Novel Human Cancer Cell Isolation System

描述(由申请人提供)：细胞系是许多癌症研究的关键试剂。人们可能会直觉地想象，癌细胞凭借其在体内的选择性生长优势，在培养中将相对容易建立。然而，培养癌细胞株却出人意料地困难。裸鼠和严重联合免疫缺陷(SCID)小鼠是支持人类癌细胞生长的宝贵工具。当癌细胞被收获用于体外细胞系生产时，成纤维细胞和其他基质细胞通常会在培养皿中过度生长，在大多数情况下会阻止恶性细胞的分离。侵袭和转移(癌症的特征)的研究同样也因为无法恢复完成这一过程的少量癌细胞而受到阻碍。为了绕过这些问题，我们正在培育HPRT缺陷的免疫缺陷小鼠，并已经构建了一只HPRT裸鼠。与其他免疫缺陷小鼠类似，它们将支持许多异种移植癌的生长。在肿瘤生长过程中，小鼠细胞取代了人类的基质细胞。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。在这项计划中，我们计划：建立SCID HPRT缺失小鼠，展示生化缺陷小鼠在细胞系生产方面的优势，并展示增强细胞从自发转移中恢复的能力。具体目标#1：制造生物化学可选择的免疫缺陷小鼠，专为恢复人类癌细胞而设计。具体地说，就是产生SCID HPRT缺失的小鼠。具体目标#2：证明HPRT缺失小鼠相对于标准裸鼠和SCID小鼠的优势。具体地说，记录小鼠基质细胞的替换，并从患者来源的原发癌症(乳腺癌、前列腺癌、肺癌、胰腺癌和神经胶质癌)建立的异种移植中分离出纯人类癌细胞株。比较补充基质细胞和不补充基质细胞的恢复情况。具体目标#3：恢复少量转移性癌细胞的文献效用。具体地说，确定从添加的样本中恢复的效率，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。老鼠将被存放在杰克逊实验室，分发给包括美国国立卫生研究院在内的非营利性机构的研究人员。

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.