Clonal dynamics and single cell transcriptomics of prostate luminal epithelial cells in homeostasis and aging

项目摘要 最常见的前列腺疾病，良性前列腺肥大和前列腺癌与 衰老，并归因于激素失衡和腺体稳态的丧失。考虑到前列腺 由于病理学对男性健康的影响及其对医疗保健系统的重大负担，重要的是进一步 阐明了正常的内稳态对失调的克隆生长造成的障碍。到目前为止，大多数研究都集中在 阐明各种前列腺干细胞群体的身份，并验证其干细胞在前列腺中的潜力， 培养测定。然而，对成年人中起作用的细胞和群体动力学的精确理解， 前列腺稳态，以及它的精细平衡如何随着衰老而变化，并有助于与年龄相关的细胞 过度增殖，迄今为止还没有出现。这一领域的进展将非常有益于确定 老年组织中去调节的前列腺群体和设计预防性治疗。在初步研究中， 使用体内谱系追踪和单细胞转录组学，我发现了一个“管腔中间”转录 具有独特的Wnt/p63信号传导，其构成癌细胞群的大部分 在肿瘤形成的早期阶段扩大。 在该提案的F99阶段，我将研究LumI细胞状态作为癌症起源的首选细胞 模型和Wnt信号和p63控制的潜在生长程序。具体来说，我将雇用 功能性类器官测定以研究Wnt信号传导在体外促进LumI细胞生长中的作用， 描述体内消除Wnt/p63信号传导的作用。在提案的K 00阶段，我将利用 我的方法，并产生新的小鼠模型，以描绘细胞水平的异质性和生长模式， 在成人和老年前列腺腔层中的作用，并探讨其分子调控机制。 具体来说，我将利用创新的数学建模，整合从遗传学获得的克隆数据， 谱系追踪以表征前列腺细胞所采用的成年和老化稳态生长的模式。我会 还产生了一种新的小鼠模型：Nkx3.1CreERT2/+-Confetti-CARLIN小鼠，用于同时体内谱系 跟踪和条形码，这将提供增加的谱系祖先分辨率。单细胞RNAseq和单 cell ATACseq将用于评估细胞状态并揭示前列腺的动态分子异质性， 在谱系追踪小鼠模型中的管腔细胞。K 00 Aim将提供一个全面的功能图， 在成人和老化前列腺中的管腔前列腺细胞，并鉴定谱系和相关的主调节基因 用Luminal克隆。 总之，我的研究将提供一个全面的了解人口增长动态， 前列腺组织的腔层，并可能导致识别新的治疗靶点， 前列腺过度增生性疾病。

PROJECT SUMMARY The most common prostate disorders, benign prostate enlargement and prostate cancer are associated with aging and attributed to hormonal imbalances and loss of glandular homeostasis. Given the major impact prostate pathologies have on men’s health and their significant burden on healthcare systems, it is important to further elucidate the barriers posed by normal homeostasis to deregulated clonal growth. To date, most studies focused on elucidating the identity of various stem cell prostate populations and on validating their stem cell potential in culture assays. However, a precise understanding of the cellular and population dynamics at play in adult prostate homeostasis, and how its fine balances change with aging and contribute to age-related cellular hyperproliferations, has not emerged so far. Advances in this area would be extremely beneficial for identifying deregulated prostate populations in aged tissues and for designing prophylactic therapies. In preliminary studies, using in vivo lineage-tracing and single cell transcriptomics, I uncovered a “luminal intermediate” transcriptional state (LumI) with unique Wnt/p63 signaling which constitutes a great portion of the cancer cell populations expanding in early stages of tumorigenesis. In the F99 phase of the proposal, I will investigate the LumI cell state as the preferred cell of origin in cancer models and the underlying growth program controlled by Wnt signaling and p63. Specifically, I will employ functional organoid assays to investigate the role of Wnt signaling in promoting LumI cell growth in vitro and delineate the effects of eliminating the Wnt/p63 signaling in vivo. In the K00 phase of the proposal, I will leverage my methods and generate new mouse models to delineate the cellular level heterogeneity and growth patterns in adult and aged prostate luminal layer and explore the underlying molecular regulatory mechanisms. Specifically, I will utilize innovative mathematical modeling to integrate the clonal data obtained from genetic lineage tracing to characterize the modes of adult and aging homeostatic growth adopted by prostate cells. I will also generate a novel mouse model: Nkx3.1CreERT2/+-Confetti-CARLIN mouse for simultaneous in vivo lineage tracing and barcoding which will provide increased lineage ancestry resolution. Single cell RNAseq and Single cell ATACseq will be used to assess cellular states and uncover the dynamic molecular heterogeneity of prostate luminal cells in the lineage tracing mouse model. The K00 Aim will provide a comprehensive functional map of luminal prostate cells in adult and aging prostate and identify lineages and master regulator genes associated with luminal clones. Taken together, my studies will provide a comprehensive understanding of population growth dynamics in the luminal layer of prostate tissue and may lead to identification of novel therapeutic targets for age-related prostate hyperproliferative disorders.