The collagen gel samples were prepared as described in previous section and then were mounted on AFM sample plate and a liquid cell with PBS was used. as potential BC metastasis marker. rich protein (PINCH-1) [7], which in turn binds to Integrin-Linked Kinase (ILK), and alpha-parvin (PARVA) forming a stable ternary protein complex that promotes cell survival [8C10]. Although, RSU-1 was originally identified as suppressor of Ras-dependent oncogenic transformation [11], little is known regarding its expression and role in cancer. From the studies currently published on RSU-1 and cancer, there is consensus on the fact SR 3576 that RSU-1 has anti-tumorigenic effects suppressing cancer cell growth [11C14]. Regarding its expression in various precancerous or cancer tissues though, results are limited and sometimes contradictory. A study in familial adenomatous polyposis involving a small number of samples showed a reduction in RSU-1 protein expression in polyposis samples compared to normal colonic mucosa [15] while another study showed RSU-1 mRNA expression to be dramatically up-regulated in metastatic colon cancer samples compared to healthy controls as well as compared to primary colon cancer samples [16]. Furthermore, a somatic copy number variation (CNV) analysis in hepatocellular carcinoma samples showed that the gene exhibited a high frequency of CNVs with 7 deletions and 3 amplifications [17] indicating that is frequently deleted in human liver cancer. Moreover, it was recently shown that RSU-1 expression is significantly elevated both at the mRNA and protein level in BC samples compared to respective adjacent normal tissue with the increase being more obvious in metastatic samples compared to non-metastatic [18]. Consistent with this finding, RSU-1 was demonstrated to be significantly upregulated in the aggressive MDA-MB-231 breast cancer cells compared to less aggressive MCF-7 cells [18], as well as in the aggressive HepG2 hepatocellular carcinoma cells compared to the less invasive PLC/PRF/5 (Alexander) hepatoma cells [19]. Interestingly, an alternatively-spliced variant of was identified in 30% of high grade gliomas and 2/3 of oligodendrogliomas but not in other brain, bladder, colon tumors of normal tissue [20] while rare RSU-1 deletion were also identified in three cancer types from the Cancer Genome Atlas [21]. Hence, RSU-1 seems to have the potential of being both promising and clinically relevant novel marker and therapeutic target of cancer cell metastasis. Apart from the involvement of cell-ECM adhesion proteins, it has also been shown that mechanical cues can promote cancer metastasis [22, 23]. In fact, cancer tissues often contain a larger amount of ECM proteins than normal tissues and thus, are typically stiffer, expressed with a larger value of Young’s modulus. Tumor stiffening is the only mechanical aspect that patients and clinicians can feel as in many cases Rabbit polyclonal to LPA receptor 1 tumors become stiffer compared to the surrounding tissue. Because of their increased ECM stiffness, cancer tissues restrict more the movement of cancer cells, exerting larger mechanical compressive forces on them. Thus, mechanical compression can, not only reduce cancer cell proliferation and induce apoptosis but it can also increase the invasive and metastatic potential of cancer cells [6, 22C30]. In the current study, we set out to investigate the role of cell-ECM adhesion proteins in relation to matrix stiffness with regard to cell invasion. Traditional two-dimensional (2D) monolayer cultures could not be used, as they cannot take into account the ECM stiffness of the tumor microenvironment [31]. Thus, in order to better approximate the real tumor setting [38]. As shown in Figure ?Figure2,2, MCF-7 (Figure 2A-2D), MDA-MB-231 (Figure 2E-2H) and MDA-MB-231-LM2 cells SR 3576 (Figure 2I-2L) were indeed embedded in the gels growing at different levels in all three dimensions within the 3D collagen matrix. The different levels of focus, seen in the pictures, involving cells grown in the gels confirm our observations (Figure 2B-2D, Figure 2F-2H, Figure 2J-2L). Open in a separate window Figure 2 BC cells grown in 3D collagen gels in conditions of increasing matrix stiffness(A-D) Morphology of MCF-7 cells grown in 2D culture (A), or embedded in Collagen gels of 0.5mg/ml (B), 1.0 mg/ml (C) and 3.0 mg/ml (D). (E-H) Morphology of MDA-MB-231 cells grown in 2D culture (E), or embedded in SR 3576 Collagen gels of 0.5 mg/ml (F), 1.0 mg/ml (G) and 3.0.