In vivo characterization of keratinocytes in the melanoma microenvironment

项目摘要 黑色素瘤是最致命的皮肤癌，其进展为局部浸润和转移，导致不良的预后。 患者的结果，强调需要更好地了解黑色素瘤的进展。在黑色素瘤期间 在肿瘤进展过程中，致瘤细胞必须克服微环境所施加的生长限制。 角质形成细胞虽然角质形成细胞对正常黑素细胞的调控作用已知之甚多， 它们在黑色素瘤中的相互作用。我们的初步数据表明黑色素瘤诱导上皮细胞- 间充质转化（EMT）计划在相邻的角质形成细胞在目的1，我将研究的作用， 角质形成细胞EMT对黑色素瘤起始的影响。在本研究中，我将以斑马鱼为动物模型进行研究 角质形成细胞和黑色素瘤细胞之间的体内相互作用。我会诱导黑色素瘤自发形成 在具有GFP标记的角质形成细胞的转基因斑马鱼系中，并使用成像来确认形态学变化 表明肿瘤相关角质形成细胞（TAK）中的EMT。然后，我将评估他们的EMT转录因子 和粘附蛋白的变化。我们假设黑色素瘤诱导的角质形成细胞EMT将导致 角质形成细胞对黑色素瘤增殖的调节作用。我们会通过击倒急救队来验证这个假设 角质形成细胞中的转录因子，并评估对斑马鱼黑色素瘤起始和增殖的影响 模型此外，我们的初步数据还强调了TAK的旁分泌信号的上调 参与黑色素瘤的迁移和侵袭。在目标2中，我将确定角质形成细胞如何分泌 影响新生黑色素瘤迁移和侵袭的因素。为了研究黑色素瘤在体内的迁移，我将 首先优化我们实验室中现有的成像管道，通过对细胞迁移的成像来定量跟踪细胞迁移。 斑马鱼皮然后，我可以评估敲除角质细胞衍生因子（如内皮素）的效果， Wnt 5A和BDNF使用细胞类型特异性CRISPR编辑来确定它们对迁移的影响。我们 假设这些旁分泌因子的迁移作用来自体外数据，我们将看到减少 黑色素瘤迁移和侵袭到邻近组织的迁移跟踪和组织学通过敲除这些 角质形成细胞中的因子。通过描述肿瘤相关角质形成细胞在黑色素瘤中的作用， 微环境，这项建议旨在了解黑色素瘤如何改变其微环境，以克服 它的自然生长抑制，并确定新的目标，以限制黑色素瘤的进展。

PROJECT SUMMARY Melanoma is the most lethal of skin cancers, with progression to local invasion and metastasis leading to poor patient outcomes, highlighting the need for better understanding of melanoma progression. During melanoma progression, tumorigenic cells must overcome growth restraints imposed by the microenvironmental keratinocytes. Although much is known of keratinocyte regulatory controls on normal melanocytes, less is known about their interactions in melanoma. Our preliminary data suggests that melanoma induces an epithelial-to- mesenchymal transition (EMT) program in adjacent keratinocytes In Aim 1, I will investigate the role of keratinocyte EMT on melanoma initiation. For this study, I will use the zebrafish as an animal model to study in vivo interactions between keratinocytes and melanoma cells. I will induce spontaneous melanoma formation in transgenic zebrafish lines with GFP labeled keratinocytes and use imaging to confirm morphological changes indicative of EMT in tumor-associated keratinocytes (TAKs). Then, I will assess them for EMT transcription factor and adhesion protein changes. We hypothesize that melanoma-induction of keratinocyte EMT will result in loss of keratinocyte regulation on melanoma proliferation. We will test this hypothesis by knocking out EMT transcription factors in keratinocytes and assess effects on melanoma initiation and proliferation in the zebrafish model. In addition, our preliminary data has also highlighted an upregulation of paracrine signals from TAKs involved in melanoma migration and invasion. In Aim 2, I will determine how keratinocyte-derived secreted factors affect migration and invasion of nascent melanoma. To study migration of melanoma in vivo, I will first optimize existing imaging pipelines in our lab to quantitatively to track cell migration by imaging of the zebrafish skin. I can then assess the effect of knocking out keratinocyte-derived factors such as endothelin, Wnt5A and BDNF using cell-type specific CRISPR-editing to determine their effects on migration. We hypothesize that given the migratory role of these paracrine factors from in vitro data, we will see reduced melanoma migration and invasion into adjacent tissues on migration tracking and histology by knocking-out these factors in keratinocytes. By characterizing the role of tumor-associated keratinocytes in the melanoma microenvironment, this proposal seeks to understand how melanoma modifies its microenvironment to overcome its natural growth restraints and identify new targets to limit melanoma progression.