Development and Characterization of 3D Hybrid Spheroids for the Investigation of the Crosstalk Between B-Cell Non-Hodgkin Lymphomas and Mesenchymal Stromal Cells

Abstract

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.

Formation of Lymphoma Hybrid Spheroids and Drug Testing in Real Time with the Use of Fluorescence Optical Tweezers

“…Importantly, any other external elements were entered into the experimental setup to stabilize the patterned structure. These spheroids formed in real time reflected the self-aggregation of lymphoma and stromal cells in the hydrogel culture, as we recently described [19].…”

“…Given that tumors are composed of multiple cell types, 3D co-culturing increases the complexity of tumor models [15][16][17][18]. We recently established that lymphoma cells and MSCs, when co-cultured on agarose hydrogels, self-aggregate into stabile 3D spheroids [19]. Interestingly, we observed that stromal cells form the core of the spheroid and are evenly surrounded by layered lymphoma cells.…”

“…The optimal time and dose of ibrutinib (IBR) and doxorubicin (DOX) on Ri-1 and Raji cell lines have been previously investigated [19]. The ibrutinib IC 50 for inhibition of Ri-1 and Raji cells proliferation was 0.514 µM and 6.18 µM, respectively, while the values of IC50 for DOX were 0.833 µM and 0.174 µM for Ri-1 and Raji cell lines, respectively.…”

Emerging Strategies in 3D Culture Models for Hematological Cancers

“…The presence of a stromal population enabled the manipulation and processing of spheroids for histological analyses, because stromal cells are able to produce ECM networks that can both physically trap and biologically attract non-adherent cells. 1,38,40 Furthermore, the stromal component added a higher level of complexity to the model, mimicking the crosstalk that takes place within the microenvironment. Another approach that could be exploited for the generation of more compact models, besides adding ECM-producing cells to the culture, is encapsulation of self-assembling spheroids in hydrogel shells to trap the aggregates in a defined space.…”

“…1,3,37 The creation of a co-culture adding a stromal component is an efficient solution to obtain a more compact and cohesive spheroid. 38,39 Duś-Szachniewicz et al, exploiting a non-adhesive agarose microwell system, developed a 3D model of hybrid spheroids to study the crosstalk between B-cell non-Hodgkin lymphoma and MSC cells. The presence of a stromal population enabled the manipulation and processing of spheroids for histological analyses, because stromal cells are able to produce ECM networks that can both physically trap and biologically attract non-adherent cells.…”