Drivers of cancer cell invasion and metastasis

Abstract

Metastatic spread of cancer cells from their primary site requires invasion into the vasculature, extravasation at the distant organ site and colonization of the distant organ. Here we studied mechanisms of attachment and invasion into endothelial monolayers by cancer cells to identify driver molecules and signaling pathways that are crucial for the invasive phenotype.

We found that human cancer cell invasion of an endothelial monolayer is altered by the density at which the cancer cells are grown in tissue culture. Gene expression analysis showed that immune response cytokines such as IL6, IL8, CXCL1, 2 & 3 are upregulated and serve as drivers of transendothelial invasion of subconfluent MDA-MB-231 cells. Blocking the receptors of these cytokines inhibited cancer cell migration and endothelial invasion in vitro. Conditioned media (CM) from invasive MDA-MB-231 cells restored the invasive phenotype to non-invasive MDA-MB-231 cell subpopulations, and CM from non-invasive cells inhibited the invasive phenotype suggesting that the invasive phenotype is dependent on factors released from the cells.

MDA-MB-231 cell subpopulations with different invasive phenotypes was also studied in live zebrafish embryos. These in vivo studies corroborated the distinctly invasive phenotype of the cancer cell subpopulations in vitro. CXCR2 blocking antibodies inhibited MDA-MB-231 invasion and changed their homing pattern in the zebrafish model. Additionally, the non-invasive MDA-MB-231 showed reduced lung colonization when injected in the tail vein of nude mice. We conclude that autocrine and paracrine acting cytokine networks contribute to cancer cell invasion.

In an independent functional genomics approach to dissect driver pathways of malignant progression different human and mouse cancer cell lines were transduced with lentiviral-based genome or kinome-targeted shRNA libraries. Cancer cell subpopulations were then selected for their loss-of-function in an endothelial invasion assay in vitro. After several rounds of selection, the respective shRNA targeted genes were identified from the selected cancer cell populations. Two to four distinct shRNAs targeted each identified gene.

We conclude that cancer cell invasion of endothelial monolayers can model one of the hallmarks of invasive cancer and can be used to identify driver pathways of malignant progression that may translate into targets for therapy.