Explicit Separation of Growth and Motility in a New Tumor Cord Model

We investigate a new model of tumor growth in which cell motility is considered an explicitly separate process from growth. Bulk tumor expansion is modeled by individual cell motility in a density-dependent diffusion process. This model is implemented in the context of an in vivo system, the tumor cord. We investigate numerically microscale density distributions of different cell classes and macroscale whole tumor growth rates as functions of the strength of transitions between classes. Our results indicate that the total tumor growth follows a classical von Bertalanffy growth profile, as many in vivo tumors are observed to do. This provides a quick validation for the model hypotheses. The microscale and macroscale properties are both sensitive to fluctuations in the transition parameters, and grossly adopt one of two phenotypic profiles based on their parameter regime. We analyze these profiles and use the observations to classify parameter regimes by their phenotypes. This classification yields a novel hypothesis for the early evolutionary selection of the metastatic phenotype by selecting against less motile cells which grow to higher densities and may therefore induce local collapse of the vascular network.     

Mathematical Modeling of Cellular Cross-Talk Between Endothelial and Tumor Cells Highlights Counterintuitive Effects of VEGF-Targeted Therapies

Tumor growth and progression are critically dependent on the establishment of a vascular support system. This is often accomplished via the expression of pro-angiogenic growth factors, including members of the vascular endothelial growth factor (VEGF) family of ligands. VEGF ligands are overexpressed in a wide variety of solid tumors and therefore have inspired optimism that inhibition of the different axes of the VEGF pathway—alone or in combination—would represent powerful anti-angiogenic therapies for most cancer types. When considering treatments that target VEGF and its receptors, it is difficult to tease out the differential anti-angiogenic and anti-tumor effects of all combinations experimentally because tumor cells and vascular endothelial cells are engaged in a dynamic cross-talk that impacts key aspects of tumorigenesis, independent of angiogenesis. Here we develop a mathematical model that connects intracellular signaling responsible for both endothelial and tumor cell proliferation and death to population-level cancer growth and angiogenesis. We use this model to investigate the effect of bidirectional communication between endothelial cells and tumor cells on treatments targeting VEGF and its receptors both in vitro and in vivo. Our results underscore the fact that in vitro therapeutic outcomes do not always translate to the in vivo situation. For example, our model predicts that certain therapeutic combinations result in antagonism in vivo that is not observed in vitro. Mathematical modeling in this direction can shed light on the mechanisms behind experimental observations that manipulating VEGF and its receptors is successful in some cases but disappointing in others.     

Antibiotic bedaquiline effectively targets growth,survival and tumor angiogenesis of lung cancer through suppressing energy metabolism

Tumor angiogenesis plays essential roles during lung cancer progression and metastasis. Therapeutic agent that targets both tumor cell and vascular endothelial cell may achieve additional anti-tumor efficacy. We demonstrate that bedaquiline, a FDA-approved antibiotic drug, effectively targets lung cancer cells and angiogenesis. Bedaquiline dose-dependently inhibits proliferation and induces apoptosis of a panel of lung cancer cell lines regardless of subtypes and molecular heterogeneity. Bedaquiline also inhibits capillary network formation of human lung tumor associated-endothelial cell (HLT-EC) on Matrigel and its multiple functions, such as spreading, proliferation and apoptosis, even in the presence of vascular endothelial growth factor (VEGF). We further demonstrate that bedaquiline acts on lung cancer cells and HLT-EC via inhibiting mitochondrial respiration and glycolysis, leading to ATP reduction and oxidative stress. Consistently, oxidative damage on DNA, protein and lipid were detected in cells exposed to bedaquiline. Importantly, the results obtained in in vitro cell culture are reproducible in in vivo xenograft lung cancer mouse model, confirming that bedaquiline suppresses lug tumor growth and angiogenesis, and increases oxidative stress. Our findings demonstrating that energy depletion is effectively against lung tumor cells and angiogenesis. Our work also provide pre-clinical evidence to repurpose antibiotic bedaquiline for lung cancer treatment.     

Influence of interstitial fluid dynamics on growth and therapy of angiogenic tumor. Analysis by mathematical model

We have developed a spatially distributed mathematical model of angiogenic tumor growth in tissue with account of interstitial fluid dynamics and bevacizumab monotherapy. In this model the process of neovascularization is initiated by tumor cells in a state of metabolic stress, vascular endothelial growth factor (VEGF) being its main mediator. The model takes into consideration the convection flows arising in dense tissue due to active proliferation and migration of tumor cells as well as interstitial fluid inflow from blood vascular system, its outflow through lymphatic system and redistribution in the area of tumor growth. The work considers the diffusive approximation of interstitial fluid dynamics in tumor and normal tissue. Numerical study of the model showed that in absence of therapy a peritumoral edema is formed due to the increase of interstitial fluid inflow from angiogenic capillaries. In the case of rapid interstitial fluid outflow through lymphatic system and its fast transport from necrotic zone to normal tissue the regimes of full growth stop are observed in case of low-invasive tumor. Under bevacizumab monotherapy the peritumoral edema vanishes and low-invasive tumor may not only decelerate its growth, but also start shrinking for a large range of parameters.     

Opposing roles for CD34 in B16 melanoma tumor growth alter early stage vasculature and late stage immune cell infiltration

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34(-/-) mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34(-/-) mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34(-/-) mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.     

Predictive Modeling of Neuroblastoma Growth Dynamics in Xenograft Model After Bevacizumab Anti-VEGF Therapy

Neuroblastoma is the leading cause of cancer death in young children. Although treatment for neuroblastoma has improved, the 5-year survival rate of patients still remains less than half. Recent studies have indicated that bevacizumab, an anti-VEGF drug used in treatment of several other cancer types, may be effective for treating neuroblastoma as well. However, its effect on neuroblastoma has not been well characterized. While traditional experiments are costly and time-consuming, mathematical models are capable of simulating complex systems quickly and inexpensively. In this study, we present a model of vascular tumor growth of neuroblastoma IMR-32 that is complex enough to replicate experimental data across a range of tumor cell properties measured in a suite of in vitro and in vivo experiments. The model provides quantitative insight into tumor vasculature, predicting a linear relationship between vasculature and tumor volume. The tumor growth model was coupled with known pharmacokinetics and pharmacodynamics of the VEGF blocker bevacizumab to study its effect on neuroblastoma growth dynamics. The results of our model suggest that total administered bevacizumab concentration per week, as opposed to dosage regimen, is the major determining factor in tumor suppression. Our model also establishes an exponentially decreasing relationship between administered bevacizumab concentration and tumor growth rate.     

Antisense Oligonucleotide against hTERT (Cantide) Inhibits Tumor Growth in an Orthotopic Primary Hepatic Lymphoma Mouse Model

Background

Human xenograft models, resulting from orthotopic transplantation (implantation into the anatomically correct site) of histologically intact tissue into animals, are important for investigating local tumor growth, vascular and lymphatic invasion at the primary tumor site and metastasis.

Methodology/Principal Findings

We used surgical orthotopic transplantation to establish a nude mouse model of primary hepatic lymphoma (PHL), HLBL-0102. We performed orthotopic transfer of the HLBL-0102 tumor for 42 generations and characterized the tumor cells. The maintenance of PHL characteristics were supported by immunohistochemical and cytogenetic analysis. We also report the antitumor effect of Cantide, an antisense phosphorothioate oligonucleotide against hTERT, on the growth of HLBL-0102 tumors. We showed a significant, dose-dependent inhibition of tumor weight and serum LDH activity in the orthotopically transplanted animals by Cantide. Importantly, survival was prolonged in Cantide-treated HLBL-0102 tumor-bearing mice when compared to mock-treated mice.

Conclusions/Significance

Our study provided the basis for the development of a clinical trial protocol to treat PHL.     

Nonlinear simulation of tumor necrosis,neo-vascularization and tissue invasion via an adaptive finite-element/level-set method

We present a multi-scale computer simulator of cancer progression at the tumoral level, from avascular stage growth, through the transition from avascular to vascular growth (neo-vascularization), and into the later stages of growth and invasion of normal tissue. We use continuum scale reaction-diffusion equations for the growth component of the model, and a combined continuum-discrete model for the angiogenesis component. We use the level set method for describing complex topological changes observed during growth such as tumor splitting and reconnection, and capture of healthy tissue inside the tumor. We use an adaptive, unstructured finite element mesh that allows for finely resolving important regions of the computational domain such as the necrotic rim, the tumor interface and around the capillary sprouts. We present full nonlinear, two-dimensional simulations, showing the potential of our virtual cancer simulator. We use microphysical parameters characterizing malignant glioma cells, obtained from recent in vitro experiments from our lab and from clinical data, and provide insight into the mechanisms leading to infiltration of the brain by the cancer cells. The results indicate that diffusional instability of tumor mass growth and the complex interplay with the developing neo-vasculature may be powerful mechanisms for tissue invasion.     

Antiangiogenic and proapoptotic activity of a novel glycoprotein from U. indica is mediated by NF-kappaB and Caspase activated DNase in ascites tumor model

We have identified a novel glycoprotein from Urginea indica bulbs with potent in vivo antitumor activity against growth of an ascites tumor, mouse mammary carcinoma. In this paper we report characterization of a 29 kDa glycoprotein from U. indica and demonstrate the mechanism of antiangiogenic and proapoptotic activity. N-terminal sequence of the high performance liquid chromatography (HPLC) pure glycoprotein showed sequence homology to an extent of 40-50% with known angiogenesis inhibitor and apoptosis-inducing protein from C. elegans and G. gallus respectively. Our results on antiangiogenic property of the glycoprotein include inhibition of in vivo angiogenesis assays, decreased micro vessel density count and CD31 antigen staining in 29 kDa glycoprotein treated mice peritoneum. In vitro inhibition of vascular endothelial growth factor induced proliferation of human umbilical vein endothelial cells (HUVECs) by the glycoprotein further supports its antiangiogenic activity. The mechanism of antiangiogenesis involved inhibition of translocation of nuclear factor kappa B to the nucleus resulting in decreased expression of vascular endothelial growth factor gene as is demonstrated by our results on quantification of vascular endothelial growth factor levels in the glycoprotein treated tumor bearing mice. Our results on activation of Caspase-3 with concomitant translocation of caspase activated DNase to the tumor cell nuclei resulting in DNA fragmentation induced by the glycoprotein in vivo clearly demonstrated a parallel proapoptotic activity of the glycoprotein.     

VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations

K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras^LA1 model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations.     

VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations

K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras^LA1 model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations.     

3D Multi-Cell Simulation of Tumor Growth and Angiogenesis

We present a 3D multi-cell simulation of a generic simplification of vascular tumor growth which can be easily extended and adapted to describe more specific vascular tumor types and host tissues. Initially, tumor cells proliferate as they take up the oxygen which the pre-existing vasculature supplies. The tumor grows exponentially. When the oxygen level drops below a threshold, the tumor cells become hypoxic and start secreting pro-angiogenic factors. At this stage, the tumor reaches a maximum diameter characteristic of an avascular tumor spheroid. The endothelial cells in the pre-existing vasculature respond to the pro-angiogenic factors both by chemotaxing towards higher concentrations of pro-angiogenic factors and by forming new blood vessels via angiogenesis. The tumor-induced vasculature increases the growth rate of the resulting vascularized solid tumor compared to an avascular tumor, allowing the tumor to grow beyond the spheroid in these linear-growth phases. First, in the linear-spherical phase of growth, the tumor remains spherical while its volume increases. Second, in the linear-cylindrical phase of growth the tumor elongates into a cylinder. Finally, in the linear-sheet phase of growth, tumor growth accelerates as the tumor changes from cylindrical to paddle-shaped. Substantial periods during which the tumor grows slowly or not at all separate the exponential from the linear-spherical and the linear-spherical from the linear-cylindrical growth phases. In contrast to other simulations in which avascular tumors remain spherical, our simulated avascular tumors form cylinders following the blood vessels, leading to a different distribution of hypoxic cells within the tumor. Our simulations cover time periods which are long enough to produce a range of biologically reasonable complex morphologies, allowing us to study how tumor-induced angiogenesis affects the growth rate, size and morphology of simulated tumors.     

The Vascular Basement Membrane as “Soil” in Brain Metastasis

Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Paget''s “seed and soil” concept. However, there is little direct evidence for this “neurotropic” growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the “soil” for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM) as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesion and invasion of malignant cells and was sufficient for tumor growth prior to any evidence of angiogenesis. Blockade or loss of the β1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies.     

MicroRNA-101 inhibits angiogenesis via COX-2 in endometrial carcinoma

Abnormal angiogenesis is critically involved in tumor progression and metastasis including endometrial cancer and is regulated by microRNAs such as microRNA-101 (miR-101). We hypothesize that miR-101 expression is disrupted in endometrial cancer and modulation of miR-101 levels is sufficient to regulate tumor growth through angiogenesis. We examined the expression levels of miR-101 and factors involved in angiogenesis in the patients with endometrial cancer. We also overexpressed or inhibited miR-101 in RL-95-2 cells and examined their effects on cell toxicity and tumor growth. Finally, we determined if miR-101 regulated tumorigenesis through cyclooxygenase-2 (COX-2). We found that miR-101 levels were significantly reduced. Factors involved in angiogenesis included vascular endothelial growth factor-A (VEGF-A), thrombospondin-1 (TSP-1), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and aromatase (P450arom), which were increased in endometrial carcinoma. Modulation of miR-101 level was sufficient to affect tumor growth. Finally, we found that the effects of miR-101 inhibition on tumor growth were suppressed by COX-2 inhibition. Our results suggest that modulating miR-101 and COX-2 levels or their activity may be a potential therapeutic strategy for endometrial cancer.     

Tumor Vascular Permeability to a Nanoprobe Correlates to Tumor-Specific Expression Levels of Angiogenic Markers

Background

Vascular endothelial growth factor (VEGF) receptor-2 is the major mediator of the mitogenic, angiogenic, and vascular hyperpermeability effects of VEGF on breast tumors. Overexpression of VEGF and VEGF receptor-2 is associated with the degree of pathomorphosis of the tumor tissue and unfavorable prognosis. In this study, we demonstrate that non-invasive quantification of the degree of tumor vascular permeability to a nanoprobe correlates with the VEGF and its receptor levels and tumor growth.

Methodology/Principal Findings

We designed an imaging nanoprobe and a methodology to detect the intratumoral deposition of a 100 nm-scale nanoprobe using mammography allowing measurement of the tumor vascular permeability in a rat MAT B III breast tumor model. The tumor vascular permeability varied widely among the animals. Notably, the VEGF and VEGF receptor-2 gene expression of the tumors as measured by qRT-PCR displayed a strong correlation to the imaging-based measurements of vascular permeability to the 100 nm-scale nanoprobe. This is in good agreement with the fact that tumors with high angiogenic activity are expected to have more permeable blood vessels resulting in high intratumoral deposition of a nanoscale agent. In addition, we show that higher intratumoral deposition of the nanoprobe as imaged with mammography correlated to a faster tumor growth rate. This data suggest that vascular permeability scales to the tumor growth and that tumor vascular permeability can be a measure of underlying VEGF and VEGF receptor-2 expression in individual tumors.

Conclusions/Significance

This is the first demonstration, to our knowledge, that quantitative imaging of tumor vascular permeability to a nanoprobe represents a form of a surrogate, functional biomarker of underlying molecular markers of angiogenesis.     

Aminopeptidase N (CD13): Expression,Prognostic Impact,and Use as Therapeutic Target for Tissue Factor Induced Tumor Vascular Infarction in Soft Tissue Sarcoma

Aminopeptidase N (CD13) is expressed on tumor vasculature and tumor cells. It represents a candidate for targeted therapy, e.g., by truncated tissue factor (tTF)-NGR, binding to CD13, and causing tumor vascular thrombosis. We analyzed CD13 expression by immunohistochemistry in 97 patients with STS who were treated by wide resection and uniform chemo-radio-chemotherapy. Using a semiquantitative score with four intensity levels, CD13 was expressed by tumor vasculature, or tumor cells, or both (composite value, intensity scores 1-3) in 93.9% of the STS. In 49.5% tumor cells, in 48.5% vascular/perivascular cells, and in 58.8%, composite value showed strong intensity score 3 staining. Leiomyosarcoma and synovial sarcoma showed low expression; fibrosarcoma and undifferentiated pleomorphic sarcoma showed high expression. We found a significant prognostic impact of CD13, as high expression in tumor cells or vascular/perivascular cells correlated with better relapse-free survival and overall survival. CD13 retained prognostic significance in multivariable analyses. Systemic tTF-NGR resulted in significant growth reduction of CD13-positive human HT1080 sarcoma cell line xenografts. Our results recommend further investigation of tTF-NGR in STS patients. CD13 might be a suitable predictive biomarker for patient selection.     

Real-time visualization and quantitation of vascular permeability in vivo: implications for drug delivery

The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer tissues. However, techniques for studying vascular delivery systems in vivo often require complex mammalian models and time-consuming, surgical protocols. The developing chicken embryo is a well-established model for human cancer that is easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were grown on the chorioallantoic membrane (CAM), a thin vascular membrane which overlays the growing chick embryo. The real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an interleukin-2 (IL-2) peptide fragment or vascular endothelial growth factor (VEGF). VEGF treatment increased vascular leak in the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be manipulated to improve chemotherapeutic targeting to tumors.