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Research at Mass General
Lance L. Munn, PhD Associate Professor of Radiation Oncology Harvard Medical School
Associate Biologist Edwin L. Steele Laboratory for Tumor Biology
As part of the Edwin L. Steele Laboratory for Tumor Biology, research at the Munn Laboratory focuses on blood vessel structure and function in normal and pathological conditions. Within this broad area, I have projects that address:
Flow of fluid within the lymphatic system is central to many aspects of physiology, including fluid homeostasis and immune function, and poor lymphatic drainage results in significant morbidity in millions of patients each year. We are investigating the mechanisms of lymphatic pumping, considering the nitric oxide and calcium dynamics driven by mechanobiological mechanisms.
During angiogenesis, endothelial cells abandon their normal arrangement in the vessel wall to migrate into the extravascular matrix. This process is controlled by mul-tiple signals and is necessary for tissue regeneration and tumor growth. Using in vitro models and microfluidic devices, we are investigating the biochemical and mechanical determinants of this morphogenic transformation.
To form new, patent blood vessels, angiogenic sprouts must connect. The process by which this happens -anastomosis – is poorly understood, but represents new targets for vascular therapy. Using intravital microscopy and engineered vascular devices, we are following the steps of anastomosis to identify cellular and molecular mechanisms that may eventually be targeted for enhancing wound healing or inhibiting pathological angiogenesis.
In many normal physiological responses, endothelial cells and the blood vessel networks they form undergo dramatic changes in morphology and function. Examples include angiogenesis in wound healing, vessel dilation/hyperpermeability in inflammation, and endometrial angiogenesis in the female reproductive cycle.
Endothelial cells, in cooperation with other stromal cells, have to accomplish these diverse changes by responding to a limited number of growth factors including VEGF, PlGF and bFGF. We are using a systems biology approach to understand how the various growth factors and cells cooperate to produce these seemingly diverse functions. Because tumor angiogenesis relies on many of these same growth factors and cellular mechanisms (but in an abnormal, poorly controlled way), these studies will allow a better understanding of tumor angiogenesis and anti-angiogenic therapy.
During the initial stage of metastasis, cancer cells must breach the vessel wall and enter the circulation. Despite intense research in this area, the cellular mechanisms by which this occurs are poorly understood. Some tumors seem to metastasize as single rogue cells, while others travel in groups or clusters; some seem to actively migrate into the vessel, while others may be passively pushed. Using gene array analysis and carefully designed coculture systems, we are assessing the mechanical and cellular determinants of the initiation of metastasis.
With sufficient understanding of the underlying mechanisms, mathematical models can be assembled to validate existing hypotheses and generate new ones.
Lance Munn, PhDPrincipal InvestigatorContact by email
Despina Bazou, PhDResearch FellowContact by email
Gabriel Gruionu, PhDInstructor in SurgeryContact by email
Christian Kunert, PhDContact by email
Nir Maimon, PhDResearch Fellow in Radiation OncologyContact by email
Jonathan Song, PhDContact by email
Kunert C, Baish JW, Liao S, Padera TP, Munn LL. Mechanobiological oscillators control lymph flow. Proc Natl Acad Sci USA. 2015; 112: 10938-10943
Mechanobiological signals can provide dynamic feedback to biological systems. In this work, we study the pumping of lymph by lymphatic vessels, and how it can be controlled by mechanical feedback signals.
Proc Natl Acad Sci U S A. 2011;108(37):15342-7 - PMID: 21876168 - PMCID: PMC3174629
During angiogenesis, endothelial cells (ECs) from intact blood vessels quickly infiltrate avascular regions via vascular sprouting. This process is fundamental to many normal and pathological processes such as wound healing and tumor growth, but its initiation and control are poorly understood.
Vascular endothelial cell growth factor (VEGF) can promote vessel dilation and angiogenic sprouting, but given the complex nature of vascular morphogenesis, additional signals are likely necessary to determine, for example, which vessel segments sprout, which dilate, and which remain quiescent.
Fluid forces exerted by blood and plasma are prime candidates that might co-direct these processes, but it is not known whether VEGF cooperates with mechanical fluid forces to mediate angiogenesis.
Using a microfluidic tissue analog of angiogenic sprouting, we found that fluid shear stress, such as exerted by flowing blood, attenuates EC sprouting in a nitric oxide-dependent manner and that interstitial flow, such as produced by extravasating plasma, directs endothelial morphogenesis and sprout formation.
Furthermore, positive VEGF gradients initiated sprouting but negative gradients inhibited sprouting, promoting instead sheet-like migration analogous to vessel dilation. These results suggest that ECs integrate signals from fluid forces and local VEGF gradients to achieve such varied goals as vessel dilation and sprouting.
Bazou D, Ng MR, Song JW, Chin SM, Maimon N, Munn LL. Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting. Scientific Reports. 2016; 6:34046.
By performing proteomic analyses on endothelial cell exposed to fluid shear forces, we determined that HDAC1 phosphorylation is a key step in the mechanotransduction of signals involved in angiogenic morphogenesis. Bazou D, Ng MR, Song JW, Chin SM, Maimon N, Munn LL. Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting. Scientific Reports. 2016; 6:34046.
Dupin MM, Halliday I, Care CM, Alboul L, Munn LL. Modeling the flow of dense suspensions of deformable particles in three dimensions. Phys Rev E Stat Nonlin Soft Matter Phys. 2007; 75:066707
Despite more than a century of investigations, the anomalous behavior of flowing blood and how immune cells are transported in the blood stream are still not well-understood. Because blood is a dense suspension of deformable cells suspended in plasma, it has been difficult to mathematically describe and analyze all the simultaneous cell interactions that collectively result in the unusual bulk flow characteristics of blood in vessels. We use numerical simulations to study the flow aberrations that result from interactions between blood cells in the flow, including leukocytes. These interactions, and the mechanical properties of the cells, greatly affect the bulk properties of blood (e.g. viscosity and plasma-skimming).
Lab Chip. 2011;11(17):2941-7 - PMID: 21773633 - PMCID: PMC3743538
Blood cells naturally auto-segregate in postcapillary venules, with the erythrocytes (red blood cells, RBCs) aggregating near the axis of flow and the nucleated cells (NCs)—which include leukocytes, progenitor cells and, in cancer patients, circulating tumor cells—marginating toward the vessel wall.
We have used this principle to design a microfluidic device that extracts nucleated cells (NCs) from whole blood. Fabricated using polydimethylsiloxane (PDMS) soft lithography, the biomimetic cell extraction device consists of rectangular microchannels that are 20-400 µ wide, 11 µ deep and up to 2 cm long.
The key design feature is the use of repeated expansions/contractions of triangular geometry mimicking postcapillary venules, which enhance margination and optimize the extraction. The device operates on unprocessed whole blood and is able to extract 94 ± 4.5% of NCs with 45.75 ± 2.5-fold enrichment in concentration at a rate of 5 nl s(-1).
The device eliminates the need to preprocess blood via centrifugation or RBC lysis, and is ready to be implemented as the initial stage of lab-on-a-chip devices that require enriched nucleated cells. The potential downstream applications are numerous, encompassing all preclinical and clinical assays that operate on enriched NC populations and include on-chip flow cytometry.
Not all tumor vessels are equal. Tumor-associated vasculature includes immature vessels, regressing vessels, transport vessels undergoing arteriogenesis and peritumor vessels influenced by tumor growth factors.
Current techniques for analyzing tumor blood flow do not discriminate between vessel subtypes and only measure average changes from a population of dissimilar vessels. We developed methodologies for simultaneously quantifying blood flow (velocity, flux, hematocrit and shear rate) in extended networks at single-capillary resolution in vivo.
Our approach relies on deconvolution of signals produced by labeled red blood cells as they move relative to the scanning laser of a confocal or multiphoton microscope and provides fully resolved three-dimensional flow profiles within vessel networks.
Using this methodology, we show that blood velocity profiles are asymmetric near intussusceptive tissue structures in tumors in mice. Furthermore, we show that subpopulations of vessels, classified by functional parameters, exist in and around a tumor and in normal brain tissue.
Cheng G, Liao S, Kit Wong H, Lacorre DA, di Tomaso E, Au P, Fukumura D, Jain RK, Munn LL. Engineered blood vessel networks connect to host vasculature via wrapping-and-tapping anastomosis. Blood. 2011; 118:4740-9
During angiogenesis, new vessels need to connect to existing vasculature to become perfused. This process, known as anastomosis, is virtually unexplored, even though it plays a central role in development, tissue repair and pathological angiogenesis. We used intravital microscopy in mouse models to study the process by which engrafted vessels connect to host vasculature and discovered that the connections are made by a process of “wrapping and tapping” in which the engrafted endothelium ensheaths the existing vessels before degrading the underlying endothelium and redirecting blood flow into the previously unperfused network. Cheng G, Liao S, Kit Wong H,
Bazou D, Maimon N, Gruionu G, Munn LL. Self-assembly of vascularized tissue to support tumor explants in vitro. Integrative Biology. 2016; 8:1301-11.
Testing the efficacy of cancer drugs requires functional assays that recapitulate the cell populations, anatomy and biological responses of human tumors. We have developed methodology for maintaining harvested tumor tissue in vitro by placing them in a support bed with self-assembled stroma and vasculature. The harvested biopsy or tumor explant integrates with the stromal bed and vasculature, providing the correctextracellular matrix (collagen I, IV, fibronectin), associated stromal cells, and a lumenized vessel network.Our system provides a new tool that will allow ex vivo drug-screening and can be adapted for the guidance of patient-specific therapeutic strategies. Bazou D, Maimon N, Gruionu G, Munn LL. Self-assembly of vascularized tissue to support tumor explants in vitro. Integrative Biology. 2016; 8:1301-11.
Gruionu GG, Bazou D, Maimon N, Onita-Lenco M, Gruionu LG, Huang P, Munn LL. Implantable Tissue Isolation Chambers for Analyzing Tumor Dynamics In Vivo. Lab Chip. 2016; 16:1840-51
In this paper, we demonstrated how devices microfabricated from polydimethyl siloxane (PDMS) can be used to study tumor development in vivo. By partially confining tumor growth within chambers molded in various shapes, we were able to clearly track the infiltration of host cells and vasculature into the tumor, and monitor the integration of the tumor with the host collagen matrix. This project forms a bridge between the disciplines of microfluidics and tumor biology, creating new opportunities to study tumor progression in vivo using optical microscopy. Gruionu GG, Bazou D, Maimon N, Onita-Lenco M, Gruionu LG, Huang P, Munn LL. Implantable Tissue Isolation Chambers for Analyzing Tumor Dynamics In Vivo. Lab Chip. 2016; 16:1840-51
In this collaborative project with the Tarbell lab at City College New York, we removed heparan sulfate from the glycocalyx surface of cancer cells and studied their phenotype in vitro and their ability to metastasize in vivo. We found that the loss of heparan sulfate disrupted the mechanobiological signaling normally initiated by fluid shear forces. Depletion of heparan sulfate also prevented local invasion and distant metastasis, suggesting it might be a key mediator of mechanically-induced metastasis. Qazi H, Shi ZD, Song JW, Cancel LM, Huang P, Zeng Y, Roberge S, Munn LL*, Tarbell JM.* Heparan Sulfate Proteoglycans Mediate Renal Carcinoma Metastasis. International Journal of Cancer. 2016; 139:2791-2801. *Equal contributions.
Proc Natl Acad Sci U S A. 2011;109(3):911-6 - PMID: 22203958 - PMCID: PMC3271885
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.
Munn LL. Dynamics of tissue topology during cancer invasion and metastasis. Physical Biology. 2013; 10(6):065003 (13pp).
During tumor progression, cancer cells mix with other cell populations including epithelial and endothelial cells. Although potentially important clinically as well as for our understanding of basic tumor biology, the process of mixing is largely a mystery.
Furthermore, there is no rigorous, analytical measure available for quantifying the mixing of compartments within a tumor. We develop mathematical models of tissue repair and tumor growth based on collective cell migration that simulate a wide range of observed tumor behaviors with correct tissue compartmentalization and connectivity.
The resulting dynamics are analyzed in light of the Euler characteristic, which describes key topological features such as fragmentation, looping and cavities.
The analysis predicts a number of regimes in which the cancer cells can encapsulate normal tissue, form a co-interdigitating mass, or become fragmented and encapsulated by endothelial or epithelial structures. Key processes that affect the topological changes are the production of provisional matrix in the tumor, and the migration of endothelial or epithelial cells on this matrix..
Bazou D, Maimon N, Gruionu G, Munn LL. Self-assembly of vascularized tissue to support tumor explants in vitro. Integrative Biology. 2016; 8:1301-11
Qazi H, Shi ZD, Song JW, Cancel LM, Huang P, Zeng Y, Roberge S, Munn LL*, Tarbell * Heparan Sulfate Proteoglycans Mediate Renal Carcinoma Metastasis. International Journal of Cancer. 2016; 139:2791-2801. *Equal contributions.
Gruionu GG, Bazou D, Maimon N, Onita-Lenco M, Gruionu LG, Huang P, Munn LL. Implantable Tissue Isolation Chambers for Analyzing Tumor Dynamics In Vivo. Lab Chip. 2016; 16:1840-51.
Munn LL, Jain RK. The Forces of Cancer. Scientist (2016) 30: 52-57.
Kunert C, Baish JW, Liao S, Padera TP, Munn LL. Mechanobiological oscillators control lymph flow. Proc Natl Acad Sci U S A. 2015; 112:10938-43.
Song JW, Munn LL. Fluid forces control endothelial sprouting. Proc Natl Acad Sci U S A. 2011; 108: 15342-7.
Edwin L. Steele Laboratory for Tumor Biology
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