Question

Describe an experimental approach to identify invadopodia in a migratory tumor cell.

Answer 1

Question -Describe an experimental approach to identify invadopodia in a migratory tumor cell.

Invadopodia, cancer cell protrusive structures with associated proteolytic activity, provide cancer cells with the ability to remodel the extracellular matrix. Invadopodia facilitate invasive migration and their formation correlates with cancer cell invasiveness and metastatic potential. The unambiguous identification of invadopodia is an important step to undergo studies on invadopodia regulatory inputs, functional outputs, as well as the prevalence and significance of invadopodia for cancer cells and human tumors. The adaptor protein TKS5 is a known invadopodia regulatory protein, which is necessary for invadopodia formation and activity. TKS5 is highly enriched at invadopodia and, unlike other commonly used invadopodia markers, it does not accumulate significantly in other types of cellular protrusions.

1.TKS5 immunofluorescence staining in cultured cancer cells

Here, we describe a protocol for the analysis of TKS5 localization at invadopodia in the human pancreatic adenocarcinoma line BxPC-3. This cell line requires the presence of an extracellular matrix component(such as gelatin)to fully induce invadopodia formation in vitro. Other cell lines may form invadopodia when grown directly on glass, or require additional matrices or growth factor supplements to readily form invadopodia. The adequate conditions to induce invadopodia formation, therefore, are cell-type dependent and need to be identified experimentally. Detection of invadopodia using TKS5 staining can be combined with the detection of F-actin using a phalloidin fluorescent conjugate, or with the detection of focal proteolysis using a gelatin fluorescent conjugate as a substrate to grow cells. Here, we described the staining of TKS5 and F-actin in BxPC-3 cells growing on unlabeled gelatin B. Preparation of fluorescent gelatin coverslips and analysis of invadopodia activity can be used in combination with TKS5 immunofluorescence.

Cancer cell lines are incubated for 24 h on gelatin-coated coverslips, and stained with anti-TKS5 monoclonal antibody to identify invadopodia . Samples are co-stained with phalloidin conjugated with Alexa 568 to reveal F-actin. Slides were mounted in medium containing DAPI to visualize nuclei. Cancer cells stained with the indicated markers.

When using a cancer cell line for the first time to identify invadopodia formation, it is advisable to stain TKS5 along with F-actin , and also to stain TKS5 in cells growing on fluorescent gelatin to verify extracellular matrix degrading invadopodia activity

2.TKS5 immunofluorescence staining in paraffin embedded tumor sections

This method has been valuable to identify for the first time invadopodia inside formalin-fixed paraffin-embedded archived tumor samples.

Formalin fixed paraffinembedded invasive carcinomas were processed for immunofluorescence staining with an antibody against human TKS5.Images were obtained at 100 X magnification.

TKS5 staining pattern is expected to be membranous and cytoplasmic, but the intensity of the staining will be variable depending on TKS5 protein abundance in each particular tumor sample. In order to visualize invadopodia-like structures, tumor areas need to be carefully analyzed at 100X magnification. TKS5-positive puncta corresponding to invadopodia-like structures, often appear close to the nuclei and display a polarized distribution.TKS5 immunofluorescence of human surgical tumor specimens in combination with Cortactin staining is used.