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

Abstract

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.

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

“…In particular, we investigated the behavior of LoVo-S and LoVo-R cells growing in ECM substrates covering Millipore filters, which did not mechanically allow them to invade after 24 h. We found that when low aggressive LoVo-S cells were hindered to cross the Millipore filter and only tried to invade a concentrated type I Collagen substrate (3.5 mg/mL) they developed a morphological and biomolecular EMT phenotype. Surprisingly, the more aggressive doxorubicin-resistant LoVo-R cells appeared independent on TME, because they did not change their morphological phenotype, EMT markers, and HPSE expression in all the different tested ECM substrates [26].…”

“…Data obtained after 24 h from these free-migrating cells were compared with the results reported in a previous study, where LoVo cells were cultivated for 24 h on the same ECM substrates covering a Millipore filter whose pore size stopped cell migration [26]. After 24 h, the freedom of movement attenuated LoVo-S cells' aggressiveness by reducing all the MMPs in both LoVo-S and -R cells cultivated in any substrate.…”

“…In contrast, the more aggressive and resistant to doxorubicin LoVo-R CRC cells did not change their morphological phenotype or epithelial to mesenchymal transition (EMT) markers' expression in different concentrations of different ECM substrates. [26]. With the aim to better understand the dynamic behavior of LoVo-S and LoVo-R CRC cell invasion of the ECM, in this study we investigated the behavior and phenotype of these cancer cells when they were free to move and invade Matrigel, mimicking the BM or type I collagen meshwork, reproducing the collagen array of the bowel desmoplastic lamina propria.…”

Characteristics of the Colorectal Cancer Microenvironment—Role in Cancer Progression and Therapeutic Possibilities

“…Their role includes maintaining hypoxia in the TME and cross-linking collagen fibers, causing ECM stiffness and, consequently, its degradation. The consequence is the proliferation of cancer cells, their release from the niche, and migration [242,243]. The ECM reorganization induced by reduced elasticity of fibers is mainly a consequence of reprogrammed niche cells depositing larger amounts of collagen, primarily type I as well as II, III, IV, and IX.…”

Cellular and Molecular Mechanisms of the Tumor Stroma in Colorectal Cancer: Insights into Disease Progression and Therapeutic Targets

“…Furthermore, MMPs, including MMP9 and MMP14, may confer resistance to various cancer therapies by altering tumor vasculature to impact drug delivery, promoting survival pathways in cancer cells, or driving the acquisition of more aggressive phenotypes through EMT [ 173 , 174 , 175 ]. The specific MMPs implicated in therapy resistance may depend on the type of treatment administered.…”