The organization of the extracellular matrix has a profound impact on cancer development and progression. The matrix becomes aligned throughout tumor progression, providing “highways” for tumor cell invasion. Aligned matrix is associated with breast density and is a negative prognostic factor in several cancers; however, the underlying mechanisms regulating this reorganization remain poorly understood. Deletion of the tumor suppressor Pten in the stroma was previously shown to promote extracellular matrix expansion and tumor progression. However, it was unknown if PTEN also regulated matrix organization. To address this question, a murine model with fibroblast-specific Pten deletion was used to examine how PTEN regulates matrix remodeling. Using second harmonic generation microscopy, Pten deletion was found to promote collagen alignment parallel to the mammary duct in the normal gland and further remodeling perpendicular to the tumor edge in tumor-bearing mice. Increased alignment was observed with Pten deletion in vitro using fibroblast-derived matrices. PTEN loss was associated with fibroblast activation and increased cellular contractility, as determined by traction force microscopy. Inhibition of contractility abrogated the increased matrix alignment observed with PTEN loss. Murine mammary adenocarcinoma cells cultured on aligned matrices derived from Pten−/− fibroblasts migrated faster than on matrices from wild-type fibroblasts. Combined, these data demonstrate that PTEN loss in fibroblasts promotes extracellular matrix deposition and alignment independently from cancer cell presence, and this reorganization regulates cancer cell behavior. Importantly, stromal PTEN negatively correlated with collagen alignment and high mammographic density in human breast tissue, suggesting parallel function for PTEN in patients.

Matrix metalloproteinases (MMPs) are important for many different types of cancer-related processes, including metastasis. Understanding the functional impact of changes in MMP activity during cancer treatment is an important facet not typically evaluated as part of preclinical research. With MMP activity being a critical component of the metastatic cascade, we designed a 3D hydrogel system to probe whether pharmacological inhibition affected human melanoma cell proteolytic activity; metastatic melanoma is a highly aggressive and drug-resistant form of skin cancer. The relationship between MMP activity and drug treatment is unknown, and therefore we used an in situ fluorogenic MMP sensor peptide to determine how drug treatment affects melanoma cell MMP activity in three dimensions. We encapsulated melanoma cells from varying stages of progression within PEG-based hydrogels to examine the relationship between drug treatment and MMP activity. From these results, a metastatic melanoma cell line (A375) and two inhibitors that inhibit RAF (PLX4032 and sorafenib) were studied further to determine whether changes in MMP activity led to a functional change in cell behavior. A375 cells exhibited increased MMP activity despite an overall decrease in metabolic activity with PLX4032 treatment. The changes in proteolytic activity correlated with increased cell elongation and increased single-cell migration. In contrast, sorafenib did not alter MMP activity or cell motility, showing that the changes induced by PLX4032 were not a universal response to small-molecule inhibition. Therefore, we argue the importance of studying MMP activity with drug treatment and its possible implications for unwanted side effects.hydrogel | cancer | matrix metalloproteinase | cell motility | BRAF/MEK P roteolytic cleavage of the tumor microenvironment is an important mediator of cancer metastasis, which is the primary cause of mortality in cancer patients. Remodeling of the local environment involves a complex balance of cellularly secreted enzymes that promote and inhibit matrix degradation (reviewed in ref. 1). As part of this process, the matrix metalloproteinase (MMP) family of zinc-dependent enzymes plays a key role in this remodeling by degrading extracellular matrix (ECM) proteins, which promotes tumor cell invasion and colonization of distant metastatic sites, stimulates blood vessel infiltration, and releases numerous growth factors (2-6). MMP expression is elevated in nearly all solid tumors, including breast, colon, pancreas, and melanoma, and increased expression is often correlated with decreased survival (7). Additionally, targeted overexpression of MMP-3, -7, or -14 in vivo results in increased mammary carcinogenesis (8), and several knockout mouse models have decreased tumor incidence, angiogenesis, and metastasis (9).Although clearly important during cancer progression, MMP activity is not routinely evaluated during in vitro preclinical screening for potential cancer therapeutics. This is due partly to the many layers of regulat...

Metastatic melanoma is highly drug resistant, though the exact mechanisms of this resistance are not completely understood. One method to study melanoma drug responsiveness in vitro is through the use of multicellular spheroids, which have been found to exhibit decreased drug sensitivity compared to traditional 2D culture on various substrates. Because it is unclear whether dimensionality, cell-matrix interactions, and/or cell-cell contacts may influence melanoma drug responsiveness, we utilized a synthetic PEG-based hydrogel to compare the responses of cells cultured on top of or encapsulated within matrices with the same adhesive ligand density, polymer density, and material properties. We found that depending on the stage of progression with which the melanoma cells were derived, the cells responded differently to PLX4032 treatment, a commercially available melanoma drug. Specifically, early stage WM35 cells were insensitive to dimensionality (i.e., 2D versus 3D culture), while metastatic A375 cells exhibited decreased responsiveness in 3D compared to 2D. To further understand the role of the microenvironment on early stage melanoma cells, we tested single WM35 cells and multicellular WM35 spheroids in 3D. Results revealed that the spheroids were similarly sensitive to PLX4032 treatment compared to single cell encapsulations. Collectively, this study implicates the role that 3D microenvironments (i.e., dimensionality) may play in observed melanoma drug responsiveness, and the potential lack of influence of cell-matrix interactions over cell-cell contacts in early stages of melanoma resistance to PLX4032-induced apoptosis.