PO.CL01.23 · 临床研究
建立具有免疫细胞逃逸策略的循环肿瘤细胞 3D 培养系统
Establishment of 3D culture system of circulating tumor cells with immune cell evasion strategy
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摘要 Abstract
中文摘要
引言
循环肿瘤细胞（CTC）是从原发肿瘤脱落的癌细胞，通过脉管系统迁移并形成转移灶。与其他液体活检成分（如游离 DNA）相比，CTC 作为诊断工具具有更多优势，因为它们包含所有细胞组分，能够提供更多关于癌细胞的信息。然而，血液中 CTC 的数量非常少，在应用各类分析方面存在局限性。因此，我们开发了 CTC 3D 培养系统，用于在使用自动化 Smart Biopsy™ 细胞分离器富集 CTC 后扩增 CTC 的数量。在本研究中，我们使用癌细胞系构建了一个 CTC 模拟模型，以建立适合 CTC 生长的 3D 培养环境，并使用临床样本进行了 CTC 3D 培养的初步试验。
方法
为使用癌细胞系建立 3D 培养系统，我们将 H358（肺癌）、HCT116（结肠癌）与外周血单个核细胞（PBMC）掺入（细胞:PBMC 比例= 1:10 ~ 1:250），并在 3D 条件下启动培养。为仅保留生长所需的最少癌细胞数量，我们测试了多种方法，包括动态培养和静态培养。随后将混合细胞悬液包埋入 matrigel 中并孵育至生长。在获得梨花女子大学木洞医院机构审查委员会批准后，使用前列腺癌临床样本进行了 CTC 3D 培养的初步试验。
结果
我们观察到，当凝胶内存在数量更多的 PBMC 时，免疫细胞的运动在频率和速度上均增加。因此，在大量 PBMC 迁移出穹顶后，癌细胞逐渐扩增并形成小簇。以每个穹顶 100 个细胞起始，到第 2 周癌细胞簇的直径达到约 100-150 μm。通过在体外再现临床样本中遇到的关键挑战，如 CTC 数量极低及其易受免疫介导清除的影响，我们的模型能够系统评估这些局限性并识别克服它们的策略。此外，使用临床血液样本对该系统进行的初步应用产生了有前景的初步结果。
结论
我们建立了 CTC 3D 培养系统，并通过多种体外试验来确定使 CTC 样细胞能够在免疫压力下存活的培养条件，且我们的培养系统通过前列腺癌临床样本的离体培养成功生长 CTC 而得到验证。
查看英文原文 English abstract
Introduction
Circulating tumor cells (CTCs), are cancer cells shed from primary tumors, travel through the vasculature and give rise to metastatic lesions. CTCs have more advantages as a diagnostic tool compared with other liquid biopsy components such as cell-free DNA, because they contain all cellular components could provide more information of cancer cells. However, the number of CTC in the blood is very small, there are limitations in applying various kinds of analyses. Therefore, we have developed CTC 3D culture system for expanding number of CTCs after the enrichment of CTC with automated Smart Biopsy TM Cell Isolator. In this study, a CTC-mimicking model with the cancer cell lines to establish a 3D culture environment suitable for CTC growth and a pilot test of CTC 3D culture with clinical samples were performed.
Method
To establish the 3D culture system with cancer cell lines, we spiked H358 (lung cancer), HCT116 (colon cancer) with peripheral blood mononuclear cells (PBMCs) (cell:PBMC ratio= 1:10 ~ 1:250) and initiated culture under 3D conditions. To retain only the minimal number of cancer cells required for growth we tested various approaches including dynamic culture and static culture. Then mixed cell suspension was embedded into the matrigel and incubated until growth. A pilot test for CTC 3D culture was performed with clinical samples of prostate cancer after the approval of institutional review board of Ewha Womans University Mokdong Hospital.
Result
We observed that immune cell movement increased both in frequency and speed when higher numbers of PBMCs were present within the gel. As a result, following significant PBMC migration out of the dome, cancer cells progressively expanded and formed small clusters. Starting with 100 cells per dome, cancer cell clusters reached approximately 100-150 μm in diameter by week 2. By recapitulating in vitro the key challenges encountered in clinical samples, such as extremely low number of CTCs and their susceptibility to immune-mediated elimination, our model enables systematic evaluation of these limitations and the identification of strategies to overcome them. Furthermore, pilot application of this system using clinical blood samples yielded promising preliminary results.
Conclusions
We have established the 3D CTC culture system with various kinds of in vitro tests for defining culture conditions that allow CTC-like cells to survive immune pressure and our culture system was validated with successful growth of CTC with ex vivo culture of clinical samples of prostate cancer.
利益披露 Disclosure
J. Moon,
CytoGen Employment.
J. Lee,
CytoGen Employment.
S. Kim,
CytoGen Employment.
M. Hwang,
CytoGen Employment.
J. Kim,
CytoGen Employment.