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

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.

Recent Advances in Implantation-Based Genetic Modeling of Biliary Carcinogenesis in Mice

“…With regard to GBC, only a few studies have reported orthotopic implantation of the GB and resultant tumor development. However, there is an opposing view of the feasibility of orthotopic tumor development by direct inoculation into the GB, and an organoid-based two-step inoculation method was recently established to model orthotopic GBC development [ 24 ]. In this subsection, we highlight the technical aspects and limitations of the implantation approach ( Figure 4 ).…”

“…To address these issues, a novel two-step model was recently documented for GBC. Transduced organoid-derived subcutaneous tumors in syngeneic mice were subjected to orthotopic implantation, as fragmented “tumor bud.” This approach, termed implantation of organoid-derived tumor buds (IoTB), enabled the establishment of a fine GBC model without injection-associated cell leakage [ 24 ].…”

“…Specifically, subcutaneous tumors were first generated in syngeneic mice by implanting Kras G12D -expressing GB organoids that were subjected to CRISPR/Cas9-based deletion of Trp53 , p19 Arf , and Smad4 . Subsequently, the minced fragments of derived tumors, called tumor buds (TBs), were sutured on the outside of the GB [ 24 ]. Orthotopic tumors highly resemble human GBC in terms of macroscopic and microscopic features and transcriptional profiles.…”

“…From the pool of three–four subcutaneous tumors, 20 TBs were generated, eventually leading to a cohort of 20 tumor-bearing mice. Notably, mice were open-inspected for accurate evaluation of the tumor volume and divided into two subgroups of equal tumor burden for treatment and non-treatment with gemcitabine [ 24 ].…”

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“…We adopted the Matrigel-based organoid culture technique because it enables long-term and physiological propagation of tissue stem cells. By integrating lentiviral gene transduction of murine orga-Editorial noids and their subsequent allograft implantation in immunodeficient mice, we assessed the tumorigenicity of each oragnoid [2][3][4]. In most cases, the outcomes were essentially similar to those observed in GEM models, even in the absence of an organ-specific microenvironment or cellular immunity, highlighting the validity and robustness of this ex vivo model [5,6].…”