Barkan Dalit, Green Jeffrey E
Department of Biology, University of Haifa.
J Vis Exp. 2011 Aug 11(54):2914. doi: 10.3791/2914.
Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time. The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression and reside growth-arrested in the patients' bone marrow, blood and lymph nodes. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems. In vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo. We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.
乳腺癌复发通常会经历很长的潜伏期，在此期间没有癌症迹象，而且转移灶可能在原发肿瘤切除及辅助治疗多年后才会在临床上显现出来。对这一现象的一种可能解释是，肿瘤细胞已在转移部位播种，对传统疗法具有抗性，并能长时间保持休眠状态。先前已将继发性位点休眠癌细胞的存在描述为既不增殖也不发生凋亡的静止单个细胞。此外，这些单个细胞已被证明在疾病进展的早期就从原发肿瘤播散出来，并停滞生长于患者的骨髓、血液和淋巴结中。因此，了解调节休眠或向增殖状态转变的机制对于发现预防疾病复发的新靶点和干预措施至关重要。然而，由于缺乏可用的模型系统，揭示调节从肿瘤休眠向转移生长转变的机制受到了阻碍。先前已描述了用于研究肿瘤细胞转移进展的体内和体外模型系统。然而，这些模型系统并未以实时且高通量的方式提供关于是什么触发单个休眠肿瘤细胞增殖成为转移性疾病的机制性见解。我们最近开发了一种三维体外系统，以模拟在体内表现出休眠（D2.OR、MCF7、K7M2 - AS.46）或增殖（D2A1、MDA - MB - 231、K7M2）转移行为的细胞的体内生长特征。我们证明，在转移部位在体内表现出休眠的肿瘤细胞在三维（3D）基底膜提取物（BME）中培养时仍保持静止，而在体内具有高转移性的细胞在经过不同但相对较短的静止期后在3D培养中很容易增殖。重要的是，通过利用三维体外模型系统，我们首次证明细胞外基质（ECM）组成在调节休眠肿瘤细胞是否会转变为增殖状态方面起着重要作用，并已在体内研究中得到证实。因此，本报告中描述的模型系统提供了一种体外方法来模拟肿瘤休眠并研究由微环境诱导的向增殖生长的转变。
