Fig 2: CXCR4 expression on PCs correlates with the percentage of transferred mitochondria.A Calcein AM labeled HMCLs were cultured alone or with MitoTracker red stained HS-5. Mitochondrial red fluorescence internalization was detected in HMCLs in time-lapse by using operetta. B Quantification of the area under the curve (A.U.C.) ± SD. **** vs U266 and NCI-H929 cell lines. C Flow cytometry analysis of mitochondrial red fluorescence internalized by HMCLs (CD45+ gated) cocultured with MitoTracker red labeled HS-5 cells for 24 h. D Flow cytometry analysis for CXCR4 expression of HMCLs. Representative histograms with the corresponding isotype control (dotted line) are shown. ****vs U266 and NCI-H929 cell lines. CXCR4 expression levels were correlated with the percentage of transferred mitochondria in HMCLs (E) and primary CD138+ cells (F). The data are presented as means ± SD of three independent experiments (**p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig 3: CXCR4 colocalizazion with CD138+ cells is higher in MM patients who failed to respond to BTZ.The amount of the percentage of CD138+CD38+CXCR4+ cells was assessed in BM (A) and PB (B) samples from MM at diagnosis (n = 30/19) and resistant/refractory patients (n = 18/8). C Analysis of CXCR4 colocalization with CD138+ cells in BM biopsy specimens from patients who responded to BTZ (n = 3) and patients who failed to respond (n = 3). The left panel shows representative pictures of a responder vs a non-responder patient. Scale bar: 10 µm. Bars indicate the standard error means (*p < 0.05).

Fig 4: MSC plasticity in MM microenvironment.Pro-tumor phenotype of MM-MSCs is associated to a metabolic rewiring and an increased ability to donate mitochondria. Furthermore, myeloma PCs increase the expression of CX43 and CXCL12 proteins in stromal cells with consequent activation of the corresponding receptor CXCR4, which favors mitochondrial uptake from MSCs. The high mitochondrial trafficking in MM microenvironment supports tumor to escape PI-induced intracellular oxidative stress and mitochondrial damage.