Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging,confocal microscopy,and histological tracking of triple-labeled albumin

Increased expression of vascular endothelial growth factor (VEGF) has been associated with increased lymph node metastases. The aim of this work was to determine whether VEGF-induced hyperpermeability affects peritumor interstitial convection and lymphatic drain, thus linking this growth factor with lymphatic function. Noninvasive imaging of lymphatic function induced by vascular hyperpermeability was achieved by following the distribution of albumin triple-labeled with biotin, fluorescein, and gadolinium-diethylene triamine pentaacetic acid. This contrast material allowed for multimodality imaging using magnetic resonance imaging (MRI), confocal microscopy, and histology. Overexpression of VEGF in C6-pTET-VEGF165 tumors, inoculated in hind limbs of nude mice, elevated vascular permeability, interstitial convection, and lymphatic drain. These were manifested in dynamic MRI measurements by outward flux of the contrast material, the rate of which correlated with tumor volume followed by directional flow toward the popliteal lymph node. Avidin-chase, namely i.v. administration of avidin, was applied for inducing rapid clearance of the intravascular biotinylated contrast material, thus allowing early experimental separation between vascular leak and lymphatic drain. Repeated MRI measurements of the same mice were conducted 48 h after withdrawal of VEGF by addition of tetracycline to the drinking water. VEGF withdrawal decreased tumor blood-plasma volume fraction by 43%, reduced tumor permeability by 75%, and abolished interstitial convection of the contrast material. Histological sections and whole-mount confocal microscopy confirmed VEGF-induced changes in permeability and interstitial accumulation of the contrast material, as well as uptake of the contrast material into peritumor lymphatic vessels. These results revealed a direct link between expression of VEGF165 and peritumor lymphatic drain, thus suggesting a possible role for tumor-derived VEGF in metastatic spread to sentinel lymph nodes.     

Sequencing surgery,radiotherapy and chemotherapy: insights from a mathematical analysis

We present results from a mathematical analysis that is aimed at finding the best way to sequence the three traditional cancer treatments: surgery (S), chemotherapy (C), and radiotherapy (R). The mathematical model tracks the temporal evolution of the primary tumor and its associated metastases, and incorporates the primary tumor's effect on the dormancy and growth of the metastases. We show that the SCR schedule (i.e., surgery followed by chemotherapy followed by radiotherapy) achieves a higher cure probability than SRC if the primary tumor is sufficiently large or if the metastatic population is sufficiently large relative to the primary tumor. We also show that a novel schedule, SRCR, which splits the radiotherapy regimen into two disjoint portions, is optimal among all schedules, provided that the patient's dormant metastatic tumors do not become vascularized within about 40 days after surgery.     

Predicting success or failure of immunotherapy for cancer: insights from a clinically applicable mathematical model

The objective of this study was to create a clinically applicable mathematical model of immunotherapy for cancer and use it to explore differences between successful and unsuccessful treatment scenarios. The simplified predator-prey model includes four lumped parameters: tumor growth rate, g; immune cell killing efficiency, k; immune cell signaling factor, λ; and immune cell half-life decay, μ. The predator-prey equations as functions of time, t, for normalized tumor cell numbers, y, (the prey) and immunocyte numbers, ×, (the predators) are: dy/dt = gy - kx and dx/dt = λxy - μx. A parameter estimation procedure that capitalizes on available clinical data and the timing of clinically observable phenomena gives mid-range benchmarks for parameters representing the unstable equilibrium case in which the tumor neither grows nor shrinks. Departure from this equilibrium results in oscillations in tumor cell numbers and in many cases complete elimination of the tumor. Several paradoxical phenomena are predicted, including increasing tumor cell numbers prior to a population crash, apparent cure with late recurrence, one or more cycles of tumor growth prior to eventual tumor elimination, and improved tumor killing with initially weaker immune parameters or smaller initial populations of immune cells. The model and the parameter estimation techniques are easily adapted to various human cancers that evoke an immune response. They may help clinicians understand and predict certain strange and unexpected effects in the world of tumor immunity and lead to the design of clinical trials to test improved treatment protocols for patients.     

Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice

BACKGROUND: Angiogenesis is essential for tumor growth and progression. Therefore, inhibition of angiogenesis is being studied as a new anticancer therapy. Because cytotoxic chemotherapy is more effective on rapidly growing tumors than on slowly growing tumors, it has been assumed that antiangiogenic therapy will also be effective only on rapidly growing, highly vascularized tumors. We compared the effects of two angiogenesis inhibitors, TNP-470 and angiostatin, on slowly growing, poorly vascularized and rapidly growing, highly vascularized human tumors in mice. METHODS: Slowly growing (RT-4) and rapidly growing (MGH-U1) human bladder carcinoma cell lines were grown in severe combined immunodeficiency mice. Established tumors were treated with one of the two angiogenesis inhibitors. Tumor volumes, vascularity, and proliferation indices were determined. The in vitro effects of TNP-470 and of angiostatin on the proliferation of RT-4 and MGH-U1 cells were also investigated. All statistical tests were two-sided. RESULTS: RT-4 and MGH-U1 tumor growth was statistically significantly inhibited by both angiogenesis inhibitors (P<.001). Both inhibitors decreased the blood vessel density in both tumor types but did not alter the in vivo proliferation indices of the tumors. TNP-470, but not angiostatin, marginally decreased the in vitro proliferation of MGH-U1 cells. CONCLUSION: Slowly growing, poorly vascularized tumors in animal models respond as well as rapidly growing, highly vascularized tumors to therapy with the angiogenesis inhibitors TNP-470 and angiostatin.     

Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor‐D

Lymphatic spread is an important clinical determinant in the prognosis of many human cancers. The lymphangiogenic factor vascular endothelial growth factor‐D (VEGF‐D) is implicated in the promotion of lymphatic metastasis through the development of lymphatic vessels in some human cancers. In this study, we developed an anti‐VEGF‐D monoclonal antibody, cVE199, and investigated its in vitro properties, in vivo effects against tumors and possible target indications to evaluate its potential as a therapeutic antibody. The cVE199 molecule was revealed to have a specific binding reactivity against human VEGF‐D, as well as a specific inhibitory activity against the binding of human VEGF‐D to VEGFR‐3. In addition, cVE199 was found to inhibit the biological activity of VEGF‐D against lymphatic cells in vitro. Because we determined that a neuroblastoma cell line, SK‐N‐DZ, abundantly expressed VEGF‐D, an in vivo efficacy study was performed using a xenograft model of SK‐N‐DZ. We found that cVE199 significantly decreased lymphatic metastasis of SK‐N‐DZ as well as lymphangiogenesis in primary lesions. Finally, we investigated VEGF‐D expression in human neuroblastoma, finding that the molecule was expressed in 11 of 29 human neuroblastoma specimens (37.9%). In conclusion, we found that a novel anti‐VEGF‐D monoclonal antibody, cVE199, with specific reactivity against human VEGF‐D, prevents lymphatic metastasis of neuroblastoma through the inhibition of lymphangiogenesis in an animal model. In addition, our results show that VEGF‐D is expressed in some cases of human neuroblastomas, which suggests that cVE199 is a potential anti‐metastasis therapeutic antibody in neuroblastoma treatment.     

Sunitinib inhibits lymphatic endothelial cell functions and lymph node metastasis in a breast cancer model through inhibition of vascular endothelial growth factor receptor 3

Introduction  

Metastasis is a common event and the main cause of death in cancer patients. Lymphangiogenesis refers to the formation of new lymphatic vessels and is thought to be involved in the development of metastasis. Sunitinib is a multi-kinase inhibitor that blocks receptor tyrosine kinase activity, including that of vascular endothelial growth factor receptors (VEGFRs). Although sunitinib is a clinically available angiogenesis inhibitor, its effects on lymphangiogenesis and lymph node metastasis remain unclear. The purpose of this study was to investigate the effects of sunitinib on vascular endothelial growth factor receptor 3 (VEGFR-3) and a related event, lymphangiogenesis.     

Imaging steps of lymphatic metastasis reveals that vascular endothelial growth factor-C increases metastasis by increasing delivery of cancer cells to lymph nodes: therapeutic implications

Preclinical and clinical studies positively correlate the expression of vascular endothelial growth factor (VEGF)-C in tumors and the incidence of lymph node metastases. However, how VEGF-C regulates individual steps in the transport of tumor cells from the primary tumor to the draining lymph nodes is poorly understood. Here, we image and quantify these steps in tumors growing in the tip of the mouse ear using intravital microscopy of the draining lymphatic vessels and lymph node, which receives spontaneously shed tumor cells. We show that VEGF-C overexpression in cancer cells induces hyperplasia in peritumor lymphatic vessels and increases the volumetric flow rate in lymphatics at the base of the ear by 40%. The increases in lymph flow rate and peritumor lymphatic surface area enhance the rate of tumor cell delivery to lymph nodes, leading to a 200-fold increase in cancer cell accumulation in the lymph node and a 4-fold increase in lymph node metastasis. In our model, VEGF-C overexpression does not confer any survival or growth advantage on cancer cells. We also show that an anti-VEGF receptor (VEGFR)-3 antibody reduces both lymphatic hyperplasia and the delivery of tumor cells to the draining lymph node, leading to a reduction in lymph node metastasis. However, this treatment is unable to prevent the growth of tumor cells already seeded in lymph nodes. Collectively, our results indicate that VEGF-C facilitates lymphatic metastasis by increasing the delivery of cancer cells to lymph nodes and therapies directed against VEGF-C/VEGFR-3 signaling target the initial steps of lymphatic metastasis.     

Effect of combination therapy with a novel bisphosphonate, minodronate (YM529), and docetaxel on a model of bone metastasis by human transitional cell carcinoma.

PURPOSE: Transitional cell carcinoma (TCC) of the urinary tract is a chemosensitive tumor. Most deaths from TCC of the urinary tract are caused by metastasis, which is resistant to conventional chemotherapy. Frequent sites of metastases from TCC of the urinary tract are regional lymph nodes, liver, lung, and bone. Of these distant metastases, bone metastasis is consistently resistant to cisplatin-based conventional chemotherapy. Therefore, in this study, we investigated whether or not a newly developed minodronate, YM529, could prevent osteolytic bone metastasis of human TCC and also enhance the effect of docetaxel in a bone tumor model of athymic nude mice. EXPERIMENTAL DESIGN: In the present study, we evaluated the effect of in vitro treatment with minodronate and/or docetaxel on the proliferation by cell count, the induction of apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and the biological activity of osteoclast by pit formation assay in human bladder cancer cell line, UMUC-14, and mouse osteoclast cells. In vivo, we examined the effect of minodronate in a bone tumor model of athymic nude mice, in which the percutaneous intraosseal injection in the tibia of UMUC-14, leads to osteolytic bone tumor, as a bone metastasis model. To examine whether or not minodronate could inhibit tumorigenicity and enhance the effect of the chemotherapeutic agent, docetaxel, we gave minodronate i.p. and/or docetaxel i.p. to nude mice 3 days after an intraosseal tumor implantation. Moreover, proliferation and the induction of apoptosis of cancer cells and osteoclasts in bone tumors were determined by immunohistochemistry and the TUNEL assay. RESULTS: In vitro: In vitro treatment with docetaxel inhibited proliferation and resorption pit-forming activity and induced apoptosis of mouse osteoclast cells and UMUC-14 cells. In vitro treatment with minodronate inhibited proliferation and activity and induced apoptosis of mouse osteoclast cells but not UMUC-14 cells. The treatment with minodronate enhanced the inhibition of proliferation and activity by docetaxel in osteoclasts. In vivo: In vivo combination therapy with docetaxel and minodronate significantly reduced the tumor incidence compared with the control (P < 0.05) and also growth of intraossal TCC in athymic nude mice compared with the control (P < 0.001), single therapy with docetaxel (P < 0.01), and minodronate (P < 0.05). Drug-induced body weight loss was not significantly different in any treatment group. Therapy with minodronate significantly enhanced inhibition of proliferation by docetaxel in osteoclasts of bone tumors compared with the control (P < 0.01), single therapy with docetaxel (P < 0.01), and minodronate (P < 0.05). CONCLUSIONS: These studies indicate that combination therapy with minodronate and docetaxel may be beneficial in patients with bone metastasis of human TCC in the urinary tract.     

Enhancement of interstitial photodynamic therapy by mitomycin C and EO9 in a mouse tumour model

The tumoricidal effect of interstitial photodynamic therapy (IPDT) using Photofrin was found to increase when combined with the bioreductive alkylating agent mitomycin C (MMC) and, to a lesser extent, with the indoloquinone EO9. When MMC was given prior to IPDT of RIFI tumours, the light dose required for a given regrowth time or for 50% cure was reduced by a factor of 2 compared with IPDT alone. MMC given immediately after illumination did not increase the effects of IPDT, although MMC plus illumination without photosensitizer produced a significant increase in regrowth time compared with MMC or light alone. Combination of IPDT with EO9, given directly before illumination, only marginally increased the tumour regrowth times at non-toxic doses. These results demonstrate that combining IPDT with MMC greatly improves the tumour response. Factors such as PDT-induced hypoxia, pH changes, temperature in-creases and production of toxic reactive oxygen species by both drugs may play a role in the enhanced MMC cvtotoxicity.     

Deguelin—An inhibitor to tumor lymphangiogenesis and lymphatic metastasis by downregulation of vascular endothelial cell growth factor‐D in lung tumor model

Deguelin, a rotenoid of the flavonoid family, has been reported to possess antiproliferative and anticarcinogenic activities in several cell lines and tumor models. However, it is still unclear whether deguelin effectively inhibits tumor‐associated lymphangiogenesis and lymphatic metastasis. Since tumor production of vascular endothelial cell growth factor (VEGF)‐D was associated with tumor lymphangiogenesis and lymphatic metastasis, we established the mouse lymphatic metastasis model by transfecting high expression VEGF‐D into LL/2 Lewis lung cells (VEGF‐D‐LL/2) and explored the effects of deguelin on lymphatic metastasis in the immunocompetent C57BL/6 mice. Our results indicated that deguelin inhibited proliferation, migration of VEGF‐D‐LL/2 cells via downregulating AKT and mitogen‐activated protein kinase pathway and interfered tube formation of lymphatic vascular endothelial cells on matrigel at nanomolar concentrations. Deguelin significantly downregulated the expression of VEGF‐D both at mRNA and protein levels in VEGF‐D‐LL/2 cells in a dose‐dependent manner. In the in vivo study, intraperitoneal administration of deguelin (4 mg/kg) remarkably inhibited the tumor‐associated lymphangiogenesis and lymphatic metastasis. The rates of lymph node and lung metastasis in deguelin‐treated mice were 0 and 16.7% compared with 58.3 and 83.3% in control group mice, respectively. Deguelin also resulted in a remarkable delay of tumor growth and prolongation of life span. Immunohistochemical staining with antibodies against VEGF‐D, LYVE‐1 and VEGFR‐3 revealed fewer positive vessel‐like structures in deguelin‐treated mice compared with control group mice. Taken together, we demonstrate for the first time that deguelin suppresses tumor‐associated lymphangiogenesis and lymphatic metastasis by downregulation of VEGF‐D both in vitro and in vivo.     

Effect of UFT by oral administration using a murine hepatic metastasis model

We examined the antitumor effect of UFT, a 5-FU derivative using a hepatic metastasis model in mice. BALB/c and CDF1 mice were given orally UFT (30 mg/kg, 50 mg/kg daily or 120 mg/kg every other day) for 20 days after inoculation of colon-26 tumor cells into the portal vein. The number of metastatic nodules and the weight of the liver decreased significantly in all groups of UFT treatment. The therapeutic effect with 50 mg/kg UFT was better than those with the other doses. NK and LAK activities of spleen cells obtained from UFT-treated tumor bearing mice were not significantly different from the untreated control. IL-1 and IL-2 production of the spleen cells were also not significantly changed by the treatment with UFT. These results suggested that the oral administration of UFT represents an antitumor effect against hepatic metastasis in mice without sever suppression of the host immune system.     

An antiangiogenic urokinase-derived peptide combined with tamoxifen decreases tumor growth and metastasis in a syngeneic model of breast cancer

Expression of urokinase (uPA) and its receptor (uPAR) is associated with increased tumor-cellinvasion and metastasis in several malignancies including breast cancer.An 8-mer peptide derived from the nonreceptor-binding domain of urokinase (A6) has been shown to have antiangiogenic and proapoptotic effects to block the progression of breast cancer in vivo. In the present study, we evaluated the effects of A6 and the antiestrogen tamoxifen (TAM) alone and in combination on estrogen-receptor-positive Mat B-III rat breast cancer cells in vitro and in vivo. Treatment of Mat B-III cells with A6 and TAM resulted in a dose-dependent decrease in tumor-cell invasion through Matrigel; these effects were more marked when A6 and TAM were tested in combination. In addition, treatment of Mat B-III cells with either A6 or TAM resulted in a significant reduction of vascular endothelial growth factor receptor (flk-1) expression and in transforming growth factor beta activity, effects that were significantly higher after combined treatment with A6 and TAM. For in vivo studies, female Fischer rats were inoculated with Mat B-III cells (1 x 10(6)) into the mammary fat pad. These orthotopic tumors were staged to 30-40 mm(3) in volume and then treatment was initiated with A6 (75 mg/kg/day) and TAM (3 mg/kg/day) alone or in combination. Both A6 and TAM caused a significant reduction in tumor volume; however, these antitumor effects were significantly greater in animals receiving both A6 and TAM, which demonstrated a 75% reduction in tumor growth as compared with control animals. The number of macroscopic tumor foci was significantly reduced in A6-treated animals, whereas TAM failed to exhibit any antimetastatic effects. Histological analysis of primary tumors from different groups showed a decrease in new blood-vessel density and increased tumor-cell death in A6- and TAM-treated animals, and these effects were greater in experimental animals receiving A6 and TAM in combination. Collectively, these studies demonstrate that the addition of novel antiangiogenic/antimetastatic agents like A6 to hormone therapy can enhance the antitumor effects of hormone therapy through increased inhibition of angiogenesis and induction of tumor-cell death.     

口腔癌VEGF-CmRNA表达和血管及淋巴管生成与淋巴道转移关系的研究

背景与目的VEGF-C与癌周的淋巴管血管生成以及肿瘤的淋巴道转移可能有密切的关系,本研究探讨口腔鳞癌组织血管、淋巴管密度与VEGF-CmRNA表达及淋巴道转移的关系.方法VEGF-C逆转录PCR(RT-PCR),血管、淋巴管酶组化染色、光镜及图像分析观察血管、淋巴管总体面数密度(TNa).结果VEGF-CmRNA表达阳性的淋巴管TNa(26.42±5.85)明显高于阴性淋巴管TNa(17.34±6.48)(P<0.01);VEGF-CmRNA表达阳性的血管TNa(35.16±15.55)略高于阴性的血管TNa(33.49±13.73)(P>0.05).淋巴结转移组血管TNa(44.19±14.29)比无淋巴结转移组TNa(30.61±11.82)增加(P<0.01)、淋巴结转移组淋巴管TNa(30.67±5.76)比无淋巴结转移组TNa(21.94±5.84)增加(P<0.01).结论VEGF-C主要介导了癌周淋巴管生成,对血管生成有一定影响;血管、淋巴管密度的同时增加可能与VEGF、VEGF-C及其受体的协同表达有一定关系.     

Tumor necrosis factor superfamily 15 promotes lymphatic metastasis via upregulation of vascular endothelial growth factor‐C in a mouse model of lung cancer

Lymphatic metastasis is facilitated by lymphangiogenic growth factor vascular endothelial growth factor‐C (VEGFC) that is secreted by some primary tumors. We previously identified tumor necrosis factor superfamily 15 (TNFSF15), a blood vascular endothelium‐derived cytokine, in lymphatic endothelial cells, as a key molecular modulator during lymphangiogenesis. However, the effect of TNFSF15 on tumor lymphatic metastasis and the underlying molecular mechanisms remain unclear. We report here that TNFSF15, which is known to inhibit primary tumor growth by suppressing angiogenesis, can promote lymphatic metastasis through facilitating lymphangiogenesis in tumors. Mice bearing tumors induced by A549 cells stably overexpressing TNFSF15 exhibited a significant increase in densities of lymphatic vessels and a marked enhancement of A549 tumor cells in newly formed lymphatic vessels in the primary tumors as well as in lymph nodes. Treatment of A549 cells with TNFSF15 results in upregulation of VEGFC expression, which can be inhibited by siRNA gene silencing of death domain‐containing receptor‐3 (DR3), a cell surface receptor for TNFSF15. In addition, TNFSF15/DR3 signaling pathways in A549 cells include activation of NF‐κB during tumor lymphangiogenesis. Our data indicate that TNFSF15, a cytokine mainly produced by blood endothelial cells, facilitates tumor lymphangiogenesis by upregulating VEGFC expression in A549 cells, contributing to lymphatic metastasis in tumor‐bearing mice. This finding also suggests that TNFSF15 may have potential as an indicator for prognosis evaluation.     

Effect of antiangiogenic therapy on tumor growth, vasculature and kinase activity in basal- and luminal-like breast cancer xenografts

Several clinical trials have investigated the efficacy of bevacizumab in breast cancer, and even if growth inhibiting effects have been registered when antiangiogenic treatment is given in combination with chemotherapy no gain in overall survival has been observed. One reason for the lack of overall survival benefit might be that appropriate criteria for selection of patients likely to respond to antiangiogenic therapy in combination with chemotherapy, are not available. To determine factors of importance for antiangiogenic treatment response and/or resistance, two representative human basal- and luminal-like breast cancer xenografts were treated with bevacizumab and doxorubicin alone or in combination. In vivo growth inhibition, microvessel density (MVD) and proliferating tumor vessels (pMVD = proliferative microvessel density) were analysed, while kinase activity was determined using the PamChip Tyrosine kinase microarray system. Results showed that both doxorubicin and bevacizumab inhibited basal-like tumor growth significantly, but with a superior effect when given in combination. In contrast, doxorubicin inhibited luminal-like tumor growth most effectively, and with no additional benefit of adding antiangiogenic therapy. In agreement with the growth inhibition data, vascular characterization verified a more pronounced effect of the antiangiogenic treatment in the basal-like compared to the luminal-like tumors, demonstrating total inhibition of pMVD and a significant reduction in MVD at early time points (three days after treatment) and sustained inhibitory effects until the end of the experiment (day 18). In contrast, luminal-like tumors only showed significant effect on the vasculature at day 10 in the tumors having received both doxorubicin and bevacizumab. Kinase activity profiling in both tumor models demonstrated that the most effective treatment in vivo was accompanied with increased phosphorylation of kinase substrates of growth control and angiogenesis, like EGFR, VEGFR2 and PLCγ1. This may be a result of regulatory feedback mechanisms contributing to treatment resistance, and may suggest response markers of value for the prediction of antiangiogenic treatment efficacy.     

Tumor vasculature and microenvironment normalization: a possible mechanism of antiangiogenesis therapy

Tumor antiangiogenesis therapy has been in application for more than 30 years; however, its mechanism remains obscure. An intriguing hypothesis, which has recently gained acceptance, explores the possibility that antiangiogenesis therapy may transiently normalize tumor vasculature and its microenvironment, thus enhancing chemoradiotherapy efficacy. As the equilibrium between proangiogenesis and antiangiogenesis factors is perturbed in the tumor and tips to the former, tumor vasculature tends to exhibit abnormal structure and function. Abnormal vasculature is tightly associated with an uncharacteristic microenvironment, including uneven perfusion, hypoxia, and increased interstitial fluid pressure: This malignant microenvironment hinders the delivery of chemotherapeutics to tumor cells and desensitizes the malignant cells to radiation. Antiangiogenesis therapy can reverse the imbalance and transiently normalize this microenvironment and gives a new perspective for combining antiangiogenesis therapy and traditional chemoradiotherapy.