AML-MSCs characterization in 3D model. (A) Scheme of the in vitro 3D culture procedure. Hydroxyapatite/collagen scaffolds were seeded with stromal cells and cultured for 7 days before the addition of primary AML cells (t = 0). Differential analysis was conducted at days 7, 14, and 21. (B) Representative z projections of AML-MSCs and primary AML cells cocultured in a 3D scaffold at different time points stained with Hoechst (blue), Calcein-AM (green), and ECD-CD45 (red). The z projection is the sum of intensity fluorescence of 100 µm z stack, ×20 magnification; scale bar, 100 µm. Graph shows the ratio AML cells per volume in the 3D scaffold, which is representative of AML cell viability, in the presence of stromal cells (+AML-MSCs) or not (stroma free [SF]), at different time points (n = 4). (C) Representative image of heterotypic nanotubes (arrowheads) that interconnected leukemia cells (red) and AML-MSCs (green) during coculture in 2D (left) and 3D (right). The gray signal in 3D corresponds to the second harmonic generation from collagen and hydroxyapatite of the scaffold. AML cells membrane were labeled with Vybrant-DiI directly before seeding. Wider area is reported in supplemental Movie 2. Scale bar, 20 µm; n = 3. (D) Calcein-AM dye transfer assay showing that calcein-AM dye (green) was transferred to contacting AML cells in 2D coculture, whereas it was absent in distant AML cells (red); n = 3; scale bar, 20 µm. (E) Immunofluorescence staining of AML cells contacting MSCs (red) in 2D coculture. Contact area between arrowheads showed enrichment in CX43 Alexa 488 signal (green); scale bar, 20 µm. The graph shows the corresponding fluorescence intensity profile. **P < .01, ***P < .001, ****P < .0001.