Novel Human Cancer Cell Isolation System

新型人类癌细胞分离系统

• 批准号: 7680212
• 负责人: JAMES R. ESHLEMAN
• 金额: $ 30.63万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7680212
• 关键词: Abbreviations; Aminopterin; Biochemical; Cancer Cell Growth; Cancer cell line; Cell Line; Cell Separation; Cells; Deposition; Disseminated Malignant Neoplasm; Distal; Fibroblasts; Genes; Growth; HSV-Tk Gene; Harvest; Human; Human Cell Line; Hypoxanthines; Immunodeficient Mouse; In Vitro; Institution; Interleukin 2 Receptor Gamma; Knockout Mice; Lung; Malignant Neoplasms; Modeling; Monoclonal Antibodies; Mouse Cell Line; Mucinous Neoplasm; Mus; Mutation; Natural Killer Cells; Neoplasm Metastasis; Non obese; Nonsense-Mediated Decay; Nude Mice; Organ; Pancreas; Pancreatic Intraepithelial Neoplasia; Papillary; Pathway interactions; Patients; Population; Problem Solving; Process; Production; Prostate; Reagent; Recovery; Research Personnel; SCID Mice; Sampling; Severe Combined Immunodeficiency; Site; Stromal Cells; Supplementation; System; Tail; Thioguanine; Thymidine; Transferase; United States National Institutes of Health; Veins; War; Work; Xenograft procedure; anticancer research; cancer cell; design; diabetic; implantation; in vivo; malignant breast neoplasm; mouse model; mutant; novel; nucleotide metabolism; prevent; public health relevance; tissue culture; tool; tumor; tumor growth

DESCRIPTION (provided by applicant): Cell lines are critical reagents for much of cancer research. One might intuitively imagine that cancer cells, with their in vivo selective growth advantage, would be relatively easy to establish in culture. However, generating cancer cell lines is surprisingly difficult. Athymic ("nude") and severe combined immunodeficient (SCID) mice are valuable tools that support human cancer cell growth. When the cancer is harvested for in vitro cell line production, fibroblasts and other stromal cells commonly overgrow the culture dish and in most cases prevent isolation of the malignant cells. The study of invasion and metastasis (hallmarks of cancer) is similarly hindered by the inability to recover small numbers of cancer cells that have completed the process. To circumvent these problems, we are generating immunodeficient mice that are hprt defective, and have already constructed a nude hprt-null mouse. Similar to other immunodeficient mice, they will support growth of many xenografted cancers. During tumor growth, mouse cells replace the human stromal cells. When we wish to recover the human cells, we will eliminate the mouse stromal cells by growing the culture in the classic selective media, HAT. In this proposal, we plan to: generate SCID hprt-null mice, demonstrate superiority of the biochemically defective mice for cell line production, and demonstrate enhanced recovery of cells from spontaneous metastases. Specific Aim #1: Generate biochemically selectable immunodefective mice designed for human cancer cell recovery. Specifically, generate SCID hprt-null mice. Specific Aim #2: Document superiority of the hprt-null mice over standard nude and SCID mice. Specifically, document mouse stromal cell replacement and isolate pure populations of human cancer cell lines from xenografts established from patient-derived primary cancers (breast, prostate, lung, pancreatic and glial cancers). Compare recovery with and without stromal cell supplementation. Specific Aim #3: Document utility in recovering small numbers of metastatic cancer cells. Specifically, determine recovery efficiency from spiked samples, and isolate organ-specific metastases from experimental metastases and from spontaneous metastases after orthotopic implantation. Mice will be deposited at Jackson Labs for distribution to investigators at non-profit institutions, including the NIH. PUBLIC HEALTH RELEVANCE: Cell lines from human cancers are invaluable tools, especially from specific patients with cancers bearing known genetic defects, however generating cell lines is surprisingly difficult due to non-cancer cell overgrowth. We propose to make biochemically defective mice that support human cancer growth, but when these cancers are transferred to tissue culture, the mouse cells can be easily eliminated. We plan to demonstrate that the new mice are superior in producing cell lines and recovery of metastases.

描述（由申请人提供）：细胞系是许多癌症研究的关键试剂。人们可能会直观地认为，癌细胞具有体内选择性生长的优势，在培养中相对容易建立。然而，产生癌细胞系是非常困难的。胸腺（裸）和严重联合免疫缺陷（SCID）小鼠是支持人类癌细胞生长的有价值的工具。当收集癌细胞用于体外细胞系生产时，成纤维细胞和其他基质细胞通常在培养皿中过度生长，在大多数情况下阻碍了恶性细胞的分离。侵袭和转移（癌症的特征）的研究同样受到无法恢复已经完成这一过程的少量癌细胞的阻碍。为了避免这些问题，我们正在产生hprt缺陷的免疫缺陷小鼠，并且已经构建了裸hprt缺失小鼠。与其他免疫缺陷小鼠类似，它们将支持许多异种移植癌症的生长。在肿瘤生长过程中，小鼠细胞取代了人类基质细胞。当我们希望恢复人类细胞时，我们将通过在经典的选择性培养基HAT中培养来消除小鼠基质细胞。在这个提议中，我们计划：产生SCID hprt缺失的小鼠，证明生化缺陷小鼠在细胞系生产方面的优势，并证明自发转移细胞的恢复能力增强。具体目标#1：产生生物化学选择性免疫缺陷小鼠设计用于人类癌细胞恢复。具体来说，生成SCID hprt缺失小鼠。具体目标2：证明hprt缺失小鼠优于标准裸小鼠和SCID小鼠。具体来说，记录小鼠间质细胞替代，并从患者来源的原发性癌症（乳腺癌、前列腺癌、肺癌、胰腺癌和神经胶质癌）建立的异种移植物中分离出纯人类癌细胞系。比较补充基质细胞和不补充基质细胞的恢复情况。特定目标#3：记录在恢复少量转移癌细胞中的效用。具体来说，确定加标样品的恢复效率，并从实验转移和原位植入后的自发转移中分离出器官特异性转移。小鼠将存放在杰克逊实验室，分发给包括NIH在内的非营利机构的研究人员。公共卫生相关性：来自人类癌症的细胞系是非常宝贵的工具，特别是来自患有已知遗传缺陷的特定癌症患者的细胞系，然而，由于非癌细胞过度生长，产生细胞系非常困难。我们建议制造生化缺陷的小鼠来支持人类癌症的生长，但是当这些癌症转移到组织培养中时，小鼠细胞很容易被消除。我们计划证明新小鼠在产生细胞系和转移恢复方面具有优势。