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

Over the past 20 years, huge advances have been made in modelling human diseases such as cancer using genetically modified mice. Accurate in vivo models are essential to examine the complex interaction between cancer cells, surrounding stromal cells, tumour-associated inflammatory cells, fibroblast and blood vessels, and to recapitulate all the steps involved in metastasis. Elucidating these interactions in vitro has inherent limitations, and thus animal models are a powerful tool to enable researchers to gain insight into the complex interactions between signalling pathways and different cells types. This review will focus on how advances in in vivo models have shed light on many aspects of cancer biology including the identification of oncogenes, tumour suppressors and stem cells, epigenetics, cell death and context dependent cell signalling

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease

Intestinal homeostasis and neoplasia studied using conditional transgenesis

Constitutive mouse models of intestinal neoplasia, such as the Apcmin/+ (multiple intestinal neoplasia) mouse have proven valuable tools both for furthering our understanding of tumorigenesis and for the development of therapeutic strategies. However, the in vivo study of a number of genes has been precluded by their absolute requirement during embryonic development. This has led to the development of conditional strategies that allow gene regulation in vivo. This review describes the principal techniques used to achieve conditional transgenesis within the mouse intestine, with a particular focus upon the Cre–Lox and Tet-regulable systems. Further, we discuss how these techniques are being used to dissect the mechanisms governing both normal homeostasis and neoplastic development within the intestine

Origin and maintenance of the intestinal cancer stem cell

Colorectal cancer is one of the most common cancers in the western world and its incidence is steadily increasing. Understanding the basic biology of both the normal intestine and of intestinal tumorigenesis is vital for developing appropriate and effective cancer therapies. However, relatively little is known about the normal intestinal stem cell or the hypothetical intestinal cancer stem cell, and there is much debate surrounding these areas. This review briefly describes our current understanding of the properties of both the intestinal stem cell and the intestinal cancer stem cell. We also discuss recent theories regarding the origin of the intestinal cancer stem cell, and the signals required for its maintenance and proliferation. Finally, we place the relevance of cancer stem cell research into context by discussing potential clinical applications of targeting the intestinal cancer stem cell

PTEN loss and KRAS activation cooperate in murine biliary tract malignancies

Carcinomas of the biliary tract are aggressive malignancies in humans. Loss of the tumour suppressor PTEN has previously been associated with cholangiocarcinoma development in a murine model. Activation of KRAS is reported in up to one-third of human cholangiocarcinomas and 50% of gall bladder carcinomas. In this study we aimed to test the potential interaction between PTEN and KRAS mutation in biliary tract malignancy. We used an inducible Cre–LoxP-based approach to coordinately delete PTEN and activate KRAS within the adult mouse biliary epithelium. We found that activation of KRAS alone has little effect upon biliary epithelium. Loss of PTEN alone results in the development of low-grade neoplastic lesions, following long latency and at low incidence. Combination of both mutations causes rapid development of biliary epithelial proliferative lesions, which progress through dysplasia to invasive carcinoma. We conclude that activation of the PI3′K pathway following loss of PTEN is sufficient to drive slow development of low-grade biliary lesions in mice. In contrast, mutational activation of KRAS does not result in a similar phenotype, despite a prediction that this should activate both the RAF–MEK–ERK and PI3′-kinase pathways. However, mutation of both genes results in rapid tumourigenesis, arguing that PTEN normally functions as a ‘brake’ on the PI3′-kinase pathway, limiting the influence of KRAS activation. Mutation of both genes creates a ‘permissive’ environment, allowing the full effects of both mutations to be manifested. These data reveal an in vivo synergy between these mutations and provides a new mouse model of biliary tract malignanc

PI3'-kinase inhibition forestalls the onset of MEK1/2 inhibitor resistance in BRAF-mutated melanoma

Phosphatidylinositide 3′ (PI3′)-lipid signaling cooperates with oncogenic BRAFV600E to promote melanomagenesis. Sustained PI3′-lipid production commonly occurs via silencing of the PI3′-lipid phosphatase PTEN or, less commonly, through mutational activation of PIK3CA, encoding the 110-kDa catalytic subunit of PI3′-kinase-α (PI3Kα). To define the PI3K catalytic isoform dependency of BRAF-mutated melanoma, we used pharmacologic, isoform-selective PI3K inhibitors in conjunction with melanoma-derived cell lines and genetically engineered mouse (GEM) models. Although BRAFV600E/PIK3CAH1047R melanomas were sensitive to the antiproliferative effects of selective PI3Kα blockade, inhibition of BRAFV600E/PTENNull melanoma proliferation required combined blockade of PI3Kα, PI3Kδ, and PI3Kγ, and was insensitive to PI3Kβ blockade. In GEM models, isoform-selective PI3K inhibition elicited cytostatic effects, but significantly potentiated melanoma regression in response to BRAFV600E pathway–targeted inhibition. Interestingly, PI3K inhibition forestalled the onset of MEK inhibitor resistance in two independent GEM models of BRAFV600E-driven melanoma. These results suggest that combination therapy with PI3K inhibitors may be a useful strategy to extend the duration of clinical response of patients with BRAF-mutated melanoma to BRAFV600E pathway–targeted therapies. Significance: Although BRAFV600E pathway–targeted therapies elicit melanoma regression, the onset of drug resistance limits the durability of response. Here, we show that combined treatment with PI3K inhibitors significantly forestalled the onset of MEK1/2 inhibitor–resistant disease in BRAF-mutated GEM melanoma models. These results provide a conceptual framework for the combined deployment of BRAFV600E plus PI3K pathway–targeted inhibitors in the treatment of a subset of patients with BRAF-mutated melanoma

Characterizing the cellular uptake of neural stem-cell derived exosomes using live-cell imaging techniques

Introduction: Neural stem cell derived exosomes (“ExoPr0”); purified from the conditioned medium of a GMP manufactured, conditionally-immortalized human neural stem cell line (“CTX0E03”), demonstrates a unique biodistribution profile in mice compared to exosomes derived from a control producer cell line. We have previously shown that ExoPr0 is able to cross the blood brain barrier, and to further explicate these findings, we investigated the uptake of ExoPr0 at the cellular level using live-cell imaging techniques. Methods: We employed live-cell confocal microscopy to directly visualize uptake of fluorescently labelled exosomes. A quantitative image analysis protocol was developed and applied to assess the uptake of exosomes in a number of cell types. Results: Time course incubations of cells treated with ExoPr0 produced data that revealed heterogeneity in uptake between cell types. ExoPr0 was compared to exosomes derived from a control producer cell line, highlighting source-specific differences in uptake kinetics. Uptake was observed to occur through more than one pathway resulting in trafficking through endo-lysosomal compartments. The effect of cell cycle on the uptake of ExoPr0 was investigated, but was not observed as having a significant influence. Summary/conclusion: Findings from this study have eluded to the specificity of ExoPr0 towards different cell types and work is ongoing to further elucidate the delivery mechanism of ExoPr0 and understand the subcellular trafficking in recipient cells

PTEN loss and KRAS activation leads to the formation of serrated adenomas and metastatic carcinoma in the mouse intestine

Mutation or loss of the genes PTEN and KRAS have been implicated in human colorectal cancer (CRC), and have been shown to co-occur despite both playing a role in the PI3' kinase (PI3'K) pathway. We investigated the role of these genes in intestinal tumour progression in vivo, using genetically engineered mouse models, with the aim of generating more representative models of human CRC. Intestinal-specific deletion of Pten and activation of an oncogenic allele of Kras was induced in wild-type (WT) mice and mice with a predisposition to adenoma development (Apcfl/+). The animals were euthanized when they became symptomatic of a high tumour burden. Histopathological examination of the tissues was carried out, and immunohistochemistry used to characterize signalling pathway activation. Mutation of Pten and Kras resulted in a significant life-span reduction of mice predisposed to adenomas. Invasive adenocarcinoma was observed in these animals, with evidence of activation of the PI3'K pathway but no metastasis. However, mutation of Pten and Kras in WT animals not predisposed to adenomas led to perturbed homeostasis of the intestinal epithelium and the development of hyperplastic polyps, dysplastic sessile serrated adenomas and metastasizing adenocarcinomas with serrated features. These studies demonstrate synergism between Pten and Kras mutations in intestinal tumour progression, in an autochthonous and immunocompetent murine model, with potential application to preclinical drug testing. In particular, they show that Pten and Kras mutations alone predispose mice to the spectrum of serrated lesions that reflect the serrated pathway of CRC progression in humans

Vitamin D3 suppresses morphological evolution of the cribriform cancerous phenotype

Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH)2D3,theactive form of vitamin D. CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ (phosphatase and tensin homolog, cell division cycle 42 and protein kinase C zeta). Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted “Swiss cheese-like” cribriform morphology (CM) comprising multiple abnormal “back to back” lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors

Epithelial-specific loss of PTEN results in colorectal juvenile polyp formation and invasive cancer

Cowden syndrome (CS) is a rare autosomal dominant cancer-prone disorder caused by germ-line mutation of the phosphatase and tensin homolog mutated on chromosome 10 (PTEN) tumor-suppressor gene. Affected patients commonly develop juvenile polyps, and show an elevated risk of developing colorectal cancers. The etiology of these peculiar polyps remains unclear, although previous work has suggested somatic PTEN alterations in the stroma of juvenile polyps. After a long latency period, we find epithelial-specific PTEN deletion to cause formation of juvenile polyps in the colorectum without stromal PTEN loss. More important, we find that these lesions closely recapitulate all of the characteristic histopathological features of juvenile polyps seen in patients with CS, including stromal alterations and dysplastic transformation to colorectal carcinoma. The stromal alterations we identify after epithelial-specific PTEN loss suggest that PTEN may be involved in altered epithelial-mesenchymal cross talk, which, in turn, predisposes to colorectal neoplasia and polyposis. Our transgenic model is the first to recapitulate colorectal juvenile polyposis in patients with CS. We conclude that stromal PTEN loss is not a prerequisite for the formation of juvenile polyps, and that colorectal juvenile polyps in CS are bona fide neoplastic precursor lesion