Culturing viably captured circulating tumor cells using conditional reprogramming

摘要 转移的存在与否是预后的最重要决定因素， 癌症的管理。循环肿瘤细胞（CTC）通过外周血的扩散是 显示为后期明显转移的预后指标。目前，我们只有非常有限的 对CTC的总体基本生物学理解，以及CTC在 个体患者的疾病进展和治疗反应。有几种方法 尽管已经描述了用于从血液中捕获或分离CTC的方法，但是大多数具有局限性，并且非常不稳定。 很少允许在分离时培养CTC。因此，我们不得不研究一个有限的， 在静态时间点捕获的CTC产生有限的细胞和分子数据，排除了我们的 能够动态地研究CTC的功能方面。我们开发了一种独特的 精密设计的微过滤器平台，可有效分离较大CTC和较小CTC 血细胞，并已将其修改为可行的CTC捕获。与此同时，我们还开发了一个 一种新的肿瘤细胞培养方法，我们称之为条件性重编程细胞（CRC） 系统，使用ROCK抑制剂处理的饲养层。 我们建议整合这两个新平台以实现可行的CTC捕获和培养， 具体目标有两个：1）将微滤技术和CRC技术联合收割机相结合， 在模型系统中可靠地从血液中培养癌细胞，以及2）使用组合技术 从转移性前列腺癌、乳腺癌、结肠癌和肺癌患者中捕获并培养CTC。 我们有大量的初步数据表明，我们的捕获技术可以有效地 从同系小鼠模型中的血液中捕获活的肿瘤细胞， 培养的细胞保持了原始的表型。在一个关键的进展中，我们还 证明了有效分离活的CTC并成功建立培养物的能力 其可用于下游功能和分子表征。启联的 技术可以有效和可靠地生长肿瘤细胞，甚至从少量的肿瘤细胞开始， 创始人细胞，CRC显示出对肿瘤的形态、表型和遗传保真度 混元我们的数据还表明，从临床样品中建立的CRC显示出功能性 通过使用它们来预测实际患者对药物的反应。因此， 这里提出的用于捕获和培养稀有CTC的微过滤器和CRC系统具有高的 成功的可能性，并可以改变我们评估和管理癌症患者的方式。 我们还认为，成功的反恐委员会文化将导致重要和必要的洞察力 转移到目前的癌症模型系统不能提供的转移过程中。

Abstract The presence or absence of metastasis is the most important determinant of prognosis and management of cancer. Spread of circulating tumor cells (CTC) via the peripheral blood is shown to be prognostic indicator of later overt metastases. Currently we have a very limited overall fundamental biological understanding of CTCs, and of the clinical significance of CTCs in individual patients' disease progression and treatment response. Several approaches have been described for capture or isolation of CTC from the blood, most have limitations and a very few allow culture of the CTC upon isolation. We are thus forced to study a finite, small number of CTC captured at static time points to yield limited cellular and molecular data, precluding our ability to study CTC dynamically for their functional aspects. We have developed a unique precisely engineered microfilter platform that effectively separates larger CTC from smaller blood cells, and have modified it for viable CTC capture. In parallel, we have also developed a novel method for tumor cell culture, which we refer to as conditionally reprogrammed cell (CRC) system, using a ROCK-inhibitor-treated feeder layer. We propose to integrate the two novel platforms to achieve viable CTC capture and culture, following two Specific Aims: 1) To combine the microfilter and CRC technologies to capture and reliably grow cancer cells from blood in a model system, and 2) To use combined technologies to capture and grow CTC from patients with metastatic prostate, breast, colon and lung cancer. We have substantial preliminary data which shows that our capture technology can efficiently capture viable tumor cells from blood in a syngeneic mouse model which can be cultured such that the cultured cells preserve the original phenotype. In a key advance, we have also demonstrated the ability to efficiently isolate viable CTCs and successfully establish cultures that can be available for downstream functional and molecular characterization. Our CRC technology can efficiently and reliably grow tumor cells, starting even from small numbers of founder cells, and that the CRC show morphologic, phenotypic and genetic fidelity to the tumor of origin. Our data also shows that the CRC established from clinical samples show functional utility through their use to predict response to drugs in actual patients. Thus, the combination of the microfilter and CRC systems proposed here for capture and culture of rare CTC has a high likelihood of success, and can transform the way we evaluate and manage patients with cancer. We also believe that successful CTC cultures will lead to an important and necessary insight into metastatic process that the present cancer model systems cannot provide.