EphB2 and ERK signaling are required for heterotypic contact inhibition of locomotion to drive cell sorting

Abstract

SummaryInteractions between different cell-types can induce distinct contact inhibition of 1 locomotion (CIL) responses that are hypothesized to control population-wide 2 behaviors during embryogenesis [1, 2]. However, our understanding of the signals 3 that lead to cell-type specific repulsion, and the precise capacity of heterotypic CIL 4 responses to drive emergent behaviors is lacking. Using a new in vitro model of 5 heterotypic CIL between epithelial and mesenchymal cells, we show that 6 fibrosarcoma cells (HT1080), but not fibroblasts (NIH3T3), are actively repelled by 7 epithelial cells in culture. We show that knocking down EphB2 in fibrosarcoma cells 8 specifically leads to disruption of the repulsion phase of CIL in response to 9 interactions with epithelial cells. Furthermore, this heterotypic interaction requires 10 ERK activation, downstream of EphB2 signaling. We also examine the population-11 wide effects when these various cell combinations, and their specific heterotypic CIL 12 responses, are allowed to interact in culture. Mixtures of fibrosarcoma and epithelial 13 cells -unlike fibroblasts and epithelial cells -lead to complete sorting and 14 segregation of the two populations, and inhibiting their distinct CIL response by 15 knocking down EphB2 or ERK in fibrosarcoma cells disrupts this emergent sorting 16 behavior. Our understanding of the mechanisms underlying developmental 17All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/373696 doi: bioRxiv preprint first posted online Jul. 20, 2018; 2 behaviors such as cell sorting is lacking as predominant sorting hypotheses, such as 18 differential adhesion, have recently been found inadequate in predicting the sorting 19 of mesenchymal cells [3, 4]. These data suggest that heterotypic CIL responses, in 20 conjunction with processes such as differential adhesion, may aid the sorting of cell 21 populations during embryogenesis. 22 23 Results and Discussion 24 25 Fibroblasts and fibrosarcoma cells exhibit distinct responses upon collision 26with an epithelial cell monolayer 27To study heterotypic cell-cell collisions, we developed a confrontation assay 28 whereby two different cell-types are separated by a barrier, which upon removal, 29 creates a uniform gap into which the different cell populations migrate and collide. 30Following a screen of a range of different epithelial versus mesenchymal cell-types, 31 an interesting and unexpected phenomenon was revealed. When a population of 32 migrating epithelial cells (HaCaT) encountered a population of migrating fibroblasts 33 (NIH3T3), both populations ceased their forward migration, forming a sharp border 34 ( Figure 1A, B and Video S1). This is in stark contrast to fibrosarcoma cells (HT1080) 35 which, upon collision with epithelial cells, seemed to undergo a complete repulsion 36 ( Figure 1A, B and Video ...