Chronic lymphocytic leukemia (CLL) is a slow progressing hematologic cancer, characterized by the abnormal maturation of B-lymphocytes and their accumulation in peripheral blood, bone marrow, and lymph nodes (LNs). In patients, LNs provide a microenvironment for CLL cell activation, by interaction with T-cells expressing the ligand CD40L and a hyaluronic acid-enriched extracellular matrix (ECM). While CLL cells are quiescent in circulation, upon interaction with the LN microenvironment, they acquire an activated phenotype, inducing proliferation and therapy resistance. Various approaches have been developed to study such interactions, using both scaffolding materials and cell-based co-culture systems, however, these systems are prone to high variability and produce results that are difficult to analyse.

In this project we developed a synthetic lymph node (SynLN) model that allows us to deepen mechanistic understanding of the role of LN matrix and cellular microenvironment in CLL, and the pathways involved in the disease progression and therapeutic evasion in a controlled and reproducible manner. We combined artificial ECM and synthetic cell-based artificial tissues with two CLL cell lines to isolate and quantify the impact of the CD40 activation, as well as the role of the 3D matrix in CLL proliferation and response to treatments. We characterized the SynLN structure by optical coherence tomography and confocal microscopy, measured marker expression by flow cytometry and performed RNA sequencing to decipher functional mechanisms promoting microenvironmental-driven therapy resistance.

Molecular profiling showed that both cell lines exhibit differential expression of critical markers when cultured in 2D vs SynLN, which underscores the relevance of our 3D setup. We further assessed the role of B cell receptor (BCR) activation during this process, mimicking the presence of follicular dendritic cells in the LNs. Stimulation with anti-IgM did not show significant differences in the metabolic activity of the cells, however, a clear shift in marker expression was observed. Our latest RNA sequencing data proves that all components in our model, from the three-dimensional structure itself to the matrix composition and added signals, contribute to transcriptome changes in both cell lines. This data will be crucial to resolve functional mechanisms between inter- and extracellular signalling.

Our setup is a first example of synthetic cell-based tissues to systematically dissect the relevance of individual signalling events leukemic diseases, specifically allowing to resolve the contribution of biophysical and biochemical signalling cues.