The Co-Culture Of Primary CLL Cells With Bone Marrow Mesenchymal Cells, CD40 Ligand and CpG ODN Promotes The Proliferation Of Chemoresistant CLL Cells Phenotypically Comparable To Those From The Proliferative Centers
Authors: Purroy Noelia , Abrisqueta Pau , Calpe Eva , Carpio Cecilia , Palacio Carles , Gallur Laura , Crespo Marta , Bosch Francesc 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation and proliferation of monoclonal CD5+ mature B-cells in peripheral blood (PB), lymph nodes (LN), and bone marrow (BM). The microenvironment found in BM and LN, where CLL cells interact with different accessory cells, induces active proliferation of CLL cells and protects them from spontaneous and chemotherapy-induced apoptosis. This may explain why the BM and secondary lymphoid organs are preferential sites for minimal residual disease persistence and for relapses observed in all patients after treatment. Therefore, further characterization of primary CLL cells found in the proliferative niches can facilitate the discovery and study of new targets specifically expressed by this proliferative and resistant subset of primary CLL cells.
In order to further characterize the proliferative subset of primary CLL cells we analyzed the characteristics of proliferating subclones of CLL cells found in PB and compared it to the phenotype of primary CLL cells co-cultured ex vivo in conditions mimicking the microenvironment of the proliferative niches. Firstly, we compared actively proliferating CLL cells from 40 patients diagnosed with CLL with their quiescent counterpart. For this, we analyzed by flow cytometry (FC) the differential expression of ZAP-70, CD38, and the chemokine receptors CXCR4, CXCR5 and CCR7 in Ki-67 positive vs. negative CLL cells. Ki-67 positive CLL cells had higher expression levels of ZAP-70 and CD38, while the expression levels of all the chemokine receptors analyzed were significantly lower (Figure 1). This phenotype indicates that these cells may have an increased capability to respond to different survival and migration signals provided by the cellular microenvironment, since high expression of ZAP-70 and CD38 has been related to increased response to this kind of signaling. Moreover, downregulation of chemokine receptors would indicate recent stimulation by chemoatracting cytokines. Furthermore, with the aim of ex vivo mimic the microenvironment found in the proliferative centers, we co-cultured primary CLL cells from 27 patients with the BM-derived stromal cell line UE6E7T-2, 1μg/mL soluble CD40L and 1.5μg/mL CpG ODN, and analyzed the effects in terms of proliferation, modulation of surface molecules and development of chemoresistance. Of note, already 24 hours after co-culture, CLL cells increased their size, resembling stimulated B cells, and tended to be located in sparse clusters that included dividing cells, as observed after immunocytochemistry studies. In this setting, proliferative responses assessed by Ki-67 expression analyzed by FC were already observed after 24 hours (mean % Ki-67 positive cells: 1.48±0.28 in co-culture vs. 0.56±0.11 in suspension, P<0.01) and further increased after 48 hours (mean % Ki-67 positive cells: 2.89±0.51 in co-culture vs. 0.25±0.10 in suspension, P<0.01) and 72 hours (mean % Ki-67 positive cells: 7.68±2.11 in co-culture vs. 0.81±0.24 in suspension, P<0.001). Next, we analyzed the expression levels of ZAP-70, CD38 and the chemokine receptors CXCR4, CXCR5 and CCR7. Interestingly, the modulation of the expression of these molecules in co-cultured CLL cells correlated with the previously mentioned differential expression observed between the Ki-67 positive vs. negative compartments, indicating that the ex vivo co-culture highly resembles the conditions found in proliferative niches (Figure 1). Finally, in order to assess the role of microenvironment in chemoresistance, CLL cells were cultured in suspension and in co-culture for 48 hours and subsequently treated with increasing doses of fludarabine for 24 hours. Interestingly, the co-culture of CLL cells inhibited at such extend the capacity of fludarabine to induce apoptosis that it was not possible to calculate its lethal dose 50, whereas lethal dose 50 for fludarabine in CLL cells in suspension was 416μM (95%CI 125.5-1379).
In conclusion, these results suggest that culturing CLL cells in the previously described conditions promotes proliferation and the acquisition of a phenotype which bears many similarities to the one found in actively proliferating CLL cells circulating in PB. This study provides a model for a co-culture system which might serve as a basis for the development and testing of new drugs that target the proliferative and drug resistant CLL cell compartment.

Disclosures:
No relevant conflicts of interest to declare.