Lama4^−/− niche confers AML cell chemoresistance to Ara-C via modulating ROS levels associated with increased mitochondrial transfer from MSCs to AML cells. (A) AML onset and survival of Lama4^+/+ and Lama4^−/− mice following injection of DsRed-expressing MLL-AF9⁺ cells. The mice were irradiated with 6 Gy prior to the AML cell injection. The AML engraftment in PB was analyzed by FACS 14 days after AML cell injection. The mice were then injected with normal saline (NS) or Ara-C for 5 days starting at day 15. (B) Spleen size in Lama4^+/+ and Lama4^−/− mice 1 to 3 days post-Ara-C or NS treatment. (C) The frequency of residual total AML cells (left) and enrichment of chemoresistant CD36⁺ AML cells (right) in Lama4^+/+ and Lama4^−/− mouse BM at 1 day after the last injection of Ara-C. The percent of CD36⁺ cells were presented as fold changes relative to the average percent of the cells in NS-treated mice. (D) ROS levels in the AML cells from Lama4^+/+- and Lama4^−/−-recipient PB at 14 days post-AML cell injection. The left panel shows MFI of ROS in the AML cells, and the right panel is representative FACS histograms of ROS levels. (E) ROS levels in the AML cells from Lama4^+/+- and Lama4^−/−-recipient BM at 1-day post-Ara-C treatment. The data shown are the MFIs of ROS (left) and fold changes (right) in ROS levels in the AML cells post-Ara-C treatment relative to the mean values in NS-treated mice in each experiment. (F) Experimental setup for testing mitochondrial transfer from MSCs to AML cells. The MLL-AF9⁺ AML cells were cocultured with Lama4^+/+ and Lama4^−/− MSCs prelabeled with MitoTracker green. The MSC-derived mitochondria were analyzed for the MFI of MitoTracker in AML cells at 24 hours postcoculture by FACS and confocal microscopy. (G) Simultaneous detection of ROS, mitochondrial mass (Mitotracker⁺), and mitochondrial membrane potential (MMP) Tetramethylrhodamine methyl ester perchlorate (TMRM)⁺ in Lama4^+/+ and Lama4^−/− MSCs prior to the cocultures. (H) Representative FACS histograms showing MFIs of MitoTracker, TMRM, and ROS in the MSCs prior to the cocultures. (I) Increased mitochondrial mass in Lama4^−/− MSCs cocultured with AML cells. Data shown are fold changes in MitoTracker MFI in Lama4^−/− MSCs compared with that in Lama4^+/+ MSCs. (J) Mitochondrial transfer from BM MSCs to AML cells in the cocultures with and without Ara-C treatment. Data shown are relative MitoTracker MFI in the AML cells cocultured with Lama4^−/− MSCs to that with Lama4^+/+ MSCs. (K) Representative FACS profiles showing mitoTracker⁺ AML cells 24 hours postcoculture with Lama4^+/+ and Lama4^−/− MSCs. (L) Confocal images showing MSC-derived mitochondria (green) in the AML cells at 24 hours postcoculture. The data shown are from 2 to 4 independent experiments, and horizontal bars show median values. The P values shown in the panels were determined by 2-tailed or 1-tailed unpaired Student t test. See also in supplemental Figure 10-11.