Abstract Heterogeneity in phenotypes of malignantly transformed cells and aberrant glycan expression on their surface are two prominent hallmarks of cancers that have hitherto not been linked to each other. In this paper, we identify heterogeneity in a specific glycan linkage: α2,6-linked sialic acids within breast cancer cells in vivo and in culture. Upon sorting out two populations with moderate and relatively higher cell surface α2,6-linked sialic acid levels from the triple negative breast cancer cell line MDA-MB-231, both populations (denoted as medium and high-2,6-Sial cells respectively) stably retained their levels in early passages. Upon continuous culturing, medium 2,6-Sial cells recapitulated the heterogeneity of the unsorted line whereas high 2,6-Sial cells showed no such tendency. Compared with the high 2,6-Sial, the medium 2,6-Sial cells showed greater adhesion to reconstituted extracellular matrices (ECM) as well as invaded faster as single cells. The level of α2,6-linked sialic acids in the two sublines was found to be consistent with the expression of a specific glycosyl transferase, ST6GAL1 . Stably knocking down ST6GAL1 in the high 2,6-Sial cells, enhanced their invasiveness. When cultured together, medium 2,6-Sial cells differentially migrated to the edge of growing tumoroid-like cultures, whereas high 2,6-Sial cells formed the central bulk. Simulations in a Cellular Potts model-based computational environment that is calibrated to our experimental findings suggest that the heterogeneity of cell-ECM adhesion, likely regulated by α2,6-linked sialic acids facilitates niches of highly invasive cells to efficiently migrate centrifugally as the invasive front of a malignant tumor. Significance Statement Cell-surface sugars are aberrantly expressed in cancer but their contributions to tumor heterogeneity are not known. In this study, we uncover and separate breast cancer populations with distinct α2,6-linked sialic acid levels. The moderately expressing population shows stronger adhesion to extracellular matrix than the high expressing population. It also invades faster through the matrix as single cells. Combining experiments with computational modelling, we show that the heterogeneity in matrix adhesion is vital to accentuating cell invasion. In some conditions, invasion of heterogeneous populations may compare with, or exceed that of, homogeneous moderately expressing populations. Our findings are vital to furthering our understanding of how cancers spread and potentially qualify efforts to manage the disease through glycan-editing or immunotherapeutic approaches.

ST6GAL1-mediated heterogeneity in sialic acid levels potentiates invasion of breast cancer epithelia