Abstract
BackgroundCancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Several widely used chemotherapeutic agents that target the cytoskeleton microtubules and cause cell cycle arrest at mitosis have been reported to modulate CTC number or the size of CTC clusters.ResultsIn this study, we investigated in vitro the impact of mitotic arrest on the ability of breast tumor cells to form clusters. By using live imaging and quantitative image analysis, we found that MCF-7 cancer cell aggregation is compromised upon incubation with paclitaxel or vinorelbine, two chemotherapeutic drugs that target microtubules. In line with these results, we observed that MCF-7 breast cancer cells experimentally synchronized and blocked in metaphase aggregated poorly and formed loose clusters. To monitor clustering at the single-cell scale, we next developed and validated an in vitro assay based on live video-microscopy and custom-designed micro-devices. The study of cluster formation from MCF-7 cells that express the fluorescent marker LifeAct-mCherry using this new assay allowed showing that substrate anchorage-independent clustering of MCF-7 cells was associated with the formation of actin-dependent highly dynamic cell protrusions. Metaphase-synchronized and blocked cells did not display such protrusions, and formed very loose clusters that failed to compact.ConclusionsAltogether, our results suggest that mitotic arrest induced by microtubule-targeting anticancer drugs prevents cancer cell clustering and therefore, could reduce the metastatic potential of circulating tumor cells.
Highlights
Cancer cell aggregation is a key process involved in the formation of tumor cell clusters
To assess their effect on MCF-7 cell ability to compact and form clusters, cells incubated with paclitaxel or vinorelbine for 24 h were subjected to a previously described aggregation assay in which the progressive aggregation and compaction of 500 cells seeded in non-adherent 96-well plates are monitored by video-microscopy for 5 h [15, 16] (Fig. 1a)
We found that clustering of MCF-7 cells incubated with paclitaxel or vinorelbine was altered, and that at 5 h, compaction was reduced by about 25% compared with untreated cells (Fig. 1c, d)
Summary
Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Circulating tumor cells (CTCs) are detected in patients’ blood samples, and CTC clusters have been associated with higher metastatic potential [3, 4]. Formation of tumor cell clusters prevents anoikis in the absence of anchorage. It has been shown that breast cancer metastases arise from keratin 14-expressing tumor cell clusters [1]. We recently reported, using time-lapse microscopy-based clustering that E-cadherins and desmoglein and desmocolin, two desmosomal proteins, are involved in cancer cell aggregation [15]. We demonstrated the involvement of gap junction intercellular communication in regulating the earliest step of tumor cell clustering [16]
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