Ceccato Jessica, Piazza Maria, Pizzi Marco, Manni Sabrina, Piazza Francesco, Caputo Ilaria, Cinetto Francesco, Pisoni Lorena, Trojan Diletta, Scarpa Riccardo, Zambello Renato, Tos Angelo Paolo Dei, Trentin Livio, Semenzato Gianpietro, Vianello Fabrizio
Hematology Unit, Department of Medicine, University of Padua, Padua, Italy.
Laboratory of Myeloma and Lymphoma Pathobiology, Veneto Institute of Molecular Medicine (VIMM) and Foundation for Advanced Biomedical Research (FABR), Padua, Italy.
Front Oncol. 2022 Oct 18;12:947823. doi: 10.3389/fonc.2022.947823. eCollection 2022.
About 30% of patients with diffuse large B-cell lymphoma (DLBCL) relapse or exhibit refractory disease (r/r DLBCL) after first-line immunochemotherapy. Bone marrow (BM) involvement confers a dismal prognosis at diagnosis, likely due to the interaction between neoplastic cells and a complex tumor microenvironment (TME). Therefore, we developed a 3D model from human decellularized femoral bone fragments aiming to study the role of mesenchymal stromal cells (MSC) and the extracellular matrix (ECM) in the adaptation, growth, and drug resistance of DLBCL lymphoma cells. The 3D spatial configuration of the model was studied by histological analysis and confocal and multiphoton microscopy which allowed the 3D digital reproduction of the structure. We proved that MSC adapt and expand in the 3D scaffold generating niches in which also other cell types may grow. DLBCL cell lines adhered and grew in the 3D scaffold, both in the presence and absence of MSC, suggesting an active ECM-lymphocyte interaction. We found that the germinal center B-cell (GCB)-derived OCI-LY18 cells were more resistant to doxorubicin-induced apoptosis when growing in the decellularized 3D bone scaffold compared to 2D cultures (49.9% +/- 7.7% Annexin V cells in 2D condition compared to 30.7% + 9.2% Annexin V 3D adherent cells in the ECM model), thus suggesting a protective role of ECM. The coexistence of MSC in the 3D scaffold did not significantly affect doxorubicin-induced apoptosis of adherent OCI-LY18 cells (27.6% +/- 7.3% Annexin V 3D adherent cells in the ECM/MSC model after doxorubicin treatment). On the contrary, ECM did not protect the activated B-cell (ABC)-derived NU-DUL-1 lymphoma cell line from doxorubicin-induced apoptosis but protection was observed when MSC were growing in the bone scaffold (40.6% +/- 5.7% . 62.1% +/- 5.3% Annexin V 3D adherent cells . 2D condition). These data suggest that the interaction of lymphoma cells with the microenvironment may differ according to the DLBCL subtype and that 2D systems may fail to uncover this behavior. The 3D model we proposed may be improved with other cell types or translated to the study of other pathologies with the final goal to provide a tool for patient-specific treatment development.
约30%的弥漫性大B细胞淋巴瘤（DLBCL）患者在一线免疫化疗后会复发或出现难治性疾病（r/r DLBCL）。骨髓（BM）受累在诊断时预后不佳，这可能是由于肿瘤细胞与复杂的肿瘤微环境（TME）之间的相互作用所致。因此，我们利用人脱细胞股骨片段构建了一个3D模型，旨在研究间充质基质细胞（MSC）和细胞外基质（ECM）在DLBCL淋巴瘤细胞的适应、生长和耐药性中的作用。通过组织学分析、共聚焦显微镜和多光子显微镜研究了该模型的3D空间构型，这些技术实现了该结构的3D数字再现。我们证明了MSC在3D支架中适应并扩增，形成了其他细胞类型也可生长的生态位。DLBCL细胞系在有或没有MSC存在的情况下，都能在3D支架上黏附并生长，这表明ECM与淋巴细胞之间存在活跃的相互作用。我们发现，与二维培养相比，生发中心B细胞（GCB）来源的OCI-LY18细胞在脱细胞3D骨支架中生长时，对阿霉素诱导的凋亡更具抗性（二维条件下Annexin V阳性细胞为49.9%±7.7%，而在ECM模型中3D黏附细胞为30.7%+9.2%），这表明ECM具有保护作用。3D支架中MSC的共存并未显著影响阿霉素诱导的黏附OCI-LY18细胞的凋亡（阿霉素处理后，ECM/MSC模型中3D黏附细胞的Annexin V阳性率为27.6%±7.3%）。相反，ECM不能保护活化B细胞（ABC）来源的NU-DUL-1淋巴瘤细胞系免受阿霉素诱导的凋亡，但当MSC在骨支架中生长时可观察到保护作用（3D黏附细胞的Annexin V阳性率为40.6%±5.7%. 62.1%±5.3%，二维条件下）。这些数据表明，淋巴瘤细胞与微环境的相互作用可能因DLBCL亚型而异，二维系统可能无法揭示这种行为。我们提出的3D模型可以通过添加其他细胞类型进行改进，或转化用于其他病理学研究，最终目标是为个性化治疗开发提供一个工具。
