Multiplexed dissection of metastatic ability, organ tropism, and immune evasion

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a frequent and almost uniformly fatal malignancy. PDAC metastasizes to many different organs and is largely refractory to conventional chemo- and immuno-therapies. PDAC cells exist in diverse cell states, however, the extent to which these states dictate metastatic ability, organ tropism, and immune evasion remains largely unexplored. The critical molecular drivers underlying metastatic fitness phenotypes that drive success at each step of the metastatic cascade are likely highly diverse and remain largely unknown. To bridge this knowledge gap, we will integrate genetic barcoding with single cell profiling to uncover the determinants of the fundamental properties of metastasis. We are uniquely positioned to conduct this multidisciplinary systems-level proposal which employs innovative methods for multiplexed dissection of the metastatic process. We will employ molecular barcoding and high-throughput barcode sequencing to quantify the metastatic ability of an extensive panel of >30 PDAC cell lines across time, organ site, and immune environment. Our preliminary data from our pooled and barcoded cell line panel demonstrate highly heterogeneous metastatic phenotypes and cell states, and that our approach provides both cell line-level and clonal resolution of metastatic ability. In Aim 1, we will pool our barcoded cell lines for several types of transplantations that mimic modes of metastatic spread and quantify the metastatic ability of highly diverse cell states across different time durations, organ sites, and immune environments. Similar approaches will be employed to investigate cancer adaptation for immune evasion. In Aim 2, we will relate metastatic phenotypes to the molecular programs underlying diverse cell states and determine how cell state influences metastatic fitness and immune evasion. By performing scRNA-seq on the multi-component barcode in our cell lines following intrasplenic and intravenous injection at multiple time points and immune environments, we will determine the cell states that confer selection for successful metastasis and adaptation to immune response. In Aim 3, we will investigate key regulators of metastatic phenotypes using multiplexed functional approaches. We will prioritize genes that are highly expressed or strongly induced in cell lines with enhanced metastatic ability and inactivate each candidate driver of metastasis in each cell line. By transplanting this pool of genetically altered cell lines, followed by Perturb-seq, we will determine the impact and transcriptional effects of each candidate driver on metastatic ability and organ tropism. Our study will uncover fundamental determinants of PDAC metastasis using comprehensive and systems-level approaches.