Abstract
Abstract Purpose: Cancer cell cluster formation is a key process involved during primary tumor growth, metastatic cells dissemination and secondary tumor evolution. We previously reported that cell-cell adhesion proteins such as E-Cadherin and desmosomal proteins are involved in cell aggregation independently of cell migration or matrix adhesion (Saias et al. 2015). Here we investigate the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells. Experimental Design: Live cell imaging and image processing was used to evaluate MCF7 clustering in the presence of pharmacological inhibitors and to perform a LOPAC® small bioactive compound library screening. Flow cytometry was performed to monitor calcein transfer assay and to evaluate GJIC. Results: We first demonstrate that functional GJIC are established in the early phase of aggregation. We next show that pharmacological inhibition of GJIC using Tonabersat and Meclofenamate both reduces calcein transfer and delays clustering of MCF7 cells. We found that treatment with Latrunculin A, an actin cytoskeleton-disrupting agent, inhibits MCF7 clustering and impairs calcein transfer. In a screen for inhibitors of cell aggregation using a small-compound chemical library we identified Brefeldin A, an inhibitor of vesicular trafficking, and showed that it also impairs calcein transfer. Conclusion: Our results demonstrate that GJIC are involved at the earliest stages of the anchorage-independent clustering process. They shed new light on the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.
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