Intravital multiphoton microscopy has provided powerful mechanistic insights into health and disease, and has become a common instrument in the modern biological laboratory. The requisite high numerical aperture and exogenous contrast agents that enable multiphoton microscopy, however, limit ability to investigate substantial tissue volumes or to probe dynamic changes repeatedly over prolonged periods. Here, we introduce optical frequency domain imaging (OFDI) as an intravital microscopy that circumvents the technical limitations of multiphoton microscopy and, as a result, provides unprecedented access to previously unexplored, critically important aspects of tissue biology. Using novel OFDI-based approaches and entirely intrinsic mechanisms of contrast, we present rapid and repeated measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy, thereby demonstrating the potential of OFDI to facilitate the exploration of physiological and pathological processes and the evaluation of treatment strategies. †Authors to whom correspondence should be addressed: R.K.J (jain@steele.mgh.harvard.edu) or B.E.B (bouma@helix.mgh.harvard.edu). * Authors contributed equally to this work Author Contributions BJV developed OFDI technology, designed and performed most of the experiments, developed methodology, headed all data analysis and wrote the manuscript. RML designed and performed most of the experiments, developed methodology, headed all data analysis and wrote the manuscript. JAT contributed to vascular tracing of OFDI data. TPP performed lymphangiography experiments and contributed to data analysis and manuscript preparation. LAB performed VEGF-R2 blockade in vivo experiments. TS developed and performed fractal characterization and contributed to manuscript preparation. LLM contributed to vascular data analysis. GJT contributed to OFDI technology development. DF contributed to experimental design and manuscript preparation. RKJ and BEB contributed to the design of experiments, preparation of the manuscript, and supervised the project.

Uncontrolled growth in a confined space generates mechanical compressive stress within tumors, but little is known about how such stress affects tumor cell behavior. Here we show that compressive stress stimulates migration of mammary carcinoma cells. The enhanced migration is accomplished by a subset of "leader cells" that extend filopodia at the leading edge of the cell sheet. Formation of these leader cells is dependent on cell microorganization and is enhanced by compressive stress. Accompanied by fibronectin deposition and stronger cell-matrix adhesion, the transition to leader-cell phenotype results in stabilization of persistent actomyosin-independent cell extensions and coordinated migration. Our results suggest that compressive stress accumulated during tumor growth can enable coordinated migration of cancer cells by stimulating formation of leader cells and enhancing cell-substrate adhesion. This novel mechanism represents a potential target for the prevention of cancer cell migration and invasion.mechanobiology | solid stress | collective migration | metastasis

Tissue engineering requires formation of a de novo stable vascular network. Because of their ability to proliferate, differentiate into endothelial cells, and form new vessels, blood-derived endothelial progenitor cells (EPCs) are attractive source of cells for use in engineering blood vessels. However, the durability and function of EPC-derived vessels implanted in vivo are unclear. To this end, we directly compared formation and functions of tissue-engineered blood vessels generated by peripheral blood-and umbilical cord blood-derived EPCs in a model of in vivo vasculogenesis. We found that adult peripheral blood EPCs form blood vessels that are unstable and regress within 3 weeks. In contrast, umbilical cord blood EPCs form normalfunctioning blood vessels that last for more than 4 months. These vessels exhibit normal blood flow, perm-selectivity to macromolecules, and induction of leukocyte-endothelial interactions in response to cytokine activation similar to normal vessels.