ERrrr...Where are the progenitors? Hormone receptors and mammary cell heterogeneity

The mammary epithelium is a highly heterogenous and dynamic tissue that includes a range of cell types with varying levels of proliferative capacity and differentiation potential, from stem to committed progenitor and mature cells. Generation of mature cells through expansion and specification of immature precursors is driven by hormonal and local stimuli. Intriguingly, although circulating hormones can be directly sensed only by a subset of mammary cells, they also regulate the behaviour of cells lacking their cognate receptors through paracrine mechanisms. Thus, mapping the hormonal signalling network on to the emerging mammary cell hierarchy appears to be a difficult task. Nevertheless, a first step towards a better understanding is the characterization of the hormone receptor expression pattern across individual cell types in the mammary epithelium. Here we review the most relevant findings on the cellular distribution of hormone receptors in the mammary gland, taking into account differences between mice and humans, the methods employed to assess receptor expression as well as the variety of approaches used to resolve the mammary cell heterogeneity

Cas proteins: dodgy scaffolding in breast cancer

The members of the Cas protein family (p130Cas/ BCAR1, Nedd9/HEF1, EFS and CASS4) are scaffold proteins required for the assembly of signal transduction complexes in response to several stimuli, such as growth factors, hormones and extracellular matrix components. Given their ability to integrate and coordinate multiple signalling events, Cas proteins have emerged as crucial players in the control of mammary cell proliferation, survival and differentiation. More importantly, it has been found that alterations of their expression levels result in aberrant signalling cascades, which promote initiation and progression of breast cancer. Based on the increasing data from in vitro, mouse model and clinical studies, in this review we will focus on two Cas proteins, p130Cas/BCAR1 and Nedd9, and their coupled signalling pathways, to examine their role in mammary cell transformation and in the acquirement of invasiveness and drug resistance of breast cancer cells

Caveolin-1 implicated as a pro-invasive gene in high-grade glioma cell models: implementation of a 3d spheroid matrix invasion assay

INTRODUCTION: The poor prognosis associated with Glioblastoma multiforme (GBM) is multifactorial but includes the capacity of residual tumour cells not removed by surgery and resistant to radio-/chemo-therapy undergoing diffuse invasion into the surrounding brain tissue. Caveolin-1 (Cav-1) is the major structural and functional component of caveolae. In a number of tumour types Cav-1 is recognised to participate in cytoskeletal rearrangement, integrin-mediated adhesion and/or matrix remodelling. We proposed Cav-1 serves to promote invasion of GBM cells. To investigate this we have employed in an in-vitro 3D cellular invasion assay. METHOD: The human GBM cell lines, UP007 and UP029 established from primary cultures of biopsy-derived brain tumours (University of Portsmouth), U-87 MG (ECACC) and U-373 MG (ECACC) were genetically modified to stably knock-out Cav-1 using a Lentiviral Cav-1 shRNA approach; corresponding stably transfected non-target (NT) shRNA cell lines were generated as controls. Neuropheres were formed and embedded within an extracellular matrix (Matrigel™). Over a two-/four-day period (depending on cell line) the migration of cells away from the neurophere core (CORE) was quantified by image capture and processing (Image J) using a custom-developed MatLab script for pixel density analysis indicative of the density of migrating cellular material. RESULTS: Cav-1 knockout resulted in significant (P0.05) towards reduced invasion. Depending upon the cell line the Cav-1 knockdown also resulted in reduced size and cellular density of the neurosphere core (UP007 and UP029) indicative of reduced proliferation and/or cell survival capacity. CONCLUSION: Using an in-vitro 3D cellular invasion assay we have found Cav-1 expression in a series of three GBM cell lines to promote cellular invasion capacity. Ongoing studies are addressing signalling mechanisms and the influence of the microenvironment

Droplet microfluidics for tumor drug-related studies and programmable artificial cells

Anticancer drug development is a crucial step toward cancer treatment, that requires realistic predictions of malignant tissue development and sophisticated drug delivery. Tumors often acquire drug resistance and drug efficacy, hence cannot be accurately predicted in 2D tumor cell cultures. On the other hand, 3D cultures, including multicellular tumor spheroids (MCTSs), mimic the in vivo cellular arrangement and provide robust platforms for drug testing when grown in hydrogels with characteristics similar to the living body. Microparticles and liposomes are considered smart drug delivery vehicles, are able to target cancerous tissue, and can release entrapped drugs on demand. Microfluidics serve as a high-throughput tool for reproducible, flexible, and automated production of droplet-based microscale constructs, tailored to the desired final application. In this review, it is described how natural hydrogels in combination with droplet microfluidics can generate MCTSs, and the use of microfluidics to produce tumor targeting microparticles and liposomes. One of the highlights of the review documents the use of the bottom-up construction methodologies of synthetic biology for the formation of artificial cellular assemblies, which may additionally incorporate both target cancer cells and prospective drug candidates, as an integrated “droplet incubator” drug assay platform

The receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis

PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here we demonstrate that PTPRB negatively regulates branching morphogenesis in the mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in estrogen receptor positive luminal cells. During mammary development Ptprb expression is down-regulated during puberty, a period of extensive of ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of FGFR activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology

NOTCH and AKT signalling interact to drive mammary tumour heterogeneity

A better understanding of the mechanisms generating tumour heterogeneity will allow better targeting of current therapies, identify potential resistance mechanisms and highlight new approaches for therapy. We have previously shown that in genetically modified mouse models carrying conditional oncogenic alleles, mammary tumour histotype varies depending on the combination of alleles, the cell type to which they are targeted and, in some cases, reproductive history. This suggests that tumour heterogeneity is not a purely stochastic process; rather, differential activation of signalling pathways leads to reproducible differences in tumour histotype. We propose the NOTCH signalling pathway as one such pathway. Here, we have crossed conditional knockout Notch1 or Notch2 alleles into an established mouse mammary tumour model. Notch1/2 deletion had no effect on tumour-specific survival; however, loss of Notch alleles resulted in a dose-dependent increase in metaplastic adenosquamous carcinomas (ASQCs). ASQCs and adenomyoepitheliomas (AMEs) also demonstrated a significant increase in AKT signalling independent of Notch status. Therefore, the NOTCH pathway is a suppressor of the ASQC phenotype, while increased PI3K/AKT signalling is associated with ASQC and AME tumours. We propose a model in which PI3K/AKT and NOTCH signalling act interact to determine mouse mammary tumour histotype

INSIDIA:a FIJI macro delivering high-throughput and high-content spheroid invasion analysis

Time-series image capture of in vitro 3D spheroidal cancer models embedded within an extracellular matrix affords examination of spheroid growth and cancer cell invasion. However, a customizable, comprehensive and open source solution for the quantitative analysis of such spheroid images is lacking. Here, the authors describe INSIDIA (INvasion SpheroID ImageJ Analysis), an open-source macro implemented as a customizable software algorithm running on the FIJI platform, that enables high-throughput high-content quantitative analysis of spheroid images (both bright-field gray and fluorescent images) with the output of a range of parameters defining the spheroid “tumor” core and its invasive characteristics

Caveolin-1, a driver of invasive phenotype in in-vitro 3D-spheroid assays comprised of high grade GBM cells association with an AKT-inhibited phenotype

INTRODUCTION Glioblastoma multiforme (GBM) cells display a highly invasive phenotype, a hallmark which counters effective surgical and radiotherapy strategies. Caveolin-1 (Cav-1) is the main structural and functional component of caveolae. The impact of the expression of Cav-1 within a range of tumour and tumour-associated stromal cells is variable with both oncogenic and tumour suppressive roles reported which appear to be both disease-specific and context-dependent. Our hypothesis is that Cav-1 serves as promoter of invasion of GBM cells. MATHERIALS AND METHODS To investigate our hypothesis we used a lentiviral shRNA approach to silence Cav-1 in three GBM cell lines (U87, UP007, UP029) derived from adult brain tumours. We employed an in-vitro 3D cell-sprouting invasion assay with GBM cell spheres embedded in Matrigel. Quantification of invasion was undertaken using a novel image analysis tool or 3D systems, INSIDIA (ImageJ Macro for High-throughput Spheroid Invasion Analysis). Parallel migration and invasion studies were performed using a Boyden Chamber approach, as well as cell-cell adhesion assays. Activation of signalling pathways in 2D and 3D cultures were performed by proteomic array and Western Blot analysis. RESULTS AND CONCLUSION GBM cells expressing Cav-1 (Cav-1 +ve) displayed a higher invasive capacity compared cells where Cav-1 had been silenced Cav-1 –ve), the latter also showing increased cell-cell adhesion. A significant finding from the signalling analysis was an inverse association between Cav-1 silencing and activation of AKT evidenced by increased phosphorylation at both Ser473 and Thr308 sites. Ongoing studies are exploring this signalling axis and its relationship to the invasive phenotype. CM and MG acknowledge Cancer Research Wales support. GP and HF acknowledge Brain Tumour Research support

p130Cas promotes invasiveness of three dimensional ErbB2-transformed mammary acinar structures by enhanced activation of mTOR/p70S6K and Rac1

ErbB2 over-expression is detected in approximately 25% of invasive breast cancers and is strongly associated with poor patient survival. We have previously demonstrated that p130Cas adaptor is a crucial mediator of ErbB2 transformation. Here, we analysed the molecular mechanisms through which p130Cas controls ErbB2-dependent invasion in three-dimensional cultures of mammary epithelial cells. Concomitant p130Cas over-expression and ErbB2 activation enhance PI3K/Akt and Erk1/2 MAPK signalling pathways and promote invasion of mammary acini. By using pharmacological inhibitors, we demonstrate that both signalling cascades are required for the invasive behaviour of p130Cas over-expressing and ErbB2 activated acini. Erk1/2 MAPK and PI3K/Akt signalling triggers invasion through distinct downstream effectors involving mTOR/p70S6K and Rac1 activation, respectively. Moreover, in silico analyses indicate that p130Cas expression in ErbB2 positive human breast cancers significantly correlates with higher risk to develop distant metastasis, thus underlying the value of the p130Cas/ErbB2 synergism in regulating breast cancer invasion. In conclusion, high levels of p130Cas favour progression of ErbB2-transformed cells towards an invasive phenotype