Two Distinct Co-Culture Systems, Designed to Mimic the Tumor Microenvironment, Induce Remarkably Similar Phenotypic Changes in Primary CLL Cells.
Authors: Buggins Andrea Gail Sherman , Pearce Laurence , Patten Piers EM , Morgan Liam , Brennan Paul , Fegan Chris , Devereux Stephen , Pepper Chris J 
Published: October 2019

Abstract
Abstract 2362
Poster Board II-339
There is growing evidence that interactions in the tumour microenvironment promote the survival, proliferation and drug resistance of primary chronic lymphocytic leukaemia (CLL) cells. Furthermore, progressive CLL is frequently associated with lymphadenopathy, pointing to a crucial role for signals emanating from the tissue microenvironment in the accumulation of malignant B-cells. Proliferation of CLL cells appears to be confined to specialized structures called pseudofollicles, which contain a number of cell types including CLL cells, T-cells, vascular endothelial cells and stromal cells. Using two separate model systems designed to mimic this microenvironment, we characterised the phenotypic changes induced in primary CLL cells and assessed both viability and proliferation when compared with corresponding control cultures. In the first model system we co-cultured CLL cells with mouse embryonic fibroblasts transfected with human CD40L supplemented with soluble IL-4. In the second model system we utilised the microvascular endothelial cell line HMEC-1 with and without the addition of stimulated autologous T-cells. Microarray analysis of this cell line confirmed that it expresses high levels of the CD38 ligand CD31 as well as the cytokines IL-2, IL-4, IL-6 and the chemokines CXCL1 and CXCL2. Both model systems resulted in enhanced CLL cell survival when compared to control cultures (P<0.0001). Furthermore, these conditions induced significant cell division as evidenced by increasing absolute cell counts, significant reductions in mean fluorescence intensity (MFI) of CFSE-loaded cells beyond day 4 (P<0.0001) and the presence of mitotic cells on cytospins. Morphologically, the CLL cells showed a marked increase in size together with cytoplasmic projections. In the CD40L expressing fibroblast model this was associated with a modest increase in CD11c expression (P = 0.02) but no increase in CD103 or the plasmacytoid marker CD138 (P = 0.23 and P = 0.45 respectively). We showed a consistent increase in CD38 MFI in both culture systems (P = 0.0003) and this was further increased by the addition of activated T-cells (P = 0.007). Importantly, in the CD40L transfected fibroblast system, this increase in CD38 expression was strongly associated with an increase in the expression of ZAP-70 (r2 = 0.43) adding further weight to the suggestion that these parameters are in some way biologically linked. Extended immunophenotyping revealed marked increases in the expression of CD69 and CD44 (P<0.0001 and P = 0.0005 respectively). Both of these molecules are NF-κB regulated genes suggesting a role for this transcription factor in generating the altered phenotype observed. Taken together, we have demonstrated that two very different co-culture systems induce remarkably similar changes in primary CLL cells. It seems likely that the further characterisation of these model systems will reveal important new information about the nature of the key interactions between CLL cells and accessory cells in the tissue microenvironment. They also represent a more realistic, clinically relevant, drug testing platform.

Disclosures:
No relevant conflicts of interest to declare.