Development of Next-Generation Blood-to-barcode (B2B) chip for In Vivo CRISPR-Based Discovery of Metastasis Regulators

开发下一代血液转条形码 (B2B) 芯片，用于体内基于 CRISPR 的转移调节因子发现

• 批准号: 10577058
• 负责人: Shana O Kelley
• 金额: $ 62.59万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-07 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10577058
• 关键词: Acceleration; Algorithms; Antimetastatic Agent; Automobile Driving; Bar Codes; Bioinformatics; Biological Assay; Biological Models; Biology; Blood; Blood Circulation; Blood specimen; CRISPR screen; CRISPR-mediated transcriptional activation; Cancer Biology; Cancer Patient; Cancerous; Cause of Death; Cell Line; Cell Separation; Cells; Cessation of life; Chemicals; Circulation; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Coupled; Cues; Development; Disease; Disseminated Malignant Neoplasm; Exhibits; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic; Genetic Drift; Genetic study; Genomics; Goals; Guide RNA; Heterogeneity; Human; Human Biology; Human Genome; In Vitro; Individual; Invaded; Knock-out; Lead; Lesion; Malignant Neoplasms; Malignant neoplasm of prostate; Mesenchymal; Metastatic Prostate Cancer; Microfluidic Microchips; Microfluidics; Migration Assay; Modeling; Molecular; Mus; Mutation; Neoplasm Circulating Cells; Neoplasm Metastasis; Pathway interactions; Patient-Focused Outcomes; Performance; Phenotype; Population; Primary Neoplasm; Process; Production; Protein Secretion; Proteins; Proteomics; Reporting; Reproducibility; Research Personnel; Resistance; Role; Route; Sampling; Series; Signal Transduction; Site; Solid Neoplasm; Specimen; System; Technology; Testing; Therapeutic; Therapeutic Agents; Tissue-Specific Gene Expression; Travel; Triage; Tumor Promotion; Validation; Whole Blood; Xenograft Model; Xenograft procedure; cancer cell; cell motility; clinically relevant; combat; comparative; design; drug discovery; drug testing; drug-like compound; druggable target; effective therapy; enhancing factor; functional genomics; genetic information; genome-wide; human disease; improved; in vivo; inhibitor; knockout gene; lead candidate; loss of function; multidisciplinary; neoplastic cell; new technology; new therapeutic target; next generation; next generation sequencing; novel; novel therapeutics; overexpression; pharmacologic; prevent; programs; prostate cancer cell; prostate cancer cell line; prostate cancer model; rare cancer; scale up; screening; targeted treatment; therapeutic target; transcriptomics; tumor; tumor heterogeneity; tumor progression; tumor xenograft; whole genome

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.

项目总结： 广泛传播与出现肿瘤细胞对现有的耐药 治疗药物对近90%的癌症死亡负有最终责任。然而， 促进肿瘤转移的遗传程序和潜在的信号网络 对转移性疾病进程的协调仍然没有明确的定义。 为了开发有效的抗转移治疗药物，提高患者的生活质量 结果，需要进一步阐明转移的生物学基础。 值得注意的是，到目前为止，大多数比较遗传学研究都依赖于初级 肿瘤和转移灶便于鉴定遗传学因素， 推动肿瘤的进展和扩散。然而，破译转移的驱动因素 仅基于实体肿瘤的遗传信息是有限的，因为 分歧性和肿瘤异质性。因为转移的肿瘤细胞必须离开 原发肿瘤，从原发肿瘤中分离出来的循环肿瘤细胞(CTCs)和 种子转移灶更适合于更全面地了解 转移疾病的过程。然而，CTCs的有效捕获和无偏 由于CTCs的稀有和脆弱，基因组扩增具有极大的挑战性。 因此，需要新的技术和平台来有效地利用生物 CTCs用于系统识别促进转移的遗传因素。 最近，我们报道了第一个体内基因组规模的CTC CRISPR基因敲除屏幕 专门设计用来识别导致肿瘤细胞扩散的遗传因素。 移植瘤接种CRISPR编辑的转移性前列腺癌 细胞，每个细胞都有覆盖所有蛋白质的单基因功能丧失的遗传改变 人类基因组的编码基因。使用高性能微流控芯片 直接从小鼠体内高效捕获CTC的免疫磁性细胞分选法 血液与条码引导RNA的下一代测序(NGS) 通过浓缩分析，我们论证了使用四氯化碳的可行性和可靠性 用于鉴定促进肿瘤细胞扩散的关键遗传因素 照亮抑制转移驱动通路的靶向路线。 在这个项目中，我们将开发下一代血液到条形码(B2B)芯片(Aim1)， 加速基于CRISPR的体内发现工作，以确定关键遗传因素 影响转移潜能。B2B芯片将驱动一系列体内CRISPR 一组人和小鼠转移性前列腺的激活筛选(AIM2) 为建立大范围肿瘤转移模型而策略性选择的癌细胞系 体内的潜力以及转移性肿瘤的来源。总的来说，这些屏幕是 预计将揭示可以在治疗上靶向的遗传因素，以限制 转移性肿瘤的发展。通过系统的临床相关性优先排序 以及使用一组体外和体内方法对前列腺癌进行验证 模型系统(Aim3)，我们的铅遗传因子作为靶向抗肿瘤的临床应用 转移性媒介将被建立。