Mitochondria Are Transferred from Non-Malignant Stroma Cells to Malignant Plasma Cells, Via Tunnelling Nanotubules in a CD38 Dependent Mechanism, a Process That Promotes Myeloma Proliferation

Background

Survival and proliferation of multiple myeloma (MM) is dependent on the bone marrow microenvironment where the malignant plasma cells rely on both glycolysis and oxidative phosphorylation to generate ATP. Recent work by our group and others have shown that malignant cells can acquire mitochondria from benign non-malignant cells in the tumor micro-environment (Marlein at al. 2017 Blood). Furthermore others have shown that CD38 controls mitochondrial transfer between astrocytes and neurons after stroke (Hayakawa et al. 2016 Nature). Therefore, since mitochondria are essential for the survival, proliferation and chemotherapy resistance of leukemia together with the knowledge that CD38 controls mitochondrial transfer in brain injury we hypothesised that mitochondria are transferred to MM from BMSC via CD38 dependent mechanism.

Methods

Bone marrow containing primary MM cells was obtained from patients following informed consent, in accordance with the Declaration of Helsinki and under approval from the United Kingdom National Research Ethics Service. Malignant plasma cells were purified using magnetic-activated cell sorting with CD138+ microbeads. Primary BMSC were also purified from patient bone marrow, using adherence and characterised using flow cytometry for expression of CD90+, CD73+, CD105+ and CD45-. MM-derived cell lines were obtained from the European Collection of Cell Cultures where they are authenticated by DNA fingerprinting. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining and an in vivo MM NSG xenograft model. Visualisation of tunnelling nanotubes (TNT) was achieved using fixed cell confocal microscopy, quantification of these was carried out by quantification of TNT-anchor points [1]. CD38 expression on primary MM and cell lines was analysed using flow cytometry and qPCR. To inhibit CD38 we used a blocking antibody and lentivirus targeted shRNA. In vivo MM progression in an NSG MM xenograft model was monitored through bioluminescent live animal imaging over time and by overall survival.

Results

First we stained BMSC with MitoTracker Green stain and cultured these with primary MM cells. Using flow cytometry and confocal microscopy we detected MitoTracker fluorescence in the MM cells after co-culture, showing that stained mitochondria are transferred from BMSC to MM cells in-vitro. In a human MM cell line into NSG mouse xenograft model, we detect murine mitochondrial DNA in sorted human MM1S and U266 cells post-transplant. Through fixed cell confocal microscopy we found that mitochondria move through TNTs. Using our BMSC with MitoTracker Green stain MM co-culture assay, we found that CD38 knockdown (Kd) of MM1S cells reduced mitochondrial transfer in-vitro. CD38 Kd improved survival in our NSG xenograft model and moreover blocking CD38 expression reduced mitochondrial transfer between BMSC and MM cells in-vivo. Finally the number of tumor derived TNT-anchor points on BMSC is significantly reduced after co-culture with CD38 knockdown MM cells compared to control knockdown MM cells, highlighting that CD38 is important for TNT formation and binding to BMSC.

Conclusion

Here we show for the first time that mitochondrial transfer occurs between BMSC and malignant plasma cells which supports the growth and proliferation of MM in-vivo and in-vitro. Moreover we report that the mitochondria move through TNTs and that CD38 expression on MM is functionally important for this pro-tumoral mitochondrial transfer to occur.