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Fig. 1 | BMC Cancer

Fig. 1

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

Fig. 1

DEspR modulates survival and functionality of Panc1-CSCs, cscTCs, and TCs. a DEspR-protein diagram showing epitope-1 and epitope-2 with corresponding anti-DEspR mAbs 7c5, 5g12, 6g8, hu-6g8; consensus sequences for glycosylation (green), internalization recognition sequence (navy), O-glycosylation (purple), and phosphorylation (yellow). b Representative images showing internalization of fluorescent AF568-labeled 7c5 and AF568-IgG4 isotype-control in Panc1-TCs after 1- and 2-h, and associated apoptotic (Apop.) and necroptotic (Necrop.) cell morphology changes. Chi-square test for independence, two-tailed t-test p<0.0001; 7c5-treated n=280 cells, isotype-treated n=242 cells. c Flow-cytometry showing [Top]: DEspR cell-surface expression on Panc1-TCs (65–75%) and Panc1-CSCs (60–80%) [dashed-line isotype vs. red AF-568-7c5]; [Middle]: ALDEFLUOR activity/ expression in Panc1-CSCs (68–72%) [dashed-line DEAB control vs green ALDEFLUOR] and ALDEFLUOR expression of DEspR+ Panc1-CSCs (58.2–60.4%); [Bottom]: CD133 expression on Panc1 CSCs (28–34%) [dashed-line isotype vs. green AF-488-CD133] and DEspR+/CD133+ CSCs (24.1%). d Flow-cytometry showing DEspR cell-surface expression on MiaPaca2-TCs (58–65%) and Capan-1-TCs (31–40%) [dashed-line isotype vs. green AF-488-7c5]. e Panc1-CSC tumorspheres (Bar: 100 μm) with staining of DEspR+ Panc1-CSCs from dispersed tumorspheroids. After 30-min 7c5-AF568 binding, then MoFlo-sorting and plating in low-adherence cultures, phase contrast image shows viable DEspR[−] CSCs (7c5-Tx DEspR[−]) but no spheroid-formation; in contrast to minimal to non-thriving 7c5-sorted/inhibited DEspR+ CSCs (7c5-Tx DEspR+). f Panc1-cscTCs co-express αSMA (red) and Col1A1(green), merged (yellow) (top bar: 20 μm, bottom bar: 50 μm). g Fluorescent microscopy comparing αSMA (red) expression in control (top) vs. 6g8-treated (bottom) cscTCs. Anti-DEspR- reduced αSMA-expression in Panc1-cscTCs (14.4 ± 4.3%); non-treated controls (75.2 ± 13.7%); 6g8: n = 7 high-power-fields (HPFs); control: n = 8 HPFs, ≥40 cells/HPF, p < 0.0001 two-tailed t-test. h Anti-DEspR mAbs reduced Panc1-CSC Col1A1- secretion [6g8-treated: 97 ± 16 pg/ml, 7c5-treated: 54.5 ± 2.5 pg/ml, control: 487 ± 52 pg/ml]; n = 4 replicate wells/group, ***p < 0.001, ANOVA multiple-comparisons test. i Representative double-immunofluorescence of Panc1-CDX PPC tumor: human-specific DEspR+(red), human/rat Col1A1 + (green) cscTCs within TC-islands, DEspR+Col1A1+ co-expressing cscTCs (yellow), and DEspR[−]/Col1A1+ cscTCs and stromal cells (green) in merged panel. Representative Masson-Trichrome (MT)-stained [LEFT, MIDDLE] and H&E-stained [RIGHT] sections of PPC tumors, showing collagen-deposition (MT:blue, H&E:bright pink) surrounding cscTCs. Bar: 50 μm

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