Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity

SIMPLE SUMMARY: Predicting metastatic bone tropism in breast cancer by simply analyzing cell morphology would be highly desirable, in order to identify breast cancer patients at high risk for bone metastasis or to develop innovative bone-targeting drugs. In the current in vitro study, we show that MDA-MB-231 breast cancer cells are morphologically heterogeneous and exhibit a wide range of stable morphologies on a single cell level. Using the highly osteotropic derivative cell lines of MDA-MB-231 (MDA-MET, MDA-BONE), we demonstrate that single cell morphology reflects the molecular, migratory and biophysical nature of a given breast cancer cell and is specifically altered upon adoption of a bone-tropic phenotype. Thus, we propose that cell morphology could be an informative readout for understanding breast cancer heterogeneity and for predicting bone-metastases in breast cancer patients. ABSTRACT: Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes.