Factors that influence age-dependent Ras mutant cancer development in mice

本研究旨在了解衰老的组织环境如何影响肿瘤的发生和发展。我们假设涉及衰老癌症启动细胞的细胞自主机制和涉及衰老免疫系统的细胞非自主机制在这一过程中发挥作用。我们计划对比两种由ras驱动的小鼠癌症模型——由角质形成细胞（源自外胚层）的Kras激活驱动的皮肤皮肤癌，以及由肺泡II型细胞（源自内胚层）的Kras激活驱动的肺腺癌——以了解在相同遗传背景下，它们在年轻和年老小鼠之间的发展差异。我们相信这些模型是检验年龄诱导的肿瘤起始细胞和巨噬细胞变化的理想模型，可以阐明年轻和年老宿主中肿瘤发展的明显差异。目的1。在小鼠肿瘤模型中通过Ras癌基因和下游Ras信号通路定义年龄依赖性肿瘤起始。目的1。比较年轻和年老小鼠皮肤和肺部肿瘤的发生。我们将比较年轻小鼠（8周）和老年小鼠（12个月）肺和皮肤肿瘤起始率和肿瘤生长率。目标1 b。比较体外ras转化的年轻和年老小鼠肿瘤起始细胞的致瘤性。我们将对原代皮肤角质形成细胞和肺泡2型（AT2）细胞进行短期体外培养，并使用腺病毒- cre激活Ras癌基因。所产生的细胞的致瘤性将通过将其移植到同基因的年轻人和老年人中，然后监测肿瘤的生长来评估。目标1 c。年轻和年老小鼠肿瘤细胞的磷酸化蛋白质组学和转录组学分析。在Aim 1A中使用来自年轻和年老小鼠的肿瘤，在Aim 1B中使用来自年轻和年老小鼠的原代细胞培养，我们将使用Cantley实验室最近开发的软件进行磷酸化蛋白质组学分析，以描述kinome活性，该软件可以根据来自年老和年轻小鼠的肿瘤之间的磷酸化肽变化预测最被激活或抑制的激酶。同时，我们将进行单细胞RNA测序，以分析肿瘤、基质和免疫细胞聚集的年龄诱导变化。目标2。定义衰老巨噬细胞在Ras肿瘤发展中的作用。目标2。肿瘤相关巨噬细胞（TAM）功能分析。使用Aim 1中描述的模型，将分析年轻小鼠与老年小鼠肿瘤中TAM的丰度和定位。我们还将对这些tam进行单细胞RNA测序，以了解其基因表达谱的差异。目标2 b。巨噬细胞耗竭和靶向治疗对肺转移的影响。我们将在年轻和年老小鼠中短暂地消耗巨噬细胞，并测试同基因Kras突变细胞对肺转移的影响。2 c目标。骨髓移植对肿瘤发展的影响。我们将把来自年轻小鼠的骨髓来源的骨髓细胞移植到同基因年老小鼠身上，反之亦然，以测试肿瘤的发展是否会改变。

This proposal aims to understand how the aging tissue environment can influence tumor initiation and tumor development. We hypothesize that both cell-autonomous mechanisms involving the aging cancer initiating cells and cell non-autonomous mechanisms involving the aging immune system play a role in this process. We plan to contrast two Ras-driven mouse cancer models - cutaneous skin cancer driven by Kras activation that arise from keratinocytes (ectoderm origin), and lung adenocarcinomas driven by Kras activation that arise from alveolar type II cells (endoderm origin) - to understand how their developments differ between young and old mice in identical genetic backgrounds. We believe these models are ideal for examining age-induced change in tumor initiating cells and in macrophages to illuminate distinct differences in tumor development in a young vs. old host. Aim 1. Defining age-dependent tumor initiation by the Ras oncogene and down-stream Ras signaling pathway in mouse tumor models. Aim 1A. Compare skin and lung tumor initiation in young vs. old mice. We will compare the rate of tumor initiation and tumor growth in young (8 weeks) and old (12 months) mice for tumor initiation in the lung and in the skin. Aim 1B. Compare the tumorigenicity of in vitro Ras-transformed young vs. old tumor-initiating cell in young vs. old mice. We will generate short-term in vitro culture of primary skin keratinocytes and lung alveolar type 2 (AT2) cells and activate the Ras oncogenes using adenoviral-Cre. The tumorigenicity of the resulting cells will be assessed by transplanting them into syngeneic young and old recipients orthotopically and then monitoring tumor growth. Aim 1C. Phosphoproteomic and transcriptomic profiling of tumor cells from young and old mice. Using tumors from young and old mice in Aim 1A and primary cell culture from young and old mice in Aim 1B, we will conduct phospho-proteomic analysis to profile kinome activity using the software recently developed in the Cantley lab that predicts the kinases that are most activated or inhibited based on phospho-peptide changes between tumors from old vs. young mice. In parallel, we will conduct single cell RNA sequencing to profile age-induced changes in the clustering of tumor, stroma, and immune cells. Aim 2. Define a role of the aging macrophage in Ras tumor development. Aim 2A. Analysis of tumor associated macrophage (TAM) function. Using models described in Aim 1, TAM abundance and localization will be analyzed in tumors from young vs. old mice. We will also carry out single cell RNA sequencing of these TAMs to understand differences in their gene expression profiles. Aim 2B. Impact of macrophage depletion and therapeutic targeting on lung metastasis. We will transiently deplete macrophage in young vs. old mice and test the impact on lung metastasis of syngeneic Kras mutant cells. Aim 2C. Impact of bone marrow transplant on tumor development. We will transplant bone-marrow-derived myeloid cells from young mice to syngeneic old mice, and vice versa, to test whether tumor development will be altered.