Invadopodia enable cooperative invasion and metastasis of breast cancer cells

Abstract Multiple clones of cancer cells can seed metastases via collective invasion and dissemination. While it is known that cancer clones can cooperate during invasion, the events leading to it and the effects it may have on metastasis are not known. In this study, we demonstrate that, when mixed in 3D spheroids, the isogenic 4T1 and 67NR breast cancer cells sort from each other, followed by their cooperative invasion. By time-lapse microscopy of heterogenous spheroids embedded in collagen I, we show that the invasive 4T1 cells that reach the spheroid edge remain there, while the non-invasive 67NR cells move randomly. This results in cell sorting and enrichment of invasive 4T1 cells at the spheroid periphery. Following cell sorting, 4T1 cells lead the 67NR cells in cooperative invasion. Elimination of invadopodia in 4T1 cells, by knockdown of the protein Tks5, blocks invasion and demonstrates that invasion requires invadopodia only in leader cells and not in follower cells. Importantly, using syngeneic mouse model, we demonstrate that cells with and without invadopodia can also engage in cooperative metastasis. Altogether, our results suggest that a few clones with invadopodia could drive the metastasis of cell clusters from heterogeneous tumors.