Figure 5.
Lama4-deficient MSCs promote AML cell proliferation and chemoresistance in vitro. (A) Experimental layout for assessing AML LSCs by cobblestone area-forming cell (CAFC) assay using Lama4+/+ and Lama4−/− BM MSCs. The expanded BM MSCs were seeded 24 hours before plating 150 to 300 MLL-AF9 AML cells. Cytarabine (Ara-C) was added to reach a final concentration of 0.5 μM 2 days after the initiation of the coculture to allow for maximal cell-cell adhesion and migration. The CAFCs were counted on day 7 and collected on day10 for FACS analysis. (B) Representative image of CAFCs from AML cells after coculture with the MSCs. (C) The number of CAFCs generated from the AML cells after coculture with the MSCs in the presence or absence of Ara-C (left) and the reduction of CAFCs after Ara-C treatment (right). The reduction was shown as a percent of the corresponding control cultures without Ara-C. (D) The total number of AML cells in the cultures with MSCs. (E) The number of AML LSCs (KIT+ cells) in the cocultures. (F) The number of chemoresistant AML LSCs (CD36+) in the cocultures. The data shown are from 3 independent experiments, and horizontal bars show median values. The P values shown in the panels were determined by an unpaired Student t test. (G) ROS levels were detected by FACS in the MSCs (left) and AML cells (right) on days 7 to 10 in the coculture. The data shown are fold changes in ROS MFI relative to that in the control culture with Lama4+/+ MSCs, and from 3 independent experiments. Each dot indicates the average value of triplicate measurement in each experiment. The P values shown in the panels were determined by an unpaired Student t test. (H) Experimental layout for testing the impact of LAMA4 on patient-derived AML cell proliferation and drug response using antibody inhibition. The monoclonal antibody against human LAMA4 was used to neutralize LAMA4 in human BM MSCs in the coculture with human AML cells (THP1). The antibodies were added 1 hour prior to seeding THP1 cells to achieve LAMA4 neutralization in the MSCs. Ara-C was added at 4 to 6 hours postseeding THP1 cells to allow efficient interactions of the cells with the MSCs. (I) Fold changes in THP1 cell counts after anti-LAMA4 antibody treatment (left) and in combination with Ara-C (right). (J) Experimental design for testing the impact of LAMA4 on human AML cell proliferation and drug response using LAMA4 knockdown strategy. LAMA4 knockdown in human MSCs was generated by lentiviral LAMA4 shRNA transduction. The selected LAMA4 knockdown MSCs were then used for coculture with THP1 cells with or without Ara-C. (K) LAMA4 mRNA expression was reduced in the human MSCs by lentiviral shRNA directed against LAMA4. (L) Fold changes in THP1 cell counts in the coculture with LAMA4 knockdown MSCs and the scrambled controls.

Lama4-deficient MSCs promote AML cell proliferation and chemoresistance in vitro. (A) Experimental layout for assessing AML LSCs by cobblestone area-forming cell (CAFC) assay using Lama4+/+ and Lama4−/− BM MSCs. The expanded BM MSCs were seeded 24 hours before plating 150 to 300 MLL-AF9 AML cells. Cytarabine (Ara-C) was added to reach a final concentration of 0.5 μM 2 days after the initiation of the coculture to allow for maximal cell-cell adhesion and migration. The CAFCs were counted on day 7 and collected on day10 for FACS analysis. (B) Representative image of CAFCs from AML cells after coculture with the MSCs. (C) The number of CAFCs generated from the AML cells after coculture with the MSCs in the presence or absence of Ara-C (left) and the reduction of CAFCs after Ara-C treatment (right). The reduction was shown as a percent of the corresponding control cultures without Ara-C. (D) The total number of AML cells in the cultures with MSCs. (E) The number of AML LSCs (KIT+ cells) in the cocultures. (F) The number of chemoresistant AML LSCs (CD36+) in the cocultures. The data shown are from 3 independent experiments, and horizontal bars show median values. The P values shown in the panels were determined by an unpaired Student t test. (G) ROS levels were detected by FACS in the MSCs (left) and AML cells (right) on days 7 to 10 in the coculture. The data shown are fold changes in ROS MFI relative to that in the control culture with Lama4+/+ MSCs, and from 3 independent experiments. Each dot indicates the average value of triplicate measurement in each experiment. The P values shown in the panels were determined by an unpaired Student t test. (H) Experimental layout for testing the impact of LAMA4 on patient-derived AML cell proliferation and drug response using antibody inhibition. The monoclonal antibody against human LAMA4 was used to neutralize LAMA4 in human BM MSCs in the coculture with human AML cells (THP1). The antibodies were added 1 hour prior to seeding THP1 cells to achieve LAMA4 neutralization in the MSCs. Ara-C was added at 4 to 6 hours postseeding THP1 cells to allow efficient interactions of the cells with the MSCs. (I) Fold changes in THP1 cell counts after anti-LAMA4 antibody treatment (left) and in combination with Ara-C (right). (J) Experimental design for testing the impact of LAMA4 on human AML cell proliferation and drug response using LAMA4 knockdown strategy. LAMA4 knockdown in human MSCs was generated by lentiviral LAMA4 shRNA transduction. The selected LAMA4 knockdown MSCs were then used for coculture with THP1 cells with or without Ara-C. (K) LAMA4 mRNA expression was reduced in the human MSCs by lentiviral shRNA directed against LAMA4. (L) Fold changes in THP1 cell counts in the coculture with LAMA4 knockdown MSCs and the scrambled controls.

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