Fibroblasts in Nodular Sclerosing Classical Hodgkin Lymphoma Are Defined by a Specific Phenotype and Protect Tumor Cells From Brentuximab-Vedotin Induced Injury

Several studies have described a crosstalk between the tumour cells of cHL, the Hodgkin- and Reed-Sternberg (HRS) cells, and cancer-associated fibroblasts (CAF). However, to date a deep molecular characterization of these fibroblasts is lacking. Aim of the present study therefore was a comprehensive characterization of these fibroblasts. Gene expression of fibroblasts isolated from primary lymph node suspensions (LA = lymphoadenitis, n=7, cHL_MC = classical Hodgkin lymphoma mixed cellularity subtype, n=5; cHL_NS = classical Hodgkin lymphoma nodular sclerosing subtype, n=8; Fib = primary fibroblasts culture at passage 4-6 in vitro, KB = arbitrary sample ID prefix, FST = arbitrary sample ID prefix). As previously shown for other types of cancer-associated fibroblasts, treatment by luteolin could reverse this fibroblast phenotype. Working concentration of 30 µM luteolin was added to sample KB33 and harvested 48 h post treatment for comparative analysis with Fib_cHL_NS samples. Three biological replicates were applied. The cHL specific adherence-based interaction of both cell fractions revealed an initiation of inflammatory key regulators. The classical Hodgkin lymphoma cell line L-428 and adherent activated fibroblasts cells from primary nodular sclerosis cHL were used. Floating cell fractions were removed by repeated washing. Adherent interacting coculture layer was harvested for comparative gene expression analysis to mock coculture. Mock coculture was set up separated in equivalent ratios and pooled before RNA analysis. Three biological replicates were applied.

已有多项研究描述了经典霍奇金淋巴瘤（classical Hodgkin lymphoma, cHL）的肿瘤细胞——霍奇金与里-施（Hodgkin- and Reed-Sternberg, HRS）细胞——与癌症相关成纤维细胞（cancer-associated fibroblasts, CAF）之间的细胞串扰。但截至目前，针对这类成纤维细胞的深度分子特征解析仍存在研究空白。因此本研究的核心目标是对这类成纤维细胞开展全面的特征分析。本研究分析了从原发性淋巴结悬液中分离得到的成纤维细胞的基因表达情况：其中LA为淋巴结炎（lymphoadenitis），样本量n=7；cHL_MC为经典霍奇金淋巴瘤混合细胞型亚型，样本量n=5；cHL_NS为经典霍奇金淋巴瘤结节硬化型亚型，样本量n=8；Fib为体外传代4-6代的原代成纤维细胞培养物；KB与FST为自定义样本ID前缀。正如此前针对其他类型癌症相关成纤维细胞的研究所示，木犀草素（luteolin）处理可逆转这类成纤维细胞的异常表型。本实验向样本KB33中加入30 μM的木犀草素处理浓度，并在处理48小时后收集样本，以与Fib_cHL_NS样本开展对比分析。本次实验设置了3次生物学重复。针对两种细胞组分开展的cHL特异性黏附相互作用实验显示，炎症关键调控因子的表达被激活启动。本实验使用了经典霍奇金淋巴瘤细胞系L-428，以及来自原发性结节硬化型cHL的黏附活化成纤维细胞。通过反复洗涤去除悬浮细胞组分，收集黏附的共培养相互作用层，以与模拟共培养样本开展对比基因表达分析。模拟共培养以等比例设置并分离，在RNA分析前进行混合。本次实验同样设置了3次生物学重复。