Thursday, November 21, 2024
11/21/2024
Project Summary: Widespread dissemination together with emergence of tumor cell resistance to existing therapeutic agents is ultimately responsible for almost 90% of cancer deaths. However, the metastasis-promoting genetic programs and the underlying signaling networks orchestrating the progression of the metastatic disease process remain poorly defined. In order to develop effective anti-metastatic therapeutic agents and improve patient outcomes, further progress elucidating the fundamental biology of metastasis is needed. Notably, majority of the comparative genetic studies to date have relied on the primary tumors and the metastatic lesions to facilitate the identification of genetics factors that drive tumor progression and dissemination. However, deciphering drivers of metastasis solely based on the genetic information from solid tumors is limiting due to genetic divergence and tumor heterogeneity. Since metastatic tumor cells must leave the primary tumor, circulating tumor cells (CTCs) that break free from primary tumors and seed metastatic lesions are better suited to facilitate comprehensive understanding of the metastatic disease process. However, efficient capture of CTCs and unbiased genomic amplification are extremely challenging due to the rarity and fragility of CTCs. Thus, new technologies and platforms are needed to effectively utilize the biology of CTCs for systematic identification of metastasis-promoting genetic factors. Recently we reported the very first genome-scale in vivo CTC CRISPR knockout screen specifically designed to identify genetic-factors contributing to tumor cell dissemination. Xenografted tumors were seeded with pooled CRISPR-edited metastatic prostate cancer cells, each harboring single gene loss-of-function genetic alterations covering all protein coding genes of the human genome. Using a high-performance microfluidic immunomagnetic cell sorting approach for efficient CTC capture directly from mouse blood coupled with next-generation sequencing (NGS) for barcoded guide RNA enrichment analysis, we demonstrated the feasibility and reliability of the use of CTCs for the identification of critical genetic factors promoting tumor cell dissemination thereby illuminating targeted routes for inhibiting metastasis driving pathways. In this project, we will develop a next-generation blood-to-barcode (B2B) chip (Aim1) that accelerates in vivo CRISPR-based discovery efforts to identify critical genetic factors impacting metastatic potential. The B2B chip will power a series of in vivo CRISPR activation screens (Aim2) across a panel of human and mouse metastatic prostate cancer cell lines strategically selected for modeling broad range of tumor metastatic potential in vivo as well as origins of metastatic tumors. Collectively, these screens are anticipated to reveal genetic factors that could be targeted therapeutically to limit the development of metastatic tumors. Through systematic clinical relevancy prioritization and validations using a battery of in vitro and in vivo approaches in prostate cancer model systems (Aim3), clinical utility of our lead genetic factors as targeted anti- metastatic agents will be established.
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