RT Journal Article SR Electronic T1 In vitro lymph node-mimicking 3D model displays long-term T cell-dependent CLL proliferation and survival JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.04.03.535388 DO 10.1101/2023.04.03.535388 A1 Marco Vincent Haselager A1 Bianca Francisca van Driel A1 Eduard Perelaer A1 Dennis de Rooij A1 Danial Lashgari A1 Remco Loos A1 Arnon P. Kater A1 Perry D. Moerland A1 Eric Eldering YR 2023 UL http://biorxiv.org/content/early/2023/04/05/2023.04.03.535388.abstract AB Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental cells and signals. The lymph node (LN) is the critical site of in vivo CLL proliferation and development of resistance to both chemotherapy and targeted agents. We present a new model that incorporates key aspects of the CLL LN which enables investigation of CLL cells in the context of a protective niche. We describe a 3D in vitro culture system utilizing ultra-low attachment (ULA) plates to create spheroids of CLL cells derived from peripheral blood (PB). Starting from CLL:T cell ratios as observed in LN samples, CLL activation was induced by either direct stimulation and/or indirectly via T cells. Compared to 2D cultures, 3D cultures promoted CLL proliferation in a T cell-dependent manner, and enabled expansion for up to 7 weeks, including the formation of follicle-like structures after several weeks of culture. Addition of LN-derived stromal cells further enhanced the proliferative capacity. This model enables high-throughput drug screening, of which we describe response to Btk inhibition, venetoclax resistance, and T cell-mediated cytotoxicity as examples. In summary, we present the first LN-mimicking in vitro 3D culture for primary CLL, which enables readouts such as real-time drug screens, kinetic growth assays and spatial localization. This is the first in vitro CLL system that allows testing of response and resistance to venetoclax and Btk inhibitors in the context of the tumor microenvironment, thereby opening up new possibilities for clinically useful applications.Competing Interest StatementR. Loos is an employee and equity holder of Bristol Myers Squibb.