A novel in vivo model system of melanoma using a human-mouse neural crest cell chimera

 描述(申请人提供)：转移性黑色素瘤是增长最快的癌症之一，仅在2009年就导致超过8,650人死亡，而且这个数字预计还会随着时间的推移而增加。研究黑色素瘤的模型有多种局限性，包括大多数转基因系统无法准确复制癌症等复杂疾病表型，以及人类胚胎干细胞(HES)和人类诱导多能干细胞(HIPS)技术无法复制宿主微环境。然而，已有研究表明，侵袭性黑色素瘤细胞上调神经脊(NC)细胞引导和分化标记的子集，并且是NC细胞来源的癌症。我们假设可以利用人类NC细胞嵌合体建立一个研究黑色素瘤发展、侵袭和转移的体内平台。这项研究有两个具体的目标：1)优化HES和HIPS细胞来源的NC细胞在小鼠系统中的整合；2)在体内模拟人类黑色素瘤的发展和转移。这些嵌合体将通过宫内注射HES或HIPS细胞来源的NC细胞到胚胎8.5天(E8.5)发育中的小鼠胚胎中而产生。人类NC供体细胞将被标记EGFP标记，注入的胚胎将在孕期和出生后进行分析，以定位注入的细胞并建立优化其存活和定植的方法，例如通过慢病毒转染法过表达NC细胞生存基因。然后，我们将利用这个嵌合系统来过度表达在黑色素瘤中具有已知作用的常见基因，并将这些NC细胞注射到E8.5的子宫中，在小鼠模型系统中模拟黑色素瘤的发生和发展。此外，我们将促进HES或HIPS细胞衍生的NC细胞在皮下注射到免疫低下的小鼠体内后形成肿瘤，形成的肿瘤细胞可以在E8.5培养并重新注射到发育中的小鼠胚胎中，以研究肿瘤在发育期的迁移和转移。总体而言，我们建议将胚胎发育研究与癌症发展联系起来，重点放在黑色素瘤上。我们将使用HES/HIPS细胞技术结合活体小鼠建模来分析黑色素瘤的进展。这一新的系统不仅将架起胚胎发育和癌症生物学知识的桥梁，还将提供黑色素瘤的体内模型，用于测试潜在的治疗药物。

 DESCRIPTION (provided by applicant): Metastatic melanoma is one of the fastest growing cancers, causing more than 8,650 deaths in 2009 alone, and this number is projected to increase over time. Models for the study of melanoma have multiple limitations, including the inability of most transgenic systems to accurately reproduce complex disease phenotypes such as cancer, and the inability of human embryonic stem (hES) and human induced pluripotent stem (hiPS) cell technology to reproduce the host microenvironments. It has been shown, however, that aggressive melanoma cells up-regulate a subset of neural crest (NC) cell guidance and differentiation markers as well as being a NC cell-derived cancer. We hypothesize that an in vivo platform for the study of melanoma development, invasion, and metastasis can be created using human NC cell chimeras. There are two specific goals of this study: 1) to optimize the incorporation of hES and hiPS cell-derived NC cells into a murine system, and 2) to model human melanoma development and metastasis in vivo. These chimeras will be created by in utero injections of hES or hiPS cell-derived NC cells into the developing mouse embryo at embryonic day 8.5 (E8.5). Human NC donor cells will be labeled with the eGFP marker and injected embryos will be analyzed during gestation and post-natally to localize the injected cells and establish methods optimizing their survival and colonization, such as the over-expression of NC cell survival genes via lentiviral transfection. We will then utilize this chimeric system to over-express common genes with known roles in melanoma, and inject these NC cells in utero at E8.5 to model melanoma initiation and development in vivo in a murine model system. Additionally, we will promote tumor formation of the hES or hiPS cell-derived NC cells after subcutaneous injection into an immune-compromised mouse, and the tumor cells formed can be cultured and re-injected at E8.5 into the developing mouse embryo to study tumor migration and metastasis over the developmental period. Overall, we propose to bridge the studies of embryonic development with cancer development, focusing on melanoma. We will analyze the progression of melanoma using hES/hiPS cell technology coupled with in vivo murine modeling. This novel system will not only bridge the knowledge of embryonic development and cancer biology, but will provide an in vivo model of melanoma to be used for testing potential therapeutic agents.