应用EpCAM/PAN-CK荧光标记流式细胞术检测肿瘤浸润淋巴细胞中肿瘤细胞残留的研究

摘要/Abstract

摘要： 作为新兴的肿瘤免疫治疗管线之一，过继性细胞免疫疗法（adoptive cell therapy/ACT）已日趋成熟；其中最直接的手段是从实体瘤组织中提取靶向性与特异性俱佳的肿瘤浸润淋巴细胞（tumor-infiltrating lymphocyte/TIL），经体外激活、扩增和优化后回输患者而达到安全、有效的临床疗效。具体的TIL工艺流程除了要保证淋巴细胞的充分扩增（数量）和免疫学活性（质量）以外，还需要解决肿瘤细胞残留问题。虽然在TIL培养过程中肿瘤细胞因为营养成分的相对缺乏和淋巴细胞的持续攻击而逐渐凋亡，在理论上不能完全排除癌细胞残留的可能性。为充分解决这一切实问题，本研究比较、分析了多种有关肿瘤细胞残留的检测方法，并根据灵敏度、可靠性、器材要求和检测效率等参数综合权衡利弊，最后将流式细胞术作为优选方案，并在检测肿瘤细胞系和源自乳腺癌患者和肺癌患者的TIL产品（N=60）时得到进一步验证。这一方法依赖荧光标记的特异性抗体有效识别上皮细胞普遍携带的标志物，即泛细胞角蛋白 (pan-cytokeratin)；其检测灵敏度<0.05%，而且有进一步提升空间，因此可以作为今后TIL和同类产品放行前有必要采纳的质量检测备选方案。

关键词: font-size:medium, ">过继性免疫细胞疗法；肿瘤浸润淋巴细胞；流式细胞术；上皮细胞粘附分子；泛细胞角蛋白

Abstract: Adoptive cell therapy (ACT) offers a promising option for treating solid tumors， and various ACT pipelines now rely on the use of tumor-infiltrating lymphocytes (TIL) that are activated and expanded in vitro. Before TIL products are infused back into cancer patients， multiple mandatory tests must be done to ensure adequate cell numbers， as well as other parameters that gauge TIL quality and safety， especially the a quantitative assessment of residual tumor cells. Although cancer cells gradually die out (through apoptosis) due to a relative lack of nutrients and a continuous attack by lymphocytes in the process of cell culture， the possibility of residual cancer cells cannot be ruled out. To fully address this important and practical issue， the study compared various detection methods in terms sensitivity， reliability， efficiency and requirement for special equipment. In the end， flow cytometry was used for testing TIL products (N=60) from patients with breast cancer or lung cancer. Experimental results revealed that cancer cells， which are of epithelial origin in these patients， universally carry pan-cytokeratin on cell surface， and can be readily quantified using fluorescence-labelled antibodies against pan-cytokeratin. Overall， the sensitivity of this assay is <0.05% and can be improved further through the use of exclusion markers， being a backup method for control of quality analysis as product releasing when there is a need to rapidly and reliably distinguish unwanted cancer cells from TIL products.

Key words: font-size:medium, ">Adoptive cell therapy；Tumor-infiltrating lymphocyte； Flow cytometry； Epithelial cell adhesion molecule； Pan-cytokeratin

中图分类号:

王涛, 周足力, 初明, 王星星, 董兆惠, 唐建明, 王铁山, 陈萌, 张秀军. 应用EpCAM/PAN-CK荧光标记流式细胞术检测肿瘤浸润淋巴细胞中肿瘤细胞残留的研究[J]. 中国医药导刊, 2023, 25(2): 132-139.

WANG Tao, ZHOU Zuli, CHU Ming, WANG Xingxing, DONG Zhaohui, TANG Jianming, WANG Tieshan, CHEN Meng, ZHANG Xiujun. Study on the Tumor Cell Residue of Tumor-Infiltrating Lymphocyte by Flow Cytometry with EpCAM/PAN-CK Fluorescent Labeling[J]. CHINESE JOURNAL OF MEDICINAL GUIDE, 2023, 25(2): 132-139.

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