Abstract PO-028: A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma
Authors: Zadro Alex , Arribas Alberto , Colombo Maria Vittoria , Cannas Eleonora , Spriano Filippo , Cascione Luciano , Arrigoni Chiara , Candrian Christian , Moretti Matteo , Bertoni Francesco 
Published: June 2024

Abstract
Background. Small molecules targeting the B-cell receptor (BCR) signaling, including PI3K and BTK inhibitors, have represented a step forward for managing lymphoma patients. However, resistance often develops, negatively impacting the benefit of patients exposed to drugs. Thus, developing preclinical tools to identify modalities to overcome this phenomenon is important. The bone marrow is commonly involved in marginal zone lymphoma (MZL) patients, and it might represent a safe environment where lymphoma cells escape therapy. Bone marrow stromal cells (BMSCs) induce proliferation and drug resistance in several tumor entities, mainly through the release of cytokines. To study potential resistance mechanisms to BCR targeting agents, we aimed to develop a 3D co-culture system in which primary BMSCs were used to model the bone marrow tumor microenvironment while exposing an MZL cell line to the PI3K inhibitor copanlisib.
Methods. The MZL cell line VL51 was cultured in 3D fibrin gel (10mg/mL), either alone or with primary BMSCs, in 96-well plates using the OT-2 liquid handler (Opentrons) for high-throughput seeding purposes. Copanlisib treatment started on day 3. The effects of the drug were assessed through morphological observations and quantitative measurements of images taken on days 3, 5, and 7 in the presence or absence of BMSCs. On day 7, supernatants were analyzed for 105 secreted factors using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems / Bio-Techne).
Results. We observed differences in VL51 growth pattern in the different conditions. Most importantly, the co-culture with BMSCs significantly (p=0.025) decreased VL51 sensitivity to copanlisib. When not treated, VL51 cells formed significantly larger clusters (p&lt;0.001) in the presence of BMSCs. However, upon copanlisib treatment, VL51 cells formed smaller and more distributed clusters comparable in size to those in the mono-culture. Subsequent analyses of the supernatants from the mono- or co-cultures following DMSO or copanlisib treatment identified 14 differentially secreted cytokines among the different conditions. Validation experiments were done using recombinant proteins in 2D cultures for 6 factors. Among these, recombinant IGFBP-3, serpin E1, and PTX-3 significantly decreased VL51 sensitivity to copanlisib in 2D cultures (p=0.05, 0.01, and 0.04, respectively). Validation experiments are ongoing with additional in vitro models and using BTK inhibitors.
Conclusions. We created a high-throughput 3D co-culture MZL system and found that BMSCs reduce sensitivity to copanlisib in MZL models. Secretion of IGFBP-3, Serpin E1, and PTX-3 reduced response to copanlisib. This 3D-model will be used to assess resistance mechanisms to clinically relevant compounds and to evaluate alternative therapeutic approaches to improve the treatment of lymphoma patients.
Citation Format: Alex Zadro, Alberto Arribas, Maria Vittoria Colombo, Eleonora Cannas, Filippo Spriano, Luciano Cascione, Chiara Arrigoni, Christian Candrian, Matteo Moretti, Francesco Bertoni. A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-028.