Abstract
BackgroundCancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells. We previously reported that cell-cell adhesion proteins, such as E-cadherin, and desmosomal proteins are involved in cell aggregation to form clusters independently of cell migration or matrix adhesion. Here, we investigated the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells.MethodsWe used live cell image acquisition and analysis to monitor the kinetics of MCF7 cell clustering in the presence/absence of GJIC pharmacological inhibitors and to screen a LOPAC® bioactive compound library. We also used a calcein transfer assay and flow cytometry to evaluate GJIC involvement in cancer cell clustering.ResultsWe first demonstrated that functional GJIC are established in the early phase of cancer cell aggregation. We then showed that pharmacological inhibition of GJIC using tonabersat and meclofenamate delayed MCF7 cell clustering and reduced calcein transfer. We also found that brefeldin A, an inhibitor of vesicular trafficking, which we identified by screening a small compound library, and latrunculin A, an actin cytoskeleton-disrupting agent, both impaired MCF7 cell clustering and calcein transfer.ConclusionsOur results demonstrate that GJIC are involved from the earliest stages of anchorage-independent cancer cell aggregation. They also give insights into the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.
Highlights
Cancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells
Functional gap junctions are established during clustering of MCF7 cancer cells As already published [10] and illustrated in Fig. 1a, when seeded in anchorage-free conditions that prevent cell adhesion to the substrate, breast adenocarcinoma MCF7 cells progressively clustered to form a solid shaped aggregate within 5 h
After mixing calcein-positive and –negative MCF7 cells at the time 0 of the clustering assay, we monitored by flow cytometry analysis calcein fluorescence level in the population over time (Fig. 1b)
Summary
Cancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells. Cancer cell dissemination and invasion have been prominently associated with the activation of the epithelial-to-mesenchymal transition (EMT) program This program induces cancer cell morphological and motility changes and leads to the inhibition of the expression of cell-cell adhesion molecules [1]. It was shown that increased expression of E-cadherin, a major component of cell adherens junctions, enhances the formation of cell aggregates [7]. This is consistent with the finding that loss of functional E-cadherin is associated with cancer cell invasiveness and metastasis formation [1]. On the other hand, increased cancer cell adhesion properties have been associated with higher experimental metastatic potential in vivo [8, 9]
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