NG2-PG in tumor vascularization and progression

描述（由申请方提供）：在乳腺癌的MMTV-PyMT小鼠模型中，NG 2蛋白聚糖不被乳腺肿瘤细胞表达，但被肿瘤间质中的至少三种重要细胞类型表达：肿瘤脉管系统中的周细胞、从循环侵入肿瘤的髓样细胞和乳腺脂肪垫中的脂肪细胞。 通过几个标准，包括肿瘤潜伏期，生长速率和转移，NG 2的消融大大减缓了MMTV-PyMT模型中的乳腺肿瘤进展，强调了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，我们专注于NG 2支持周细胞和髓样细胞促肿瘤活性的机制。 该提议的具体目的是在MMTV-PyMT模型中检查周细胞NG 2和髓样细胞NG 2对乳腺肿瘤进展的各自作用。 出于这些目的，我们将利用这两个群体中NG 2的细胞类型特异性消融来分析自发性和同种异体移植乳腺肿瘤的进展。 在目标1中，我们将比较对照小鼠和通过将NG 2 floxed小鼠与Pdgfrb/Cre转基因小鼠杂交产生的周细胞特异性NG 2缺失小鼠中的肿瘤进展和肿瘤血管化。 肿瘤血管系统的表征将包括确定内皮细胞的周细胞鞘、周细胞和内皮细胞的成熟、基底层的组装、血管通畅性、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NG 2支持周细胞/内皮细胞串扰的机制。 在目标2中，我们将比较对照小鼠和通过将NG 2 floxed小鼠与LysM/Cre转基因小鼠杂交产生的骨髓特异性NG 2缺失小鼠中的骨髓细胞功能。 NG 2消融效应的表征将包括确定M1与M2极化，评估关键骨髓细胞群的大小和分化状态的变化，以及这些细胞群在关键部位（如血管和肿瘤边缘）的定位。 肿瘤细胞和巨噬细胞的体外共培养将用于探索NG 2影响巨噬细胞/肿瘤细胞相互作用的机制。 在目标3中，我们将使用周细胞特异性和骨髓特异性NG 2缺失小鼠来研究NG 2在乳腺肿瘤转移到肺中的重要性。 我们将使用荧光标记的乳腺肿瘤细胞来将转移过程分解为其组成阶段，包括肿瘤细胞向脉管系统的内渗、肿瘤细胞从脉管系统向肺的外渗以及肺中转移前小生境的建立。

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.