NG2-PG in tumor vascularization and progression

描述（由申请人提供）：在MMTV-PyMT乳腺癌小鼠模型中，乳腺肿瘤细胞不表达NG2蛋白多糖，但在肿瘤基质中至少有三种重要细胞类型表达NG2蛋白多糖：肿瘤血管中的周细胞、从循环侵入肿瘤的髓系细胞和乳腺脂肪垫中的脂肪细胞。在MMTV-PyMT模型中，通过肿瘤潜伏期、生长速度和转移等几个标准，消融NG2可大大减缓乳腺肿瘤的进展，强调微环境因素在促进肿瘤发生中的作用。由于我们对肿瘤血管化和转移的兴趣，我们将重点关注NG2支持周细胞和髓细胞促肿瘤活性的机制。本提案的具体目的是在MMTV-PyMT模型中研究周细胞NG2和骨髓细胞NG2对乳腺肿瘤进展的各自影响。为此，我们将在这两个人群中利用细胞类型特异性的NG2消融来分析自发性和同种异体移植乳腺肿瘤的进展。在目的1中，我们将比较对照小鼠和通过与Pdgfrb/Cre转基因小鼠杂交产生的周细胞特异性NG2缺失小鼠的肿瘤进展和肿瘤血管化。肿瘤血管系统的特征将包括确定内皮细胞的周细胞嵌套、周细胞和内皮细胞的成熟、基底层的组装、血管通畅、血管渗漏和肿瘤缺氧。周细胞和内皮细胞的体外共培养将用于进一步阐明NG2支持周细胞/内皮细胞串扰的机制。在目的2中，我们将比较对照小鼠和通过将NG2粘接小鼠与LysM/Cre转基因小鼠杂交产生的骨髓特异性NG2缺失小鼠的骨髓细胞功能。NG2消融影响的表征将包括确定M1与M2极化，评估关键髓细胞群大小和分化状态的变化，以及这些群体在关键部位（如脉管系统和肿瘤边缘）的定位。通过肿瘤细胞和巨噬细胞的体外共培养，探讨NG2影响巨噬细胞/肿瘤细胞相互作用的机制。在Aim 3中，我们将使用周细胞特异性和髓细胞特异性NG2缺失小鼠来研究NG2在乳腺肿瘤肺转移中的重要性。我们将使用荧光标记的乳腺肿瘤细胞将转移过程分解为其组成阶段，包括肿瘤细胞内渗到脉管系统，肿瘤细胞从脉管系统外渗到肺部，以及在肺部建立转移前壁龛。

DESCRIPTION (provided by applicant): The NG2 proteoglycan is not expressed by mammary tumor cells in the MMTV-PyMT mouse model of breast cancer, but is expressed by at least three important cell types in the tumor stroma: pericytes in the tumor vasculature, myeloid cells that invade the tumors from the circulation, and adipocytes in the mammary fat pad. By several criteria, including tumor latency, growth rate, and metastasis, ablation of NG2 greatly slows mammary tumor progression in the MMTV-PyMT model, emphasizing the power of microenvironmental factors in promoting tumorigenesis. Due to our interest in tumor vascularization and metastasis, we are focusing on mechanisms by which NG2 supports the tumor-promoting activities of pericytes and myeloid cells. The specific aims of this proposal will be to examine the respective effects of pericyte NG2 and myeloid cell NG2 on mammary tumor progression in the MMTV-PyMT model. For these purposes we will utilize cell type-specific ablations of NG2 in these two populations to analyze the progression of both spontaneous and allografted mammary tumors. In Aim 1 we will compare tumor progression and tumor vascularization in control mice and in pericyte-specific NG2 null mice produced by crossing NG2 floxed mice with Pdgfrb/Cre transgenic mice. Characterization of the tumor vasculature will include determinations of pericyte ensheathment of endothelial cells, maturation of pericytes and endothelial cells, assembly of the basal lamina, vessel patency, vessel leakiness, and tumor hypoxia. In vitro co-cultures of pericytes and endothelial cells will be used to further elucidate mechanisms by which NG2 supports pericyte/endothelial cell crosstalk. In Aim 2 we will compare myeloid cell function in control mice and in myeloid-specific NG2 null mice produced by crossing NG2 floxed mice with LysM/Cre transgenic mice. Characterization of the effects of NG2 ablation will include determination of M1 versus M2 polarization, assessment of changes in the size and differentiation state of key myeloid populations, and localization of these populations to critical sites such as vasculature and tumor margins. In vitro co-cultures of tumor cells and macrophages will be used to explore mechanisms by which NG2 affects macrophage/tumor cell interaction. In Aim 3 we will use both the pericyte-specific and myeloid-specific NG2 null mice to study the importance of NG2 in mammary tumor metastasis to the lungs. We will use fluorescent-labeled mammary tumor cells to dissect the metastatic process into its component stages, including intravasation of tumor cells into the vasculature, extravasation of tumor cells from the vasculature into the lungs, and establishment of pre-metastatic niches in the lungs.