Organoid-based ex vivo reconstitution of Kras-driven pancreatic ductal carcinogenesis

Abstract

The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model.

Establishment and characterization of patient‐derived organoids from a young patient with cervical clear cell carcinoma

“…To date, it has been applied to various research fields, including infectious disease models, developmental biology, and epithelial regeneration . We also reported the establishment of murine organoid‐based carcinogenesis models for intestine, lung, hepatobiliary tract, and pancreas . These organoid culture techniques have now become common for patient‐derived samples from diverse types of cancer, which revealed that tumor‐derived organoids basically retained the morphology and genetic aberrations of the original tumors .…”

Organoid research in digestive system tumors (Review)

“…As the technology has advanced, organoids have been extensively used as a powerful tool of tumor research, particularly regarding the digestive system (Fig. 1) (7)(8)(9)(10)(11).…”

“…The organoid model is more representative of typical physiological conditions compared with other models (4). Currently, the organoids for normal tissues, including the stomach (7), small intestine (8), liver (9), pancreas (10) and prostate (11), have been established successfully. Compared with PDCs, the patient-derived organoids (PDOs) not only reflect histopathological characteristics and the genomic and transcriptomic profiles of the original tumor, but also recapitulate the components of the tumor microenvironment (12).…”

Establishment and Molecular Phenotyping of Organoids from the Squamocolumnar Junction Region of the Uterine Cervix

“…Such tissue samples were collected in a hospital immediately after surgery, and processed the next morning following overnight transfer to the lab while maintained in a cold media ( Figure 1A). As a culture media, we tested the standard culture media supplemented with EGF, R-spondin-1, Noggin, Jagged-1, and Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor Y27632, which we have confirmed robustly applicable to organoid culture of murine primary cells from various organs and human gynecological neoplasms [20,22,23,30,32], to facilitate future cross-referencing of organoids from various organs.…”

“…It has been applied to various research fields, including infectious diseases [17], developmental biology [18], and tissue regeneration [19]. By taking advantage of propagating normal stem cells in vitro, we have established murine organoid-based ex vivo carcinogenesis models for the intestine [20], lungs [21], hepatobiliary tract [22], and pancreas [23], by in vitro lentiviral gene transduction [24] or chemical treatment [25], followed by inoculation in the dorsal skin of nude mice. More recently, organoid culture techniques have been further applied to patient-derived tumor samples of diverse organs, which revealed that organoids basically retained the histological features and genetic aberrations of the original tumors [26][27][28].…”