Figure 2.
Mitochondrial transfer from MSCs to AML cells is induced by OxPhos inhibition. (A) OCI -AML3 and MOLM13 cells were stably transfected with mitochondria-targeted PDHA1-GFP, and MSCs were transfected with PDHA1-dsRed (upper left panel). Confocal microscopy images at ×40 magnification. Representative confocal images of dual fluorescence (GFP and dsRed)–positive OCI-AML3 cells cocultured with MSCs in the presence of 30 nM IACS-010759 for 72 hours (lower left panel; yellow arrow: GFP and dsRed dual-positive cell). To quantitatively determine the rate of mitochondrial transfer, OCI-AML3 cells cocultured with MSCs were separated from the MSC monolayer by careful pipetting, and the GFP and dsRed dual-positive recipient cells per 100 GFP+ cells (n > 5) were counted at ×40 magnification by live-cell imaging with confocal microscopy (right panel). Laser scanning was used to obtain images under a confocal microscope. (B) To compare direct-contact to noncontact conditions, OCI-AML cells were cocultured with PDHA1-dsRed–transfected MSCs in direct contact or separated by a transwell insert and treated with IACS-010759 (30 nM) for 72 hours. OCI-AML3 cells cultured in direct-contact conditions were separated from the MSC monolayer by careful pipetting. dsRed-positive OCI-AML3 cells per 100 OCI-AML3 cells (n > 5) were counted at ×40 magnification by live-cell imaging with confocal microscopy. (C) PDHA1-GFP–transfected OCI-AML3 cells were treated with the OxPhosi IACS-010759 (30 nM) for 72 hours in the presence or absence of PDHA1-dsRed transfected MSCs. The rate of MSC-derived mitochondrial transfer in OCI-AML3 cells that adhered to MSCs was determined by flow cytometric analysis after depletion of MSCs with MACS as described in supplemental Materials and Methods. Representative flow cytometry plots are shown on the right . (D) Representative confocal microscopy images show the formation of TNTs and protrusion formation in OCI-AML3 cells after treatment with IACS-010759 under MSC coculture conditions. The red arrows indicate the transfer of MSC-derived mitochondria along TNTs. Confocal microscopy images at ×63 magnification. (E) OCI-AML3 cells were treated with the OxPhosi IACS-010759 (30 nM) for 48 hours in the presence of MSCs. Representative electron microscopy images show the IACS-010759–treated OCI-AML3 cells migrating to cocultured MSCs with mitochondrial transport to the leading edge of a protrusion, which was not observed in untreated (control) cells. Red arrows indicate mitochondria. Error bars in the graphs show the means ± SDs of the results from 3 independent experiments. **P < .01. SSC, side scatter.

Mitochondrial transfer from MSCs to AML cells is induced by OxPhos inhibition. (A) OCI -AML3 and MOLM13 cells were stably transfected with mitochondria-targeted PDHA1-GFP, and MSCs were transfected with PDHA1-dsRed (upper left panel). Confocal microscopy images at ×40 magnification. Representative confocal images of dual fluorescence (GFP and dsRed)–positive OCI-AML3 cells cocultured with MSCs in the presence of 30 nM IACS-010759 for 72 hours (lower left panel; yellow arrow: GFP and dsRed dual-positive cell). To quantitatively determine the rate of mitochondrial transfer, OCI-AML3 cells cocultured with MSCs were separated from the MSC monolayer by careful pipetting, and the GFP and dsRed dual-positive recipient cells per 100 GFP+ cells (n > 5) were counted at ×40 magnification by live-cell imaging with confocal microscopy (right panel). Laser scanning was used to obtain images under a confocal microscope. (B) To compare direct-contact to noncontact conditions, OCI-AML cells were cocultured with PDHA1-dsRed–transfected MSCs in direct contact or separated by a transwell insert and treated with IACS-010759 (30 nM) for 72 hours. OCI-AML3 cells cultured in direct-contact conditions were separated from the MSC monolayer by careful pipetting. dsRed-positive OCI-AML3 cells per 100 OCI-AML3 cells (n > 5) were counted at ×40 magnification by live-cell imaging with confocal microscopy. (C) PDHA1-GFP–transfected OCI-AML3 cells were treated with the OxPhosi IACS-010759 (30 nM) for 72 hours in the presence or absence of PDHA1-dsRed transfected MSCs. The rate of MSC-derived mitochondrial transfer in OCI-AML3 cells that adhered to MSCs was determined by flow cytometric analysis after depletion of MSCs with MACS as described in supplemental Materials and Methods. Representative flow cytometry plots are shown on the right . (D) Representative confocal microscopy images show the formation of TNTs and protrusion formation in OCI-AML3 cells after treatment with IACS-010759 under MSC coculture conditions. The red arrows indicate the transfer of MSC-derived mitochondria along TNTs. Confocal microscopy images at ×63 magnification. (E) OCI-AML3 cells were treated with the OxPhosi IACS-010759 (30 nM) for 48 hours in the presence of MSCs. Representative electron microscopy images show the IACS-010759–treated OCI-AML3 cells migrating to cocultured MSCs with mitochondrial transport to the leading edge of a protrusion, which was not observed in untreated (control) cells. Red arrows indicate mitochondria. Error bars in the graphs show the means ± SDs of the results from 3 independent experiments. **P < .01. SSC, side scatter.

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