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

项目摘要/摘要 髓系淋巴祖细胞在乳腺癌淋巴管诱导中的新作用 乳腺癌(BC)中常见的淋巴结转移是最重要的预后因素。 结果不佳的指标。为了到达局部区域的淋巴结，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。 船只。因此，了解肿瘤的致癌机制具有重要的临床意义。 淋巴管生成，即形成新的淋巴管。 目前认为，导致新淋巴管形成的主要机制是通过 旁分泌淋巴管生成因子-C激活其受体VEGFR-3在淋巴管中的表达 内皮细胞。我们最近发现了一种根本不同的肿瘤淋巴管生成机制 这与当前的观点相辅相成。这种机制是由肿瘤动员的骨髓(BM)介导的。 受炎性肿瘤环境影响的来源的单核细胞祖细胞分化为 淋巴样细胞。这些细胞在这里被称为单核细胞来源的淋巴管内皮细胞前体细胞或M- LECP有两个主要特征：(1)髓系和淋巴系特异性蛋白的共表达 通常被分离成不同的谱系；以及(2)整合到先前存在的淋巴管的能力 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BC患者的血液和肿瘤中M-LECP水平非常高，以及 来自人类和小鼠的各种转移性原位乳腺肿瘤。所有含有M-的肿瘤 LECP还显示淋巴管髓系特异性标记物阳性，这是一种公认的现象 表明M-LECP的血管整合是发芽所必需的。重要的是，我们最近建立了 肿瘤募集的M-LECP水平与肿瘤淋巴密度和淋巴结显著相关 临床上的BC患者的状况。我们在BC模型中的研究表明，M-LECP起源于BM来源 CD11b+细胞，淋巴标记物泊多普宁(Pdpn)高度阳性。领养转让 CD11b和Pdpn鉴定的转移性荷瘤小鼠不同的BM亚群 与低转移肿瘤相比，淋巴管密度和淋巴转移明显增加。 初步数据还表明，通过激活M-LECP可以在体外忠实地复制分化 人外周血单核细胞Toll样受体-4(TLR4)的表达这个过程是受控的 通过核因子-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。 依赖TLR4的NF-kB和c-Maf上调导致VEGFR-3通路激活 成为获得淋巴表型的关键里程碑。基于这些发现，我们假设 肿瘤淋巴管的生长是转移的基本前提，M-R强烈促进肿瘤淋巴生长。 TLR4激活因子对LECP与骨髓髓系前体细胞的分化作用 为了验证这一假设，我们提出了以下具体目标： (1)验证M-LECP与肿瘤诱导的淋巴管生成直接和显著相关的假设 和临床BC中的淋巴转移；(2)描述了骨髓重新编程的分子机制 确定TLR4在CD11b+/Pdpn+骨生成中的作用 骨髓亚群及其在肿瘤淋巴管形成和转移中的直接作用。 影响和翻译相关性： 我们期望，这些研究将建立一种新的机制，通过它来招募骨髓来源的单核细胞 乳腺肿瘤可促进淋巴管生成和向淋巴结转移。对这一新概念的验证 将加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- 滞留和血液循环的M-LECP可能会识别患有更具侵袭性肿瘤的BC患者 先发制人的治疗，以抑制局部地区的传播。这样的研究结果可以支持目前的临床 范型，从而降低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.