Invadopodia Sample Clauses

Invadopodia. A special protrusion in mediating metastasis 26

Invadopodia. A special protrusion in mediating metastasis Extracellular matrix degradation and invasion is a crucial part of the metastatic cascade (Xxxxxxxxx and Xxxxxxxx, 2011). Xxxxx is a well characterised transcriptional factor promoting metastasis. Twist has been shown to upregulate expression of platelet derived growth factor receptor α (PDGFRα) which in turn stimulates Src activity ultimately resulting in the formation of actin rich membrane protrusions which can target the secretion of MMPs and degrade the extracellular matrix in human mammary epithelial HMLE cells(Xxxxxx et al., 2011), (figure 1.3). These structures are known as invadopodia and have been implicated in the local invasion, intravasation and metastasis of breast cancer cells to the lung (Xxxxxx et al., 2011; Xxxxxxxxxxxx et al., 2012). In fact, recent advances in intravital microscopy have also allowed the visualisation of extravasating MDA-MB-231LN cells extending invadopodia into the extravascular stroma of the chorioallantoic membrane (CAM) of the chicken embryo (Xxxxx et al., 2014). Invadopodia are complex structures incorporating adhesion proteins, actin binding proteins, metalloproteases with Rho GTPases and their effectors contributing to their dynamic formation and activity (Xxxxxx and Xxxxxxxxxxx, 2011). Extracellular signals stimulate the formation of invadopodia via the activation of plasma membrane receptors initiating signalling cascades (Xxxxxxx et al., 2013). In breast cancer cells activation of EGFR, Met and TGFβ receptors has been shown to induce formation of invadopodia (Xxxxxx et al., 2008; Xxxxxxxxx et al., 2012; Xxxxxxxxx et al., 2005). Similar results have been reported for head and neck squamous carcinoma cells when VEGF becomes activated (Xxxxx et al., 2010). Activation of these signalling cascades meet at stimulating a set of kinases, particularly Src but also ERK and PAKs that induce the formation of invadopodia (Xxxxxx et al., 2016). Src phosphorylates various substrates including cortactin and Tks5 which constitute hallmark elements of invadopodia formation (Xxxxxx and Courtneidge, 2011), (figure 1.3). Tks5 is an adaptor protein shown to be crucial for invadopodia formation by specifically localising early and marking invadopodia sites as well as acting as a scaffold for other invadopodia elements such as cortactin (Xxxxxxxx et al., 2009; Xxxxxx and Xxxxxxxxxxx, 2011; Xxxxx et al., 2005). PAK1 mediated phosphorylation of cortactin Ser113 has been shown to be important...

Invadopodia. Formation of invadopodia is considered indicative of invasive potential in vitro and in vivo (Xxxxxx et al., 2011; Xxxxx et al., 2014). We sought to examine the invasive potential of our cell lines knowing that MB-231 cells are widely used in invadopodia research (Xxxxx et al., 2011) . In order to detect invadopodia formation in vitro, cells were seeded on glass coverslips previously coated with TRITC-conjugated gelatin. To allow invadopodia formation to occur cells were incubated at 37°C for 3h and were subsequently fixed with PFA and stained with Phalloidin 488 resulting in green stained cells over a red stained matrix (Hashim et al., 2013). Cells were considered able to make invadopodia when the actin puncta revealed by the phalloidin stain indicating the protrusive structure of invadopodia coincided with black holes in the red gelatin indicating matrix degradation (Xxxxxx et al., 2013). Out of the 4 cell lines that were subjected to the assay the HCC38 and MB-436 cells did not make any invadopodia (figure 3.7). Interestingly, reviewing the literature revealed that there is no published evidence of HCC38 or MB-436 cells being used in invadopodia assays. Together, these observations suggest that the HCC38 and MB-436 cells do not make invadopodia. As expected, the MB-231 cells exhibited definitive evidence of invadopodia formation and activity (figure 3.7A, 3.7B) which is consistent with their widespread use in invadopodia research. The BT-549 cells showed very little evidence of invadopodia presence at the 3h incubation as only a few very small invadopodia were detected. This observation suggested that the BT-549 cells might require longer incubations on the gelatin matrix to form active invadopodia. Further characterisation of the invadopodia capacity of the BT-549 cells was thus warranted. A Invadopodia formation at 3 hours 30 % of cells making invadopodia B 20 10 0 MB-231 BT-549 HCC38 MB-436 Cell lines Figure 3.7 Invadopodia assay at 3h. (A) Cells were seeded on TRITC conjugated gelatin coated coverslips and incubated for 3h at 37°C. Coverslips were then fixed and stained with Phalloidin 488 (green) and cells were imaged. Scale bar corresponds to 10μm (B) Percentage of cells making invadopodia was determined for each individual experiment (N=3). Bars represent the mean percentage of cells making invadopodia and error bars represent the SEM.