Identification of invadopodia by TKS5 staining in human cancer lines and patient tumor samples.

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. However, the use of TKS5 as a marker of invadopodia has not been generalized, in part due to the availability of suitable antibodies against the human protein. We have evaluated two commercial antibodies raised against human TKS5. Here, we detail protocols for the detection of invadopodia-associated TKS5 in human cells in culture and in paraffin-embedded archived tumor surgical specimens using commercial antibodies. These methods should facilitate the identification and study of human invadopodia. •TKS5 staining identifies invadopodia in human cancer cell lines and archived surgical tumor specimens.

TKS5-positive invadopodia-like structures in human tumor surgical specimens.

Invadopodia, cancer cell protrusions with proteolytic activity, are functionally associated with active remodeling of the extracellular matrix. Here, we show that the invadopodia-related protein TKS5 is expressed in human pancreatic adenocarcinoma lines, and demonstrate that pancreatic cancer cells depend on TKS5 for invadopodia formation and function. Immunofluorescence staining of human pancreatic cancer cells reveals that TKS5 is a marker of mature and immature invadopodia. We also analyze the co-staining patterns of TKS5 and the commonly used invadopodia marker Cortactin, and find only partial co-localization of these two proteins at invadopodia, with a large fraction of TKS5-positive invadopodia lacking detectable levels of Cortactin. Whereas compelling evidence exist on the role of invadopodia as mediators of invasive migration in cultured cells and in animal models of cancer, these structures have never been detected inside human tumors. Here, using antibodies against TKS5 and Cortactin, we describe for the first time structures strongly resembling invadopodia in various paraffin-embedded human tumor surgical specimens from pancreas and other organs. Our results strongly suggest that invadopodia are present inside human tumors, and warrants further investigation on their regulation and occurrence in surgical specimens, and on the value of TKS5 antibodies as pathological research and diagnostic tools.

Experimental supporting data on TKS5 and Cortactin expression and localization in human pancreatic cancer cells and tumors.

In this article, using human pancreatic cancer cell lines and tumor specimens, we analyze the expression and localization of the invadopodia-related proteins TKS5 and Cortactin. Specifically, we present data on: a) TKS5 expression and localization by immunofluorescence in human pancreatic tumors, b) Cortactin expression by western blotting in various human pancreatic adenocarcinoma cell lines, c) TKS5 and Cortactin localization at invadopodia in BxPC-3 pancreatic adenocarcinoma cells, and d) TKS5 and Cortactin localization by co-immunofluorescence in human pancreatic cancer specimens. Data presented here is related to and supportive of the research article by Chen et al., "TKS5-positive invadopodia-like structures in human tumor surgical specimens" (Chen et al., 2019), where interpretation of the research data presented here is available.