The Natural Progression of Microvasculature in Primary Tumor and Lymph Node Metastases in a Breast Carcinoma Model: Relationship between Microvessel Density, Vascular Endothelial Growth Factor Expression and Metastatic Invasion

Abstract

The natural course of tumor microvascularity in rat MTLn3 mammary adenocarcinomas was studied. The relationship between microvessel density (MVD), vascular endothelial growth factor (VEGF) expression, and histopathology was compared in primary and metastatic axillary (ALN) and inguinal lymph node (ILN) tumors over 5-6 tumor doublings. Excised tumors were examined for (i) MVD assessed by immunostaining with anti-CD31 antibody, (ii) VEGF expression assessed by immunostaining with anti-VEGF antibody, and (iii) histopathologic extent of metastatic lymph node invasion. MVD and VEGF scores rose asymptotically with increasing tumor weight in both primary and metastatic tumors. The MVD saturation level was significantly greater for primary tumors (MVD = 22) than for ALNs or ILNs (MVD = 14). Maximal VEGF score was not statistically different between the three kinds of tumors, however the rate of rise in VEGF expression was different. Near-maximal VEGF expression occurred early in tumor growth, preceding microvessel development. Both MVD and VEGF expression in lymph nodes were proportional to the pathology score characterizing increasing metastatic invasion. LNMs limited to the subcapsular sinus had the lowest MVD, indicating an ability to survive without significant vasculature. These findings underscore the differences in angiogenesis between primary tumors and LNMs and have implications for therapy of metastatic cancer. INTRODUCTIONThe development of new blood vessels, a multi-step process known as angiogenesis, is required for both tumor growth and metastatic spread of tumor cells. 1,2 Angiogenesis is initiated by tumor cell-secreted angiogenic factors, particularly members of the vascular endothelial growth factor (VEGF) family. 3 VEGF-A is essential for the normal development and differentiation of the vascular system 4,5 and also induces pathological endothelial cell proliferation and angiogenesis. 3,4,6 VEGF-C and VEGF-D are important in lymphangiogenesis and lymphatic metastasis. [7][8][9] Blocking VEGF ligand-receptor interactions significantly inhibits tumor growth 10-16 thus providing a promising target for therapeutic intervention. [17][18][19] Hypoxia is an important regulator of VEGF. [20][21][22] Since hypoxic areas are commonly present in tumors, upregulation of VEGF leads to angiogenesis and provides a mechanism for tumors to maintain a vascular supply insuring oxygen and nutrients. 23 The transition from a quiescent tumor to an invasive tumor, reflecting a change in the balance between cell death and proliferation, is accompanied by the acquisition of angiogenic properties. 24 This so-called angiogenic switch requires not only upregulation of promoters of angiogenesis, but also downregulation of suppressors. 25 Thus, tumor dormancy and control may be achieved by endogenous angiogenesis inhibitors such as angiostatin, 26 endostatin, 27 and metronomic chemotherapy. 28 Tumor cells can also be stimulated in a paracrine manner by growth factors and other cytokines produced by stromal cells, particula...

The mechanisms on apoptosis by inhibiting VEGF expression in human breast cancer cells

“…Via its binding to tyrosine kinase receptors, particularly VEGFR-2, VEGF induces mitogenesis and chemotaxis of endothelial cells, which lead to new blood vessel formation, tumor growth, and metastasis. In addition to their typical expression on endothelial cells, VEGF and VEGFR-2 are expressed in certain solid tumors [5,6]. This finding has led to the hypothesis that an autocrine loop exists by which VEGF stimulates tumor growth not only via its actions on endothelial cells but also by directly stimulating VEGFR-2 on tumor cells.…”

“…Evidences support the correlation between levels of VEGF tumor progression and invasion in breast cancer, which is the leading cause of death in women 30 to 70 years of age in the world [5,8,9]. VEGFR-2 and VEGF are coexpressed in primary breast carcinomas, and their expression is increased when tumors shift to an angiogenic phenotype [10].…”

“…It is well known that VEGF is secreted by most solid tumors [3][4][5][6] and, via its interaction with its receptor VEGFR-2, mediates many key components of angiogenesis, including endothelial cell proliferation, invasion, migration, and survival, as well as vessel permeability [3][4][5][6][7]. Interestingly, near-maximal VEGF expression happens earlier than microvessel development during tumor growth, suggesting potential VEGF might have other important functions.…”

EGFR and microvessel density in canine malignant mammary tumours

“…High levels of angiogenic factors and histologic evidence of increased tumour neovascularization are considered to be associated with malignancy and prognosis in human breast cancer (Hanahan and Folkman, 1996;Locopo et al, 1998;Gasparini, 2000Gasparini, , 2001Leek, 2001) and different studies have indicated similar results in CMT (Restucci et al, 2000;Lavalle et al, 2009;Queiroga et al, 2011). EGFR signaling and angiogenesis have been independently evaluated as targets for therapy in human studies (Fox et al, 1994;Weidner and Gasparini, 1994;Nieto et al, 2007;Nickerson et al, 2012) and several factors support the potential interaction between them: tumour cells can be stimulated in a paracrine manner by growth factors; endothelial cells and tumour cells can stimulate each other's growth in the tumour microenvironment (Rowe et al, 2004); both EGF and TGF-a (ligands for EGFR) induce angiogenesis (Perrotte et al, 1999); EGFR expression and function in tumour-associated endothelial cells have also been described (Rak et al, 1995).…”

Altered Pigment Epithelium–Derived Factor and Vascular Endothelial Growth Factor Levels in Lymphangioma Pathogenesis and Clinical Recurrence

“…[11][12][13] Expression of VEGF and its receptors (VEGFR-2 and VEGFR-3) have extensively been correlated with malignant behavior, metastasis, and worsened prognosis in many different tumor types including colorectal cancer, prostate cancer, head and neck squamous cell cancer, breast cancer, ovarian cancer, and lung cancer. [14][15][16][17][18][19] In addition, anti-VEGF receptor antibodies have been demonstrated to decrease tumor growth, decrease angiogenesis, and increase hypoxia in preclinical studies. 20,21 Clinically, they also have been shown to increase patient survival when combined with chemotherapy.…”