Biomedical subjects
Publications and source records attributed to Lance L Munn.
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Defective fibrillar collagen polymerization in primary tumors has been correlated with increased metastasis. However, it is unclear how collagen organization influences tumor invasion. In this study, we show that collagen I polymerized without telopeptides (the flanking regions of collagen molecules) can differentially affect the three-dimensional migration of mammary carcinoma cells. MDA-MB-231 cells capable of proteolytic degradation and mesenchymal motion, invaded telopeptide-intact and telopeptide-free collagen gels to the same extent. In contrast, MDA-MB-435S cells, with typical features of amoeboid cells (poor collagenolytic activity, rounded cell morphology), were 5-fold more invasive in telopeptide-free than telopeptide-intact collagen. A fraction of the MDA-MB-435S cells that invaded telopeptide-intact or telopeptide-free collagen had a rounded morphology; however, in telopeptide-free collagen, a significant fraction of the cells switched from a rounded to elongated morphology (protrusion formation). The dynamic changes in cellular shape facilitated MDA-MB-435S locomotion through the narrow interfiber gaps, which were smaller than cell diameters. Based on the spherical morphology of MDA-MB-435S cells, we tested if the changes in cell shape and invasion were related to RhoA-ROCK activity; GTP-bound RhoA was measured in pull-down assays. RhoA activity was 1.8-fold higher for MDA-MB-435S cells seeded on telopeptide-free than telopeptide-intact collagen. Y27632 inhibition of ROCK, a Rho effector, significantly reduced the changes in cellular morphodynamics and the invasion of MDA-MB-435S cells but did not alter the invasion of MDA-MB-231 cells. Thus, the higher RhoA activity of MDA-MB-435S cells in telopeptide-free collagen enhances the changes in cellular morphodynamics associated with motility and invasion.
Endothelial cells of blood vessels in tumors may be thin, fragile, and defective in barrier function. We found previously that the endothelium of vessels in human colon carcinoma xenografts in mice is a mosaic structure. Approximately 85% of tumor vessels have uniform CD31 and/or CD105 immunoreactivity, but the remainder have focal regions that lack these common endothelial markers. The present study assessed the ultrastructure of the vessel lining and the integrity of the basement membrane in these regions. Using immunolabeling and confocal microscopy, we identified blood vessels that lacked CD31 and CD105 immunoreactivity and then analyzed the ultrastructure of these vessels by transmission electron microscopy. Eleven percent of vessels in orthotopic tumors and 24% of vessels in ectopic tumors had defects in CD31 and CD105 staining measuring on average 10.8 microm (range, 1-41.2 microm). Ultrastructural studies identified endothelial cells at 92% of CD31- and CD105-negative sites in orthotopic tumors and 70% of the sites in ectopic tumors. Thus, most regions of tumor vessels that lack CD31 and CD105 immunoreactivity represent attenuated endothelial cells with abnormal expression of endothelial cell markers, but some are gaps between endothelial cells. More than 80% of the defects lacked immunoreactivity for multiple basement membrane proteins.
NO has been shown to mediate angiogenesis; however, its role in vessel morphogenesis and maturation is not known. Using intravital microscopy, histological analysis, alpha-smooth muscle actin and chondroitin sulfate proteoglycan 4 staining, microsensor NO measurements, and an NO synthase (NOS) inhibitor, we found that NO mediates mural cell coverage as well as vessel branching and longitudinal extension but not the circumferential growth of blood vessels in B16 murine melanomas. NO-sensitive fluorescent probe 4,5-diaminofluorescein imaging, NOS immunostaining, and the use of NOS-deficient mice revealed that eNOS in vascular endothelial cells is the predominant source of NO and induces these effects. To further dissect the role of NO in mural cell recruitment and vascular morphogenesis, we performed a series of independent analyses. Transwell and under-agarose migration assays demonstrated that endothelial cell-derived NO induces directional migration of mural cell precursors toward endothelial cells. An in vivo tissue-engineered blood vessel model revealed that NO mediates endothelial-mural cell interaction prior to vessel perfusion and also induces recruitment of mural cells to angiogenic vessels, vessel branching, and longitudinal extension and subsequent stabilization of the vessels. These data indicate that endothelial cell-derived NO induces mural cell recruitment as well as subsequent morphogenesis and stabilization of angiogenic vessels.
Leukocytes comprise less than 1% of all blood cells. Enrichment of their number, starting from a sample of whole blood, is the required first step of many clinical and basic research assays. We created a microfluidic device that takes advantage of the intrinsic features of blood flow in the microcirculation, such as plasma skimming and leukocyte margination, to separate leukocytes directly from whole blood. It consists of a simple network of rectangular microchannels designed to enhance lateral migration of leukocytes and their subsequent extraction from the erythrocyte-depleted region near the sidewalls. A single pass through the device produces a 34-fold enrichment of the leukocyte-to-erythrocyte ratio. It operates on microliter samples of whole blood, provides positive, continuous flow selection of leukocytes, and requires neither preliminary labeling of cells nor input of energy (except for a small pressure gradient to support the flow of blood). This effortless, efficient, and inexpensive technology can be used as a lab-on-a-chip component for initial whole blood sample preparation. Its integration into microanalytical devices that require leukocyte enrichment will enable accelerated transition of these devices into the field for point-of-care clinical testing.
Historically, predicting macroscopic blood flow characteristics such as viscosity has been an empirical process due to the difficulty in rigorously including the particulate nature of blood in a mathematical representation of blood rheology. Using a two-dimensional lattice Boltzmann approach, we have simulated the flow of red blood cells in a blood vessel to estimate flow resistance at various hematocrits and vessel diameters. By including white blood cells (WBCs) in the flow, we also calculate the increase in resistance due to white cell rolling and adhesion. The model considers the blood as a suspension of particles in plasma, accounting for cell-cell and cell-wall interactions to predict macroscopic blood rheology. The model is able to reproduce the Fahraeus-Lindqvist effect, i.e., the increase in relative apparent viscosity as tube size increases, and the Fahraeus effect, i.e., tube hematocrit is lower than discharge hematocrit. In addition, the model allows direct assessment of the effect of WBCs on blood flow in the microvasculature, reproducing the dramatic increases in flow resistance as WBCs enter short capillary segments. This powerful and flexible model can be used to predict blood flow properties in any vessel geometry and with any blood composition.
At the time of transplantation, tumor fragments contain "passenger" cells: endothelial cells and other stromal cells from the original host. Here, we investigated the fate of genetically labeled endothelial and nonendothelial stromal cells after transplantation in syngeneic mice. We report that angiogenic stroma associated with tumor or adipose tissue persists when transplanted, remains functional, and governs the initial neovascularization of grafted tissue fragments for more than 4 weeks after implantation. Surprisingly, the passenger endothelial cells survive longer than other stromal cells, which are replaced by host-activated fibroblasts after 3 weeks. The transplantability of tumor stroma suggests that the angiogenic potential of a tumor xenograft, which determines its viability, depends on the presence of passenger endothelial cells and other stromal cells within the xenograft. These studies of tumor tissue transplantation provide a platform for exploring the role of epithelial-stromal interactions in studies of tumor heterogeneity and drug resistance.
We developed a novel orthotopic mouse tumor model of renal cell carcinoma to collect and characterize cells spontaneously shed from SN12C (renal cell carcinoma) and SN12L1 (high metastatic variant of SN12C) tumors grown in kidneys of severe combined immunodeficient mice. Viability of the shed cell population was greater for SN12L1 tumors (25%) compared with SN12C tumors (11%, P < 0.05). Gene array analysis of 23 genes involved in metastasis showed that CD44, alpha3 integrin, and caveolin were down-regulated in the shed tumor cells compared with their primary counterparts, and blocking alpha3 integrin or CD44 function inhibited attachment and migration of both cell lines. These results suggest that cohesion of the cells within the primary tumor mediated by CD44 and alpha3 integrins hinders metastasis and that shedding is a passive process not necessarily mediated by cell migration in these tumors. Furthermore, resistance to apoptosis may enhance metastasis in the higher metastatic tumor.
The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF blockade has a direct and rapid antivascular effect in human tumors.
The recent landmark Phase III clinical trial with a VEGF-specific antibody suggests that antiangiogenic therapy must be combined with cytotoxic therapy for the treatment of solid tumors. However, there are no guidelines for optimal scheduling of these therapies. Here we show that VEGFR2 blockade creates a "normalization window"--a period during which combined radiation therapy gives the best outcome. This window is characterized by an increase in tumor oxygenation, which is known to enhance radiation response. During the normalization window, but not before or after it, VEGFR2 blockade increases pericyte coverage of brain tumor vessels via upregulation of Ang1 and degrades their pathologically thick basement membrane via MMP activation.
Blood-borne therapeutics, which rely on diffusion and convection for delivery, often do not accumulate in effective concentrations distant from vasculature and are therefore unable to eradicate all cells within a tumor. Motile bacteria have the potential to overcome the diffusion and pressure gradients that prevent passive materials from penetrating into poorly perfused regions of tumors. Here, we test several proposed mechanisms of Salmonella typhimurium accumulation in tumors, including: (a) entrapment in the chaotic vasculature of tumors; (b) attraction to specific tumor microenvironments; and (c) preferential replication within specific microenvironments. After systemic injection of S. typhimurium into tumor-bearing mice, we used intravital microscopy and histological techniques to quantify their interaction with tumor vasculature. Immediately after injection, few S. typhimurium (<4 in 10,000) adhered to tumor vasculature; most remained passively suspended in the blood. Despite this low initial adhesion, approximately 10,000-fold more S. typhimurium accumulated in tumors than any other organ 1 week after the injection, thus demonstrating their specificity. However, within the tumors, we found that most bacteria were located in necrotic tissue as large colonies far (750 micro m) from functional vasculature. Together, these results suggest that S. typhimurium has limited ability to adhere to tumor vasculature and migrate within tumors and only survives in tissue that becomes necrotic. Although S. typhimurium is a promising delivery vehicle because of its tumor specificity, increasing its intra-tumoral motility should improve its therapeutic effectiveness.
The traditional view of angiogenesis emphasizes proliferation and migration of vessel wall-associated endothelial cells. However, circulating endothelial progenitor cells have recently been shown to contribute to tumor angiogenesis. Here we quantify the relative contributions of endothelial and endothelial progenitor cells to angiogenesis using a mathematical model. The model predicts that during the early stages of tumor growth, endothelial progenitors have a significant impact on tumor growth and angiogenesis, mediated primarily by their localization in the tumor, not by their proliferation. The model also shows that, as the tumor grows, endothelial progenitors adhere preferentially near the tumor periphery, coincident with the location of highest vascular density, supporting their potential utility as vectors for targeted delivery of therapeutics. Model simulations of various antiangiogenic strategies show that those therapies that effectively target both endothelial and endothelial progenitor cells, either by restoring the balance between angiogenic stimulators and inhibitors or by targeting both types of cells directly, are most effective at delaying tumor growth. The combination of continuous low-dose chemotherapy and antiangiogenic therapy is predicted to have the most significant effect on therapeutic outcome. The model offers new insight into tumor angiogenesis with implications for the rational design of antiangiogenic therapy.
The vasculature is not simply a collection of hollow tubes through which blood flows - it is an organ in its own right, consisting of several cell types organized in a specific manner, and it mediates many unique biological and physiological functions such as selective filtration and angiogenesis. In tumors, blood vessels have structural and functional abnormalities that, to date, have generally hindered conventional therapies: the malformed networks make it difficult to deliver drugs uniformly to all cancer cells. Furthermore, flow in these vessels is chaotic, with intermittent stagnation followed by high-flow or even flow reversal in isolated segments. Cancer cells in stagnated or low-flow regions will receive suboptimal drug levels during chemotherapy. However, as more is learned about the formation of tumor vasculature and how it differs from that in normal tissue, more effective and directed therapies to fight cancer might be developed.
Leukocyte rolling on the vascular endothelium requires initial contact between leukocytes circulating in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte-endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms also play a role. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells (RBCs) and leukocytes as they enter postcapillary expansions, but the details of the fluid dynamics have not been elucidated. We have analyzed the interactions of red and white blood cells as they flow from a capillary into a postcapillary venule using a lattice Boltzmann approach. This technique provides the complete solution of the flow field and quantification of the particle-particle forces in a relevant geometry. Our results show that capillary-postcapillary venule diameter ratio, RBC configuration, and RBC shape are critical determinants of the initiation of cell rolling in postcapillary venules. The model predicts that an optimal configuration of the trailing red blood cells is required to drive the white blood cell to the wall.
The unchecked growth of a solid tumor produces solid stress, causing deformation of the surrounding tissue. This stress can result in clinical complications, especially in confined environments such as the brain, and may also be responsible for pathophysiological anomalies such as the collapse of blood and lymphatic vessels. High stress levels may also inhibit further cell division within tumors. Unfortunately, little is known about the dynamics of stress accumulation in tumors or its effects on cell biology. We present a mathematical model for tumor growth in a confined, elastic environment such as living tissue. The model, developed from theories of thermal expansion using the current configuration of the material element, allows the stresses within the growing tumor and the surrounding medium to be calculated. The experimental observation that confining environments limit the growth of tumor spheroids to less than the limit imposed by nutrient diffusion is incorporated into the model using a stress dependent rate for tumor growth. The model is validated against experiments for MU89 tumor spheroid growth in Type VII agarose gel. Using the mathematical model and the experimental evidence we show that the tumor cell size is reduced by solid stress inside the tumor spheroid. This leads to the interesting possibility that cell size could be a direct indicator of solid stress level inside the tumors in clinical setting.
Tumor vessels possess unique physiological features that might be exploited for improving drug delivery. In the present study, we investigate the possibility of modifying polyethylene glycol-ylated liposome cationic charge of polyethylene glycol coated liposomes to optimize delivery to tumor vessels using biodistribution studies and intravital microscopy. The majority of liposomes accumulated in the liver, and increasing charge resulted in lower retention in the spleen and blood. Although overall tumor uptake was not affected by charge in the biodistribution studies, intravital microscopy showed that increasing the charge content from 10 to 50 mol % doubled the accumulation of liposomes in tumor vessels, suggesting a change in intratumor distribution; no significant effect of charge on interstitial accumulation could be detected, possibly attributable to spatial heterogeneity. Increased vascular accumulation of cationic liposomes was similar in two different tumor types and sites. Our results suggest that optimizing physicochemical properties of liposomes that exploit physiological features of tumors and control the intratumor distribution of these drug carriers should improve vascular-specific delivery.
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.
Leukocyte rolling and arrest on the vascular endothelium is a central event in normal and pathological immune responses. However, rigorous estimation of the fluid and surface forces involved in leukocyte-endothelial interactions has been difficult due to the particulate, non-Newtonian nature of blood. Here we present a Lattice-Boltzmann approach to quantify forces exerted on rolling leukocytes by red blood cells in a "virtual blood vessel." We report that the normal force imparted by erythrocytes is sufficient to increase leukocyte binding and that increases in tangential force and torque can promote rolling of previously adherent leukocytes. By simulating changes in hematocrit we show that a close "envelopment" of the leukocyte by the red blood cells is necessary to produce significant changes in the forces. This novel approach can be applied to a large number of biological and industrial problems involving the complex flow of particulate suspensions.