Size-based sorting of cancer cells reveals functional heterogeneity among subpopulations

Cancer cells display marked heterogeneity in size and morphology, traits long recognized in pathology as indicators of pleomorphism and poor prognosis. Yet, the functional significance and phenotypic consequences of these morphological variations within a single cancer cell population remain poorly understood. Here, we employed a deterministic lateral displacement (DLD) microfluidic device with a circular micro-pillar array to fractionate MDA-MB-231 breast cancer cells into distinct small and large subpopulations based on size. Following sorting, the isolated fractions were expanded in culture and subjected to assays to measure proliferation, migration and invasion. We found that these subpopulations maintained their characteristic sizes over several days and exhibited distinct functional behaviors that differentially contributed to the overall invasive phenotype of MDA-MB-231 cells. Notably, while the two subpopulations proliferated at the same rate, the small-cell fraction displayed enhanced migratory capacity and invasion both in 2D and 3D assays, thus representing the primary drivers of the invasive phenotype of MDA-MB-231 cells. Together, our findings demonstrate that label-free, size-based sorting reveals biologically meaningful functional heterogeneity within morphologically diverse cancer cell populations.