NG2-PG in tumor vascularization and progression

描述(申请人提供)：在MMTV-PYMT乳腺癌小鼠模型中，NG2蛋白多糖不在乳腺肿瘤细胞中表达，但在肿瘤间质中至少表达三种重要的细胞类型：肿瘤血管系统中的周细胞，从循环中侵入肿瘤的髓系细胞，以及乳房脂肪垫中的脂肪细胞。在MMTV-PYMT模型中，通过包括肿瘤潜伏期、生长速度和转移在内的几个标准，NG2的消融显著减缓了乳腺肿瘤的进展，强调了微环境因素在促进肿瘤发生中的力量。由于我们对肿瘤血管形成和转移的兴趣，我们将重点放在NG2支持周细胞和髓系细胞促肿瘤活性的机制上。这项建议的具体目的将是检查周细胞NG2和髓细胞NG2在MMTV-PYMT模型中对乳腺肿瘤进展的各自影响。出于这些目的，我们将利用这两个人群中特定细胞类型的NG2消融来分析自发性和同种异体乳腺肿瘤的进展情况。在目标1中，我们将比较对照小鼠和周细胞特异性NG2缺失小鼠的肿瘤进展和肿瘤血管形成，这些小鼠是通过将NG2基因突变的小鼠与PDGFRb/Cre转基因小鼠杂交而产生的。肿瘤血管的特征将包括测定内皮细胞的周细胞包膜、周细胞和内皮细胞的成熟、基底板的组装、血管通畅性、血管渗漏和肿瘤缺氧。在体外，周细胞和内皮细胞的共培养将被用来进一步阐明NG2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NG2基因缺失小鼠的髓系细胞功能，这些小鼠是由NG2系小鼠与LysM/Cre转基因小鼠杂交产生的。NG2消融效果的特征将包括确定M1与M2极化，评估关键髓系群体的大小和分化状态的变化，以及将这些群体定位于关键部位，如血管系统和肿瘤边缘。肿瘤细胞和巨噬细胞的体外共培养将被用来探索NG2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髓系特异性NG2缺失小鼠来研究NG2在乳腺肿瘤肺部转移中的重要性。我们将使用荧光标记的乳腺肿瘤细胞将转移过程分成不同的阶段，包括肿瘤细胞进入血管系统，肿瘤细胞从血管系统渗出到肺，以及在肺中建立转移前的壁龛。 公共卫生相关性：乳腺癌治疗的改进将需要更好地了解与乳腺肿瘤细胞本身和肿瘤所在的宿主间质相关的促癌因素。NG2蛋白多糖存在于两种基质细胞上，即微血管周细胞和肿瘤髓系细胞，这两种细胞通过支持肿瘤血管形成和转移而促进乳腺肿瘤的进展。我们对NG2在刺激这些细胞的促肿瘤活性中的作用的研究将加深我们对血管形成和转移过程的理解，并将确定蛋白多糖作为乳腺癌治疗的多焦点靶点。

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. PUBLIC HEALTH RELEVANCE: Improvements in the treatment of breast cancer will require a better understanding of tumor-promoting factors associated with both the mammary tumor cells themselves and the host stroma in which the tumors reside. The NG2 proteoglycan is present on two stromal cell types, microvascular pericytes and tumor myeloid cells, that promote mammary tumor progression by supporting tumor vascularization and metastasis. Our studies on the role of NG2 in stimulating the tumor-promoting activities of these cells will enhance our understanding of the vascularization and metastatic processes and will also identify the proteoglycan as a multifocal target for breast cancer therapy.