M2-like Macrophages Transfer Mitochondria to Acute Myeloid Leukaemia Cells Via Tunnelling Nanotubes Promoting Therapy Resistance

Introduction: The bone marrow microenvironment actively drives therapy resistance in AML. Leukaemic cells interact with bone marrow (BM) resident stromal cells and immune cells, such as macrophages (Mφs). M2-like Mφs are elevated in the BM of AML patients, and transfer their mitochondria to primary AML cells enhancing AML cell proliferation (Weinhäuser et al. Science Advances 2022). Mitochondrial transfer from BM stromal cells (BMSCs) to AML cells is increased by cytarabine (Ara-C), with transfer driving BMSC-mediated protection from ara-C-induced apoptosis (Moschoi et al. Blood 2016). We have previously shown that M2-like Mφs, via cell-to-cell contact, protect AML cells from therapy-induced apoptosis (Miari et al. HemaSphere 2023). However, it is unknown if mitochondrial transfer from Mφs to AML cells drives therapy resistance in AML.

Aims: First, we determined if mitochondrial transfer from M2-like Mφs to AML cells is enhanced by the standard chemotherapeutics daunorubicin (DNR) and Ara-C. We further investigated whether blocking tunnelling nanotube (TNT)-mediated mitochondrial transfer re-sensitised AML cells to DNR-induced apoptosis. Finally, we examined protein expression in AML cells cultured on and off Mφs in order to identify proteins potentially involved in mitochondrial transfer and subsequently therapy resistance.

Methods: Healthy blood donor CD14⁺ monocyte derived-Mφs and iPSC-derived Mφs were generated, with mature Mφs polarised into M2-like Mφs with 72hrs of treatment with 100 ng/mL of M-CSF. Mitochondrial transfer was assessed in CFSE-stained AML cell lines (U937, THP-1 and KG1a) and primary cells after co-culture with M2-like Mφs in the presence of DNR (0.25 µM, 0.125 µM and 3 µM respectively) or DNR and Ara-C (2.5µM) combination therapy, or vehicle control. Co-culture experiments were also performed in the presence of cytochalasin B (1µM) to block TNT formation. Mitochondrial transfer and cellular apoptosis were then assessed following 24-72h treatment, via flow cytometric analysis of MitoTracker Deep Red (MTDR) staining for mitochondrial content and Annexin V/eFluor 450 staining for AML cell viability. Additionally, total levels of reactive oxygen species (ROS) were evaluated in U937 cells under normal and DNR-induced stress conditions via flow cytometry for the ROS probe CellROX Deep Red. Moreover, tandem mass tag mass spectroscopy (TMT-MS) was utilised to assess differentially expressed proteins in U937 cells cultured on and off Mφs. Immunoblotting was performed to assess levels of phosphorylated STAT3.

Results: Both DNR treatment and the DNR and Ara-C combination enhanced the capacity of monocyte and iPSC-derived M2-like Mφs to transfer mitochondria to AML cells. Functionally, AML cells in co-culture exhibited lower ROS generation at steady state and under DNR-induced conditions versus AML cells in monoculture. Either the treatment of AML cells with cytochalasin B or physical separation of AML cells from Mφs, via transwell inserts both abolished DNR-enhanced mitochondrial transfer, highlighting that mitochondrial transfer likely occurs via TNTs and requires cell-to-cell contact. Importantly, cytochalasin B treatment re-sensitised AML cells to DNR-induced apoptosis. TMT-MS results show that Ras homolog gene family member C (RhoC) was upregulated in U937 cells co-cultured with Mφs versus monocultures. Immunoblot analysis revealed an increase in phosphorylated-STAT3^tyr705 (downstream target of RhoC) in U937-Mφ co-cultures versus monocultures. This is interesting as activated STAT3 has been implicated in TNT formation in Mφs (Souriant et al. Cell Reports 2019).

Conclusions: These findings demonstrate for the first time that M2-like Mφs protect AML cells from the killing effects of standard of care AML therapeutics by transferring their mitochondria, via TNTs, and that by blocking mitochondrial transfer AML cells can be re-sensitised to DNR-induced apoptosis. These findings provide a rationale for further studies investigating blockade of TNT formation and/or RhoC/STAT3, as a strategy to circumvent M2-like Mφ-driven therapy resistance in AML.