Precision modeling of gall bladder cancer patients in mice based on orthotopic implantation of organoid-derived tumor buds

Shingo Kato,Tetsuya Matsuura,Tomohiko Tamura,Makiko Enaka,Noritoshi Kobayashi,Yoshiaki Maru,Yuichiro Yabuki,Daisuke Kurotaki,Yoshitaka Hippo,Atsushi Nakajima,Kentaro Fushimi,Sho Hasegawa,Akihiro Suzuki,Takuma Higurashi,Masato Yoneda

doi:10.1038/s41389-021-00322-1

Shingo Kato, Tetsuya Matsuura + Show 13 more

Open Access

https://doi.org/10.1038/s41389-021-00322-1

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Abstract

Genetically engineered mice (GEM) are the gold standard for cancer modeling. However, strict recapitulation of stepwise carcinogenesis from a single tumor-initiating epithelial cell among genetically intact cells in adults is not feasible with the currently available techniques using GEM. In previous studies, we partially overcame this challenge by physically isolating organs from adult animals, followed by genetic engineering in organoids and subcutaneous inoculation in nude mice. Despite the establishment of suitable ex vivo carcinogenesis models for diverse tissues, tumor development remained ectopic and occurred under immunodeficient conditions. Further refinement was, therefore, necessary to establish ideal models. Given the poor prognosis and few models owing to the lack of gall bladder (GB)-specific Cre strain, we assumed that the development of authentic models would considerably benefit GB cancer research. Here, we established a novel model using GB organoids with mutant Kras and Trp53 loss generated in vitro by lentiviral Cre transduction and CRISPR/Cas9 gene editing, respectively. Organoid-derived subcutaneous tumor fragments were sutured to the outer surface of the GB in syngeneic mice, which developed orthotopic tumors that resembled human GB cancer in histological and transcriptional features. This model revealed the infiltration of similar subsets of immune cells in both subcutaneous and orthotopic tumors, confirming the appropriate immune environment during carcinogenesis. In addition, we accurately validated the in vivo efficacy of gemcitabine, a common therapeutic agent for GB cancer, in large cohorts. Taken together, this model may serve as a promising avatar of patients with GB cancer in drug discovery and precision medicine.

Highlights

Carcinogenesis is primarily driven by accumulated genetic alterations in epithelial cells but is influenced by mutual interactions among non-malignant epithelial cells, stromal cells, and immune cells in the tissue microenvironment
Histological analysis demonstrated that both Korg and K/53org comprised monolayer cells expressing the epithelial cell marker CK19 and a gall bladder (GB) cell marker CK7; more Ki-67-positive cells were detected in K/53org, indicating higher proliferation potential (Fig. 1B)
As patient-derived cancer models, xenografts in immunodeficient mice and organoids in culture have recently drawn attention as promising modalities for preclinical studies owing to their advantages over cancer cell lines that may have undergone extensive alterations from original tumors[18]

Summary

Introduction

Carcinogenesis is primarily driven by accumulated genetic alterations in epithelial cells but is influenced by mutual interactions among non-malignant epithelial cells, stromal cells, and immune cells in the tissue microenvironment. Tissueor cell lineage-specific genetic engineering in floxed conditional alleles may not always be achieved with high accuracy. Cre expression is usually driven by the promoters of genes that are expressed in a limited number of tissue types or cell lineages, highly specific markers are yet to be identified in many organs. Only a subset of cells develops tumors amid seemingly normal cells that have already undergone gene recombination. This is not the case in the pathogenesis of sporadic cancer, wherein a single cell eventually develops a full-blown tumor among genetically intact epithelial cells

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