Leader cell development and function in Breast Tumor Collective Migration

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Project Summary for Parent Grant Accumulated evidence in human breast cancer and mouse models of breast cancer have shown that tumor cells invade collectively through the basement membrane (BM) and continue as collective groups to traverse the collagen-rich ECM to access lymphatic and vascular vessels. Rather than single cells, in the circulation clusters of heterogeneous circulating tumor cells (CTCs), that also contain tumor-associated stromal cells such as cancer associated fibroblasts (CAFs), account for >90% of metastases. To move collectively requires coordinated cell–cell and cell–matrix interactions. Hallmarks of collective cell migration include: 1) Cells remain physically and functionally connected such that the integrity of cell–cell junctions are preserved during movement. 2) A subgroup of cells typically defines the leading edge, and thus, the direction of collective migration. These are known as “leader “cells and differ in function from “follower” cells. 3) Collective movement also involves intimate interaction with accessory stromal cells that release polarity-inducing and pro-migratory factors as well as contribute to path finding by physically remodeling the surrounding ECM. Several hypotheses have been proposed to explain cancer leader cell development during collective migration. Yet how these leader cells develop, arrive and define the front edge, then lead directed collective migration, and whether this phenomenon is necessary and sufficient to effect directed collective migration are largely unknown. We have developed novel microfluidic devices in which to study the collective migration of primary breast tumor organoids in response to multiple environmental signals In the present proposal we propose to use primary breast tumor organoids with their inherent cellular heterogeneity to determine how leader cells develop and function, in response to multiple environmental signals, so as to direct collective migration. To do so we propose two specific aims. Specific Aim 1. To determine how K14 leader cells within primary breast tumor organoids polarize to the leading edge and then function to direct collective migration. Specific Aim 2: To understand chemo-mechanical feedback between CAF-based ECM remodeling and leader-based invasion. Project Summary for Supplement In breast tumor invasion, clustered cells invade through surrounding stromal collagen to enter circulation. This process is regulated by leader cells that come to the front of a breast tumor collective and “lead” the entire organoid to invade collagen. The supplement project asks whether there is an optimal balance of cellular forces and collagen density and crosslinking. This is a multivariate problem that requires modeling predictions; it would otherwise be much more difficult to straightaway perform numerous experiments to try all possible combinations of cell types and collagen compositions. This supplement project studies how dysfunctional cell- cell communication can alter cell invasion modes in 3D environments of varying stiffness and fiber architecture. Cell populations are captured in a cellular Potts lattice-based modeling framework and collagen as a 3D field that can evolve by cellular forces. Modeling parameters corresponding to cell protrusions (regulated by P- cadherin and Rac), cell-cell adhesions (α-catenin), and cellular contractility (RhoA) are varied in a given cell population. In parallel, collagen concentration, degradation rate, crosslinking rate, and long-distance force propagation are also varied. Simulations are performed to measure how invasion modes of these different populations vary according to fiber microstructure of 3D collagen matrices. The modeling predictions from this supplement project, particularly relating to the roles of P-cadherin, Rac, α-catenin and RhoA in collective cell 3D invasion, inform other projects in the parent grant.