CXCL12/CXCR4 Axis Drives Mitochondrial Trafficking in Tumor Myeloma Microenvironment
Authors: Giallongo Cesarina , Dulcamare Ilaria , Tibullo Daniele , Del Fabro Vittorio , Vicario Nunzio , Parrinello Nunziatina Laura , Romano Alessandra , Scandura Grazia , Barbato Alessandro , La Spina Enrico , Cambria Daniela , Lazzarino Giacomo , Conticello Concetta , Li Volti Giovanni , Musumeci Giuseppe , Palumbo Giuseppe A. , Di Raimondo Francesco 
Published: November 2021

Abstract
Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) also transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. As metabolic rewiring is involved in the regulation of MSC phenotype, we first analyzed metabolic profile of healthy control (HC-) and MM-MSCs. NAD +/NADH ratio was decreased in MM-MSCs (n=8) as compared with HC-MSCs (n=4, p&lt;0.05), meanwhile ATP/ADP ratio was not significantly different between the two groups. This led us to analyze whether MM-MSCs were much prone in transferring mitochondria than HC-MSCs. We first labeled HC- and MM-MSCs with Mitotracker Red CMXRos before co-culture with MM cells. After 24h of coculture, we quantified mitochondria transfer by flow cytometry. The obtained values were significantly higher in MM cells co-cultured with MM-MSCs (n=10) as compared to PCs co-cultured with HC-MSCs (n=5, p&lt;0.01).
In the cell-to-cell contact the gap junction-forming protein CX43 has been found critical for mitochondria uptake in lung and brain injury and it also can regulate CXCL12 secretion by MSCs. We found that MM-MSCs showed a significantly up-regulated CXCL12 expression as compared to HC-MSCs (p&lt;0.001). Therefore, we co-cultured HS-5 cells with myeloma cell lines and observed that significantly increased CXCL12-CX43 colocalization in healthy MSCs. To evaluate the selective PC-induced activation of CXCL12 expression via CX43 in MSCs, we co-cultured HS-5 cells with MM cell lines and exposed cocultures to ioxynil octanoate (IO), a selective inhibitor of CX43-based gap junctions. We found that the up-regulation of CXCL12 induced by MM cells was reverted by exposition to the CX43 inhibitor, thereby indicating that CX43 activated by PCs regulates CXCL12 production in MSCs. Given that CX43 is involved in mitochondria trafficking, we subsequently cocultured MM cells with HS-5 in presence or not of IO. Our data showed that mitochondrial transfer was abolished by CX43 inhibitor. Given that MM PCs induced increased CX43 and CXCL12 colocalization in HS-5 cells, we supposed that CXCL12/CXCR4 signaling could regulate mitochondria trafficking throughout this axis. For this reason, we analyzed the kinetic of mitochondria uptake of several HMCLs and related their CXCR4 expression with the percentage of transferred mitochondria. Our data demonstrated that HMCLs with higher expression of CXCR4 had also higher percentage of transferred mitochondria both in time lapse and flow cytometry. The correlation between CXCR4 expression and the percentage of mitochondria uptake in HMCLs was also confirmed in primary myeloma PCs. Furthermore, plerixafor, a selective inhibitor of CXCR4, significantly reduced mitochondrial transfer from MSCs to myeloma PCs further establishing mechanistically that CXCR4/CXCL12 is directly involved in mitochondrial trafficking. Next, we investigated whether combination of plerixafor with bortezomib or carfilzomib interferes with mitochondrial transfer from MSCs to PCs. Interestingly, we found that the proteasome inhibitors promoted mitochondrial transfer while their combination with plerixafor inhibited mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM.
In conclusion, we have shown that MM-MSCs are relatively low dependent on mitochondria metabolism and are inclined to transfer mitochondria to MM tumor cells. Furthermore, tumor PCs increase the expression of CX43 in MSCs leading to an increased levels of CXCL12 and stimulation of its corresponding receptor expressed on MM cells. The resulting CX43/CXCL12/CXCR4 interplay enhances mitochondrial trafficking from MSCs to myeloma PCs and can protect cancer cells against anti-myeloma agents. Understanding pro-tumorigenic phenotype of MSCs and mechanisms of adhesion and heterocellular communication favoring their interaction with cancer PCs, will allow to manipulate critical pathways, including CXCL12/CXCR4 axis, thus improving disease outcome.

Disclosures
Di Raimondo: Pfizer: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Jazz Pharmaceutical: Honoraria; Janssen Pharmaceuticals: Honoraria; Amgen: Honoraria.