The Inhibitory Effect of Propylene Glycol Alginate Sodium Sulfate on Fibroblast Growth Factor 2-Mediated Angiogenesis and Invasion in Murine Melanoma B16-F10 Cells In Vitro.

Melanoma is one of the most malignant and aggressive types of cancer worldwide. Fibroblast growth factor 2 (FGF2) is one of the critical regulators of melanoma angiogenesis and metastasis; thus, it might be an effective anti-cancer strategy to explore FGF2-targeting drug candidates from existing drugs. In this study, we evaluate the effect of the marine drug propylene glycol alginate sodium sulfate (PSS) on FGF2-mediated angiogenesis and invasion. The data shows that FGF2 selectively bound to PSS with high affinity. PSS inhibited FGF2-mediated angiogenesis in a rat aortic ring model and suppressed FGF2-mediated invasion, but not the migration of murine melanoma B16-F10 cells. The further mechanism study indicates that PSS decreased the expression of activated matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9), and also suppressed their activity. In addition, PSS was found to decrease the level of Vimentin in B16-F10 cells, which is known to participate in the epithelial-mesenchymal transition. Notably, PSS did not elicit any changes in cancer cell viability. Based on the results above, we conclude that PSS might be a potential drug to regulate the tumor microenvironment in order to facilitate the recovery of melanoma patients.

Endothelial Kruppel-like factor 4 regulates angiogenesis and the Notch signaling pathway.

Regulation of endothelial cell biology by the Notch signaling pathway (Notch) is essential to vascular development, homeostasis, and sprouting angiogenesis. Although Notch determines cell fate and differentiation in a wide variety of cells, the molecular basis of upstream regulation of Notch remains poorly understood. Our group and others have implicated the Krüppel-like factor family of transcription factors as critical regulators of endothelial function. Here, we show that Krüppel-like factor 4 (KLF4) is a central regulator of sprouting angiogenesis via regulating Notch. Using a murine model in which KLF4 is overexpressed exclusively in the endothelium, we found that sustained expression of KLF4 promotes ineffective angiogenesis leading to diminished tumor growth independent of endothelial cell proliferation or cell cycling effects. These tumors feature increased vessel density yet are hypoperfused, leading to tumor hypoxia. Mechanistically, we show that KLF4 differentially regulates expression of Notch receptors, ligands, and target genes. We also demonstrate that KLF4 limits cleavage-mediated activation of Notch1. Finally, we rescue Notch target gene expression and the KLF4 sprouting angiogenesis phenotype by supplementation of DLL4 recombinant protein. Identification of this hitherto undiscovered role of KLF4 implicates this transcription factor as a critical regulator of Notch, tumor angiogenesis, and sprouting angiogenesis.

Transscleral delivery of Nd: YLF laser at 1,047 nm causes vascular occlusion in experimental pigmented choroidal melanoma.

PURPOSE: The aims of this study were to determine the scleral attenuation of focused neodymium: yttrium-lanthanum-fluoride laser at 1,047 nm applied transsclerally and whether transscleral delivery can close the vascular supply at the base of experimental choroidal melanoma in rabbits. METHODS: Fifty-two New Zealand albino rabbits were included. Scleral laser attenuation was measured across fresh sclera. B16F10 melanomas were established in the subchoroidal space of 49 rabbits. Twenty-one animals were killed immediately after transscleral treatment, 14 were followed for 2 weeks to 4 weeks, and 14 were followed without treatment. Ophthalmoscopy, fundus photographs, and fluorescein angiography were performed before treatment, immediately after, and weekly during the follow-up. Eyes were examined by light microscopy. RESULTS: Sclera attenuated laser energy by 31% ± 7%. Immediately after treatment, angiography showed diffuse hypofluorescence in 71% (15 of 21 rabbits). Light microscopy showed vascular occlusion extending at least two thirds of the tumor thickness from the base. Seven of the 14 tumors followed for 15 days ± 8 days were eradicated. There was no correlation between tumor height and eradication. CONCLUSION: Rabbit sclera attenuated 31% ± 7% of laser energy. A single transscleral treatment causes tumor vascular closure at the base and may serve as an adjuvant therapy to ensure destruction of deep and intrascleral tumor cells.

Triterpenoids amplify anti-tumoral effects of mistletoe extracts on murine B16.f10 melanoma in vivo.

PURPOSE: Mistletoe extracts are often used in complementary cancer therapy although the efficacy of that therapy is controversially discussed. Approved mistletoe extracts contain mainly water soluble compounds of the mistletoe plant, i.e. mistletoe lectins. However, mistletoe also contains water-insoluble triterpenoids (mainly oleanolic acid) that have anti-tumorigenic effects. To overcome their loss in watery extracts we have solubilized mistletoe triterpenoids with cyclodextrins, thus making them available for in vivo cancer experiments. EXPERIMENTAL DESIGN: B16.F10 subcutaneous melanoma bearing C57BL/6 mice were treated with new mistletoe extracts containing both water soluble compounds and solubilized triterpenoids. Tumor growth and survival was monitored. In addition, histological examinations of the tumor material and tumor surrounding tissue were performed. RESULTS: Addition of solubilized triterpenoids increased the anti-tumor effects of the mistletoe extracts, resulting in reduced tumor growth and prolonged survival of the mice. Histological examination of the treated tumors showed mainly tumor necrosis and some apoptotic cells with active caspase-3 and TUNEL staining. A significant decrease of CD31-positive tumor blood vessels was observed after treatment with solubilized triterpenoids and different mistletoe extracts. CONCLUSION: We conclude that the addition of solubilized mistletoe triterpenoids to conventional mistletoe extracts improves the efficacy of mistletoe treatment and may represent a novel treatment option for malignant melanoma.

In vitro and in vivo effects of an anti-mouse endoglin (CD105)-immunotoxin on the early stages of mouse B16MEL4A5 melanoma tumours.

TGF-beta superfamily co-receptors are emerging as targets for cancer therapy, acting both directly on cells and indirectly on the tumour neovasculature. Endoglin (CD105), an accessory component of the TGF-beta receptor complex, is expressed in certain melanoma cell lines and the endothelial cells of tumour neovessels. Targeting endoglin with immunotoxins is an attractive approach for actively suppressing the blood supply to tumours. Here, we report evidence indicating that endoglin is expressed in mouse melanoma B16MEL4A5 and mouse fibroblast L929 cell lines. We prepared an immunotoxin to target endoglin by coupling the rat anti-mouse MJ7/18 (IgG2a) monoclonal antibody (mAb) to the non-toxic type 2 ribosome-inactivating protein nigrin b (Ngb) with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a linker with a molar nigrin b at a MJ7/18 stoichiometry of 2:1. The MJ7-Ngb immunotoxin generated killed both cell lines, with IC50 values of 4.2 × 10(-9) M for B16MEL4A5 and 7.7 × 10(-11) M for L929 cells. For in vivo assays of the immunotoxin, B16MEL4A5 cells were injected subcutaneously into the right flanks of 6-week-old C57BL/6 J mice. When the animals developed palpable solid tumours, they were subjected to treatment with the immunotoxin. While treatment with either MJ7/18 mAb or Ngb did not affect tumour development, treatment with the immunotoxin completely and steadily blocked tumour growth up to 7 days, after which some tumours re-grew. Thus, vascular-targeting therapy with this anti-vascular immunotoxin could promote the destruction of newly created tumour vessels at early stages of B16MEL4A5 tumour development and readily accessible CD105+ B16MEL4A5 melanoma cells.

The chemotherapeutic effect of essential oil of Plectranthus amboinicus (Lour) on lung metastasis developed by B16F-10 cell line in C57BL/6 mice.

Current investigation is to evaluate the anticancer activity of the essential oil of Plectranthus amboinicus (Lour) on B16F-10 melanoma cell line injected C57BL/6 mice, and it was simultaneously treated with the essential oil of P. amboinicus (Lour) (50 µg/dose) via i.p. for 21 days. The present investigation exhibited the potent chemotherapeutic/chemopreventive effect of the essential oil of P. amboinicus (Lour) over lung metastasis that developed. To our knowledge, this is the first report in evaluating the effect of essential oil of P. amboinicus (Lour) using lung cancer model.

Antiangiogenic activity of berberine is mediated through the downregulation of hypoxia-inducible factor-1, VEGF, and proinflammatory mediators.

Berberine, a naturally occurring isoquinoline alkaloid, is present in a number of important medicinal plants. Berberine has a wide range of biochemical and pharmacological effects, including anticancer effects. In this study, we elucidated the mechanism of antiangiogenic activity of berberine using in vivo and in vitro models. In vivo antiangiogenic activity was studied using B16F-10 melanoma cells and induced capillary formation in C57BL/6 mice. Berberine, at 10 mg/kg body weight, showed significant inhibition in tumor-directed capillary formation and in various proangiogenic factors, such as vascular endothelial growth factor (VEGF), and proinflammatory mediators, such as interleukin (IL)-1ß, IL-6, tumor necrosis factor alpha (TNF-α), and granulocyte macrophage colony-stimulating factor (GM-CSF), which are involved in tumor angiogenesis. At the same time, it could also increase antitumor factors, such as IL-2 and tissue-inhibitor metalloproteinase (TIMP) levels in the serum. Berberine could also inhibit endothelial motility, migration, tube formation, and vessel sprouting from rat aortic ring in vitro. Further, berberine inhibited various transcription factors involved in tumor development and angiogenesis, such as NF-ĸB, c-Fos, CREB, and ATF-2. mRNA expression levels of proangiogenic factors, such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and hypoxia-inducible factor (HIF), were also downregulated in tumor cells after treatment with berberine. Drastically elevated expressions of HIF and VEGF mRNA by tumor cells under hypoxic conditions were also decreased after treatment with berberine. This result clearly demonstrates that the antiangiogenic activity of berberine is mainly mediated through the inhibition of various proinflammatory and pro-angiogenic factors and the major ones are HIF, VEGF, COX-2, NO, NF-ĸB, and proinflammatory cytokines.

Primo vascular system of murine melanoma and heterogeneity of tissue oxygenation of the melanoma.

Murine melanoma requires the complex development of lymphatic, vascular, and non-vascular structures. A possible relationship between the primo vascular system (PVS) and the melanoma metastasis has been proposed. In particular, the PVS may be involved in oxygen transport. Vasculogenic-like networks, similar to the PVS, have been found within melanoma tumors, but their functional relationship with the PVS and meridian structures are unclear. Herein, we report on the use of an electrochemical O(2) sensor to study oxygenation levels of melanoma tumors in mice. We consistently found higher tissue oxygenation in specific sites of tumors (n=5). These sites were strongly associated with vascular structures or the PVS. Furthermore, the PVS on the tumor surface was associated with adipose tissue. Our findings suggest that the PVS is involved in the regulation of metastasis.

Study of the primo vascular system utilizing a melanoma tumor model in a green fluorescence protein expressing mouse.

A melanoma tumor is a representative malignant tumor. Melanoma tumor growth involves vigorous angiogenesis around the tumor and a vasculogenic-like network inside an aggressive tumor. Primo vessels (PVs) are also found on the surface of the tumor and coexist alongside blood vessels (BVs), and sometimes within the BVs. We hypothesized that the primo vessels system plays a significant role in regulating the development of a melanoma tumor, and therefore has a tight coupling with BVs and angiogenesis. To prove this hypothesis, we developed a murine melanoma model by inoculating melanoma cell lines into the abdominal region. We used a green fluorescent protein (GFP) expressing mouse as a host to distinguish the endogenous source of the tumor PVs. We found strong formation of PVs on the tumor that coexisted with BVs and expression of GFP. PVs also had a tight coupling with adipose tissues, especially with white adipose tissue. These data suggest that the PVs of an induced melanoma tumor evolve endogenously from the host body and may be highly related to BVs and adipose tissue. This model of PVs in an overexpressing GFP mouse is a useful system for observing PVs, primo nodes, and primo vessel networks, and has potential to be developed as a model for examining novel treatments for cancer metastasis.

Mild elevation of body temperature reduces tumor interstitial fluid pressure and hypoxia and enhances efficacy of radiotherapy in murine tumor models.

Human and rodent solid tumors often exhibit elevated interstitial fluid pressure (IFP). This condition is recognized as a prognostic indicator for reduced responses to therapy and decreased disease-free survival rate. In the present study, we tested whether induction of a thermoregulatory-mediated increase in tissue blood flow, induced by exposure of mice to mild environmental heat stress, could influence IFP and other vascular parameters within tumors. Using several murine tumor models, we found that heating results in a sustained reduction in tumor IFP correlating with increased tumor vascular perfusion (measured by fluorescent imaging of perfused vessels, laser Doppler flowmetry, and MRI) as well as a sustained reduction in tumor hypoxia. Furthermore, when radiation therapy was administered 24 hours postheating, we observed a significant improvement in efficacy that may be a result of the sustained reduction in tumor hypoxia. These data suggest, for the first time, that environmental manipulation of normal vasomotor function is capable of achieving therapeutically beneficial changes in IFP and microvascular function in the tumor microenvironment.

17ß-estradiol-linked nitro-L-arginine as simultaneous inducer of apoptosis in melanoma and tumor-angiogenic vascular endothelial cells.

Aggressive melanoma is commonly associated with rapid angiogenic growth in tumor mass, tumor cells acquiring apoptosis resistance, inhibition of cellular differentiation etc. Designing a single anticancer molecule which will target all these factors simultaneously is challenging. In the pretext of inciting anticancer effect through inhibiting nitric oxide synthase (NOS) via estrogen receptors (ER) in ER-expressing skin cancer cells, we developed an estrogen-linked L-nitro-arginine molecule (ESAr) for inciting anticancer effect in melanoma cells. ESAr showed specific anticancer effect through diminishing aggressiveness and metastatic behavior in melanoma cells and tumor. In comparison, ESAr showed significantly higher antiproliferative effect than parent molecule L-nitroarginine methyl ester (L-NAME, a NOS inhibitor) through induction of prominent apoptosis in melanoma cells. ESAr-pretreated aggressive melanoma cells could not form tumor possibly because of transformation/differentiation into epithelial-type cells. Furthermore, its antiangiogenic effect was demonstrated through ESAr-induced antiproliferation in HUVEC cells and apoptosis-induction in tumor-associated vascular endothelial cells, thereby significantly restricting severe growth in melanoma tumor. The targeting moiety, estrogen, at the therapeutic concentration of ESAr has apparently no effect in tumor-growth reduction. Albeit, no specific NOS-inhibition was observed, but ESAr could simultaneously induce these three cancer-specific antiaggressiveness factors, which the parent molecule could not induce. Our data rationalize and establish a new use of estrogen as a ligand for potentially targeting multiple cellular factors for treating aggressive cancers.

Anti-angiogenesis effect of essential oil from Curcuma zedoaria in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma zedoaria (Berg.) Rosc., a traditional Chinese herb, was used widely but absolutely prohibited for the pregnant in clinic. Based on that there is abundant angiogenesis in endometrium and placenta during gestation period, we hypothesized that some components from it could inhibit angiogenesis and then damaged the supply of oxygen and nutrition to the embryo, which finally led to gestation failure. AIM OF THE STUDY: This study was set to demonstrate whether essential oil, major components of Curcuma zedoaria had anti-angiogenic effect. MATERIALS AND METHODS: Essential oil of Curcuma zedoaria (EO-CZ) was abstracted by steam distillation extraction. Cell proliferation assay and two angiogenic models, rat aortic ring assay and chick embryo chorioallantoic membrane assay were presented. Furthermore, melanoma growth and experimental lung metastasis assay in mice were performed to evaluate its anti-angiogenesis effect in vivo. Immunohistochemical analysis and enzyme-linked immunosorbent assay (ELISA) were used to respectively detect the expression of CD34 and matrix metalloproteinases (MMPs). RESULTS: EO-CZ exhibited anti-proliferative effect on B16BL6 and SMMC-7721 cells, the IC(50), respectively was 41.8 µg/ml and 30.7 µg/ml, and on HUVEC (Human Umbilical Vein Endothelial Cells) cells with IC(50) of far more than 120 µg/ml. Both 20 µg/ml and 40 µg/ml EO-CZ indicated significant suppression on sprouting vessels of aortic ring and formation of microvessels in chick embryo chorioallantoic membrane in vitro. Moreover, solid melanoma grown in left oxter of mice was obviously inhibited after oral intake of 100 and 200 mg/kg of EO-CZ a day for 28 days, and CD34 expression indicating angiogenesis in melanoma reduced significantly compared with control; melanoma metastatic nodules in lung were detected to be inhibited, as well as MMP-2 and MMP-9 expression in serum. CONCLUSIONS: Essential oil, a fat-soluble fraction of Curcuma zedoaria, presented anti-angiogenic activity in vitro and in vivo, resulting in suppressing melanoma growth and lung metastasis. And this was associated with down-regulating MMPs.

Histopathological follow-up by tissue micro-array in a survival study after melanoma treated by nanosecond pulsed electric fields (nsPEF).

A recent study has shown that nanosecond pulsed electric fields (nsPEF) can affect the intracellular structures of melanoma within weeks. nsPEF is a non-drug, non-thermal treatment using ultrashort, intense pulsed electric fields with nanosecond durations. In the current study we followed up melanoma histopathology and metastasis with tissue micro-array 5 months post-nsPEF. After nsPEF treatment, tumor growth, tumor histology, metastasis, peri-tumor vessel and micro-vessel density were examined for the effect of nsPEF treatment on melanoma in vivo. The 17 nsPEF-treated mice were tumor-free for 169 days, significantly longer than those 19 control mice bearing melanoma without nsPEF. Histopathology follow-up showed that melanoma did not recur to the primary injection place after complete elimination. Compared with the control tumor, nsPEF-treated tumors present decreased micro-vessel density in a time-course manner in this survival study. Treatment with nsPEF caused continuous histopathological changes in melanomas, eliminated melanoma without recurrence at the primary site and prolonged animal survival time by inhibiting tumor blood supply and leading to tumor infarction. Thus, nsPEF could be applied in a non-ionizing therapeutic approach, without other agents, to locally treat tumors within a defined boundary.

Harmine inhibits tumour specific neo-vessel formation by regulating VEGF, MMP, TIMP and pro-inflammatory mediators both in vivo and in vitro.

Harmine is a beta-carboline alkaloid present in medicinal plants such as Peganum harmala that have been used as folk medicine in anticancer therapy. In this study, we demonstrated the anti-angiogenic activity of harmine using in vivo and in vitro assay systems. In vivo anti-angiogenic activity was studied using B16F-10 melanoma cells which induced capillary formation in C57BL/6 mice. Intraperitoneal administration of harmine at 10 mg/kg body weight significantly decreased tumour directed capillary formation. A drastic elevation in serum pro-angiogenic factors such as vascular endothelial growth factor (VEGF), nitric oxide (NO) and pro-inflammatory cytokines in angiogenesis induced animals was significantly decreased by harmine treatment. At the same time harmine increased anti-tumour factors like interleukin-2 (IL-2) and tissue inhibitor metalloprotease (TIMP). Moreover nuclear factor (NF)-κB and other transcription factors like CREB, ATF-2 involved in tumour development and angiogenesis were also inhibited by harmine. Various in vitro assays also supported the anti-angiogenic activity of harmine. It reduced proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC). Direct treatment of the harmine also inhibited microvessel outgrowth from the rat aortic ring. Production of other factors by tumour cells which are involved in angiogenesis like cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS) and matrix metalloproteases (MMPs) were also decrease by the treatment with harmine. Our data suggest that harmine may be a strong angiogenic inhibitor with the ability to decrease the proliferation of vascular endothelial cells and to reduce expression of various pro-angiogenic factors.

Antiangiogenic activity of ursolic acid.

Angiogenesis, the formation of new capillaries from preexisting vessels, is essential for tumor progression. Ursolic acid inhibited the tumor-associated capillary formation in C57BL/6 mice induced by highly metastatic B16F-10 melanoma cells. The levels of serum vascular endothelial growth factor (VEGF), NO, and proinflammatory cytokines were significantly reduced in ursolic acid-treated animals compared with those in control animals. The diminished expressions of VEGF and iNOS genes in B16F-10 melanoma cells treated with nontoxic concentrations of ursolic acid support these observations; the serum TIMP-1 (tissue inhibitor of metalloproteinase-1) and IL-2 (interleukin-2) levels were significantly elevated after the ursolic acid treatment. Nontoxic concentrations of ursolic acid toward human umbilical vein endothelial cells (HUVEC) were determined by MTT (methylthiazol tetrazolium) assay, and these nontoxic concentrations were selected for the in vitro studies. Nontoxic concentrations of ursolic acid inhibited vessel growth from the rat aortic ring. (3)H-thymidine proliferation assay clearly showed the inhibitory effect of ursolic acid on the proliferation of HUVECs in vitro. Ursolic acid significantly inhibited endothelial cell migration and invasion. The role of metalloproteinases has been shown to be important in angiogenesis; therefore, gelatin zymography was performed to determine whether ursolic acid affected protease activity. Gelatin zymographic analysis showed the inhibitory effect of ursolic acid on the protein expression of matrix metalloproteinases MMP-2 and MMP-9. The above observation shows the antiangiogenic activity of ursolic acid.

Apoptosis initiation and angiogenesis inhibition: melanoma targets for nanosecond pulsed electric fields.

Many effective anti-cancer strategies target apoptosis and angiogenesis mechanisms. Applications of non-ionizing, nanosecond pulsed electric fields (nsPEFs) induce apoptosis in vitro and eliminate cancer in vivo; however in vivo mechanisms require closer analysis. These studies investigate nsPEF-induced apoptosis and anti-angiogenesis examined by fluorescent microscopy, immunoblots, and morphology. Six hours after treatment with one hundred 300 ns pulses at 40 kV/cm, cells transiently expressed active caspases indicating that caspase-mediated mechanisms. Three hours after treatment transient peaks in Histone 2AX phosphorylation coincided with terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells and pyknotic nuclei, suggesting caspase-independent mechanisms on nuclei/DNA. Large DNA fragments, but not 180 bp fragmentation ladders, were observed, suggesting incomplete apoptosis. Nevertheless, tumor weight and volume decreased and tumors disappeared. One week after treatment, vessel numbers, vascular endothelial growth factor (VEGF), platelet derived endothelial cell growth factor (PD-ECGF), CD31, CD35 and CD105 were decreased, indicating anti-angiogenesis. The nsPEFs activate multiple melanoma therapeutic targets, which is consistent with successes of nsPEF applications for tumor treatment in vivo as a new cancer therapeutic modality.

Growth of injected melanoma cells is suppressed by whole body exposure to specific spatial-temporal configurations of weak intensity magnetic fields.

PURPOSE: To measure the effect of exposure to a specific spatial-temporal, hysiologically-patterned electromagnetic field presented using different geometric configurations on the growth of experimental tumours in mice. METHODS: C57b male mice were inoculated subcutaneously with B16-BL6 melanoma cells in two blocks of experiments separated by six months (to control for the effects of geomagnetic field). The mice were exposed to the same time-varying electromagnetic field nightly for 3 h in one of six spatial configurations or two control conditions and tumour growth assessed. RESULTS: Mice exposed to the field that was rotated through the three spatial dimensions and through all three planes every 2 sec did not grow tumours after 38 days. However, the mice in the sham-field and reference controls showed massive tumours after 38 days. Tumour growth was also affected by the intensity of the field, with mice exposed to a weak intensity field (1-5 nT) forming smaller tumours than mice exposed to sham or stronger, high intensity (2-5 microT) fields. Immunochemistry of tumours from those mice exposed to the different intensity fields suggested that alterations in leukocyte infiltration or vascularisation could contribute to the differences in tumour growth. CONCLUSIONS: Exposure to specific spatial-temporal regulated electromagnetic field configurations had potent effects on the growth of experimental tumours in mice.

Small-molecule inhibitors of vascular adhesion protein-1 reduce the accumulation of myeloid cells into tumors and attenuate tumor growth in mice.

Vascular adhesion protein-1 (VAP-1) is an endothelial, cell surface-expressed oxidase involved in leukocyte traffic. The adhesive function of VAP-1 can be blocked by anti-VAP-1 Abs and small-molecule inhibitors. However, the effects of VAP-1 blockade on antitumor immunity and tumor progression are unknown. In this paper, we used anti-VAP-1 mAbs and small-molecule inhibitors of VAP-1 in B16 melanoma and EL-4 lymphoma tumor models in C57BL/6 mice. Leukocyte accumulation into tumors and neoangiogenesis were evaluated by immunohistochemistry, flow cytometry, and intravital videomicroscopy. We found that both anti-VAP-1 Abs and VAP-1 inhibitors reduced the number of leukocytes in the tumors, but they targeted partially different leukocyte subpopulations. Anti-VAP-1 Abs selectively inhibited infiltration of CD8-positive lymphocytes into tumors and had no effect on accumulation of myeloid cells into tumors. In contrast, the VAP-1 inhibitors significantly reduced only the number of proangiogenic Gr-1(+)CD11b(+) myeloid cells in melanomas and lymphomas. Blocking of VAP-1 by either means left tumor homing of regulatory T cells and type 2 immune-suppressing monocytes/macrophages intact. Notably, VAP-1 inhibitors, but not anti-VAP-1 Abs, retarded the growth of melanomas and lymphomas and reduced tumor neoangiogenesis. The VAP-1 inhibitors also reduced the binding of Gr-1(+) myeloid cells to the tumor vasculature. We conclude that tumors use the catalytic activity of VAP-1 to recruit myeloid cells into tumors and to support tumor progression. Small-molecule VAP-1 inhibitors therefore might be a potential new tool for immunotherapy of tumors.

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment.

Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana-Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.

Static magnetic fields impair angiogenesis and growth of solid tumors in vivo.

Exposure to static magnetic fields (SMFs) results in a reduced blood flow in tumor vessels as well as in activation and adherence of platelets. Whether this phenomenon may have a significant functional impact on tumors has not been investigated as yet. The aim of our study was to evaluate the effects of prolonged exposure to SMFs on tumor angiogenesis and growth. Experiments were performed in dorsal skinfold chamber preparations of Syrian Golden hamsters bearing syngenic A-Mel-3 melanomas. On 3 d following tumor cell implantation one group of animals was immobilized and exposed to a SMF of 586 mT for three h. Control animals were immobilized for the same duration without SMF exposure. Using in vivo-fluorescence microscopy the field effects on tumor angiogenesis and microcirculation were analyzed for seven days. Tumor growth was assessed by repeated planimetry of the tumor area during the observation period. Exposure to SMFs resulted in a significant retardation of tumor growth ( approximately 30%). Furthermore, histological analysis showed an increased peri- and intratumoral edema in tumors exposed to SMFs. Analysis of microcirculatory parameters revealed a significant reduction of functional vessel density, vessel diameters and red blood cell velocity in tumors after exposure to SMFs compared to control tumors. These changes reflect retarded vessel maturation by antiangiogenesis. The increased edema after SMF exposure indicates an increased tumor microvessel leakiness possibly enhancing drug-uptake. Hence, SMF therapy appears as a promising new anticancer strategy-as an inhibitor of tumor growth and angiogenesis and as a potential sensitizer to chemotherapy.