肿瘤细胞治疗研究
英文原题：Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.
在本研究中，我们建立了 EBV 感染的肿瘤浸润 B 淋巴细胞的原代细胞培养，未来研究项目可用其研究这些细胞在肿瘤生物学中的作用。
背景：亟需提出更有效的抗癌治疗新思路。实验设计缺陷是阻碍治疗开发的重要因素，会导致数据不准确、难以重复，并使研究方案无法成功转化至临床。我们此前曾从头颈部鳞状细胞癌（HNSCC）组织建立上皮细胞培养物。方法：本研究从人乳头瘤病毒（HPV）阳性HNSCC组织中分离原代细胞群，采用RT-qPCR、流式细胞术和免疫荧光染色检测标志物表达，并通过细胞活力实验评估其对MDM2抑制的敏感性。结果：原代HNSCC细胞培养物在较高传代次数时延迟形成球体，这些球体的形态和生长特性类似其他已建立的HNSCC球体模型。然而，尽管存在HNSCC干细胞标志物乙醛脱氢酶1家族成员A1，细胞中仍检测不到上皮和间质标志物表达；相反，B细胞和T细胞标志物表达较强。流式细胞分析显示，第5代细胞为异质混合群体，含CD3+、CD25+ T淋巴细胞和CD19+ B淋巴细胞，比例约为4:1；晚期传代（第12代）则出现转化淋巴细胞，表型为CD45+、CD19+、CD20+，其中约10%–20%为CD3+、CD25+、CD56+。值得注意的是，整个细胞群均为FOXP3阳性，提示其可能为调节性B细胞（Breg）。随着传代增加，球体细胞中EB病毒（EBV）特异性转录本表达增加，且该群体对MDM2抑制敏感。研究在免疫缺陷小鼠中检测到HPV阳性HNSCC细胞成瘤，但未检测到EBV阳性淋巴细胞成瘤。结论：本研究建立了EBV感染的肿瘤浸润B淋巴细胞原代培养模型，可用于今后研究这类细胞在肿瘤生物学中的作用。通过详细描述其特征，我们提醒HNSCC领域研究者在进一步实验前检测原代培养物是否混入EBV感染淋巴细胞。尤其是开展基于TIL的过继免疫治疗研究时，应在原发肿瘤模型中排除这类细胞，例如采用MDM2抑制剂处理。BI-12来源的异种移植瘤可作为体内靶向研究的合适模型。
BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages ( passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (B regs ). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.
MEMBER ACCOUNT
登录成功会直接打开下一页。
