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

摘要 卡波西肉瘤相关疱疹病毒(KSHV)导致艾滋病定义癌症卡波西肉瘤(KS)。 KSHV感染是如何导致KS的，人们对此知之甚少，这主要是因为缺乏严格定义的原发病 重现KSHV感染的KS肿瘤细胞增殖特征的人细胞培养模型。 KS肿瘤细胞很可能来源于微血管淋巴管内皮细胞。因此，LEC 为KS的研究提供了一个生理相关的模型。我们已经开发了一种治疗KSHV感染的方案 这使得我们能够测量KSHV诱导的接触性增殖抑制丧失(CIP)。 在2D文化中。CIP缺失是致癌转化的一个重要特征。因此，我们的中心假设是 KSHV可以触发原代LECs的致癌转化，其过程概括为KSHV介导的过程 卡波西肉瘤的致癌作用。我们的第一个目标是确定KSHV感染的晶状体上皮细胞(KLEC)是否完全 在免疫缺陷小鼠体内转化和形成异种移植瘤。我们还假设未知 在我们的模型中，KSHV诱导的细胞基因表达的变化导致CIP的丢失。然而，在我们的 初步的批量基因表达研究存在太多此类变化的候选因素，无法直接进行 机械学研究。因此，我们的第二个目标是确定哪些细胞基因表达发生了变化 在KLEC中驱动CIP的损失。为了验证我们的假设并实现我们的目标，我们提出了两个具体的目标， 也就是说，我们将：(1)确定KLECs是否完全转化并能在 免疫缺陷小鼠，以及(2)确定驱动KSHV诱导致癌的基因表达轨迹 KLEC的变化。拟议的研究是创新的，因为我们的模型提供了一套严格定义的 能够研究KSHV感染原代人类细胞后致癌变化的实验环境 与KS相关的类型。这项工作意义重大，因为它将确定感染KSHV的LEC是否 确实完全转化，并确定KS中病毒转化的机制。最后，结果将是 很有影响力，因为我们最初的基于人类细胞的模型及其特征将在体外实现重要的 而且，潜在的是对KS潜在机制的活体研究以及改进策略的设计 进行治疗干预。

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.