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Fig. 3 | BMC Cancer

Fig. 3

From: Humanized anti-DEspR IgG4S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis ratnu/nu model

Fig. 3

Humanized anti-DEspR mAb exhibits improved potency, retains DEspR/mAb internalization and nuclear translocation. a 3D-model of humanized anti-DEspR [Left] hu-6g8, [Right] mu-6g8: complementary determining regions (navy), heavy-chain (aqua), light-chain (pink). b-d Comparison of hu-6g8 and mu-6g8 b binding-affinity to intact DEspR on Panc1-TCs, c inhibition of Panc1-CSCs, and d HUVECs angiogenesis (EC50, IC50 values: Table S2). e Representative images of hu-6g8 and mu-6g8 angiogenesis inhibition, showing concentration-dependent decreased HUVEC tube-formation. f Confocal immunofluorescence of hu-6g8/DEspR internalization and nuclear-translocation (t = 15-min to 2-h) in Panc1 and MiaPaCa2 TCs. Bar = 15 μm. g Live-cell imaging of apoptotic changes in hu-6g8-treated Panc1-TCs. Bar = 20 μm. h Higher-magnification live-cell images of hu-6g8-treated Panc1 TCs showing normal, necroptotic, and apoptotic cell morphology. Bar = 10 μm. i, j Quantitative analysis of apoptotic (apop) and necroptotic (necrop) morphological changes in hu-6g8-treated. i Panc1-TCs (n = 208, p < 0.0001) and j MiaPaCa2 (n = 284, p < 0.0001) vs. isotype-control at 2 h; chi-squared test for independence, paired t-test. k, l Internalization of hu-6g8/DEspR complexes detected by AF568-labeled anti-human-IgG mAb (red), and colocalization with cytoplasmic-nuclear shuttling proteins: galectin-1 (gal1[aqua]) or galectin-3 (gal3[green]) in k Panc1 and l MiaPaCa2 TCs, after 15-min, and 4-h of hu-6g8-treatment. Bar = 20 μm. m, n Colocalization of DEspR/gal1(aqua) or DEspR/gal3(green) (Mander’s coefficient κ > 0.5) in Panc1 and MiaPaCa2 TCs in the cytoplasm (m) or nucleus (n), at 15-min and 4-h (Table S3). (*p < 0.05, n = 261 and 251 TCs, Panc1 and MiaPaCa2, respectively, paired two-tail t-test)

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