Colorectal Cancer Cell Invasion and Functional Properties Depend on Peri-Tumoral Extracellular Matrix.

We investigated how the extracellular matrix (ECM) affects LoVo colorectal cancer cells behavior during a spatiotemporal invasion. Epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and morphological phenotypes expressed by LoVo-S (doxorubicin-sensitive) and higher aggressive LoVo-R (doxorubicin-resistant) were evaluated in cells cultured for 3 and 24 h on Millipore filters covered by Matrigel, mimicking the basement membrane, or type I Collagen reproducing a desmoplastic lamina propria. EMT and invasiveness were investigated with RT-qPCR, Western blot, and scanning electron microscopy. As time went by, most gene expressions decreased, but in type I Collagen samples, a strong reduction and high increase in MMP-2 expression in LoVo-S and -R cells occurred, respectively. These data were confirmed by the development of an epithelial morphological phenotype in LoVo-S and invading phenotypes with invadopodia in LoVo-R cells as well as by protein-level analysis. We suggest that the duration of culturing and type of substrate influence the morphological phenotype and aggressiveness of both these cell types differently. In particular, the type I collagen meshwork, consisting of large fibrils confining inter fibrillar micropores, affects the two cell types differently. It attenuates drug-sensitive LoVo-S cell aggressiveness but improves a proteolytic invasion in drug-resistant LoVo-R cells as time goes by. Experimental studies on CRC cells should examine the peri-tumoral ECM components, as well as the dynamic physical conditions of TME, which affect the behavior and aggressiveness of both drug-sensitive and drug-resistant LoVo cells differently.

Substrate Type and Concentration Differently Affect Colon Cancer Cells Ultrastructural Morphology, EMT Markers, and Matrix Degrading Enzymes.

Aim of the study was to understand the behavior of colon cancer LoVo-R cells (doxorubicin-resistant) vs. LoVo-S (doxorubicin sensitive) in the initial steps of extracellular matrix (ECM) invasion. We investigated how the matrix substrates Matrigel and type I collagen-mimicking the basement membrane (BM) and the normal or desmoplastic lamina propria, respectively-could affect the expression of epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and phenotypes. Gene expression with RT-qPCR, E-cadherin protein expression using Western blot, and phenotypes using scanning electron microscopy (SEM) were analyzed. The type and different concentrations of matrix substrates differently affected colon cancer cells. In LoVo-S cells, the higher concentrated collagen, mimicking the desmoplastic lamina propria, strongly induced EMT, as also confirmed by the expression of Snail, metalloproteases (MMPs)-2, -9, -14 and heparanase (HPSE), as well as mesenchymal phenotypes. Stimulation in E-cadherin expression in LoVo-S groups suggests that these cells develop a hybrid EMT phenotype. Differently, LoVo-R cells did not increase their aggressiveness: no changes in EMT markers, matrix effectors, and phenotypes were evident. The low influence of ECM components in LoVo-R cells might be related to their intrinsic aggressiveness related to chemoresistance. These results improve understanding of the critical role of tumor microenvironment in colon cancer cell invasion, driving the development of new therapeutic approaches.

Extracellular Matrix-Mediated Breast Cancer Cells Morphological Alterations, Invasiveness, and Microvesicles/Exosomes Release.

Breast cancer is a leading disease in women. Several studies are focused to evaluate the critical role of extracellular matrix (ECM) in various biochemical and molecular aspects but also in terms of its effect on cancer cell morphology and therefore on cancer cell invasion and metastatic potential. ECM fibrillar components, such as collagen and fibronectin, affect cell behavior and properties of mammary cancer cells. The aim of this study was to investigate using the scanning electron microscopy (SEM) how the highly invasive MDA-MB-231 breast cancer cells, interplaying with ECM substrates during cell migration/invasion, modify their morphological characteristics and cytoplasmic processes in relation to their invasive potential. In particular we reproduced and analyzed how natural structural barriers to cancer cell invasion, such as the basement membrane (Matrigel) and fibrillar components of dermis (fibronectin as well as the different concentrations/array of type I collagen), could induce morphological changes in 3D cultures. Interestingly, we demonstrate that, even with different effects, all collagen concentrations/arrays lead to morphological alterations of breast cancer cells. Intriguingly, the elongated mesenchymal shaped cells were more prominent in 3D cultures with a dense and thick substrate (thick Matrigel, high concentrated collagen network, and densely packed collagen fibers), even though cells with different shape produced and released microvesicles and exosomes as well. It is therefore evident that the peri-tumoral collagen network may act not only as a barrier but also as a dynamic scaffold which stimulates the morphological changes of cancer cells, and modulates tumor development and metastatic potential in breast cancer.

Collagen Fiber Array of Peritumoral Stroma Influences Epithelial-to-Mesenchymal Transition and Invasive Potential of Mammary Cancer Cells.

: Interactions of cancer cells with matrix macromolecules of the surrounding tumor stroma are critical to mediate invasion and metastasis. In this study, we reproduced the collagen mechanical barriers in vitro (i.e., basement membrane, lamina propria under basement membrane, and deeper bundled collagen fibers with different array). These were used in 3D cell cultures to define their effects on morphology and behavior of breast cancer cells with different metastatic potential (MCF-7 and MDA-MB-231) using scanning electron microscope (SEM). We demonstrated that breast cancer cells cultured in 2D and 3D cultures on different collagen substrates show different morphologies: i) a globular/spherical shape, ii) a flattened polygonal shape, and iii) elongated/fusiform and spindle-like shapes. The distribution of different cell shapes changed with the distinct collagen fiber/fibril physical array and size. Dense collagen fibers, parallel to the culture plane, do not allow the invasion of MCF-7 and MDA-MB-231 cells, which, however, show increases of microvilli and microvesicles, respectively. These novel data highlight the regulatory role of different fibrillar collagen arrays in modifying breast cancer cell shape, inducing epithelial-to-mesenchymal transition, changing matrix composition and modulating the production of extracellular vesicles. Further investigation utilizing this in vitro model will help to demonstrate the biological roles of matrix macromolecules in cancer cell invasion in vivo.