Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity

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.

Two high-yield complementary methods to sort cell populations by their 2D or 3D migration speed

“…To further profile heterogeneity distribution of these populations at the single-cell level before and after sorting, we employed a phenotypic assay to quantify ephrin A (EphA) receptor activation at the single-cell level. The EphA score, which is a measure of such clustering, has been reported to correlate well with migration potential and to be relevant to describe heterogeneity in cells by providing EphA score distribution for a population ( Ravasio et al , 2019 ). Supplemental Figure 2B shows that cells collected from the base layer had EphA scores that were significantly lower ( p < 0.001) than those obtained from the migratory cells after 3 d of cell migration (HN120M-S3d-E2d-top).…”

“…Ephrin clustering assay was performed as reported previously ( Ravasio et al , 2019 ). Briefly, nine chambers (3 × 3 mm) were formed by placing a silicon gasket on a clean, hydrophilic glass coverslip.…”

Geometric Control of Cell Behavior by Biomolecule Nanodistribution

“…We used the HaloTag system to immobilize our selected protein, EphA2, onto our patterned surfaces with optimized EBL technology. As a proof of concept biomolecule, we chose EphA2 because it regulates morphological processes, including tissue patterning and spatial-temporal cell positioning during development. − Additionally, increasing evidence suggests that the Eph receptor/ephrin protein family is susceptible to a nanodistribution of molecules within the cell membrane, making this molecule an ideal candidate for our studies. , EphA2 has more than one type of ephrin A ligand; therefore, its multiple binding partners can be analyzed by surface immobilization of EphA2. − Importantly, our preliminary quantitative PCR analysis showed that, out of 22 known Eph receptor/ephrin family members, our model cell type, hPSCs, expressed several Eph receptor genes, including EPHA2 , as well as ephrin ligands, confirming the presence of this signaling pathway in hPSCs (Figure S1A). Further experiments verified the presence of the EphA2 receptor and its five ligands (i.e., ephrin A1, A2, A3, A4, and A5, Figure S1B,C) by immunofluorescence and Western blot, with ephrin A4 having the lowest expression.…”

Cancer: More Than a Geneticist’s Pandora’s Box

“…Similarly, two distinct cell populations within a melanoma tumor was observed characterized by variable expression levels of MITF (Rebecca and Herlyn, 2020;Tirosh et al, 2016). Further, subpopulations with varying differential EphA cluster morphologies and intrinsic migration potential were observed in breast cancer cells using single-cell assays (Ravasio et al, 2019). Identification of such heterogeneity has revealed that multiple cancer subtypes may co-exist within an individual tumor (Yeo and Guan, 2017).…”