevant tumor suppressor genes (Trp53, Pten, and Rb1) from normal urothelial cells with high efficiency, as described [22]. This ex vivo approach can be applied to any combination of genes of interest to generate preclinical transplant models within a few weeks.…”

Section: Discussionmentioning

confidence: 99%

“…To create a strain compatible with C57BL/6 mice, mixed background triplefloxed mice (C57BL/6:129/Sv:FVB) were bred with homogenized C57BL/6 background mice for multiple generations. The process of urothelial cells disassociation and ex vivo transduction was described [22]. Briefly, four fresh bladder tissues were collected from male triple-floxed mice for disassociation.…”

Section: Isolation Of Primary Urothelial Cellsmentioning

confidence: 99%

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

Wang

Wieczorek

et al. 2022

Cancers

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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.

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Section: Tko Is a Basal-like Bladder Cancer Modelmentioning

confidence: 56%