Local photo-crosslinking of native tissue matrix regulates cell function.

Within most tissues, the extracellular microenvironment provides mechanical cues that guide cell fate and function. Changes in the extracellular matrix such as aberrant deposition, densification and increased crosslinking are hallmarks of late-stage fibrotic diseases that often lead to organ dysfunction. Biomaterials have been widely used to mimic the mechanical properties of the fibrotic matrix and study cell function. However, the initiation of fibrosis has largely been overlooked, due to the challenges in recapitulating early fibrotic lesions within the native extracellular microenvironment. Using visible light mediated photochemistry, we induced local crosslinking and stiffening of extracellular matrix proteins within ex vivo murine and human tissue. In ex vivo lung tissue of epithelial cell lineage-traced mice, local matrix crosslinking mimicked early fibrotic lesions that increased alveolar epithelial cell spreading, differentiation and extracellular matrix remodeling. However, inhibition of cytoskeletal tension or integrin engagement reduced epithelial cell spreading and differentiation, resulting in alveolar epithelial cell dedifferentiation and reduced extracellular matrix deposition. Our findings emphasize the role of local extracellular matrix crosslinking and remodeling in early-stage tissue fibrosis and have implications for ex vivo disease modeling and applications to other tissues.

Endothelial cells metabolically regulate breast cancer invasion toward a microvessel.

Breast cancer metastasis is initiated by invasion of tumor cells into the collagen type I-rich stroma to reach adjacent blood vessels. Prior work has identified that metabolic plasticity is a key requirement of tumor cell invasion into collagen. However, it remains largely unclear how blood vessels affect this relationship. Here, we developed a microfluidic platform to analyze how tumor cells invade collagen in the presence and absence of a microvascular channel. We demonstrate that endothelial cells secrete pro-migratory factors that direct tumor cell invasion toward the microvessel. Analysis of tumor cell metabolism using metabolic imaging, metabolomics, and computational flux balance analysis revealed that these changes are accompanied by increased rates of glycolysis and oxygen consumption caused by broad alterations of glucose metabolism. Indeed, restricting glucose availability decreased endothelial cell-induced tumor cell invasion. Our results suggest that endothelial cells promote tumor invasion into the stroma due, in part, to reprogramming tumor cell metabolism.

Convergent Approaches to Delineate the Metabolic Regulation of Tumor Invasion by Hyaluronic Acid Biosynthesis.

Metastasis is the leading cause of breast cancer-related deaths and is often driven by invasion and cancer-stem like cells (CSCs). Both the CSC phenotype and invasion are associated with increased hyaluronic acid (HA) production. How these independent observations are connected, and which role metabolism plays in this process, remains unclear due to the lack of convergent approaches integrating engineered model systems, computational tools, and cancer biology. Using microfluidic invasion models, metabolomics, computational flux balance analysis, and bioinformatic analysis of patient data, the functional links between the stem-like, invasive, and metabolic phenotype of breast cancer cells as a function of HA biosynthesis are investigated. These results suggest that CSCs are more invasive than non-CSCs and that broad metabolic changes caused by overproduction of HA play a role in this process. Accordingly, overexpression of hyaluronic acid synthases (HAS) 2 or 3 induces a metabolic phenotype that promotes cancer cell stemness and invasion in vitro and upregulates a transcriptomic signature predictive of increased invasion and worse patient survival. This study suggests that HA overproduction leads to metabolic adaptations to satisfy the energy demands for 3D invasion of breast CSCs highlighting the importance of engineered model systems and multidisciplinary approaches in cancer research.

Engineering strategies to capture the biological and biophysical tumor microenvironment in vitro.

Despite decades of research and advancements in diagnostic and treatment modalities, cancer remains a major global healthcare challenge. This is due in part to a lack of model systems that allow investigating the mechanisms underlying tumor development, progression, and therapy resistance under relevant conditions in vitro. Tumor cell interactions with their surroundings influence all stages of tumorigenesis and are shaped by both biological and biophysical cues including cell-cell and cell-extracellular matrix (ECM) interactions, tissue architecture and mechanics, and mass transport. Engineered tumor models provide promising platforms to elucidate the individual and combined contributions of these cues to tumor malignancy under controlled and physiologically relevant conditions. This review will summarize current knowledge of the biological and biophysical microenvironmental cues that influence tumor development and progression, present examples of in vitro model systems that are presently used to study these interactions and highlight advancements in tumor engineering approaches to further improve these technologies.

Characterization of the Cellular Output of a Point-of-Care Device and the Implications for Addressing Critical Limb Ischemia.

Critical limb ischemia (CLI) is a terminal disease with high morbidity and healthcare costs due to limb loss. There are no effective medical therapies for patients with CLI to prevent amputation. Cell-based therapies are currently being investigated to address this unmet clinical need and have shown promising preliminary results. The purpose of this study was to characterize the output of a point-of-care cell separator (MarrowStim P.A.D. Kit), currently under investigation for the treatment of CLI, and compare its output with Ficoll-based separation. The outputs of the MarrowStim P.A.D. Kit and Ficoll separation were characterized using an automated hematology analyzer, colony-forming unit (CFU) assays, and tubulogenesis assays. Hematology analysis indicated that the MarrowStim P.A.D. Kit concentrated the total nucleated cells, mononuclear cells, and granulocytes compared with baseline bone marrow aspirate. Cells collected were positive for VEGFR-2, CD3, CD14, CD34, CD45, CD56, CD105, CD117, CD133, and Stro-1 antigen. CFU assays demonstrated that the MarrowStim P.A.D. Kit output a significantly greater number of mesenchymal stem cells and hematopoietic stem cells compared with cells output by Ficoll separation. There was no significant difference in the number of endothelial progenitor cells output by the two separation techniques. Isolated cells from both techniques formed interconnected nodes and microtubules in a three-dimensional cell culture assay. This information, along with data currently being collected in large-scale clinical trials, will help instruct how different cellular fractions may affect the outcomes for CLI patients.