Figure 3.

Idelalisib reduces FDC-induced angiogenesis and TEM in sensitive patients. FL-FDC coculture supernatants with or without idelalisib (IDELA, 500 nM, 48 hours) were used to determine VEGF-A and VEGF-C protein secretion by ELISA in sensitive (n = 6) and resistant (n = 6) patients (A) and tube formation assay of endothelial HUVEC cells cultured for 24 hours with their own media alone or mixed with the corresponding supernatants (ratio 1:1) (B) (magnification ×40). Five representative images of each condition were captured using a phase-contrast microscope and analyzed by FIJI-ImageJ (angiogenesis analyzer plug-in). Representative images from a sensitive patient are shown. Node and junction numbers from sensitive (n = 5) and resistant (n = 5) patients are displayed. (C) Heatmap displaying the regulation induced by idelalisib (IDELA) in the expression of integrins and their ligands in FL cells from FL-FDC cocultures of sensitive (FL1 and FL4) and resistant patient samples (FL2 and FL3). (D) After IDELA treatment (500 nM, 48 hours) FL cells from FL-FDC cocultures with or without idelalisib (500 nM, 48 hours) of sensitive and resistant patients (n = 8) were stained with calcein and allowed to adhere for 3 hours to HUVECs. After extensive washing, the cells that remained attached were lysed, and fluorescence was measured in a Synergy HT microplate reader. (E) FL cells (n = 12) from FL-FDC cocultures with or without idelalisib (500 nM, 48 hours) were challenged to migrate for 6 hours in a gradient of FBS through trans-wells coated with HUVECs seeded on gelatin 0.1% coated + TNF-α (10 ng/mL). CD20⁺ cells crossing the HUVEC barrier were counted by flow cytometry. *P < .05, **P < .01. ns, not significant.