Soluble CX3CL1/Fractalkine Is a New Player in the Pro-Inflammatory Microenvironment of Multiple Myeloma Patients Related to Bone Marrow Plasma Cell Infiltration and Neo-Vascularization

Multiple myeloma (MM) is characterized by the tight dependence of plasma cells (PCs) to the bone marrow (BM) microenvironment that activates the production of several pro-inflammatory and growth factors involved in MM cell survival, bone remodeling alterations and increased angiogenesis. CX3CL1/Fractalkine is a unique chemokine synthetized as a membrane-bound protein and released as soluble protein after proteolytic cleavage by two metalloproteases as ADAM10 and ADAM17. CX3CL1 interacts with its single receptor CX3CR1 being involved in several physiological and pathophysiological processes including inflammation and angiogenesis. The role CX3CL1/CX3CR1 axis in MM is still unexplored and unknown.

In this study, firstly we analyzed BM plasma levels of CX3CL1 in MM patients as compared to indolent monoclonal gammopathies and healthy controls. BM plasma was obtained from 38 MM patients (median age 70 years; 53% female and 47% male; ISS I= 32%, II= 34%, III=34%) 25 of them with newly diagnosed and 13 relapsed MM, and from sex-aged matched 14 Smoldering MM (SMM) patients and 11 monoclonal gammopathy of undetermined significance (MGUS) patients. 10 healthy donors (HD) (median age 70 years; 20% female and 80% male) were used as controls. We found that plasma levels were significantly increased in the BM plasma from MM patients compared to HD (median levels 0.88 ng/ml vs 0.56 ng/ml, p=0.004). Furthermore, increased CX3CL1 levels were observed in patients with active MM as compared to patients with asymptomatic disease, as MGUS and SMM (median levels MM vs MGUS: 0.88 ng/ml vs 0.67 ng/ml, p=0.0176; median levels MM vs SMM: 0.88 ng/ml vs 0.65ng/ml, p= 0.0329). Moreover, CX3CL1 levels increased in the advanced stages of disease (median levels ISS III vs ISS I: 1.18 ng/ml vs 0.72 ng/ml, p=0.0115; median levels ISS III vs ISS II: 1.18 ng/ml vs 0.74 ng/ml, p=0.0157). Accordingly, BM soluble CX3CL1 levels were positively correlated with the percentage of BM plasma cells in the cohort of MM patients (p=0.002). Subsequently, the possible link between soluble CX3CL1/fractalkine and BM angiogenesis has been investigated. CD34 expression was evaluated by immunohistochemistry on bone biopsies obtained from 28 MM patients, showed that BM plasma levels of CX3CL1 significantly correlated with both the number of CD34 positive vessels (p= 0.002) and the micro-vessels density (p=0.002). Thereafter, we performed experiments on chorioallantoic membranes to demonstrate the role of CX3CL1 in MM-induced angiogenesis. The incubation of BM plasma of MM patients for 12 days resulted in a significant angiogenic response in the form of numerous allantoic neovessels that were significantly inhibited by blocking anti-CX3CL1 antibody. Finally, to clarify the source of CX3CL1 into the BM of MM patients, we explored CX3CL1 expression either by CD138⁺ cells on a dataset of 133 MM patients at diagnosis (GSE16122) and 23 human myeloma cell lines (HMCLs) (GSE6205), or on a proprietary dataset of primary mesenchymal stromal cells (MSCs) and osteoblasts (OBs) of 16 MM and 7 HD. Isolated BM endothelial cells from 4 MM patients and controls were also analysed. Purified CD138⁺ cells, HMCLs and BM endothelial cells were negative for CX3CL1 mRNA expression whereas MSCs and OBs were positive for CX3CL1 even if any statistically significant difference was not observed across the different groups. On the other hand, we found that CD138⁺ cells and HMCLs expressed both ADAM10 and ADAM17, leading to the hypothesis that the expression of these metalloproteinases by MM cells is accountable for the increased levels of CX3CL1 into the BM microenvironment and consequently increase of the neo-vascularization.

In conclusion, our data indicate that CX3CL1, present at high level into the BM of MM patients, is involved in MM-induced BM neo-vascularization and potentially in MM progression.