Purpose 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. Materials and Methods 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. Results 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. Conclusion 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.

Hypoxia is a common feature in most tumors, including hematological malignancies. There is a lack of studies on hypoxia- and physioxia-induced global proteome changes in lymphoma. Here, we sought to explore how the proteome of diffuse large B-cell lymphoma (DLBCL) changes when cells are exposed to acute hypoxic stress (1% of O2) and physioxia (5% of O2) for a long-time. A total of 8239 proteins were identified by LC–MS/MS, of which 718, 513, and 486 had significant changes, in abundance, in the Ri-1, U2904, and U2932 cell lines, respectively. We observed that changes in B-NHL proteome profiles induced by hypoxia and physioxia were quantitatively similar in each cell line; however, differentially abundant proteins (DAPs) were specific to a certain cell line. A significant downregulation of several ribosome proteins indicated a translational inhibition of new ribosome protein synthesis in hypoxia, what was confirmed in a pathway enrichment analysis. In addition, downregulated proteins highlighted the altered cell cycle, metabolism, and interferon signaling. As expected, the enrichment of upregulated proteins revealed terms related to metabolism, HIF1 signaling, and response to oxidative stress. In accordance to our results, physioxia induced weaker changes in the protein abundance when compared to those induced by hypoxia. Our data provide new evidence for understanding mechanisms by which DLBCL cells respond to a variable oxygen level. Furthermore, this study reveals multiple hypoxia-responsive proteins showing an altered abundance in hypoxic and physioxic DLBCL. It remains to be investigated whether changes in the proteomes of DLBCL under normoxia and physioxia have functional consequences on lymphoma development and progression.

Photodynamic therapy (PDT) is a low-invasive method of treatment of various diseases, mainly neoplastic conditions. PDT has been experimentally combined with multiple treatment methods. In this study, we tested a combination of 5-aminolevulinic acid (5-ALA) mediated PDT with thalidomide (TMD), which is a drug presently used in the treatment of plasma cell myeloma. TMD and PDT share similar modes of action in neoplastic conditions. Using 4T1 murine breast carcinoma and 2H11 murine endothelial cells lines as an experimental tumor model, we tested 5-ALA-PDT and TMD combination in terms of cytotoxicity, apoptosis, Vascular Endothelial Growth Factor (VEGF) expression, and, in 2H11 cells, migration capabilities by wound healing assay. We have found an enhancement of cytotoxicity in 4T1 cells, whereas, in normal 2H11 cells, this effect was not statistically significant. The addition of TMD decreased the production of VEGF after PDT in 2H11 cell line. Our results reveal enhanced effectiveness of 5-ALA-PDT with TMD treatment compared to 5-ALA-PDT or TMD treatment alone. The addition of TMD may be a promising proceeding of the anti-tumor effect of PDT by decreasing the VEGF concentration in the culture medium. Further studies, including testing on different cell lines, are needed to confirm this assumption.

Interactions between stromal and lymphoma cells in the bone marrow are closely related to drug resistance and therapy failure. Physiologically relevant pre-clinical three-dimensional (3D) models recapitulating lymphoma microenvironmental complexity do not currently exist. In this study, we proposed a scheme for optically controlled hybrid lymphoma spheroid formation with the use of optical tweezers (OT). Following the preparation of stromal spheroids using agarose hydrogel, two aggressive non-Hodgkin lymphoma B-cell lines, Ri-1 (DLBCL) and Raji (Burkitt lymphoma), were used to conduct multi-cellular spheroid formation driven by in-house-developed fluorescence optical tweezers. Importantly, the newly formed hybrid spheroid preserved the 3D architecture for the next 24 h. Our model was successfully used for the evaluation of the influence of the anticancer agents doxorubicin (DOX), ibrutinib (IBR), and AMD3100 (plerixafor) on the adhesive properties of lymphoma cells. Importantly, our study revealed that a co-treatment of DOX and IBR with AMD3100 affects the adhesion of B-NHL lymphoma cells.