Angiogenesis and expression of vascular endothelial growth factor, tumour necrosis factor-α and hypoxia inducible factor-1α in canine renal cell carcinoma

The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 μm) was also significantly different from normal canine kidney (23.2 ± 3.4 μm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis.     

Lymphatic spread of ductal pancreatic adenocarcinoma is independent of lymphangiogenesis

Early lymph node metastasis is common in pancreatic ductal adenocarcinoma (PDAC). The present study has examined the relationship of lymphatic spread to lymph vessel development and the expression of lymphangiogenic cytokines in a series of well-characterized PDACs. The hot spot method revealed the intratumoural and peritumoural lymphatic vessel density (LVD) to be slightly higher in PDACs than in the normal pancreas. The average intratumoural LVD, however, was strikingly decreased. There was no overexpression of vascular endothelial growth factor (VEGF)-C and VEGF-D in PDACs compared with the normal pancreas. LVD and expression of lymphangiogenic cytokines were not related to any of the biological tumour features or to patient survival. Three orthotopic nude mouse PDAC models did not reveal any increase in tumour-associated LVD, despite a high rate of lymph node metastasis. Lymph vessel proliferation was comparable in PDAC and chronic pancreatitis, in both humans and mice. In conclusion, increased lymphangiogenic activity is not required for and does not significantly affect the lymphatic spread of PDAC. The reduced number of human and murine intratumoural lymph vessels indicates that lymphatic metastasis takes place predominantly via peritumoural lymphatic vessels. The weak expression of lymphangiogenic cytokines in neoplastic cells and lymphatic vessel proliferation in peritumoural regions and chronic pancreatitis indicate that inflammation may be the reason for the low rate of lymphangiogenesis.     

Lack of prognostic significance of angiogenesis in canine melanocytic tumours

The prognostic significance of angiogenesis in some canine tumours has been investigated, but little is known about its relevance in canine melanocytic tumours (MTs). The aim of this study was to evaluate the prognostic significance of angiogenesis in canine MTs. A total of 36 cutaneous melanocytomas (benign MTs), 40 cutaneous melanomas (malignant MTs) and 43 oral melanomas were studied. Survival data were available for a subset of 59 cases. Microvessel density (MVD) and endothelial area (EA) were determined by immunolabelling using an antibody specific for von Willebrand factor (vWF). Mean MVD (expressed as the number of microvessels per mm(2)) was 129 ± 14 in melanocytomas, 191 ± 16 in cutaneous melanomas and 208 ± 16 in oral melanomas. Mean EA (expressed as the percentage of the total area) was 1.5 ± 0.14 in melanocytomas, 2.6 ± 0.2 in cutaneous melanomas and 2.4 ± 0.3 in oral melanomas. The differences in MVD and EA between melanocytomas and melanomas were significant (P = 0.001 and P = 0.003, respectively). MVD and EA were significantly correlated between cutaneous and oral MTs (r = 0.54; P <0.001 and r = 0.63; P <0.001, respectively). MVD and EA were not related to survival in cutaneous and oral MTs. In conclusion, tumour vascularization was higher in melanomas than in melanocytomas, but it seemed to have no prognostic significance in these tumours.     

Objective assessment of blood and lymphatic vessel invasion and association with macrophage infiltration in cutaneous melanoma

The aims of this study were to investigate the role of vascular invasion (blood and lymphatic), vessel density and the presence of tumour-associated macrophages as prognostic markers in 202 cutaneous melanoma patients. Sections of primary melanoma were stained with lymphatic-specific antibody D2-40 to assess lymphatic vessel invasion and density in intratumoural and peritumoural areas; an antibody against endothelial marker CD34 was used to determine blood vessel invasion and density, and an antibody against CD68 was used to determine macrophage counts. Immunohistochemically determined vascular invasion (combined blood and lymphatic) was compared with that determined using haematoxylin and eosin (H&E) staining. The use of immunohistochemistry increased detection of vascular invasion from 8-30% of patients, and histological exam of H&E-stained tissue was associated with a false positive rate of 64%. Lymphatic vessel invasion occurred at a much higher frequency than blood vessel invasion (27 and 4% of patients, respectively). Although immunohistochemically detected vessel invasion was significantly associated with histological markers of adverse prognosis, such as increased Breslow thickness, ulceration and mitotic rate (all P<0.001), no associations with relapse-free or overall survival were observed. High macrophage counts were significantly associated with markers of aggressive disease, such as Breslow thickness, ulceration and mitotic rate (P<0.001, P<0.001, P=0.005, respectively), and lymphatic vessel invasion and high microvessel density (P=0.002 and P=0.003, respectively). These results suggest that vascular invasion is more accurately detected using immunohistochemistry and occurs predominantly via lymphatic vessels. The association of vessel characteristics with histological characteristics of the primary melanoma provides evidence for their biological importance in melanoma, but that they were not associated with clinical outcome attests to the value of existing histological prognostic biomarkers. We note that a high macrophage count may be associated with neovascularisation and primary tumour growth, and may also promote invasion through lymphatic vessels.     

Tumour lymphangiogenesis is a possible predictor of sentinel lymph node status in cutaneous melanoma: a case-control study

BACKGROUND: Cutaneous melanoma spreads preferentially through the lymphatic route and sentinel lymph node (SLN) status is regarded as the most important predictor of survival. AIMS: To evaluate whether tumour lymphangiogenesis and the expression of vascular endothelial growth factor C (VEGF-C) is related to the risk of SLN metastasis and to clinical outcome in a case-control series of patients with melanoma. METHODS: Forty five invasive melanoma specimens (15 cases and 30 matched controls) were investigated by immunostaining for the lymphatic endothelial marker D2-40 and for VEGF-C. Lymphangiogenesis was measured using computer assisted morphometric analysis. RESULTS: Peritumorous lymphatic vessels were more numerous, had larger average size, and greater relative area than intratumorous lymphatics. The number and area of peritumorous and intratumorous lymphatics was significantly higher in melanomas associated with SLN metastasis than in non-metastatic melanomas. No significant difference in VEGF-C expression by neoplastic cells was shown between metastatic and non-metastatic melanomas. Using logistic regression analysis, intratumorous lymphatic vessel (LV) area was the most significant predictor of SLN metastasis (p = 0.04). Using multivariate analysis, peritumorous LV density was an independent variable affecting overall survival, whereas the intratumorous LV area approached significance (p = 0.07). CONCLUSIONS: This study provides evidence that the presence of high peritumorous and intratumorous lymphatic microvessel density is associated with SLN metastasis and shorter survival. The intratumorous lymphatic vessel area is the most significant factor predicting SLN metastasis. The tumour associated lymphatic network constitutes a potential criterion in the selection of high risk patients for complementary treatment and a new target for antimelanoma therapeutic strategies.     

Prognostic impact of VEGF, CD31, CD34, and CD105 expression and tumour vessel invasion after radical surgery for IB-IIA non-small cell lung cancer

AIMS: To evaluate the prognostic impact of tumour angiogenesis assessed by vascular endothelial growth factor (VEGF), microvessel density (MVD), and tumour vessel invasion in patients who had undergone radical resection for stage IB-IIA non-small cell lung cancer (NSCLC). METHODS: Fifty one patients (42 men, nine women; mean age, 62.3 years; SD, 6.9) undergoing complete surgical resection (35 lobectomy, 16 pneumonectomy) of pathological stage IB (n = 43) and IIA (n = 8) NSCLC were evaluated retrospectively. No patient underwent postoperative chemotherapy or neoadjuvant treatment. Tumour specimens were stained for VEGF and specific MVD markers: CD31, CD34, and CD105. RESULTS: VEGF expression significantly correlated with high CD105 expression (p < 0.0001) and tumour vessel invasion (p = 0.04). Univariate analysis showed that those patients with VEGF overexpression (p = 0.0029), high MVD by CD34 (p = 0.0081), high MVD by CD105 (p = 0.0261), and tumour vessel invasion (p = 0.0245) have a shorter overall survival. Furthermore, multivariate Cox regression analysis showed that MVD by CD34 (p = 0.007), tumour vessel invasion (p = 0.024), and VEGF expression (p = 0.042) were significant predictive factors for overall survival. Finally, the presence of both risk factors, tumour vessel invasion and MVD by CD34, was highly predictive of poor outcome (odds ratio, 3.4; 95% confidence interval, 1.7 to 6.5; p = 0.0002). CONCLUSIONS: High MVD by CD34 and tumour vessel invasion are more closely related to poor survival than the other neoangiogenetic factors in stage IB-IIA NSCLC. This may be because these factors are more closely related to the metastatic process.     

Pregnancy promotes melanoma metastasis through enhanced lymphangiogenesis

The relationships of pregnancy and melanoma have been debatable. Our aim was to assess the influence of gestation on the course of melanoma in a classic murine model of tumor progression and in women. B16 mouse melanoma cells were injected in nonpregnant or pregnant mice on day 5 of gestation. Animals were evaluated for tumor progression, metastases, and survival. Tumor sections were analyzed for lymphatic and blood vessel number and relative surface and expression of angiogenic growth factors. Finally, primary melanomas from pregnant and nonpregnant women, matched for age and tumor thickness, were also considered. Tumor growth, metastasis, and mortality were increased in B16-injected pregnant mice. Tumors displayed an increase in intratumoral lymphangiogenesis during gestation. This increased lymphatic angiogenesis was not observed in normal skin during gestation, showing its specificity to the tumor. An analysis of melanoma from pregnant and matched nonpregnant women showed a similar increase in lymphatic vessels. Tumors from pregnant mice had increased expression of vascular endothelial growth factor A at the RNA and protein levels. The increased vascular endothelial growth factor A production by melanoma cells could be reproduced in culture using pregnant mouse serum. In conclusion, pregnancy results in increased lymphangiogenesis and subsequent metastasis. Caution should be applied in the management of patients with advanced-stage melanoma during gestation.     

The vascularity of primary cutaneous melanoma

In primary cutaneous malignant melanoma, the vascularity of the dermis immediately deep to the lesion may relate to tumour aggressiveness and to prognosis. These newly formed dermal vessels are incorporated into the melanoma to form the tumour microcirculation. We have assessed the percentage vascular volume in a series of primary melanomas in order to investigate the relationship between tumour vascularity and maximum tumour thickness. For the 64 melanomas included in this study, there appeared to be a significant relationship between the percentage vascular volume and the maximum tumour thickness. This relationship was not influenced by the presence of necrosis, vascular invasion, regression, or lymphocytic infiltrate, nor by the growth phase of the tumour. However, the percentage vascular volume was very low in the occasional thick melanoma, at least one of which was associated with prolonged survival. It seems possible that a low tumour vascularity could correlate with a relatively favourable outcome in cutaneous melanoma.     

Tumor angiogenesis as a prognostic factor in thick cutaneous malignant melanoma

The prognostic value of the extent of neovascularization in cutaneous melanoma is a highly controversial issue. The aim of the current study was to evaluate whether the morphometric analysis of tumor vascularity may be helpful in predicting the clinical outcome of patients with thick cutaneous melanomas. A series of 15 patients with melanoma (>3 mm in thickness) who did not experience disease progression after long-term follow-up (10 years) and 30 matched controls who underwent recurrence and/or metastases were selected for the study. Microvessels were immunohistochemically stained with anti-CD31 antibody. Several parameters, including vessel number, vascular density, vessel area, equivalent circle diameter, perimeter, shape factor, compactness, and the number of vascular ramifications per 100 vessel sections, were quantitatively assessed by a computer-aided semi-automatic image analysis system. Mean vessel area was 341.69 microm2 in cases without progression and 512.55 microm2 in the progressed melanomas (P=0.008, Mann-Whitney U test). The mean equivalent circle diameter was 18.95 microm in non-progressed melanomas and 22.57 microm in progressed melanomas (P=0.009). The mean number of ramifications was 0.8 in cases without progression and 1.9 in the controls (P=0.03). Microvessel count and vascular density were higher in progressed cases (17.37 vs. 11.73 and 28.94/mm2 vs. 19.55/mm2, respectively), but the difference did not reach statistical significance (P=0.06). Our results suggest that neovascularization is a critical event in the progression of thick melanoma. Its prognostic significance is better assessed by quantification of vessel area, equivalent circle diameter, and microvessel branching, whereas microvessel count and vascular density do not provide significant prognostic information.     

Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival

Malignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, and lymph node metastasis is a major determinant for the staging and clinical management of melanoma. However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear. We investigated whether tumor lymphangiogenesis occurs in human malignant melanomas of the skin and whether the extent of tumor lymphangiogenesis may be related to the risk for lymph node metastasis and to patient survival, using double immunostains for the novel lymphatic endothelial marker LYVE-1 and for the panvascular marker CD31. Tumor samples were obtained from clinically and histologically closely matched cases of primary melanomas with early lymph node metastasis (n = 18) and from nonmetastatic melanomas (n = 19). Hot spots of proliferating intratumoral and peritumoral lymphatic vessels were detected in a large number of melanomas. The incidence of intratumoral lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival. Metastatic melanomas had significantly more and larger tumor-associated lymphatic vessels, and a relative lymphatic vessel area of >1.5% was significantly associated with poor disease-free and overall survival. In contrast, no differences in the density of tumor-associated blood vessels were found. Vascular endothelial growth factor and vascular endothelial growth factor-C expression was equally detected in a minority of cases in both groups. Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma.     

Histological study of the regrowth of a human melanoma xenograft exposed to single dose irradiation

The aim of this investigation was to find the location and the histological characteristics of cells that were the source of tumour regrowth after single dose irradiation. A human melanoma xenograft was irradiated with a single dose of 25.0 Gy which gives local tumour control in nearly 50% of treated animals. Serial histological sections were made from tumours removed during the first two weeks after irradiation. During the first week the perivascular organization of tumour parenchyma disappeared and the central part of the tumour became necrotic. The occurrence of vascular stasis, thrombosis and endothelial cell changes indicated that radiation injury to the vascular system was involved in the disappearance of the tumour cords. Tumour cells that remained histologically intact were located in subcapsular areas. The incidence of normal mitotic figures increased, and the fraction of abnormal mitoses and the incidence of micronuclei decreased 5 to 7 days before macroscopical regrowth was usually detected. It is concluded that cells which are the source of tumour regrowth were located in the subcapsular areas at the time of irradiation. Radiation injury to the tumour vascular system was an important factor in the necrotization of the tumour centre after treatment.     

Reduced expression of p16 and p27 is correlated with tumour progression in cutaneous melanoma

AIMS: To determine if the cyclin dependent kinase inhibitors (CDKIs) p16 and p27 show reduced expression in the progression from benign to malignant melanocytic tumours, and to correlate these findings with patient prognosis. METHODS: Ninety-two melanocytic tumours were assessed for immunohistochemical expression of p16 and p27. These specimens included nine compound naevi, 10 dysplastic naevi, 17 thin (<1 mm) melanomas, 22 thick (>1 mm) melanomas, nine in-transit metastases, 13 lymph node metastases, and 12 soft tissue metastases. Clinicopathological information on the 39 patients with melanoma primaries was obtained from the Sydney Melanoma Unit database. The median follow up period was 43.3 months. RESULTS: A significant loss of expression of p16 and p27 was found with tumour progression. Positive expression of p27 was found in all compound and dysplastic naevi but only 43.6% of melanoma primaries. Expression of p27 was greater in lymph node and in-transit metastases (63.6%), but lower in soft tissue metastases (36.4%). Positive expression of nuclear p16 was evident in 73.7% of benign naevi, 28.2% of primary melanomas and 14.7% of metastatic melanomas. Neither p16 nor p27 expression was significantly correlated with overall survival, disease free survival or other clinicopathological markers. CONCLUSIONS: The CDKIs p16 and p27 are associated with tumour progression in melanoma, but do not reliably predict recurrence or survival.     

Regional growth of different human melanomas as metastases in the brain of nude mice.

Cells from eight different human melanomas and two murine melanomas were injected into the internal carotid artery of anesthetized nude mice. Although all were injected by the same route, particular melanomas produced lesions in different regions of the brain. Two melanoma cell lines isolated originally from brain metastases in patients produced metastases predominantly in the brain parenchyma. In contrast, melanoma cells from subcutaneous or lymph node metastases produced more lesions in the meninges, choroid plexus, and ventricles than in the brain parenchyma. All of the melanomas grew in the brain after a direct intracerebral injection. The pattern of brain metastasis did not correlate with tumorigenicity per se or with the ability of the melanomas to grow in the lungs of nude mice. Two mouse melanomas showed different patterns of experimental metastasis after internal carotid artery injection, with one growing predominantly in the parenchyma and the other more frequently in the meninges and choroid plexus. The growth pattern of human melanoma metastasis in the brain of T cell-deficient nude mice suggests that it is determined by properties unique to each tumor interacting with the host's organ microenvironment.     

Autonomous histopathological regression of primary tumours associated with specific immune responses to cancer antigens

Spontaneous histopathological regression of cancer has been reported. The involvement of the immune system in such regression has been advocated, leading to the theory of immunological surveillance against cancer. A prediction of this theory is that common tumour antigens can be recognized upon repeated exposure by cell-mediated immunity, which leads to tumour regression and the subsequent appearance of tumour antigen-loss variants. However, no direct evidence has been provided in non-viral-induced experimental animal models of primary malignancy or in human primary cancer. This study examined two groups of melanoma patients where histopathological regression of the primary tumour was observed. Many of the 23 patients with multiple (> or =3) primary melanomas showed significant regression of their last melanoma (median 33%, mean 40) compared with matched melanomas from patients with a single primary melanoma (median 0%, mean 12) (p=0.0080), or compared with their first primary melanoma (p=0.0013). Regression was consistent with an 'immunization effect' seen in murine tumour transplantation studies, where inoculation with > or =3 asynchronous tumours induces transplantation rejection on subsequent challenge. A significant decrease in the expression of the melanoma common tumour antigen MART-1 in the last primary tumour from multiple melanoma patients (median 8%, mean 24) versus matched single melanoma patients (median 79%, mean 68) (p=0.0041) and in the last versus first tumour in multiple primary patients was found (p=0.0083). Metastases from 17 patients whose primary skin melanomas had completely regressed (occult primary melanoma) also showed significant MART-1 loss (median 0%, mean 11) compared with matched metastases from patients with non-regressing primary melanoma (median 51%, mean 50) (p=0.0013). MART-1 antigen-loss variants observed in the multiple primary and occult primary patients correlated with the presence of peripheral blood MART-1-specific cytotoxic T lymphocytes (CTLs) (p=0.03). No similar effects were observed with two other melanoma antigens, gp100 and CD63. Thus, in two groups of human melanoma patients, evidence is provided for histopathological tumour regression associated with cancer immune surveillance.     

Morphology of angiogenesis in human cancer: a conceptual overview, histoprognostic perspective and significance of neoangiogenesis

This paper reviews the histomorphological aspects of angiogenesis and neoangiogenesis, quantitative and qualitative, and their applications in prognostic evaluation of neoplastic diseases. The merits and weak points of intratumoral microvessel density (MVD), a widely regarded bona fide predictor of tumour growth, metastases and patient survival, are discussed. Total microvascular area (TVA) has been found useful in recent prognostic studies utilizing newer immunohistochemical vascular markers. Of particular significance is the fact that MVD and TVA are most predictive of patient outcome in those tumours that induce significant neoangiogenesis, namely carcinomas of breast and prostate, and haematological malignancies. In contrast, carcinomas of lung and urinary bladder do not show significant associations of MVD and TVA with poor prognosis, reflecting differences in angiogenic mechanisms. In gliomas, MVD appears to correlate with outcome in high-grade, but not low-grade tumours, and does not correlate with tumour cellularity in the infiltrating portions of the tumour, reflecting a paucity of neoangiogenesis and directional vascular growth. Recent studies have found CD105, Tie-2/Tek and vascular endothelial growth factor receptors to be the best markers of neoangiogenesis. The vascular parameters so measured correlate better with overall and disease-free survival in breast, colon and lung carcinoma than panendothelial markers such as CD31. A correlation of vascular patterns with prognosis has been established in ocular melanomas, glioblastomas and squamous carcinomas of head and neck region. Vascular networks with closed loops are closely associated with mortality due to metastases in uveal melanomas. Fewer bizarre glomeruloid vessels and prominent classical capillary pattern was an independent predictor of longer survival in glioblastoma. Therefore a judicious combination of quantitative and qualitative microscopic angiogenic parameters, with emphasis on neoangiogenesis and vascular patterns wherever applicable, should be an integral component of a more consistent tumour staging system for accurate prognostic evaluation of tumours, selection of optimal anti-angiogenic therapy and pertinent research.     

Investigation of intratumoural and peritumoural lymphatics expressed by podoplanin and LYVE-1 in the hybridoma-induced tumours

Tumour-associated lymphatics contribute to a key component of metastatic spread, however, the biological interaction of tumour cells with intratumoural and peritumoural lymphatics (ITLs and PTLs) has remained unclear. To address this important issue, we have focused on the morphological and molecular aspects of newly formed lymphatics (lymphangiogenesis) and pre-existing lymphatics in the intratumoural and peritumoural tissues by using a hybridoma-induced tumour model. In the present study, ITLs with very high vessel density within the tumour mass showed small and flattened contours that varied from non-solid-to-solid tumours, whereas PTLs were relatively disorganized and tortuous, and packed with a cluster of tumour cells at the tumour periphery. Lymphatic endothelial cells (LECs) both in ITLs and PTLs were expressed with LYVE-1 and podoplanin in various tumour tissues, in which initial lymphatics were extremely extended and dilated. The tumour cells were frequently detected adhering to or penetrating lymphatic walls, especially near the open junctions. In the metastatic tissues, lymphangiogenic vasculatures occurred within the tumour matrix, and collecting PTLs represented abnormal twisty valve leaflets. The Western blot and RT-PCR analysis showed local variations of LEC proliferating potentials and lymphatic involvement in metastasis by a distinct profile of the protein and mRNA expression by LYVE-1, podoplanin, Prox-1 and vascular endothelial growth factor-3 (VEGFR-3). These findings indicated that both ITLs and PTLs, including enlarged pre-existing and newly formed lymphatics, may play a crucial role in metastasis with an active tumour cell adhesion, invasion, migration and implantation.     

Vascular volume in B16 allografts and human melanoma xenografts estimated by means of Hoechst 33342

The vasculature of B16, a murine melanoma and Mel-mo, a human melanoma, was studied using intravital staining of patent capillaries by the fluorescent bisbenzamine dye Hoechst 33342. Capillaries were numerous at the edge of tumours in both the lines studied, but were scarcer within the nodules. Vascular volume as a proportion of total tumour volume was estimated by means of point counting. In both B16 and Mel-mo, the percentage vascular volume was inversely related to log tumour weight. Tumour necrosis, which increased with tumour size, was inversely correlated with percentage vascular volume, emphasizing the central under-perfusion of these experimental tumour nodules. This pattern of perfusion, with greater density of functioning capillaries at the periphery of tumour nodules, was seen in both the tumour lines examined despite differences in the degree and pattern of necrosis.     

A combination of plasmid DNAs encoding murine fetal liver kinase 1 extracellular domain,murine interleukin-12, and murine interferon-γ inducible protein-10 leads to tumor regression and survival in melanoma-bearing mice

The vascular endothelial growth factor (VEGF) and its interaction with the vascular endothelial growth factor receptor 2 [VEGFR2/murine fetal liver kinase 1 (Flk-1), human kinase domain receptor] are an important angiogenic pathway leading to tumor vascularization. A plasmid DNA encoding the complete extracellular domain (ECD) of murine Flk-1 including the endogenous signal sequence was designed as a possible competitor of the receptor to sequester VEGF. The plasmid DNA was used to treat B16F10 cell-induced subcutaneous melanomas in syngeneic mice. The Flk-1 ECD-encoding plasmid DNA injected intramuscularly did not lead to tumor reduction. However, intratumoral injection caused a dose-dependent reduction and significant retardation of tumor growth. Blood vessels analyzed by immunohistochemistry with anti-CD31 antibodies as indicators of vascularization appeared smaller in diameter after treatment. A combination of Flk-1 ECD and DNA encoding murine interleukin-12 or murine interferon-gamma inducible protein-10 improved the effect, leading to tumor regression and long-term survival of the mice.     

Behaviour of four different B16 murine melanoma cell sublines: C57BL/6J skin

Transplantable murine melanomas are well‐established models for the study of experimental cancer therapies. The aim of this study was to explore the behaviour of four different B16 murine melanoma cell sublines after inoculation into C57BL/6J mice; and, more specifically to analyse skin changes, with respect to two specific parameters: clinical (tumour volume, melanin amount, erythema) and histological (H & E, S100, VEGF expression). Both non‐invasive and invasive analysis showed that B164A5 is the most aggressive melanoma cell line for C57BL/6J's skin, followed by B16F10 and then by diminished aggressive growth pattern by the B16GMCSF and B16FLT3 cell lines.     

Deficiency of bone marrow β3‐integrin enhances non‐functional neovascularization

β3‐Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in β3‐integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on β3‐null endothelial cells. Here we transplanted β3‐null bone marrow (BM) into wild‐type (WT) mice to dissect the role of BM β3‐integrin deficiency in pathological angiogenesis. Mice transplanted with β3‐null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow β3‐integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM β3‐integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with β3‐null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with β3‐null bone marrow a significant proportion of tumour blood vessels are non‐functional when compared with tumour blood vessels in WT‐transplanted controls. Furthermore, β3‐null‐transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT‐transplanted animals. BM β3‐integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF‐induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with β3‐null bone marrow when compared with WT‐transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in β3‐null‐transplanted mice. In conclusion, although BM β3‐integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM β3‐integrin in VEGF‐induced mobilization of bone marrow‐derived cells to the peripheral circulation and for the functionality of those vessels in which BM‐derived cells become incorporated. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.