Early vascular deficits are correlated with delayed mammary tumorigenesis in the MMTV-PyMT transgenic mouse following genetic ablation of the NG2 proteoglycan

Abstract

IntroductionThe neuron-glial antigen 2 (NG2) proteoglycan promotes pericyte recruitment and mediates pericyte interaction with endothelial cells. In the absence of NG2, blood vessel development is negatively impacted in several pathological models. Our goal in this study was to determine the effect of NG2 ablation on the early development and function of blood vessels in mammary tumors in the mammary tumor virus-driven polyoma middle T (MMTV-PyMT) transgenic mouse, and to correlate these vascular changes with alterations in mammary tumor growth.MethodsThree different tumor paradigms (spontaneous tumors, transplanted tumors, and orthotopic allografts of tumor cell lines) were used to investigate the effects of NG2 ablation on breast cancer progression in the MMTV-PyMT transgenic mouse. In addition to examining effects of NG2 ablation on mammary tumor growth, we also investigated effects on the structure and function of tumor vasculature.ResultsAblation of NG2 led to reduced early progression of spontaneous, transplanted, and orthotopic allograft mammary tumors. NG2 was not expressed by the mammary tumor cells themselves, but instead was found on three components of the tumor stroma. Microvascular pericytes, myeloid cells, and adipocytes were NG2-positive in both mouse and human mammary tumor stroma. The effect of NG2 on tumor progression therefore must be stromal in nature. Ablation of NG2 had several negative effects on early development of the mammary tumor vasculature. In the absence of NG2, pericyte ensheathment of endothelial cells was reduced, along with reduced pericyte maturation, reduced sprouting of endothelial cells, reduced assembly of the vascular basal lamina, and reduced tumor vessel diameter. These early deficits in vessel structure are accompanied by increased vessel leakiness, increased tumor hypoxia, and decreased tumor growth. NG2 ablation also diminishes the number of tumor-associated and TEK tyrosine kinase endothelial (Tie2) expressing macrophages in mammary tumors, providing another possible mechanism for reducing tumor vascularization and growth.ConclusionsThese results emphasize the importance of NG2 in mediating pericyte/endothelial cell communication that is required for proper vessel maturation and function. In the absence of normal pericyte/endothelial cell interaction, poor vascular function results in diminished early progression of mammary tumors.

NG2 proteoglycan-dependent recruitment of tumor macrophages promotes pericyte-endothelial cell interactions required for brain tumor vascularization

“…33 We have reported a similar loss of vascular localization of VEGF in B16F10 tumors grown in collagen VI null mice 11 and in MMTV-PyMT mammary tumors in NG2 null mice. 6 As in the case of Mac-NG2ko mice, the loss of vascular VEGF localization in these previous studies was correlated with greatly reduced basal lamina assembly. In each case, reduced vessel diameter is the most dramatic structural manifestation of decreased vascular VEGF localization.…”

“…These are (a) the dramatic loss of N-cadherin expression by both pericytes and endothelial cells, (b) the marked loss of pericyte association with endothelial cells, and (c) the reduced diameter of tumor vessels. Changes in pericyte-endothelial cell interaction and in vessel diameter were previously noted in tumor vessels in germline NG2 null mice, 6,10 but were not detected in tumor vessels in PC-NG2ko mice.…”

“…In the context of the mouse mammary tumor viruspolyoma middle T (MMTV-PyMT) model of breast cancer, 5 germline ablation of NG2 retards mammary tumor progression. 6 While we attributed an important part of this effect to loss of NG2 from microvascular pericytes, with resulting deficits in mammary tumor vascularization, we also noted substantial losses of both tumor-associated macrophages (TAMs) and Tie2-expressing monocytes (TEMs) in tumors in NG2 null mice. In light of the importance of these myeloid populations in tumor vascularization, [7][8][9] one of our goals has been to investigate the role of NG2 in macrophage-dependent tumor vascularization.…”

“…Based on our previous experience with germline NG2 null mice and PC-NG2ko mice, 6,10,12 we expected these structural deficits in tumor vessels to have negative effects on vessel function in Mac-NG2ko mice. We therefore quantified vessel patency, vessel leakiness, and hypoxia in tumors from control and MacNG2ko mice.…”

“…Several reports from our laboratory have focused on pericyte function during tumor vascularization, 6,[10][11][12] especially with regard to their interaction with endothelial cells. These studies were aided substantially by development of methodologies for quantifying key aspects of tumor vessel structure and function.…”