Novel role of myeloid-derived lymphatic progenitors in induction of breast cancer lymphatics

项目总结/摘要 标题：髓源性淋巴祖细胞在乳腺癌淋巴管诱导中的新作用 淋巴结转移是乳腺癌的常见现象，是最重要的预后因素。 结果不佳的指标。为了到达局部淋巴结，肿瘤细胞只使用淋巴管。 毫不奇怪，淋巴转移的程度与肿瘤淋巴管的密度成正比。 船舶.因此，了解肿瘤诱导的机制具有重要的临床意义 淋巴管生成，即新淋巴管的形成。 目前认为，引起新淋巴管形成的主要机制是由 一种旁分泌淋巴管生成因子VEGF-C，激活淋巴管中表达的受体VEGFR-3 内皮细胞我们最近发现了一个根本不同的肿瘤淋巴管生成机制 这是对当前观点的补充。这种机制是由肿瘤动员的骨髓（BM）介导的。 衍生的单核细胞祖细胞在炎性肿瘤环境的影响下分化为 类神经细胞。这些细胞在这里被称为单核细胞衍生的淋巴管内皮细胞祖细胞或M- LECP具有两个主要特征：（1）髓系特异性蛋白和髓系特异性蛋白的共表达 通常分离成不同的谱系;和（2）整合到预先存在的淋巴细胞中的能力， 血管，这是淋巴生长的早期先决条件。 使用这些标准，我们发现在BC患者的血液和肿瘤中以及在一个 来自人类和小鼠起源的各种转移性原位乳腺肿瘤。所有含有M- LECP也显示淋巴管对骨髓特异性标记物呈阳性，这是一种既定现象 表明M-LECP的血管整合是发芽所需的。重要的是，我们最近建立了 肿瘤募集的M-LECP水平与肿瘤淋巴管密度和淋巴结转移显著相关， 临床BC患者的状态。我们在BC模型中的研究表明，M-LECP起源于BM衍生的 CD 11b+细胞对淋巴标志物Podoplanin（Pdpn）高度阳性。过继转移 通过CD 11b和Pdpn从转移性荷瘤小鼠中鉴定出表型不同的BM亚群 低转移组淋巴管密度明显增加，淋巴结转移明显。 初步数据还表明，M-LECP的分化可以在体外通过激活 人原代正常血液循环单核细胞中的Toll样受体4（TLR 4）。这个过程是受控的 通过NF-kB和转录因子c-Maf，一种新发现的单核细胞淋巴重编程调节因子。 TLR 4依赖性NF-kB和c-Maf的上调导致VEGFR-3通路的激活， 成为获得淋巴表型的重要里程碑。基于这些发现，我们假设 肿瘤淋巴管生长是转移的必要先决条件，M- TLR 4激活因子介导LECP与骨髓前体细胞分化的研究 为了验证这一假设，我们提出以下具体目标： (1)验证M-LECP与肿瘤诱导的淋巴管生成直接相关的假设 （2）阐明骨髓重编程的分子机制 确定TLR 4在CD 11b +/Pdpn+骨生成中的作用 骨髓细胞亚群与肿瘤淋巴管的形成和转移有直接关系。 影响和翻译相关性： 我们预计这些研究将建立一种新的机制，通过这种机制，骨髓来源的单核细胞募集 乳腺肿瘤促进淋巴管生成和淋巴结转移。验证这一新概念 将增强对肿瘤淋巴管形成机制的理解，并提出新的 抑制M-LECP分化的分子靶点。此外，检测到更高水平的肿瘤- 驻留和血液循环M-LECP可能会识别出患有更具侵袭性肿瘤的BC患者， 预先治疗以抑制局部扩散。这样的研究结果可以支持目前的临床 因此降低了BC患者的死亡率。

PROJECT SUMMARY/ABSTRACT Title: Novel Role of Myeloid-derived Lymphatic Progenitors in Induction of Breast Cancer Lymphatics Metastasis to lymph nodes, a common occurrence in breast cancer (BC), is the most significant prognostic indicator of poor outcome. To reach locoregional lymph nodes, tumor cells exclusively use lymphatic vessels. Not surprisingly, the extent of lymphatic metastasis is directly proportional to the density of tumor lymphatic vessels. It is therefore of high clinical significance to understand the mechanisms of tumor induced lymphangiogenesis, that is, the formation of new lymphatic vessels. It is currently thought that the main mechanism causing the formation of new lymphatic vessels is mediated by a paracrine lymphangiogenic factor VEGF-C that activates its receptor VEGFR-3 expressed in lymphatic endothelial cells. We recently discovered a fundamentally different mechanism of tumor lymphangiogenesis that complements the current views. This mechanism is mediated by tumor-mobilized bone marrow (BM)- derived monocytic progenitors that upon influence of the inflammatory tumor environment differentiate into lymphatic-like cells. These cells dubbed here Monocyte-derived Lymphatic Endothelial Cells Progenitors or M- LECP are characterized by the two main traits: (1) co-expression of myeloid and lymphatic-specific proteins that are typically segregated into distinct lineages; and (2) the ability to integrate into preexisting lymphatic vessels, which is an early prerequisite for lymphatic outgrowth. Using these criteria, we found very high levels of M-LECP in blood and tumors of BC patients as well as in a variety of metastatic orthotopic breast tumors from human and mouse origins. All tumors that contained M- LECP also displayed lymphatic vessels positive for myeloid-specific markers, an established phenomenon indicative of vascular integration of M-LECP that is required for sprouting. Importantly, we recently established that the levels of tumor-recruited M-LECP significantly correlate with tumor lymphatic density and lymph node status in clinical BC patients. Our studies in BC models showed that M-LECP originate from BM-derived CD11b+ cells that are highly positive for a lymphatic marker Podoplanin (Pdpn). Adoptive transfer of a phenotypically distinct BM subset identified by CD11b and Pdpn from metastatic tumor-bearing mice to mice with low-metastatic tumors significantly increased the density of lymphatic vessels and lymphatic metastasis. Preliminary data also show that differentiation of M-LECP can be faithfully reproduced in vitro by activating Toll-like Receptor-4 (TLR4) in human primary normal blood-circulating monocytes. This process is controlled by NF-kB and a transcription factor c-Maf, a newly identified regulator of monocytic-lymphatic reprogramming. TLR4-dependent upregulation of NF-kB and c-Maf leads to activation of the VEGFR-3 pathway which appears to be a critical milestone for acquisition of the lymphatic phenotype. Based on these findings, we hypothesize that tumor lymphatic outgrowth, an essential prerequisite for metastasis, is strongly promoted by M- LECP differentiating from the bone marrow myeloid precursors by TLR4-activating factors. To test this hypothesis we propose the following Specific Aims: (1) Test the hypothesis that M-LECP are directly and significantly relate to tumor-induced lymphangiogenesis and lymphatic metastasis in clinical BC; (2) Delineate the molecular mechanisms that reprogram bone marrow myeloid precursors into M-LECP; and (3) Determine the role of TLR4 in generation of the CD11b+/Pdpn+ bone marrow subset and its direct contribution to the formation of tumor lymphatic vessels and metastasis. Impact and Translational Relevance: We anticipate that these studies will establish a novel mechanism by which BM-derived monocytes recruited by breast tumors promote lymphangiogenesis and metastasis to lymph nodes. Validation of this novel concept will enhance the mechanistic understanding of the formation of tumor lymphatic vessels and suggest new molecular targets for inhibiting differentiation of M-LECP. Additionally, detection of higher levels of tumor- residing and blood-circulating M-LECP might identify BC patients with more aggressive tumors who should be preemptively treated to suppress locoregional spread. Such study outcomes can bolster the current clinical paradigms thus reducing mortality of BC patients.