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. 2022 Jan 10;71(10):2043–2068. doi: 10.1136/gutjnl-2021-324994

Figure 3.

Figure 3

Impairment of the EVNet hampers tumour growth in PDAC orthotopic, GEMM and PDX models. (A) Experimental layout: in order to impair cancer EVs secretion, a doxycycline-inducible (Tet-On) shRNA against Rab27A was transfected into MIA PaCa-2 cells, which were then orthotopically implanted into the pancreas of Rag2−/−Il2rg−/− mice. (B) Tumour growth curve measured by ultrasound and representative photos of MIA PaCa-2 Tet-On shRab27a tumours treated with doxycycline (n=8) and control (non-treated, n=7, two-way ANOVA; ***p<0.001). Scale bar: 10 mm. (C) Liver macrometastasis quantification and representative photos of livers of the MIA PaCa-2 Tet-On shRab27a model treated with doxycycline (n=8) and control (n=7) (Mann-Whitney test, *p<0.05). (D) Representative H&E staining of orthotopic MIA PaCa-2 Tet-On shRab27a tumours (left, with zoom inset) and liver metastasis (right, dashed line) treated with doxycycline and control. (E) Experimental outline of KPC mice treated with Nexinhib20 (20 mg/kg) or dimethyl sulfoxide (DMSO) (5%) at 16 weeks of age when tumours are mature and are sacrificed at humane endpoint. Treatments were administered two times per week by intraperitoneal injection. (F) Kaplan-Meier curve of the overall survival of KPC mice treated with Nexinhib20 (n=4) vs DMSO 5% (n=6) (log-rank Mantel-Cox test, *p=0.0217). (G) Experimental layout of mice injected orthotopically in the pancreas with PDX treated with Nexinhib20 (20 mg/kg) or DMSO (5%) two times per week. Treatment was started 7.5 weeks post-tumour implantation, and mice were sacrificed 4 weeks late, at 11.5 weeks post-tumour implantation. (H) Tumour growth curve of PDX pancreas orthotopic tumours measured by ultrasound treated with Nexinhib20 (n=6) or DMSO 5% (n=6) and representative photos of tumours at the time of euthanasia (two-way ANOVA; **p<0.01, ****p<0.0001). Arrows depict timepoints where treatment was started (7.5 weeks). Scale bar: 10 mm. Data are mean±SEM. ANOVA, analysis of variance; EVs, extracellular vesicles; EVNet, Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network; GEMM, genetically engineered mouse model; PDAC, pancreatic ductal adenocarcinoma; PDX, patient-derived xenograft; TRE, tetracycline response element.