A Novel 3D Hydrogel Culture System Mimics the Lymph Node and Induces CLL Survival and Proliferation

Chronic lymphocytic leukemia (CLL) has heterogeneous biological characteristics influenced in part by the interactions of tumor cells with the tumor microenvironment (TME). The lymph node (LN) represents a critical niche for in vivo CLL proliferation and development of treatment resistance. Traditional 2D cultures fall short in replicating the TME, crucial for understanding CLL biology. Consequently, 3D models have emerged as valuable alternatives with novel biomaterials that mimic the extracellular matrix (ECM) and can provide new insights into CLL development.

We have generated a 3D CLL model using biohybrid poly(ethylene glycol) (PEG)-heparin hydrogels that simulate LNs with the objective of developing CLL patient-derived organoids (PDOs) from primary samples. Two hydrogel types were evaluated in this system: Bulk hydrogels with a large pore size distribution (25-100 µm) (Pérez del Rio E. et al, Biomaterials 2020) and inverse opal (IOPAL) hydrogels with a uniform pore size (80 µm) and an interconnected structure (Santos F. et al, Biomaterials Science 2022). Following hydrogel formation, primary CLL cells were seeded and cultured for six days in a 96-well plate. Paired peripheral blood (PB) and LN samples were used to assess cell viability and proliferation by flow cytometry and 3D-cell distribution by confocal microscopy and RNA-seq analysis.

Both hydrogel types demonstrated the capacity to support the generation of PDOs from primary CLL cells and autologous T cells. The cells were seeded with ODN2006 + IL15 to induce B cells to proliferate, whereas αCD3/αCD28 Dynabeads® were added for T-cell proliferation. The viability of both populations was approximately 80%, and proliferation reached almost 100% for both primary CLL and T cells after six days of culture. However, a trend for a decrease in the percentage of CD8⁺ cells was observed, which was detrimental to the percentage of CD4⁺ T cells. Furthermore, the cell proportion mirrored the original sample, with approximately 90% tumor cells (CD19⁺ CD5⁺) and 10% T cells (CD3⁺).

Confocal microscopy revealed clusters of proliferating (Ki-67⁺) tumor (CD19⁺) cells surrounded by T cells (CD3⁺) in both hydrogel types. These results showed that Bulk and IOPAL hydrogels effectively reproduced interactions between tumor cells and TME cell populations. Interestingly, RNA-seq analysis indicated a similar expression profile to the LN sample, even when the sample was derived from PB, confirming that hydrogel-PDO system can replicate LN tumor profiles.

In conclusion, both bulk and IOPAL hydrogels successfully mimic LN ECM, enabling B and T-cell interactions. Flow cytometry and confocal microscopy demonstrated high cell viability, significant proliferation, and B and T-cell crosstalk. RNA-seq data validated the modulation of tumor cell expression profiles towards a LN-like proliferative state upon seeding in the hydrogels.