Fig. 6: MIF inhibition attenuates extramedullary leukemia progression and affects tumor vascularization in vivo.

A Schematic representation of the experimental setup, created with BioRender. B, C Subcutaneous U937.GFP-FFLuc leukemia cell tumor growth progression in NSG mice, day 0 (cell inoculation) until day 19 (end of the experiment); black vertical arrow indicates the start of mouse treatments with vehicle (veh.), GM-CSF (GM, 3000 U/kg), 4-IPP (IPP, 80 mg/kg), and GM-CSF + 4-IPP (GM/IPP, administered at 3000 U/kg and 80 mg/kg body weight, respectively) on day 9 of tumor growth. A Curves are the mean tumor volumes ± SEM (n = 4–5 mice per group) measured by calipers. C Curves are the mean total photons flux ± SEM (n = 4–5 mice per group) measured by bioluminescence; images show radiance at the same scale of all the tumor-bearing mice from the 4 treatment groups at the end of the experiment on day 19. ****p < 0.0001 compared to the vehicle on day 19 (B, C); n = 4–5 (D). Representative whole tumor image mosaics from intravital imaging by multiphoton microscopy of mice from each treatment group show U937-GFP leukemia cells (green), dextran-labeled blood vessels, and positive cells (red), and second harmonic generation imaging identified collagen I fibers (cyan); scale bars = 500 µm. E A representative illustration of image analysis approach from IVI-MP of a tumor mosaic from a GM-CSF + 4-IPP treated mouse, same tumor as in (D); first panel shows 3-D surface rendering in the red/dextran channel, second panel shows size classification and third (right) panel shows the separation of tumor blood vessels (purple) and phagocytic macrophages (blue); scale bars = 500 µm. F, G Quantification of the numbers (F) and lengths (G) of blood vessels normalized by tumor areas; bars are means ± SEM (images analyzed from ≥3 mice per group). ****p < 0.0001.