KSHV-induced oncogenic changes in a primary human lymphatic endothelial cell model of Kaposi's Sarcoma

KSHV 诱导的卡波西肉瘤原代人淋巴内皮细胞模型的致癌变化

• 批准号: 10327223
• 负责人: Eva Henriette Gottwein
• 金额: $ 22.42万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327223
• 关键词: AIDS related cancer; Acquired Immunodeficiency Syndrome; Biology; Cell Culture Techniques; Cell model; Cells; Contact Inhibition; Data; Data Set; Gene Expression; Gene Expression Profiling; Goals; Herpesviridae Infections; Human; Human Herpesvirus 8; Immunodeficient Mouse; In Vitro; Infection; Kaposi Sarcoma; Left; Lymphatic Endothelial Cells; Malignant Neoplasms; Measures; Mediating; Modeling; Mus; Nature; Oncogenic; Pathway interactions; Physiological; Primary Infection; Process; Protocols documentation; Public Health; RNA; Sampling; Study models; Testing; Therapeutic; Therapeutic Intervention; Viral; Viral Genes; Work; base; cell type; design; experimental study; improved; in vivo; innovation; insight; neoplastic cell; novel therapeutic intervention; single-cell RNA sequencing; transcriptome sequencing; tumor; tumor xenograft; tumorigenesis; tumorigenic

SUMMARY Kaposi's Sarcoma-associated herpesvirus (KSHV) causes the AIDS-defining cancer Kaposi's Sarcoma (KS). How KSHV infection causes KS is poorly understood, largely due to a lack of a rigorously defined primary human cell culture model that recapitulates the proliferative features of the KSHV-infected tumor cells in KS. KS tumor cells most likely originate from microvascular lymphatic endothelial cells (LECs). LECs therefore represent a physiologically relevant model for studies of KS. We have developed a protocol for KSHV infection of primary human LECs that allows us to measure KSHV-induced loss of contact inhibition of proliferation (CIP) in 2D culture. Loss of CIP is a key feature of oncogenic transformation. Our central hypothesis is therefore that KSHV can trigger oncogenic transformation of primary LECs, in a process that recapitulates KSHV-mediated oncogenesis in Kaposi's Sarcoma. Our first objective is to determine if KSHV-infected LECs (KLECs) are fully transformed and form xenograft tumors in immunodeficient mice. We additionally hypothesize that unknown KSHV-induced changes in cellular gene expression drive the loss of CIP in our model. However, in our preliminary bulk gene expression studies too many candidates for such changes exist to directly proceed to mechanistic studies. Our second objective is therefore to establish which cellular gene expression changes drive loss of CIP in KLECs. To test our hypothesis and achieve our objectives, we propose two Specific Aims, i.e. we will: (1) determine whether KLECs are fully transformed and can form xenograft tumors in immunodeficient mice, and (2) define gene expression trajectories that drive KSHV-induced oncogenic changes in KLECs. The proposed study is innovative, because our model provides a set of rigorously defined experimental settings that enable the study of oncogenic changes after KSHV infection of a primary human cell type with relevance to KS. This work is significant, because it will establish whether KSHV-infected LECs are indeed fully transformed and identify mechanisms of viral transformation in KS. Finally, the results will be impactful, because our primary human cell-based model and its characterization will enable important in vitro and, potentially, in vivo studies of the mechanisms underlying KS as well as the design of improved strategies for therapeutic intervention.

概括 卡波西（Kaposi）与肉瘤相关的疱疹病毒（KSHV）导致定义艾滋病的癌症卡波西（Kaposi）的肉瘤（KS）。 KSHV感染如何引起KS的理解很少，这很大程度上是由于缺乏严格定义的主要原则 人类细胞培养模型概括了KS中KSHV感染的肿瘤细胞的增殖特征。 KS肿瘤细胞最有可能起源于微血管淋巴内皮细胞（LEC）。因此 代表了KS研究的生理相关模型。我们已经开发了KSHV感染方案 原发性人体LEC，使我们能够测量KSHV诱导的接触抑制损失（CIP） 在二维文化中。 CIP的损失是致癌转化的关键特征。因此，我们的中心假设是 在概括KSHV介导的过程中，KSHV可以触发主要LEC的致癌转化 卡波西肉瘤中的肿瘤发生。我们的第一个目标是确定受KSHV感染的LEC（KLEC）是否完全 在免疫缺陷的小鼠中转化并形成异种移植肿瘤。我们还假设该未知 KSHV诱导的细胞基因表达变化驱动了我们模型中CIP的损失。但是，在我们的 初步的批量基因表达研究太多而存在于此类变化，无法直接进行 机械研究。因此，我们的第二个目标是确定哪些细胞基因表达发生变化 KLEC中CIP的驱动损失。为了检验我们的假设并实现我们的目标，我们提出了两个具体目标， 即我们将：（1）确定KLEC是否完全转化并可以形成异种移植肿瘤 免疫缺陷小鼠，（2）定义驱动KSHV诱导致癌的基因表达轨迹 KLEC的变化。拟议的研究具有创新性，因为我们的模型提供了一组严格定义的 实验环境可以研究原代人细胞KSHV感染后的致癌性变化 与KS相关。这项工作很重要，因为它将确定受KSHV感染的LEC是否是 确实，完全转化并确定了KS病毒转化的机制。最后，结果将是 有影响力，因为我们的主要基于人类细胞的模型及其表征将使重要的体外重要 而且，可能，可能是对KS基础机制以及改进策略设计的体内研究 用于治疗干预。