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

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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.