Interrogating the Impact of Protease Activity on Tumor Progression Using 3D Spheroid Models.

Cancers have a complex relationship with the surrounding environment that regulates everything from progression to response to treatment. Cell-cell and cell-matrix interactions are heavily influenced by protease biology. Studies on the tumor microenvironment have revealed a new complexity for proteases, describing novel substrates for classic proteases, and protease-independent roles for these enzymes. The rapid expansion of 3D in vitro model systems provides excellent tools to study the intricate influence of proteases on the tumor microenvironment. Here we describe a spheroid invasion assay, providing a platform to interrogate key protease-matrix interactions in the context of early-stage breast cancer. Incorporation of pharmacological inhibition and RNAi techniques enables the elucidation of key protease-dependent pathways and can be complemented with immunofluorescence analysis to visualize matrix cleavage events and visualize cell behavior during collective cell invasion.

ADAMTS3 restricts cancer invasion in models of early breast cancer progression through enhanced fibronectin degradation.

Proteases have long been associated with cancer progression, due to their ability to facilitate invasion upon matrix remodelling. However, proteases are not simply degraders of the matrix, but also play fundamental roles in modulating cellular behaviour through the proteolytic processing of specific substrates. Indeed, proteases can elicit both pro- and anti- tumorigenic effects depending on context. Using a heterocellular spheroid model of breast cancer progression, we demonstrate the repressive function of myoepithelial ADAMTS3, with its loss directing myoepithelial-led invasion of luminal cells through a physiologically relevant matrix. Degradomic analysis, using terminal amine isotopic labelling of substrates (TAILS), combined with functional assays, implicate ADAMTS3 as a mediator of fibronectin degradation. We show further that loss of ADAMTS3 enhances levels of fibronectin in the microenvironment, promoting invasion through canonical integrin α5ß1 activation. Our data highlight a tumour suppressive role for ADAMTS3 in early stage breast cancer, and contribute to the growing evidence that proteases can restrain cancer progression.

TGFß-mediated MMP13 secretion drives myoepithelial cell dependent breast cancer progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Virtually all women with DCIS are treated, despite evidence suggesting up to half would remain with stable, non-threatening, disease. Overtreatment thus presents a pressing issue in DCIS management. To understand the role of the normally tumour suppressive myoepithelial cell in disease progression we present a 3D in vitro model incorporating both luminal and myoepithelial cells in physiomimetic conditions. We demonstrate that DCIS-associated myoepithelial cells promote striking myoepithelial-led invasion of luminal cells, mediated by the collagenase MMP13 through a non-canonical TGFß - EP300 pathway. In vivo, MMP13 expression is associated with stromal invasion in a murine model of DCIS progression and is elevated in myoepithelial cells of clinical high-grade DCIS cases. Our data identify a key role for myoepithelial-derived MMP13 in facilitating DCIS progression and point the way towards a robust marker for risk stratification in DCIS patients.

Everybody needs good neighbours: the progressive DCIS microenvironment.

Ductal carcinoma in situ (DCIS) is a pre-invasive form of breast cancer where neoplastic luminal cells are confined to the ductal tree. While as many as 70% of DCIS cases will remain indolent, most women are treated with surgery, often combined with endocrine and radiotherapies. Overtreatment is therefore a major issue, demanding new methods to stratify patients. Somewhat paradoxically, the neoplastic cells in DCIS are genetically comparable to those in invasive disease, suggesting the tumour microenvironment is the driving force for progression. Clinical and mechanistic studies highlight the complex DCIS microenvironment, with multiple cell types competing to regulate progression. Here, we examine recent studies detailing distinct aspects of the DCIS microenvironment and discuss how these may inform more effective care.

Tumour microenvironment 3D-modelling: simplicity to complexity and back again.

Tumours are surrounded by a host of noncancerous cells that fulfil both supportive and suppressive roles within the tumour microenvironment (TME). The drive to understand the biology behind each of these components has led to a rapid expansion in the number and use of 3D in vitro models, as researchers find ways to incorporate multiple cell types into physiomimetic configurations. The use and increasing complexity of these models does however demand many considerations. In this review we discuss approaches adopted to recapitulate complex tumour biology in tractable 3D models. We consider how these cell types can be sourced and combined and examine methods for the deconvolution of complex multicellular models into manageable and informative outputs.