Adrenomedullin affects Lymphatics and Immune Activation to Promote Metastasis

描述(申请人提供)：癌症仍然是世界上第二大死亡原因，主要是由于肿瘤细胞转移到远处。目前，肿瘤转移的分子机制和相关因素尚未完全阐明，因此无法预防肿瘤的转移。不仅肿瘤特异性信号会加剧转移，全身信号也会加剧转移，包括与正常妊娠相关的激素和多肽。识别和理解肿瘤来源和参与肿瘤转移的系统因素仍然是一个激烈的研究领域。其中一个因素是肾上腺髓质素(AM)，这是一种多功能多肽，有充分的记录表明在人类怀孕和许多癌症期间会升高。肿瘤过程中AM升高与远处转移和患者预后不良有关，但AM在肿瘤转移中的具体作用机制尚不清楚。已发表的报告表明，AM对淋巴发育是必不可少的，并具有抗炎特性。我们最近发现高肿瘤来源的AM与肿瘤淋巴管生成和转移有关。因此，这项资助中提议的实验将试图阐明：i)肿瘤来源的AM如何诱导肿瘤微环境中的淋巴管生成；ii)妊娠期间全身应用AM如何改变肿瘤的血管形成和转移；以及iii)全身AM是否抑制免疫监视。具体目标1将确定肿瘤来源的AM是否通过巨噬细胞间接作用于促进淋巴管生成。巨噬细胞将使用磁珠从给予AM的肿瘤中分离出来，并通过流式细胞术、免疫组织化学和定量RT-PCR来评估巨噬细胞募集、重编程和淋巴特异性生长因子的表达的变化，这些都将有助于观察到的淋巴管生成。特异性目标2将测试系统性AM是否改变淋巴管生成和/或免疫激活，这两者都可能影响转移。标记的肿瘤细胞将被植入遗传剂量为AM的小鼠体内这些小鼠将使用上述方法来表征淋巴管的生成和转移。使用来自每种基因型的处女和怀孕小鼠将确定怀孕期间高系统性AM是否在报告的怀孕期间转移增强中起重要作用。为了评估免疫激活的一个成分，AM处理的自然杀伤细胞的细胞因子的产生和细胞毒性将使用ELISA和流式细胞术来表征。这些目标的完成将有助于：1)阐明AM在肿瘤转移中的局部和全身作用；2)揭示妊娠期间高水平的AM是妊娠相关转移的一个促成因素；3)加深我们对AM在先天免疫中的作用的理解。总之，这项研究可以进一步验证AM信号系统是减少肿瘤转移的药理上容易处理的靶点。

DESCRIPTION (provided by applicant): Cancer remains the second leading cause of death in the world, mostly due the metastatic spread of tumor cells to distant sites. The molecular mechanisms and factors involved in metastasis have not been fully characterized, which leads to an inability to prevent metastasis. Metastasis is exacerbated not only by tumor- specific signals, but also systemic signals, including hormones and peptides associated with normal pregnancy. Identifying and understanding both the tumor-derived and systemic factors involved in tumor metastasis remains an intense area of research. One such factor is adrenomedullin (AM), which is a multifunctional peptide well-documented as being elevated during human pregnancy and numerous cancers. High AM during cancer is associated with distant metastasis and poor patient prognosis, but the specific mechanisms of AM on metastasis are not well understood. Published reports demonstrates that AM is essential for lymphatic development and has anti-inflammatory properties. We recently showed that high tumor-derived AM is correlated with tumor lymphangiogenesis and metastasis. Therefore, the proposed experiments in this grant will attempt to elucidate i) how tumor-derived AM induces lymphangiogenesis within the tumor microenvironment, ii) how systemic AM dosage during pregnancy alters tumor vascularization and metastasis, and iii) whether systemic AM suppresses immune surveillance. Specific aim 1 will determine if tumor-derived AM acts indirectly through macrophages to promote lymphangiogenesis. Macrophages will be isolated from AM dosed tumors using magnetic beads and assessed by flow cytometry, immunohistochemistry, and quantitative RT-PCR for changes in macrophage recruitment, reprogramming, and expression of lymphatic-specific growth factors, which would all contribute to the observed lymphangiogenesis. Specific aim 2 will test if systemic AM alters lymphangiogenesis and/or immune activation, both of which could influence metastasis. Labeled tumor cells will be implanted into mice with a genetic dosage of AM. These mice will be characterized for lymphangiogenesis and metastasis using the methods mentioned above. The use of virgin and pregnant mice from each genotype will identify if high systemic AM during pregnancy is important in the reported enhanced metastasis during pregnancy. To assess one component of immune activation, AM-treated natural killer cell's cytokine production and cytotoxicity will be characterized using ELISA and flow cytometry. The completion of these aims will help i) elucidate the local and systemic roles of AM on tumor metastasis, ii) implicate high AM levels during pregnancy as a contributing factor in pregnancy-associated metastases, and iii) further our understanding of AM's roles on innate immunity. Together, this research could further verify the AM signaling system as a pharmacologically-tractable target to reduce tumor metastasis.