Cancer and Immunogenetics Laboratory, Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK.
J Pathol. 2014 Sep;234(1):34-45. doi: 10.1002/path.4371. Epub 2014 Jul 7.
We have developed a simple procedure for deriving pure cultures of growing cancer cells from colorectal cancers, including material refrigerated overnight, for pathological characterization and cytotoxicity assays. Forty-six cancers were processed and cultures set up under varying culture conditions. Use of a Rho kinase (ROCK1) inhibitor markedly increased culture survival, resulting in 80% of samples growing in culture for at least 1 month and beyond. Overnight refrigeration of samples before culture initiation had little effect on success rates, paving the way for cultures to be established for samples collected over wide geographical areas, such as those for clinical trials. Primary cultures demonstrated good correlation for differentiation markers compared to parent cancers, and were highly dynamic in 3D culture. In Matrigel, many colonies formed central lumens, indicating the presence of stem-like cells. Viable colonies in these cultures recapitulated the in vivo generation of carcinoembryonic antigen (CEA)-positive necrotic/apoptotic debris, much of which was derived from abnormal vacuolated dynamic 'bubble cells' that have not previously been described. Although bubble cells morphologically resembled signet ring cells, a rare cancer subtype, immunostaining suggested that they were most likely derived from terminally differentiated enterocytes. Micro-assays showed that drug toxicity could be measured in these cultures within hours and with sensitivity down to a few hundred cells. Primary cultures derived by our method provide valid in vitro avatars for studying the pathology of cancers in vitro and are amenable to pre-clinical drug testing, paving the way for personalized cancer treatment.
我们开发了一种从结直肠癌中获取生长癌细胞的纯培养物的简单程序，包括冷藏过夜的材料，用于病理特征分析和细胞毒性测定。对 46 例癌症进行了处理，并在不同的培养条件下建立了培养物。使用 Rho 激酶 (ROCK1) 抑制剂显著提高了培养物的存活率，使 80%的样本至少在培养中生长 1 个月以上。在培养起始前将样本冷藏过夜对成功率影响不大，为在广泛的地理区域（如临床试验）收集的样本建立培养物铺平了道路。与亲本癌症相比，原代培养物在分化标志物方面表现出良好的相关性，并且在 3D 培养中具有高度的动态性。在 Matrigel 中，许多菌落形成中央腔，表明存在干细胞样细胞。这些培养物中存活的菌落再现了体内癌胚抗原 (CEA) 阳性坏死/凋亡碎片的生成，其中大部分来自以前未描述的异常空泡化的动态“泡状细胞”。虽然泡状细胞在形态上类似于印戒细胞，这是一种罕见的癌症亚型，但免疫染色表明它们最有可能来自终末分化的肠上皮细胞。微量分析表明，这些培养物中的药物毒性可以在数小时内测量，并且敏感性可达数百个细胞。我们的方法衍生的原代培养物为体外研究癌症病理学提供了有效的体外虚拟模型，并且适用于临床前药物测试，为个性化癌症治疗铺平了道路。
