In vitrolymph node-mimicking 3D model displays long-term T cell-dependent CLL proliferation and survival

Abstract

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.

Emerging Strategies in 3D Culture Models for Hematological Cancers

“…Interestingly, we identified different modulations of a variety of genes; for example, we observed upregulation of the CXCR3 gene, involved in leukocyte trafficking, integrin activation, and cytoskeletal and focal adhesion remodeling (ie, the HCLS1 gene, involved in the migration and homing of CLL cells 11 and prognostic factors 12,13 ). These results also suggest that, for cells usually grown in suspension in vitro, we can obtain a different phenotype in 3D culture; this offers the possibility of recreating the spatial organization and disposition found in tissues in vivo 14,15 (Figure 1). Another important aspect to consider is that cells arranged in monolayers or in suspension receive all the same amounts of oxygen, nutrients, and growth factors, and consequently more cells will be in the same phase of the cell cycle.…”

“…They obtained a scalable platform able to sustain primary CLL cell growth and simulate an in vivo-like microenvironment stimulation, which could be useful in large-scale studies as a drug screening method. 14 Recently, Araujo-Ayala et al generated a 3D spheroid model of LN TME of mantle cell lymphoma (MCL), which recapitulates in vivo responses to target therapies. They demonstrate that this model fairly represents fundamental hallmarks of MCL microenvironment features as proliferation signature, BCR signaling pathway, and immune TME exhaustion.…”

“…The principle behind scaffold-free techniques is the generation of cell aggregates (ie, spheroids and organoids) using specific platforms that can exploit gravity, such as the hanging drop method. 35 There are also agitation-based methods, ultra-low attachment-coated plates, 14 or inert hydrogel capsules. 3 Those kinds of platforms are optimal for multiple reproducible replications (which is very useful for drug screening analyses) and are generally easy to handle.…”