Enhanced Directional Migration of Cancer Stem Cells in 3D Aligned Collagen Matrices

Abstract

Directed cell migration by contact guidance in aligned collagenous extracellular matrix (ECM) is a critical enabler of breast cancer dissemination. The mechanisms of this process are poorly understood, particularly in 3D, in part because of the lack of efficient methods to generate aligned collagen matrices. To address this technological gap, we propose a simple method to align collagen gels using guided cellular compaction. Our method yields highly aligned, acellular collagen constructs with predictable microstructural features, thus providing a controlled microenvironment for in vitro experiments. Quantifying cell behavior in these anisotropic constructs, we find that breast carcinoma cells are acutely sensitive to the direction and extent of collagen alignment. Further, live cell imaging and analysis of 3D cell migration reveals that alignment of collagen does not alter the total motility of breast cancer cells, but simply redirects their migration to produce largely one-dimensional movement. However, a profoundly enhanced motility in aligned collagen matrices is observed for the subpopulation of carcinoma cells with high tumor initiating and metastatic capacity, termed cancer stem cells (CSCs). Analysis of the biophysical determinants of cell migration show that nuclear deformation is not a critical factor associated with the observed increases in motility for CSCs. Rather, smaller cell size, a high degree of phenotypic plasticity, and increased protrusive activity emerge as vital facilitators of rapid, contactguided migration of CSCs in aligned 3D collagen matrices.

The tumor cell‐secreted matricellular protein WISP 1 drives pro‐metastatic collagen linearization

“…It is well established that the presence of linearized collagen fibers in tumors facilitates metastasis by providing tracks that cancer cells exploit for invading surrounding tissues (Condeelis & Segall, 2003;Provenzano et al, 2006;Levental et al, 2009;Oudin et al, 2016). Furthermore, cancer cells have been shown to invade more efficiently through lattices of linear collagen engineered in vitro than through disorganized lattices of the same density or stiffness (Kraning-Rush et al, 2013;Riching et al, 2014;Ray et al, 2017). In agreement with these observations, we show that cancer cells are more motile on WISP1-linearized Col I lattices than on nonlinearized control Col I lattices.…”

“…Type I collagen is one of the most abundant proteins deposited in the ECM of breast tumors and of many other cancer types (Kalluri & Zeisberg, 2006;Naba et al, 2014). In particular, increased linearization of fibrillar collagen, which has been proposed to arise from the mechanical tension induced by cancer cells and cancer-associated fibroblasts (Butcher et al, 2009), facilitates tumor cell invasion and metastasis by providing tracks on which tumor cells can more easily migrate (Condeelis & Segall, 2003;Provenzano et al, 2006;Levental et al, 2009;Kraning-Rush et al, 2013;Riching et al, 2014;Oudin et al, 2016;Ray et al, 2017). In particular, increased linearization of fibrillar collagen, which has been proposed to arise from the mechanical tension induced by cancer cells and cancer-associated fibroblasts (Butcher et al, 2009), facilitates tumor cell invasion and metastasis by providing tracks on which tumor cells can more easily migrate (Condeelis & Segall, 2003;Provenzano et al, 2006;Levental et al, 2009;Kraning-Rush et al, 2013;Riching et al, 2014;Oudin et al, 2016;Ray et al, 2017).…”

“…Consequently, changes in type I collagen organization have a profound impact on tumor cell behavior and on disease progression (Kalluri & Zeisberg, 2006;Butcher et al, 2009;Joyce & Pollard, 2009;Lu et al, 2012). In particular, increased linearization of fibrillar collagen, which has been proposed to arise from the mechanical tension induced by cancer cells and cancer-associated fibroblasts (Butcher et al, 2009), facilitates tumor cell invasion and metastasis by providing tracks on which tumor cells can more easily migrate (Condeelis & Segall, 2003;Provenzano et al, 2006;Levental et al, 2009;Kraning-Rush et al, 2013;Riching et al, 2014;Oudin et al, 2016;Ray et al, 2017). On this basis, collagen linearization is considered a key feature of metastatic carcinomas such as triple-negative breast cancers (TNBCs) and is predictive of poor prognosis (Colpaert et al, 2001;Conklin et al, 2011).…”

Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments

“…Collagen matrices preparation was adapted from (Ray et al, 2017b) with modifications. Briefly, high concentration rat-tail collagen I (VWR) was neutralized with a 1:1 ratio of 100 mM HEPES (Thermo Fisher Scientific) in 2X PBS and completed to a final concentration of 3 mg/mL with DMEM (Corning) supplemented with 10% FBS (which introduces FN).…”

“…Cell migration data was collected by imaging using a custom-built multi-photon laser-scanning microscope (MPLSM) (Prairie Technologies/Bruker) using a Mai Tai Ti:Sapphire laser (Spectra-Physics) to simultaneously generate MPE and SHG to visualize cells and collagen, respectively, at an excitation wavelength of 880 nm with a custom-built temperature-controlled stage insert, as described previously (Ray et al, 2017a(Ray et al, , 2017b. Briefly, time-lapse imaging of T cells inside 3D collagen matrix and on tumor slices, was obtained by creating two-channel (T cells and collagen) or three-channel (T cells, collagen, and KPC-tissue/KPCT-carcinoma cells) Z-stacks of 75 µm depth at 5 µm steps, at each stage position, every 1.5 min over at least 45 min of imaging.…”