Developing and utilising a mouse mammary organoid model

The mammary gland is a complex organ, relying upon multiple cell types and signalling pathways to orchestrate its predominantly postnatal development. To obtain a detailed understanding of such development, and also the mechanisms whose deregulation lead to mammary tumourigenesis, a physiologically relevant, quantifiable and qualitative model system is required. In vitro, currently available mammary culture systems have allowed studies into prospective stem cell populations, cell-cell interactions, differentiation, proliferation, paracrine networks and hormonal responses, but are limited in the degree to which they can truly recapitulate in vivo mammary biology. Three dimensional organoid culture systems from many tissues have recently been shown to concurrently allow sustained stem-cell maintenance, proliferation and functional differentiation; however, no such recapitulative in vitro model currently exists for the mammary gland. This thesis therefore details the development of a novel murine mammary organoid culture system. Culture conditions described, including R-Spondin1 and Neuregulin1, enable the development and expansion of mammary epithelial organoids for up to 2.5 months in culture. Organoids possess distinct basal and luminal compartments - functional steroid receptors in the latter allowing hormonal responses - and regenerative cell populations enabling mammary gland reconstitution in vivo. Additional conditions are also described promoting the growth of abnormal, tumour-like organoids. Utilisation of the organoid model allowed the study of key signalling pathways associated with mammary development; most notably revealing the critical importance of the Wnt signalling pathway in regulating normal development, its role in mammary tumourigenesis and demonstrating its potential as a target in anti-tumour therapy. Cell-cell signalling, individual cell populations and behaviours, and tumour organoid growth were studied, the latter offering a platform in which to study potential therapeutic compounds. Evidence provided furthers the understanding of mammary biology and supports that the organoid model comprises a suitable physiologically relevant tool for use in mammary research and drug discovery

RhoB regulates cell migration through altered focal adhesion dynamics

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions

Wnt and Neuregulin1/ErbB signalling extends 3D culture of hormone responsive mammary organoids

The development of in vitro culture systems quantitatively and qualitatively recapitulating normal breast biology is key to the understanding of mammary gland biology. Current three-dimensional mammary culture systems have not demonstrated concurrent proliferation and functional differentiation ex vivo in any system for longer than 2 weeks. Here, we identify conditions including Neuregulin1 and R-spondin 1, allowing maintenance and expansion of mammary organoids for 2.5 months in culture. The organoids comprise distinct basal and luminal compartments complete with functional steroid receptors and stem/progenitor cells able to reconstitute a complete mammary gland in vivo. Alternative conditions are also described that promote enrichment of basal cells organized into multiple layers surrounding a keratinous core, reminiscent of structures observed in MMTV-Wnt1 tumours. These conditions comprise a unique tool that should further understanding of normal mammary gland development, the molecular mechanism of hormone action and signalling events whose deregulation leads to breast tumourigenesis

3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model

The Rho GTPase RhoB regulates cadherin expression and epithelial cell-cell interaction

BACKGROUND: The Rho GTPase RhoB has been proposed to be a tumor suppressor in cancer and is downregulated in various tumors including prostate. RhoB has different effects on cell migration depending on the cell type and conditions, but the molecular basis for this variability is unclear. RhoB regulates trafficking of membrane receptors and integrins. We have previously shown that RhoB depletion alters focal adhesion dynamics and reduces surface levels of β1 integrin in PC3 prostate cancer cells, correlating with increased migration speed. RESULTS: Here we show that RhoB depletion reduces cell-cell adhesion and downregulates E-cadherin levels as well as increasing internalized E-cadherin in DU145 prostate cancer cells. This is accompanied by increased migration speed. RhoB localizes to cell-cell junctions together with E-cadherin in DU145 cells. RhoB depletion also reduces N-cadherin levels in PC3 cells, which do not express E-cadherin. CONCLUSIONS: These results indicate that RhoB alters migration of cells with cell-cell adhesions by regulating cadherin levels. We propose that the relative contribution of integrins and cadherins to cell migration underlies the variable involvement for RhoB in this process and that the downregulation of RhoB in some epithelial cancers could contribute to the weakening of epithelial cell-cell junction during tumor progression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12964-015-0085-y) contains supplementary material, which is available to authorized users