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

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, contact-guided migration of CSCs in aligned 3D collagen matrices.

Cancer Stem Cell Migration in Three-Dimensional Aligned Collagen Matrices.

Cell migration is strongly influenced by the organization of the surrounding 3-D extracellular matrix. In particular, within fibrous solid tumors, carcinoma cell invasion may be directed by patterns of aligned collagen in the extra-epithelial space. Thus, studying the interactions of heterogeneous populations of cancer cells that include the stem/progenitor-like cancer stem cell subpopulation and aligned collagen networks is critical to our understanding of carcinoma dissemination. Here, we describe a robust method to generate aligned collagen matrices in vitro that mimic in vivo fiber organization. Subsequently, a protocol is presented for seeding aligned matrices with distinct carcinoma cell subpopulations and performing live cell imaging and quantitative analysis of cell migration. Together, the engineered constructs and the imaging techniques laid out here provide a platform to study cancer stem cell migration in 3-D anisotropic collagen with real-time visualization of cellular interactions with the fibrous matrix. © 2018 by John Wiley & Sons, Inc.

Dynamics of 3D carcinoma cell invasion into aligned collagen.

Carcinoma cells frequently expand and invade from a confined lesion, or multicellular clusters, into and through the stroma on the path to metastasis, often with an efficiency dictated by the architecture and composition of the microenvironment. Specifically, in desmoplastic carcinomas such as those of the breast, aligned collagen tracks provide contact guidance cues for directed cancer cell invasion. Yet, the evolving dynamics of this process of invasion remains poorly understood, in part due to difficulties in continuously capturing both spatial and temporal heterogeneity and progression to invasion in experimental systems. Therefore, to study the local invasion process from cell dense clusters into aligned collagen architectures found in solid tumors, we developed a novel engineered 3D invasion platform that integrates an aligned collagen matrix with a cell dense tumor-like plug. Using multiphoton microscopy and quantitative analysis of cell motility, we track the invasion of cancer cells from cell-dense bulk clusters into the pre-aligned 3D matrix, and define the temporal evolution of the advancing invasion fronts over several days. This enables us to identify and probe cell dynamics in key regions of interest: behind, at, and beyond the edge of the invading lesion at distinct time points. Analysis of single cell migration identifies significant spatial heterogeneity in migration behavior between cells in the highly cell-dense region behind the leading edge of the invasion front and cells at and beyond the leading edge. Moreover, temporal variations in motility and directionality are also observed between cells within the cell-dense tumor-like plug and the leading invasive edge as its boundary extends into the anisotropic collagen over time. Furthermore, experimental results combined with mathematical modeling demonstrate that in addition to contact guidance, physical crowding of cells is a key regulating factor orchestrating variability in single cell migration during invasion into anisotropic ECM. Thus, our novel platform enables us to capture spatio-temporal dynamics of cell behavior behind, at, and beyond the invasive front and reveals heterogeneous, local interactions that lead to the emergence and maintenance of the advancing front.