In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis

Abstract

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms HHS Public AccessAuthor manuscript Nat Biotechnol. Author manuscript; available in PMC 2017 November 01. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript (CRC) and metastasis that rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/ Δ;Kras G12D/+ ;Trp53Δ/ Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.Recent tumor sequencing studies have identified a large number of candidate genes that are mutated in CRCs and may contribute to carcinogenesis, tumor phenotype, and treatment responses in subsets of patients. 1,2 Traditionally, functional assessment of putative cancerassociated genes in vivo has required the development of genetically engineered mouse models (GEMMs) of CRC through extensive intercrossing or de novo generation of genetargeted mice, which is expensive and time consuming. Most GEMMs of CRC such as the Apc Min mouse 3,4 are also limited by delayed tumor onset (i.e., 2-4 months) and high tumor burden (i.e., 30-100 polyps) in the small intestine, which is a rare location for human intestinal tumors and precludes study of tumor progression beyond early adenomas or longitudinal studies using colonoscopy 5,6 . Tumorigenesis can be localized to the colon with either Apc loss driven by a colon-specific promoter, which is limited by slow tumor growth (i.e., 4-6 months) 7-9 , or somatic deletion of Apc in the distal colon of Apc fl/fl mice with rectal enema of adenoviral Cre, which is requires colonic injury and/or time-consuming surgery 10-12 .In addition to GEMMs, human and mouse cell lines are used to model CRC in vivo. Typical sites of transplantation are the mouse flank or kidney capsule, which do not recapitulate the native stroma of the colon mucosa. 6 Several groups have sought to orthotopically deliver tumor cell lines into the mouse colon, either surgically into the cecal serosa 13 (which is not the relevant tissue layer for CRC development) or into the...

Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation

“…We recently developed mouse models of CRC and metastasis based on colonoscopy-guided mucosal injection of viral vectors or tumor organoids 34 . We improved on existing mucosal injection methods by using a 33-gauge needle and a 100 μl syringe, which offers precise site-directed injection and the formation of a mucosal “bubble”, with minimal risk of colon perforation.…”

“…We improved on existing mucosal injection methods by using a 33-gauge needle and a 100 μl syringe, which offers precise site-directed injection and the formation of a mucosal “bubble”, with minimal risk of colon perforation. We applied this technique to model CRC with CRISPR-Cas9 gene editing of the colon epithelium and with orthotopic transplantation of mouse and human organoids 34 . Furthermore, we showed that these systems can be used quickly (i.e., within 2-3 months) to study putative tumor-associates genes, model the entire adenoma-carcinoma-metastasis sequence, sequentially mutate genes in established adenomas, assess tumor stem cell function, study patient-derived cancers in the native colon environment, and model normal organoid function.…”

“…In our models, engineered tumor organoids are engrafted into the colon mucosa via colonoscopy guided injection. Tumors with advanced mutations spontaneously invade into the local vasculature and metastasize to the liver 34 . Other groups have reported cancer organoid transplantation without colonoscopy, either by injection into prolapsed rectal mucosa 9 or by rectal enema into mice with colitis 10,17 .…”

Organoids, organs-on-chips and other systems, and microbiota

“…Advancement in technology has enabled culturing of the stomach [52][53][54], pancreas [55][56][57], liver [58], prostate [59,60], oesophagus [46,61], gall bladder [62,63], and taste buds [64]. Additionally, CRISPR-Cas9 technology has been exploited to induce gene editing in intestinal organoids that mimics the sequential loss and gain of functions of genes involved in human colorectal cancer [65], which, after in orthotopic engraftment, could metastasise to the liver [66,67]. Thus, organoid models can be used to study advanced aspects of cancer development in a more complexed in vivo scenario.…”

Defining key concepts of intestinal and epithelial cancer biology through the use of mouse models

“…Animal models therefore remain the most accurate way of simulating disease, allowing analysis from tumour initiation through to metastasis, and with the advent of CRISPR/Cas9 to allow cheap generation of compound mutants, including inducible compound mutants, the use of animal models of disease is entering a new and exciting phase. Indeed, very recent work has used CRISPR/Cas to mutate Apc, Kras and Trp53 in colonic organoids which developed invasive tumours after orthotopic, syngeneic transplantation into the mouse colon using a modified colonoscope, suggesting the next stage of in vivo mouse models has begun (183,184) …”