CX43-Mediated Mitochondria Transfer from Bone Marrow Stromal Cells Promotes the Stemness of Leukemia Stem Cells

Abstract

Background: Leukemia stem cells (LSCs) play an important role in the occurrence, development, chemoresistance and recurrence of leukemia, but the underlying mechanisms that maintain the stemness of LSCs are poorly understood. Bone marrow stromal cells (BMSCs) are a key component in the hematopoietic microenvironment, which support and regulate the biological behaviors of hematopoietic stem cells (HSCs). Similarly, hematopoietic microenvironment plays an important role in the development of leukemia. In this study, we aimed to uncover the mechanism of BMSCs regulated stemness of leukemia stem cells through Connexin 43 (CX43) -mediated mitochondrial transfer, which might provide a promising clinical therapy target. Methods: Primary BMSCs were isolated from bone marrow of patients with AML and cultured in BMSCs specific medium within 10% fetal bovine serum . CX43 overexpressing BMSCs (CX43-BMSCS) were constructed by lentivirus transfection. The efficiency of CX43 overexpression was verified by immunofluorescence, qPCR and Western Blot. In vitro, CX43-BMSCs and EV-BMSCs (empty virus transfection) were co-cultured with KG-1a cells, respectively. In some experiments, vincristine (1nM) was used to inhibit the formation of tunneling nanotubes (TNTs). The mitochondria transfer was detected by flow cytometry and confocal microscopy. The proliferation of KG-1a from the co-culture system was assessed by flow cytometry and EDU assay. The clone formation of KG-1a was detected by using semisolid cloning assay. Results: Compared with EV-BMSCs, the mRNA and protein expression levels of CX43 in CX43-BMSCs were significantly increased (P < 0.05). Regardless of CX43 expression level, there was no obvious mitochondrial transfer was observed in the co-culture system with 10% fetal bovine serum. In the co-culture system without fetal bovine serum for 48h, we observed mitochondria transfer from BMSCs to KG-1a through TNTs, while hardly any mitochondria from KG-1a to BMSCs. Moreover, CX43-BMSCs transferred more mitochondria to KG-1a than EV-BMSCs group (P < 0.05). In addition, overexpression of CX43 in BMSCs significantly increased the adhesion, proliferation and clone formation ability of KG-1a in co-culture system (P < 0.05). The inhibition of TNTs formation by vincristine significantly reduced the mitochondrial transfer from CX43-BMSCs to KG-1a, and decreased the adhesion of CX43-BMSCs to KG-1a, as well as the proliferation and clone formation of KG-1a after co-culture. Conclusion: Under the condition of nutrient deficiency, CX43 mediated the unidirectional transfer of mitochondria from BMSCs to LSCs by promoting the adhesion of LSCs to BMSCs and the formation of TNTs, and then enhanced the proliferation and clone formation ability of LSCs.