In vivo characterization of keratinocytes in the melanoma microenvironment

黑色素瘤微环境中角质形成细胞的体内表征

• 批准号: 10643998
• 负责人: Yilun Ma
• 金额: $ 5.27万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10643998
• 关键词: Adhesions; Affect; Animal Model; Brain-Derived Neurotrophic Factor; Cell Communication; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Electroporation; Endothelin; Epidermis; Growth; Growth Factor; Histology; Human; Image; In Vitro; Individual; Invaded; Knock-out; Label; Melanins; Melanoma Cell; Melanosomes; Methods; Modeling; Morphology; Neoplasm Metastasis; Paracrine Communication; Patient-Focused Outcomes; Physical Restraint; Physiology; Pigments; Play; Proliferating; Proteins; Quantitative Reverse Transcriptase PCR; Regulation; Resolution; Role; Skin; Skin Cancer; Stratum Basale; System; Testing; Tissue Sample; Tissues; Transgenic Organisms; Tumor Expansion; Ultraviolet Rays; Up-Regulation; Western Blotting; Zebrafish; cell motility; cell type; epithelial to mesenchymal transition; human tissue; in vivo; in vivo imaging; insight; interest; keratinocyte; melanocyte; melanoma; member; migration; paracrine; programs; promoter; restraint; tool; transcription factor; transcriptome; tumor; tumor microenvironment; tumor progression; tumorigenic

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.

项目摘要 黑色素瘤是皮肤癌中最致命的，进展为局部侵袭和转移导致不良 患者的结果，强调需要更好地了解黑色素瘤进展。在黑色素瘤期间 进展，肿瘤细胞必须克服微环境施加的生长约束 角质形成细胞。尽管对正常黑色素细胞的角质形成细胞调节对照众所周知，但已知较少 关于它们在黑色素瘤中的相互作用。我们的初步数据表明，黑色素瘤诱导上皮到 - AIM 1中相邻角质形成细胞中的间充质转变（EMT）程序，我将研究 角质形成细胞EMT在黑色素瘤启动中。对于这项研究，我将使用斑马鱼作为动物模型来研究 角质形成细胞与黑色素瘤细胞之间的体内相互作用。我将诱导自发的黑色素瘤形成 在具有GFP标记角质形成细胞的转基因斑马鱼线中，并使用成像来确认形态学变化 指示与肿瘤相关角质形成细胞（TAKS）中EMT的指示。然后，我将评估它们的EMT转录因子 和粘附蛋白变化。我们假设角质形成细胞EMT的黑色素瘤诱导将导致损失 角质形成细胞对黑色素瘤增殖的调节。我们将通过淘汰EMT来检验这一假设 角质形成细胞中的转录因子并评估斑马鱼中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Taks的旁分泌信号的上调 参与黑色素瘤迁移和入侵。在AIM 2中，我将确定角质形成细胞衍生的分泌 因素影响新生黑色素瘤的迁移和侵袭。为了研究体内黑色素瘤的迁移，我将 首先优化实验室中现有的成像管道，以定量地通过成像跟踪细胞迁移 斑马鱼皮。然后，我可以评估淘汰角质形成细胞衍生的因素，例如内皮素， WNT5A和BDNF使用细胞类型的特定CRISPR编辑来确定它们对迁移的影响。我们 假设鉴于这些旁分泌因子从体外数据中的迁移作用，我们将看到减少 黑色素瘤的迁移和入侵在迁移跟踪和组织学上的邻近组织，通过淘汰这些 角质形成细胞的因素。通过表征肿瘤相关角质形成细胞在黑色素瘤中的作用 微环境，该提案试图了解黑色素瘤如何修饰其微环境以克服 它的自然生长限制并确定了限制黑色素瘤进展的新靶标。