Ricky T. Tong

Vascular Normalization by Vascular Endothelial Growth Factor Receptor 2 Blockade Induces a Pressure Gradient Across the Vasculature and Improves Drug Penetration in Tumors

Elevated interstitial fluid pressure, a hallmark of solid tumors, can compromise the delivery of therapeutics to tumors. Here we show that blocking vascular endothelial growth factor (VEGF) signaling by DC101 (a VEGF-receptor-2 antibody) decreases interstitial fluid pressure, not by restoring lymphatic function, but by producing a morphologically and functionally “normalized” vascular network. We demonstrate that the normalization process prunes immature vessels and improves the integrity and function of the remaining vasculature by enhancing the perivascular cell and basement membrane coverage. We also show that DC101 induces a hydrostatic pressure gradient across the vascular wall, which leads to a deeper penetration of molecules into tumors. Thus, vascular normalization may contribute to the improved survival rates in tumor-bearing animals and in colorectal carcinoma patients treated with an anti-VEGF antibody in combination with cytotoxic therapies.

A genetic Xenopus laevis tadpole model to study lymphangiogenesis

Lymph vessels control fluid homeostasis, immunity and metastasis. Unraveling the molecular basis of lymphangiogenesis has been hampered by the lack of a small animal model that can be genetically manipulated. Here, we show that Xenopus tadpoles develop lymph vessels from lymphangioblasts or, through transdifferentiation, from venous endothelial cells. Lymphangiography showed that these lymph vessels drain lymph, through the lymph heart, to the venous circulation. Morpholino-mediated knockdown of the lymphangiogenic factor Prox1 caused lymph vessel defects and lymphedema by impairing lymphatic commitment. Knockdown of vascular endothelial growth factor C (VEGF-C) also induced lymph vessel defects and lymphedema, but primarily by affecting migration of lymphatic endothelial cells. Knockdown of VEGF-C also resulted in aberrant blood vessel formation in tadpoles. This tadpole model offers opportunities for the discovery of new regulators of lymphangiogenesis.

Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors

Transport parameters determine the access of drugs to tumors. However, technical difficulties preclude the measurement of these parameters deep inside living tissues. To this end, we adapted and further optimized two-photon fluorescence correlation microscopy (TPFCM) for in vivo measurement of transport parameters in tumors. TPFCM extends the detectable range of diffusion coefficients in tumors by one order of magnitude, and reveals both a fast and a slow component of diffusion. The ratio of these two components depends on molecular size and can be altered in vivo with hyaluronidase and collagenase. These studies indicate that TPFCM is a promising tool to dissect the barriers to drug delivery in tumors.

Robust 3-D Modeling of Vasculature Imagery Using Superellipsoids

This paper presents methods to model complex vasculature in three-dimensional (3-D) images using cylindroidal superellipsoids, along with robust estimation and detection algorithms for automated image analysis. This model offers an explicit, low-order parameterization, enabling joint estimation of boundary, centerlines, and local pose. It provides a geometric framework for directed vessel traversal, and extraction of topological information like branch point locations and connectivity. M-estimators provide robust region-based statistics that are used to drive the superellipsoid toward a vessel boundary. A robust likelihood ratio test is used to differentiate between noise, artifacts, and other complex unmodeled structures, thereby verifying the model estimate. The proposed methodology behaves well across scale-space, shows a high degree of insensitivity to adjacent structures and implicitly handles branching. When evaluated on synthetic imagery mimicking specific structural complexities in tumor microvasculature, it consistently produces ubvoxel accuracy estimates of centerlines and widths in the presence of closely-adjacent vessels, branch points, and noise. An edit-based validation demonstrated a precision level of 96.6% at a recall level of 95.4%. Overall, it is robust enough for large-scale application

Onset of Abnormal Blood and Lymphatic Vessel Function and Interstitial Hypertension in Early Stages of Carcinogenesis

Recent improvements in diagnostic methods have opened avenues for detection and treatment of (pre)malignant lesions at early stages. However, due to the lack of spontaneous tumor models that both mimic human carcinogenesis and allow direct optical imaging of the vasculature, little is known about the function of blood and lymphatic vessels during the early stages of cancer development. Here, we used a spontaneous carcinogenesis model in the skin of DNA polymerase eta-deficient mice and found that interstitial fluid pressure was already elevated in the hyperplastic/dysplastic stage. This was accompanied by angiogenic blood vasculature that exhibited altered permeability, vessel compression, and decreased alpha-smooth muscle actin-positive perivascular cell coverage. In addition, the lymphatic vessels in hyperplastic/dysplastic lesions were partly compressed and nonfunctional. These novel insights may aid early detection and treatment strategies for cancer.

Placenta growth factor overexpression inhibits tumor growth, angiogenesis, and metastasis by depleting vascular endothelial growth factor homodimers in orthotopic mouse models

The role of placenta growth factor (PlGF) in pathologic angiogenesis is controversial. The effects of PlGF on growth, angiogenesis, and metastasis from orthotopic tumors are not known. To this end, we stably transfected three human cancer cell lines (A549 lung, HCT116 colon, and U87-MG glioblastoma) with human plgf-2 full-length cDNA. Overexpression of PlGF did not affect tumor cell proliferation or migration in vitro. The growth of PlGF-overexpressing tumors grown orthotopically or ectopically was impaired in all three tumor models. This decrease in tumor growth correlated with a decrease in tumor angiogenesis. The PlGF-overexpressing tumors had decreased vessel density and increased vessel diameter, but vessel permeability was not different from the parental tumors. Tumors overexpressing PlGF exhibited higher levels of PlGF homodimers and PlGF/vascular endothelial growth factor (VEGF) heterodimers but decreased levels of VEGF homodimers. Our study shows that PlGF overexpression decreases VEGF homodimer formation and inhibits tumor progression.

Robust 3-D modeling of tumor microvasculature using superellipsoids

This paper presents automated methods for robust modeling and analysis of 3-D tumor microvasculature. Our methodology uses a cylindroidal superellipsoid to model localized segments of vasculature. The proposed vessel model has an explicit, low-order parameterization, allowing for joint estimation of boundary and centerline information, thereby approximating the medial axis. Further, this explicit parameterization provides a geometric framework for traversing vessels in a directed manner. Topological information like branch point location and connectivity is provided as a side effect. The proposed methodology behaves quite well across scalespace, shows a high degree of insensitivity to adjacent structures and implicitly handles branching. Exemplar results are presented from a pre-clinical study of tumor microvasculature in mice

Complexity analysis of angiogenesis vasculature

Tumor vasculature has a high degree of irregularity as compared to normal vasculature. The quantification of the morphometric complexity in tumor images can be useful in diagnosis. Also, it is desirable in several other medical applications to have an automated complexity analysis to aid in diagnosis and prognosis under treatment. e.g. in diabetic retinopathy and in arteriosclerosis. In addition, prior efforts at segmentation of the tumor vasculature using matched filtering, template matching and splines have been hampered by the irregularity of these vessels. We try to solve both problems by introducing a novel technique for vessel detection, followed by a tracing-independent complexity analysis based on a combination of ideas. First, the vessel cross-sectional profile is modeled using a continuous and everywhere differentiable family of super-Gaussian curves. This family generates rectangular profiles that can accurately localize the vessel boundaries in microvasculature images. Second, a robust non-linear regression algorithm based on M-estimators is used to estimate the parameters that optimally characterize the vessel’s shape. A framework for the quantitative analysis of the complexity of the vasculature based on the vessel detection is presented. A set of measures that quantify the complexity are proposed viz. Squared Error, Entropy-based and Minimum Description Length-based Shape Complexities. They are completely automatic and can deal with complexities of the entire vessel unlike existing tortuousity measures which deal only with vessel centerlines. The results are validated using carefully constructed phantom and real image data with ground truth information from an expert observer.