Duś-Szachniewicz Kamila, Gdesz-Birula Katarzyna, Rymkiewicz Grzegorz
Institute of General and Experimental Pathology, Department of Clinical and Experimental Pathology, Wrocław Medical University, Wrocław, Poland.
Flow Cytometry Laboratory, Department of Cancer Pathomorphology, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland.
Onco Targets Ther. 2022 Jun 17;15:683-697. doi: 10.2147/OTT.S363994. eCollection 2022.
B-cell non-Hodgkin lymphomas (B-NHLs) are the most common lymphoproliferative malignancy. Despite targeted therapies, the bone marrow involvement remains a challenge in treating aggressive B-NHLs, partly due to the protective interactions of lymphoma cells with mesenchymal stromal cells (MSCs). However, data elucidating the relationship between MSCs and B-NHLs are limited and inconclusive due to the lack of reproducible in vitro three-dimensional (3D) models. Here, we developed and described a size-controlled and stable 3D hybrid spheroids of Ri-1 (diffuse large B-cell lymphoma, DLBCL) and RAJI (Burkitt lymphoma, BL) cells with HS-5 fibroblasts to facilitate research on the crosstalk between B-NHL cells and MSCs.
We applied the commercially available agarose hydrogel microwells for a fast, low-cost, and reproducible hybrid lymphoma/stromal spheroids formation. Standard histological automated procedures were used for formalin fixation and paraffin embedding (FFPE) of 3D models to produce good quality slides for histopathology and immunohistochemical staining. Next, we tested the effect of the anti-cancer drugs: doxorubicin (DOX) and ibrutinib (IBR) on mono-cultured and co-cultured B-NHLs with the use of alamarBlue and live/dead cell fluorescence based assays to confirm their relevancy for drug testing studies.
We optimized the conditions for B-NHLs spheroid formation in both: a cell line-specific and application-specific manner. Lymphoma cells aggregate into stable spheroids when co-cultured with stromal cells, of which internal architecture was driven by self-organization. Furthermore, we revealed that co-culturing of lymphoma cells with stromal cells significantly reduced IBR-induced apoptosis compared to the 3D mono-culture.
This article provides details for generating 3D B-NHL spheroids for the studies on the lymphoma- stromal cells. This approach makes it suitable to assess in a relevant in vitro model the activity of new therapeutic agents in B-NHLs.
B细胞非霍奇金淋巴瘤（B-NHLs）是最常见的淋巴增生性恶性肿瘤。尽管有靶向治疗，但骨髓受累仍是治疗侵袭性B-NHLs的一项挑战，部分原因是淋巴瘤细胞与间充质基质细胞（MSCs）之间存在保护性相互作用。然而，由于缺乏可重复的体外三维（3D）模型，阐明MSCs与B-NHLs之间关系的数据有限且尚无定论。在此，我们开发并描述了一种大小可控且稳定的由Ri-1（弥漫性大B细胞淋巴瘤，DLBCL）和RAJI（伯基特淋巴瘤，BL）细胞与HS-5成纤维细胞组成的3D混合球体，以促进对B-NHL细胞与MSCs之间相互作用的研究。
我们应用市售的琼脂糖水凝胶微孔来快速、低成本且可重复地形成混合淋巴瘤/基质球体。使用标准组织学自动化程序对3D模型进行福尔马林固定和石蜡包埋（FFPE），以制备用于组织病理学和免疫组织化学染色的高质量玻片。接下来，我们使用alamarBlue和基于活/死细胞荧光的检测方法，测试抗癌药物阿霉素（DOX）和依鲁替尼（IBR）对单培养和共培养的B-NHLs的作用，以确认它们在药物测试研究中的相关性。
我们以细胞系特异性和应用特异性的方式优化了B-NHLs球体形成的条件。淋巴瘤细胞与基质细胞共培养时会聚集形成稳定的球体，其内部结构由自组织驱动。此外，我们发现与3D单培养相比，淋巴瘤细胞与基质细胞共培养显著降低了IBR诱导的细胞凋亡。
本文提供了生成用于淋巴瘤-基质细胞研究的3D B-NHL球体的详细方法。这种方法使其适用于在相关的体外模型中评估新型治疗药物在B-NHLs中的活性。
