TUMOR-HOST INTERACTIONS IN HUMAN MELANOMA METASTASIS

背景：我们最近发现了一个过程的证据，该过程出现 是转移的重要早期因素，即“克隆优势” 由具有转移能力的细胞变体引起的原发性肿瘤。简单地说， 这些细胞不仅拥有定植和生长的机制 距离较远的器官位点，但也比其他器官具有显着的生长优势 它们的非转移对应物位于原发部位本身。这个 该过程对肿瘤生物学有几个深远的影响 临床肿瘤学并非最不重要的，它可能是必要的 远处转移的形成。原始人类的进展 放射状生长期（RGP）或“薄”的恶性黑色素瘤 垂直生长阶段 (VGP) 阶段到较厚的 VGP 阶段提供了 转移细胞“克隆优势”模型的令人信服的例子。的 该项目的主要长期目标是确定一些 原代细胞局部环境的正常细胞可能的方式 黑色素瘤可能有助于“生长主导”性质 具有转移能力的人类黑色素瘤细胞。 最近的相关发现：正常人真皮成纤维细胞的共培养 与人类黑色素瘤细胞一起可以以某种方式调节后者的生长 这是肿瘤进展的可重复函数：RGP 或早期 VGP （无转移能力的）细胞生长受到抑制，而黑色素瘤 从晚期 VGP 原发肿瘤或远处转移瘤中获得的细胞 受到生长刺激。此外，这些结果可以通过重复 使用来自正常人真皮成纤维细胞的条件培养基，因此 暗示分泌性生长因子的参与。初步 实验表明抑制剂是一种分子量的蛋白质 28 至 48 Kda 之间，转铁蛋白 (Tf) 或 Tf 样分子是 参与成纤维细胞介导的转移性黑色素瘤刺激 细胞。 总体指导假设及意义：假设 原发性人类黑色素瘤中转移细胞的克隆优势是 部分由旁分泌生长因子的综合作用介导 成纤维细胞释放的抑制剂和刺激剂。成长 抑制剂被认为对早期（非转移性）有效 黑色素瘤细胞，但在更晚期（转移性- 有能力的）亚群——而相反的假设是 刺激生长因子活性。远处转移生长也可能 成纤维细胞作为“细胞”的存在促进了这一过程 “肥料”有助于为肿瘤创造更适宜的环境 成长。 具体目标：包括以下内容：（i）多步蛋白质纯化 并将进行分子克隆程序来鉴定 抑制剂的性质（称为人真皮成纤维细胞来源的生长 抑制剂或“hDFGI”），克隆其同源 cDNA，并研究其功能 体外黑色素瘤细胞的生长和行为； (二) 的贡献 Tf 作为成纤维细胞介导的生长刺激中可能的辅助因子 将确定具有转移能力的黑色素瘤细胞； (三) 成纤维细胞黑色素瘤生长的特异性和异质性 将评估互动； (iv) 抑制作用的贡献 和刺激生长因子活性对人类黑色素瘤的生长和 将研究体内进展。

Background: We recently uncovered evidence for a process which appears to be a vital early element of metastasis, namely 'clonal dominance' of primary tumors by metastatically-competent cell variants. Simply put, such cells not only possess the machinery to colonize and grow in distant organ sites, but also have a profound growth advantage over their non-metastatic counterparts within the primary site itself. This process has several far-reaching implications for tumor biology and clinical oncology not the least of which that it may be necessary for the formation of distant metastases. The progression of primary human malignant melanomas from the radial growth phase (RGP) or 'thin' vertical growth phase (VGP) stages to the thicker VGP stage provides a compelling example of the metastatic cell 'clonal dominance' model. The major long-term objective of this project is to determine some of the possible ways by which normal cells of the local environment of primary melanomas may contribute to the 'growth dominant' nature of metastatically-competent human melanoma cells. Recent Pertinent Findings: Coculture of normal human dermal fibroblasts with human melanoma cells can modulate the growth of the latter in a way that is reproducibly a function of tumor progression: RGP or early VGP (metastatically-incompetent) cells are growth inhibited whereas melanoma cells obtained from advanced VGP primary tumors or distant metastases are growth stimulated. Moreover, these results can be duplicated by using conditioned media from normal human dermal fibroblasts, thus implicating the involvement of secreted growth factors. Preliminary experiments indicate the inhibitor is a protein of molecular mass between 28 and 48 Kda, and that transferrin (Tf) or Tf-like molecule is involved in the fibroblast-mediated stimulation of metastatic melanoma cells. Overall Guiding Hypotheses and Significance: It is hypothesized that clonal dominance of metastatic cells in primary human melanomas is in part mediated by the combined effects of paracrine growth factor inhibitors and stimulators liberated by fibroblasts. The growth inhibitor is postulated to be effective on early-stage (non-metastatic) melanoma cells but impotent on more advanced stage (metastatically- competent) subpopulations-- whereas the reverse is postulated for the stimulatory growth factor activity. Distant metastatic growth may also be facilitated by the presence of fibroblasts acting as a 'cellular fertilizer' to help create a more congenial environment for tumor growth. Specific Aims: Include the following: (i) multistep protein purification and molecular cloning procedures will be undertaken to identify the nature of the inhibitor (called human dermal fibroblast-derived growth inhibitor or 'hDFGI'), clones its cognate CDNA, and study its function on melanoma cell growth and behavior in vitro; (ii) the contribution of Tf as a possible co-factor in fibroblasts-mediated growth stimulation of metastatically-competent melanoma cells will be determined; (iii) the specificity and heterogeneity of the fibroblast-melanoma growth interaction will be evaluated; (iv) the contribution of the inhibitory and stimulatory growth factor activities to human melanoma growth and progression in vivo will be investigated.