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.

Calprotectin (S100A8/A9), a heterodimeric complex of calcium-binding proteins S100A8 and S100A9, is encoded by genes mapping to the chromosomal locus 1q21.3 of the epidermal differentiation complex. Whereas extracellular calprotectin shows proinflammatory and antimicrobial properties by signaling through RAGE and TLR4, intracytoplasmic S100A8/A9 appears to be important for cellular development, maintenance, and survival. S100A8/A9 is constitutively expressed in myeloid cells and the stratified mucosal epithelia lining the oropharyngeal and genitourinary mucosae. While upregulated in adenocarcinomas and other cancers, calprotectin mRNA and protein levels decline in head and neck squamous cell carcinoma (HNSCC). S100A8/A9 is also lost during head and neck preneoplasia (dysplasia). Calprotectin decrease does not correlate with the clinical stage (TNM) of HNSCC. When expressed in carcinoma cells, S100A8/A9 downregulates matrix metalloproteinase 2 expression and inhibits invasion and migration in vitro. S100A8/A9 regulates cell cycle progression and decelerates cancer cell proliferation by arresting at the G2/M checkpoint in a protein phosphatase 2α-dependent manner. In HNSCC, S100A8 and S100A9 coregulate with gene networks controlling cellular development and differentiation, cell-to-cell signaling, and cell morphology, while S100A8/A9 appears to downregulate expression of invasion- and tumorigenesis-associated genes. Indeed, tumor formation capacity is attenuated in S100A8/A9-expressing carcinoma cells in vivo. Hence, intracellular calprotectin appears to function as a tumor suppressor in head and neck carcinogenesis. When compared with S100A8/A9-low HNSCC based on analysis of TCGA, S100A8/A9-high HNSCC shows significant upregulation of apoptosis-related genes, including multiple caspases. Accordingly, S100A8/A9 facilitates DNA damage responses in HNSCC, promotes apoptotic cell death, and confers sensitivity to cisplatin and X-radiation in vitro. In the tumor milieu, loss of S100A8/A9 strongly associates with poor squamous differentiation and higher tumor grading, EGFR upregulation, increased DNA methylation, and, finally, poorer overall survival for patients with HNSCC. Hence, intracellular calprotectin shows a multifaceted protective role against the development of HNSCC.

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