During tumor development and ongoing metastasis the acquisition of mesenchymal cell traits by epithelial carcinoma cells is achieved through a programmed phenotypic shift called the epithelial-to-mesenchymal transition, EMT. EMT contributes to increased cancer cell motility and invasiveness mainly through invadosomes, the adhesion structures that accompany the mesenchymal migration. The invadosomes and their associated proteases restrict protease activity to areas of the cell in direct contact with the ECM, thus precisely controlling cell invasion. Our data prove that Snail-overexpressing HT-29 cells that imitate the phenotype of colon cancer cells in the early stage of the EMT showed an increase in the expression and pericellular activity of cathepsin B. It appears that the pericellular localization of cathepsin B, also observed in colon and rectum adenocarcinoma tissue samples, plays a key role in its function.

Tumor metastasis, the major problem for clinical oncology in colon cancer treatment, is linked with an epithelial-mesenchymal transition (EMT). The observed cellular transformation in this process is manifested by cell elongation, enhanced cell migration and invasion ability, coordinated by cytoskeleton reorganization. In the present study, we examined the role of tubulin-β4 (TUBB4B) downregulation that occurs during EMT in colon cancer cells, in the modulation of the function of microtubules. Based on biochemical and behavioral analysis (transmigration) we posit that the decrease of the TUBB4B level is critical for microtubule-vimentin interaction and contributes to the maintenance of polarity in migrating cells. The microscopic studies revealed that TUBB4B decrease is accompanied by cell elongation and increased number of matured focal adhesion sites, which is a characteristic of the cell metastatic stage. We also demonstrated faster polymerization of microtubules in cells with a lower level of TUBB4B. Simultaneous TUBB3 upregulation, reported during EMT, acts additively in this process. Our studies suggest that the protein level of TUBB4B could be used as a marker for detection of the preinvasive stages of the colon cancer cells. We also concluded that chemotherapy enriched to increase TUBB4B level and/or to stabilize microtubule polymerization might more effectively prevent metastasis in colon cancer development.

The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma.

During metastasis, cancer cells undergo phenotype changes in the epithelial-mesenchymal transition (EMT) process. Extracellular vesicles (EVs) released by cancer cells are the mediators of intercellular communication and play a role in metastatic process. Knowledge of factors that influence the modifications of the pre-metastatic niche for the migrating carcinoma cells is important for prevention of metastasis. We focus here on how cancer progression is affected by EVs released from either epithelial-like HT29-cells or from cells that are in early EMT stage triggered by Snail transcription factor (HT29-Snail). We found that EVs released from HT29-Snail, as compared to HT29-pcDNA cells, have a different microRNA profile. We observed the presence of interstitial pneumonias in the lungs of mice injected with HT29-Snail cells and the percent of mice with lung inflammation was higher after injection of HT29-Snail-EVs. Incorporation of EVs released from HT29-pcDNA, but not released from HT29-Snail, leads to the increased secretion of IL-8 from macrophages. We conclude that Snail modifications of CRC cells towards more invasive phenotype also alter the microRNA cargo of released EVs. The content of cell-released EVs may serve as a biomarker that denotes the stage of CRC and EVs-specific microRNAs may be a target to prevent cancer progression.

Antiphospholipid syndrome (APS) is an immunothrombotic disorder, frequently attributed to autoantibodies that bind β2-glycoprotein I (β2GPI). A study showed that the platelet-specific chemokine, platelet factor 4 (PF4), binds to β2GPI, enhancing recognition of β2GPI by APS antibodies. APS antibodies induce the release of neutrophil extracellular traps (NETs), webs of decondensed chromatin that bind both PF4 and β2GPI. We propose that PF4 bridges β2GPI to NETs (and other PF4-targeted polyanions), leading to the formation of prothrombotic PF4:β2GPI:NET immunotargets in APS. Dynamic light-scattering studies of isolated immunoglobulin G from 4 patients with triple-positive APS show formation of PF4:β2GPI:NET complexes that bind APS antibodies. NETs released in a microfluidic system bound β2GPI, but only in the presence of PF4, forming a multimolecular APS antigenic target. Whole blood infused through a photochemically injured, endothelium-lined microfluidic channel formed platelet-, fibrin-, and complement-rich thrombi that bound APS antibodies only in the presence of PF4. Thrombi were reduced in size if either ADAMTS13 or DNase I was infused. In a murine APS model, wild-type and transgenic mice expressing human PF4 (hPF4) ± FcγRIIA developed more intense neutrophil rolling along veins and more extensive thrombus formation after laser injury to cremaster arterioles and venules, whereas mice lacking PF4 did not. Three antigenically distinct anti-hPF4 monoclonal antibodies blocked thrombosis in vitro and neutrophil rolling and thrombosis in vivo. Our studies provide new insights into the basis of APS that has mechanistic parallels to other known PF4 immunothrombotic disorders and offer potential diagnostic and nonanticoagulant therapeutic strategies for clinical management.