Yoshitaka Hippo

Establishment and characterization of multiple patient-derived organoids from a case of advanced endometrial cancer

Patient-derived organoids (PDOs) retain the original tumor's characteristics to a large degree and allow direct evaluation of the drug sensitivity, thereby emerging as a valuable resource for both basic and preclinical researches. Whereas most past studies stereotypically adopted a single PDO as an avatar of the patient, it remains to be investigated whether this assumption can be justified even for the tumor with spatial diversity. To address this issue, we established and characterized multiple PDOs originating from various sites of a patient with advanced uterine carcinosarcoma (UCS). Specifically, cancer cells were separately sampled from three sites; resected UCS tumor tissue, the peritoneal lavage fluid, and an intra-uterine brushing of the tumor. The three derived PDOs were morphologically undistinguishable, displaying typical carcinoma organoids-like appearance, but two of them proliferated at a faster rate. The primary tumor harbored mutations in TP53 and STK11 along with amplifications in CCNE1, ERBB2, and KRAS. These two mutations and the CCNE1 amplification were detected in all PDOs, while either KRAS or ERBB2 amplification was selectively observed in each PDO in a mutually exclusive manner. Observed intra-tumor heterogeneity in HER2 expression was differentially reproduced in the PDOs, which mirrored each PDO's sensitivity to HER2 inhibitors. Inter-PDO heterogeneity was also evident in sensitivity to standard cytotoxic agents. Lastly, a drug screening identified four candidate reagents commonly effective to all PDOs. Collectively, we showed that multiple PDOs could help reproduce the spatial diversity of a tumor and serve as a valuable resource in UCS research in many respects.

Probing the tumorigenic potential of genetic interactions reconstituted in murine fallopian tube organoids

AbstractGenetically engineered mice have been the gold standard in modeling tumor development. Recent studies have demonstrated that genetically engineered organoids can develop subcutaneous tumors in immunocompromised mice, at least for organs that prefer predominant driver mutations for tumorigenesis. To further substantiate this concept, the fallopian tube (FT), a major cell of origin of ovarian high‐grade serous carcinoma (HGSC), which almost invariably carries TP53 mutations, was investigated for p53 inactivation‐driven tumorigenesis. Murine FT organoids subjected to lentiviral Cre‐mediated Trp53 deletion did not develop tumors. However, subsequent suppression of Pten and simultaneous induction of mutant Pik3ca led to the development of carcinoma in situ and HGSC‐like tumors, respectively, whereas concurrent deletion of Apc resulted in the development of benign cysts, mirroring frequent activation of the PI3K/AKT axis and the marginal impact of Wnt pathway activation in HGSC. Consistent with the frequent activation of the RAS pathway in HGSC, mutant Kras cooperated with Trp53 deletion for the development of tumors, which unexpectedly contained sarcoma cells in addition to carcinoma cells, despite the epithelial origin of the inoculated organoids. This finding is in sharp contrast with the exclusive adenocarcinoma development from gastrointestinal organoids with the same genotype reported in previous studies, suggesting a tissue‐specific epithelial–mesenchymal transition program. In tumor‐derived organoids, the Cre‐mediated recombination rate reached 100% for Trp53 but not for the other genes, highlighting the advantage of p53 inactivation in FT tumorigenesis. The Trp53 wildtype FT organoids expressing the mutant Kras developed sarcoma and carcinoma upon Cdkn2a suppression and Tgfbr2 deletion, respectively, revealing novel pro‐tumorigenic genetic cooperation and critical roles of TGF‐β signaling for epithelial–mesenchymal transition in FT‐derived tumorigenesis. Collectively, the organoid‐based approach represents a shortcut to tumorigenesis and provides novel insights into the relationships among genotype, cell type, and tumor phenotype underlying tumorigenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of target cells of human papillomavirus 18 using squamocolumnar junction organoids

AbstractHuman papillomavirus 18 (HPV18) is a highly malignant HPV genotype among high‐risk HPVs, characterized by the difficulty of detecting it in precancerous lesions and its high prevalence in adenocarcinomas. The cellular targets and molecular mechanisms underlying its infection remain unclear. In this study, we aimed to identify the cells targeted by HPV18 and elucidate the molecular mechanisms underlying HPV18 replication. Initially, we established a lentiviral vector (HPV18LCR‐GFP vector) containing the HPV18 long control region promoter located upstream of EGFP. Subsequently, HPV18LCR‐GFP vectors were transduced into patient‐derived squamocolumnar junction organoids, and the presence of GFP‐positive cells was evaluated. Single‐cell RNA sequencing of GFP‐positive and GFP‐negative cells was conducted. Differentially expressed gene analysis revealed that 169 and 484 genes were significantly upregulated in GFP‐positive and GFP‐negative cells, respectively. Pathway analysis showed that pathways associated with cell cycle and viral carcinogenesis were upregulated in GFP‐positive cells, whereas keratinization and mitophagy/autophagy‐related pathways were upregulated in GFP‐negative cells. siRNA‐mediated luciferase reporter assay and HPV18 genome replication assay validated that, among the upregulated genes, ADNP, FHL2, and NPM3 were significantly associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. Among them, NPM3 showed substantially higher expression in HPV‐related cervical adenocarcinomas than in squamous cell carcinomas, and NPM3 knockdown of HPV18‐infected cells downregulated stem cell‐related genes. Our new experimental model allows us to identify novel genes involved in HPV18 early promoter activities. These molecules might serve as therapeutic targets in HPV18‐infected cervical lesions.