Lymphatic metastasis contributes to mortality from solid tumors. Whether metastasizing cancer cells reach lymph nodes via intratumor lymphatic vessels is unknown. Here, we examine functional lymphatics associated with mouse tumors expressing normal or elevated levels of vascular endothelial growth factor-C (VEGF-C), a molecule that stimulates lymphangiogenesis. Although VEGF-C overexpression increased lymphatic surface area in the tumor margin and lymphatic metastasis, these tumors contained no functional lymphatics, as assessed by four independent functional assays and immunohistochemical staining. These findings suggest that the functional lymphatics in the tumor margin alone are sufficient for lymphatic metastasis and should be targeted therapeutically.

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.

The delivery of therapeutic drugs to solid tumours may be impaired by structural and functional abnormalities in blood and lymphatic vessels. Here we provide evidence that proliferating cancer cells cause intratumour vessels to compress and collapse. By reducing this compressive mechanical force and opening vessels, cytotoxic cancer treatments have the potential to increase blood perfusion, thereby improving drug delivery.