Abstract
The Eph family of receptor tyrosine kinases is crucial for assembly and maintenance of healthy tissues. Dysfunction in Eph signaling is causally associated with cancer progression. In breast cancer cells, dysregulated Eph signaling has been linked to alterations in receptor clustering abilities. Here, we implemented a single-cell assay and a scoring scheme to systematically probe the spatial organization of activated EphA receptors in multiple carcinoma cells. We show that cancer cells retain EphA clustering phenotype over several generations, and the degree of clustering reported for migration potential both at population and single-cell levels. Finally, using patient-derived cancer lines, we probed the evolution of EphA signalling in cell populations that underwent metastatic transformation and acquisition of drug resistance. Taken together, our scalable approach provides a reliable scoring scheme for EphA clustering that is consistent over multiple carcinomas and can assay heterogeneity of cancer cell populations in a cost- and time-effective manner.
Highlights
The Eph family of receptor tyrosine kinases is crucial for assembly and maintenance of healthy tissues
We developed a simple phenotypic assay based on a general scoring scheme to quantify EphA clustering induced by ephrinA1 ligation that we standardize amongst a variety of human cancer cell lines of different origins
We show that EphA cluster morphologies, which are associated with distinct migratory state of cells, constitute a single-cell-inherited trait of cell populations
Summary
The Eph family of receptor tyrosine kinases is crucial for assembly and maintenance of healthy tissues. Recent studies on breast cancer cell lines reported that cytoskeleton remodeling and EphA2 signaling interact in a feedback loop[12] to control the clustering of EphA2, which binds to ephrinA1 ligands[13,14,15,16] Alterations in this feedback loop result in changes in receptor aggregate morphologies, which, in turn, lead to phenotypic changes in cell morphologies and behaviors[17,18,19]. We hypothesized that developing a generic scoring scheme for individual cells could provide a robust phenotypic assay to ascertain the heterogeneity of cellular functional states within a cancer cell population, which could be compared across cancers of different origin It could probe intra-tumor functional heterogeneity of EphA signaling in a cost- and time-effective manner. We validated that our assay predicts alterations in EphA activation pathway in patient-derived cells (PDCs) from primary, metastatic, and drugresistant tumor cell populations
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