In Vitro 3D Spheroid Culture System Displays Sustained 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 three-dimensional (3D) in vitro culture system using ultra-low attachment plates to create spheroids of CLL cells derived from peripheral blood. 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 with two-dimensional 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. 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 Bruton's tyrosine kinase inhibitors in the context of the tumor microenvironment, thereby opening up new possibilities for clinically useful applications.