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.

Two-dimensional (2D) cell culture is still among the most commonly used models in preclinical cancer research. However, the three-dimensional (3D) in vitro models appear to assess the in vivo environment more accurately, including a more relevant architectural morphology, microenvironment, and responses to drugs. Optical trapping uses one or more focused laser beams to non-invasively manipulate the position, motion, interaction, and dynamics of structures on nano- and microscale. Optical tweezers (OT) have found numerous applications in cell biology; however, their potential in cancer research is still undervalued. In this paper, we recommend a noninvasive protocol for lymphoma-stromal cell spheroid formation with the use of optical tweezers. This method not only constructs the hybrid spheroids de novo but also enables controlling the number of attached cells and the time of adhesion formation, providing an important source of information for tissue engineering. Furthermore, OT enables the study of the early stages of hybrid spheroid formation, which cannot be achieved with standard bulk techniques. Importantly, the described model can be used to study the minimal changes in adhesion induced by anti-cancer drugs. This protocol can be easily applied to other cell types.