Non-Uniform Plasma Leakage Affects Local Hematocrit and Blood Flow: Implications for Inflammation and Tumor Perfusion

Vessel leakiness is a hallmark of inflammation and cancer. In inflammation, plasma extravasation and leukocyte adhesion occur in a coordinated manner to enable the immune response, but also to maintain tissue perfusion. In tumors, similar mechanisms operate, but they are not well regulated. Therefore, blood perfusion in tumors is non-uniform, and delivery of blood-borne therapeutics is difficult. In order to analyze the interplay among plasma leakage, blood viscosity, and vessel geometry, we developed a mathematical model that explicitly includes blood cells, vessel branching, and focal leakage. The results show that local hemoconcentration due to plasma leakage can greatly increase the flow resistance in individual vascular segments, diverting flow to other regions. Similarly, leukocyte rolling can increase flow resistance by partially blocking flow. Vessel dilation can counter these effects, and likely occurs in inflammation to maintain blood flow. These results suggest that potential strategies for improving perfusion through tumor networks include (i) eliminating non-uniform plasma leakage, (ii) inhibiting leukocyte interactions, and (iii) preventing RBC aggregation in sluggish vessels. Normalization of tumor vessels by anti-angiogenic therapy may improve tumor perfusion via the first two mechanisms.     

Imaging angiogenesis and the microenvironment

Intravital microscopy has provided unprecedented insights into tumor pathophysiology, including angiogenesis and the microenvironment. Tumor vasculature shows an abnormal organization, structure, and function. Tumor vessels are leaky, blood flow is heterogeneous and often compromised. Vascular hyperpermeability and the lack of functional lymphatic vessels inside tumors causes elevation of interstitial fluid pressure in solid tumors. These abnormalities form physiological barriers to the delivery of therapeutic agents to tumors and also lead to a hostile microenvironment characterized by hypoxia and acidosis, which hinders the effectiveness of anti-tumor treatments such as radiation therapy and chemotherapy. In addition, host-tumor interactions regulate expression of pro- and anti-angiogenic factors, resulting in pathophysiological characteristics of the tumor. On the other hand, in a physiological setting, angiogenic vessels become mature and form long-lasting functional units. Restoring the balance of pro- and anti-angiogenic factors in tumors may "normalize" tumor vasculature and thus improve its function. Administration of cytotoxic therapy during the vascular normalization would enhance its efficacy.     

抗血管生成药物在非小细胞肺癌中的应用及研究进展

新生血管的形成是肿瘤生长、进展、转移的基础,此过程涉及各类受体介导的细胞信号通路,其中刺激血管生成作用最强的生长因子是血管内皮生长因子（VEGF）。随着VEGF作用的深入认识和血管靶向治疗的临床实践,以抗新生血管为核心的治疗策略取得显著成效。现将抗血管药物的作用机制及其在治疗非小细胞肺癌中的研究进展作简要综述。     

Mathematical modeling of brain tumors: effects of radiotherapy and chemotherapy

Gliomas, the most common primary brain tumors, are diffusive and highly invasive. The standard treatment for brain tumors consists of a combination of surgery, radiation therapy and chemotherapy. Over the past few years, mathematical models have been applied to study untreated and treated brain tumors. In an effort to improve treatment strategies, we consider a simple spatio-temporal mathematical model, based on proliferation and diffusion, that incorporates the effects of radiotherapeutic and chemotherapeutic treatments. We study the effects of different schedules of radiation therapy, including fractionated and hyperfractionated external beam radiotherapy, using a generalized linear quadratic (LQ) model. The results are compared with published clinical data. We also discuss the results for combination therapy (radiotherapy plus temozolomide, a new chemotherapy agent), as proposed in recent clinical trials. We use the model to predict optimal sequencing of the postoperative (combination of radiotherapy and adjuvant, neo-adjuvant or concurrent chemotherapy) treatments for brain tumors.     

Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved α-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated α-smooth muscle actin-expression during recovery reflected the recruitment of α-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1α expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.     

血管生成抑制因子与淋巴管生成抑制因子

应用血管或淋巴管生成抑制因子破坏或抑制肿瘤的血管和淋巴管的生成,从而有效地阻止肿瘤 的生长和转移,进而达到治疗肿瘤的目的,是近些年发展起来的肿瘤的抗血管和抗淋巴管生成疗法。该法使 肿瘤的血管和淋巴管系统成为一个崭新的、有希望的抗肿瘤的靶点。人们正致力于开发和研究安全、有效、经 济、实用的血管和淋巴管生成抑制药物,该类药物被称为肿瘤血管或淋巴管生成抑制剂。     

Interferon-α as angiogenesis inhibitor: Learning from tumor models

Interferon-α (IFN-α), a cytokine with marked therapeutic activity in transplantable tumor models, has been identified as powerful angiogenesis inhibitor. The effects of IFN-α on the vasculature have been mainly attributed to inhibition of basic fibroblast growth factor production by tumor cells or downregulation of IL-8 and vascular endothelial growth factor gene expression. Moreover, IFN-α has direct effects on endothelial cells (EC), including impairment of their proliferation and migration. The gene expression profile induced by IFN-α in EC has recently been defined, and it was found that several genes encoding negative regulators of angiogenesis are upmodulated, thus providing a potential amplification mechanism for this biological activity. The anti-angiogenic effects of IFN-α appear to be associated with increased hypoxia and ischemic necrosis in subcutaneous xenograft models, whereas in transgenic mouse models, IFN-α may simultaneously interfere with both blood vessels and tumor cell proliferation, leading to regression of tumors without necrosis. The consequences of IFN-α therapy on the invasive and metastatic behavior of tumor cells are currently unknown. Finally, as effective anti-angiogenic therapy with IFN-α demands sustained localized production of this cytokine, innovative strategies of targeted delivery of the IFN-α gene into tumors are discussed.     

Normalization of the vasculature for treatment of cancer and other diseases

New vessel formation (angiogenesis) is an essential physiological process for embryologic development, normal growth, and tissue repair. Angiogenesis is tightly regulated at the molecular level. Dysregulation of angiogenesis occurs in various pathologies and is one of the hallmarks of cancer. The imbalance of pro- and anti-angiogenic signaling within tumors creates an abnormal vascular network that is characterized by dilated, tortuous, and hyperpermeable vessels. The physiological consequences of these vascular abnormalities include temporal and spatial heterogeneity in tumor blood flow and oxygenation and increased tumor interstitial fluid pressure. These abnormalities and the resultant microenvironment fuel tumor progression, and also lead to a reduction in the efficacy of chemotherapy, radiotherapy, and immunotherapy. With the discovery of vascular endothelial growth factor (VEGF) as a major driver of tumor angiogenesis, efforts have focused on novel therapeutics aimed at inhibiting VEGF activity, with the goal of regressing tumors by starvation. Unfortunately, clinical trials of anti-VEGF monotherapy in patients with solid tumors have been largely negative. Intriguingly, the combination of anti-VEGF therapy with conventional chemotherapy has improved survival in cancer patients compared with chemotherapy alone. These seemingly paradoxical results could be explained by a "normalization" of the tumor vasculature by anti-VEGF therapy. Preclinical studies have shown that anti-VEGF therapy changes tumor vasculature towards a more "mature" or "normal" phenotype. This "vascular normalization" is characterized by attenuation of hyperpermeability, increased vascular pericyte coverage, a more normal basement membrane, and a resultant reduction in tumor hypoxia and interstitial fluid pressure. These in turn can lead to an improvement in the metabolic profile of the tumor microenvironment, the delivery and efficacy of exogenously administered therapeutics, the efficacy of radiotherapy and of effector immune cells, and a reduction in number of metastatic cells shed by tumors into circulation in mice. These findings are consistent with data from clinical trials of anti-VEGF agents in patients with various solid tumors. More recently, genetic and pharmacological approaches have begun to unravel some other key regulators of vascular normalization such as proteins that regulate tissue oxygen sensing (PHD2) and vessel maturation (PDGFRβ, RGS5, Ang1/2, TGF-β). Here, we review the pathophysiology of tumor angiogenesis, the molecular underpinnings and functional consequences of vascular normalization, and the implications for treatment of cancer and nonmalignant diseases.     

Effects of targeting the VEGF and PDGF pathways in diffuse orthotopic glioma models

Currently available compounds that interfere with VEGF-A signalling effectively inhibit angiogenesis in gliomas, but influence diffuse infiltrative growth to a much lesser extent. Development of a functional tumour vascular bed not only involves VEGF-A but also requires platelet-derived growth factor receptor-β (PDGFRβ), which induces maturation of tumour blood vessels. Therefore, we tested whether combined inhibition of VEGFR and PDGFRβ increases therapeutic benefit in the orthotopic glioma xenograft models E98 and E473, both displaying the diffuse infiltrative growth that is characteristically observed in most human gliomas. We used bevacizumab and vandetanib as VEGF(R) inhibitors, and sunitinib to additionally target PDGFRβ. We show that combination therapy of sunitinib and vandetanib does not improve therapeutic efficacy compared to treatment with sunitinib, vandetanib or bevacizumab alone. Furthermore, all compounds induced reduction of vessel leakage in compact E98 tumour areas, resulting in decreased detectability of these mostly infiltrative xenografts in Gd-DTPA-enhanced MRI scans. These data show that inhibition of VEGF signalling cannot be optimized by additional PDGFR inhibition and support the concept that diffuse infiltrative areas in gliomas are resistant to anti-angiogenic therapy.     

Combination treatment significantly enhances the efficacy of antitumor therapy by preferentially targeting angiogenesis

Radiotherapy is one of the most widely used cancer treatments, but it is often unsuccessful due to the development of radioresistance by tumor cells and endothelial cells (ECs) lining the tumor blood vessels. We have previously shown that ECs are protected against ionizing irradiation primarily via the activation of the phosphoinositide 3-kinase (PI3 K)-Akt-Bcl-2 survival pathway. Here we report that combination treatment with low doses of PI3 K inhibitor (LY294002), cisplatin and gamma-irradiation resulted in significantly higher (61%) EC death as compared to each agent used alone (17, 17 and 11%, respectively). This combination treatment was equally effective in inducing tumor cell death (72%). Combination treatment also significantly inhibited EC tube formation in Matrigel (75%) as compared to each of the agents used alone (8, 8 and 18% for LY294002, cisplatin and gamma-irradiation, respectively). In our in vivo severe combined immunodeficient mouse model of human tumor growth and angiogenesis, combination treatment with low doses of LY294002, cisplatin and irradiation significantly inhibited the growth of human oral squamous carcinoma (OSCC-3) as well as prostate cancer (LnCap). The combination therapy was also very effective in inhibiting tumor angiogenesis where it showed a greater than 90% decrease in neovascularization. In contrast, combination treatment showed only a 29% inhibition of physiological angiogenesis. Taken together, these results suggest a potentially novel strategy to overcome the resistance in ECs lining tumor blood vessels, thereby enhancing the effectiveness of the radiation and chemotherapy. Moreover, this strategy of using a combination of low doses of PI3K/Akt inhibitor, cisplatin and radiation has the potential of significantly decreasing untoward side effects associated with the maximum tolerated doses of radiation and chemotherapy while maintaining their therapeutic efficacy.     

Heterogeneity of Tie2 expression in tumor microcirculation: influence of cancer type, implantation site, and response to therapy

To evaluate the expression of the Tie2/Tek tyrosine kinase receptor in tumor blood vessels, we examined Tie2lacZ(+)/RAG1(-) mice. There was considerable heterogeneity (Tie2-negative, Tie2-positive, or Tie2-composite blood vessels) in subcutaneous xenografts of human colorectal carcinoma (HCT116; 97.5% Tie2-positive vessels) versus human melanoma (WM115; 75.9% Tie2-positive vessels). Similar patterns of Tie2 expression occurred in abdominal metastases derived from the same cell lines. Immunostaining for endothelial markers and Tie2 revealed that endogenous protein levels corresponded with transgene activity. Endothelial cells were confirmed to be of mouse origin through triple immunofluorescence staining with mouse antiserum to human nuclei, isolectin GS-IB(4), and anti-Tie2. Similar Tie2 heterogeneity was observed in clinical specimens from a variety of human cancers, including malignant melanoma and colorectal carcinoma. We also examined the effect of Tek-Delta Fc anti-angiogenic therapy on tumor growth and Tie2 expression patterns in HCT116 and WM115 subcutaneous xenografts. Tek-Delta induced extensive tumor regression in HCT116 tumors and concomitant reductions in Tie2-expressing blood vessels. However, no significant responses were seen in Tek-Delta-treated WM115 tumors. Thus, vascular heterogeneity of Tie2 expression is cancer-type specific, suggesting that the tumor microenvironment and/or direct cancer cell interactions influence Tie2 endothelial expression.     

Angiogenesis and the role of epigenetics in metastasis

The major obstacle to devising effective ways to treat cancer is its heterogeneity and genetic instability. It was originally postulated that targeting the process of tumor angiogenesis could circumvent this problem, as it involves genetically stable epigenetically controlled host stroma. Thus, anti-angiogenic approaches should be applicable across various tumor types and organ sites, including metastases. However, early clinical experience with this therapy revealed unexpectedly distinct responses between different tumors and organ sites. Here we propose that the heterogeneity of pre-clinical and clinical results obtained with anti-angiogenic agents stems from the deep functional linkage that may exist between genetic and epigenetic tumor progression. Thus, epigenetic processes regulating tumor associated host blood vessels (such as tumor microenvironment) display unstable, heterogeneous and progressive characteristics to an extent comparable with (and causally linked to) the instability of the cancer cell genome. As well, many known epigenetic factors (such as hypoxia, inflammation, expression of growth factors, etc.) may have genetic causes and consequences (e.g., oncogene expression, loss of tumor suppressor genes). This reciprocal interrelationship and heterogeneity may translate into site and stage specific changes in angiogenesis regulation, and angiogenesis dependence, ultimately to changes in the metastatic ability/efficiency of cancer cells, even in the same patient. A better understanding of the linkage between genetic and epigenetic events in growth and metastasis of various cancers may result in more effective use of anti-angiogenic therapy in future. This revised version was published online in July 2006 with corrections to the Cover Date.     

A reinforced random walk model of tumour angiogenesis and anti-angiogenic strategies.

It is now well accepted that the growth of a tumour beyond approximately 2 mm in diameter is dependent on its ability to induce the growth of new blood vessels, a process called angiogenesis. This has raised hope that an anti-angiogenic treatment may be effective in the fight against cancer. Here we formulate, using the theory of reinforced random walks, an individual cell-based mathematical model of tumour angiogenesis in response to a diffusible angiogenic factor. The early stages of angiogenesis, in which endothelial cells (EC) escape the parent vessel and invade the extra-cellular matrix, are included in the model, as are the action of a proteolytic enzyme, EC proliferation and capillary branching and anastomosis. The anti-angiogenic potential of angiostatin, a known inhibitor of angiogenesis, is also examined. The capillary networks predicted by the model are in qualitative agreement with experimental observations. Proteolysis and proliferation are shown to be crucial for vascularization, whilst angiostatin is seen to be capable of limiting capillary growth.     

Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment

Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.     

Higher vessel densities in retinoblastoma with local invasive growth and metastasis

In this study, the importance of angiogenesis (the growth of new blood vessels from existing ones) for the growth of retinoblastoma was investigated by a retrospective immunohistochemical analysis. An individual vessel index for each tumor was determined using the endothelial-specific antibody CD 31 for vessel staining. The obtained data were correlated with clinical features, pathohistological characteristics, and the presence of metastasis. In 107 retinoblastomas collected between 1980 and 1990, we found no difference in the vessel densities between uni- and bilateral retinoblastomas (P = 0.41). However, tumors that had invaded the chorioid and/or the optic nerve statistically showed higher vessel densities than tumors without local invasive growth (P = 0.05 and P = 0.024). A tendency of higher vessel densities in retinoblastomas presenting with metastasis at the time of diagnosis was observed (P = 0.11). Based on this observation, we proceeded to examine all retinoblastomas presenting with metastasis at the time of diagnosis. These included patients that were treated between 1968 and 1993. The 18 investigated retinoblastomas had significantly higher vessel densities than all other retinoblastomas presenting without metastasis (P = 0.025). Our data indicate that in retinoblastoma, blood vessels are essential for local and systemic invasive growth. Therefore, an anti-angiogenic therapy could be considered in the multimodal therapy concept for retinoblastomas with invasive growth, both locally and systemically.     

Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors

Often described as incomplete or absent, the basement membrane of blood vessels in tumors has attracted renewed attention as a source of angiogenic and anti-angiogenic molecules, site of growth factor binding, participant in angiogenesis, and potential target in cancer therapy. This study evaluated the composition, extent, and structural integrity of the basement membrane on blood vessels in three mouse tumor models: spontaneous RIP-Tag2 pancreatic islet tumors, MCa-IV mammary carcinomas, and Lewis lung carcinomas. Tumor vessels were identified by immunohistochemical staining for the endothelial cell markers CD31, endoglin (CD105), vascular endothelial growth factor receptor-2, and integrin alpha5 (CD49e). Confocal microscopic studies revealed that basement membrane identified by type IV collagen immunoreactivity covered >99.9% of the surface of blood vessels in the three tumors, just as in normal pancreatic islets. Laminin, entactin/nidogen, and fibronectin immunoreactivities were similarly ubiquitous on tumor vessels. Holes in the basement membrane, found by analyzing 1- micro m confocal optical sections, were <2.5 micro m in diameter and involved only 0.03% of the vessel surface. Despite the extensive vessel coverage, the basement membrane had conspicuous structural abnormalities, including a loose association with endothelial cells and pericytes, broad extensions away from the vessel wall, and multiple layers visible by electron microscopy. Type IV collagen-immunoreactive sleeves were also present on endothelial sprouts, supporting the idea that basement membrane is present where sprouts grow and regress. These findings indicate that basement membrane covers most tumor vessels but has profound structural abnormalities, consistent with the dynamic nature of endothelial cells and pericytes in tumors.     

Local delivery of recombinant adenovirus expressing hepatitis B virus X protein and interleukin-12 results in antitumor effects via inhibition of hepatoma cell growth and intervention of tumor microenvironment

Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. Our previous studies have demonstrated that hepatitis B virus X protein (HBx) was able to inhibit the growth of HCC cells via inducing apoptosis and inhibiting tumor angiogenesis. Interleukin-12 (IL-12) is a disulfide-linked heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. In this study, to further investigate the regulatory effect of IL-12 on HBx-mediated intervention of hepatoma microenvironment especially on intervention of neovessels and immune microenvironment, we constructed the recombinant adenovirus expressing HBx and mouse IL-12 named Ad-HBx-mIL-12. HBx-mIL-12 could effectively suppress tumor growth and induce apoptosis in vivo. Moreover, treatment with Ad-HBx-mIL-12 not only induced a massive accumulation of immune cells (CD8(+) T leukocytes, macrophages and dendritic cells) in tumors in situ, also apparently reduced the number of angiogenic blood vessels within tumor tissues. These results suggest that HBx-mIL-12 can not only induce cell cycle arrest and apoptosis in HCC cells, but also effectively shift the tumor microenvironment from pro-oncogenic to antitumor through recruitment of immune cells and inhibiting stromal cell growth, such as vascular endothelial cells.     

Anti-angiogenic alternatives to VEGF blockade

Angiogenesis is a major requirement for tumour formation and development. Anti-angiogenic treatments aim to starve the tumour of nutrients and oxygen and also guard against metastasis. The main anti-angiogenic agents to date have focused on blocking the pro-angiogenic vascular endothelial growth factors (VEGFs). While this approach has seen some success and has provided a proof of principle that such anti-angiogenic agents can be used as treatment, the overall outcome of VEGF blockade has been somewhat disappointing. There is a current need for new strategies in inhibiting tumour angiogenesis; this article will review current and historical examples in blocking various membrane receptors and components of the extracellular matrix important in angiogenesis. Targeting these newly discovered pro-angiogenic proteins could provide novel strategies for cancer therapy.     

妇科肿瘤的血管形成和抗血管形成治疗研究进展

肿瘤的生长和转移需要新生血管提供血液,抗肿瘤血管形成是一种新的抗肿瘤策略。本文对妇科肿瘤领域中有关肿瘤微血管密度的检测及其预后价值、肿瘤组织血管形成活性分子的检测、血清血管形成分子的检测、抗肿瘤血管形成治疗尝试及其在妇科肿瘤治疗中的地位等问题进行了综述。