Use of zebrafish embryos as avatar of patients with pancreatic cancer: A new xenotransplantation model towards personalized medicine

Figure 1 Protocol used for the evaluation of the chemotherapy drugs efficacy. A piece of pancreatic ductal adenocarcinoma tumor tissue is injected into the yolk sac of zebrafish embryos 2 dpf. At 2 h post injection (hpi) the embryos are imaged and exposed to chemotherapy for 2 d. A and B: On the right panel a representative image of control embryos after 2 hpi and 2 dpi; C and D: On the right panel a representative image of treated embryos after 2 hpi and 2 dpi. Qualitatively images show the increased of fluorescent area in control group versus the regression in xenotransplanted embryos treated with chemotherapy.

Figure 2 Tumor tissue was successfully engrafted in healthy zebrafish embryos in all cases. A: Surgical specimen was obtained from patient with diagnosed pancreatic ductal adenocarcinoma (PDAC); B: Two fragments of the tumor were taken; C and D: One of them was used for histological evaluation (C) revealing that the percentage of epithelial cells (mean PDAC counterpart) out of the total surface area is 31.8% ± 4.9% (n = 3) (D); E: The second fragment of the tumor was used for xenotransplantation into the yolk of zebrafish embryos 2 dpf. After 2 d post xenotransplantation, the zebrafish patient-derived xenografts (zPDX) are Formalin-Fixed Paraffin Embedded. We performed the hematoxylin and eosin staining and immunohistochemistry using anti-Human Pan-Cytokeratin antibody on zPDX sections, highlighting the presence of epithelial PDAC cells (orange arrows) and the immune-negative counterpart (black arrows) that might be associated with the microenvironment side of PDAC human tissue. PDAC: Pancreatic ductal adenocarcinoma; PDX: Patient-derived xenografts; PanCK: Pan-Cytokeratin antibody.

Figure 3 Representative hematoxylin and eosin stained sections of zebrafish patient-derived xenografts. Visible morphologic alteration of the human pancreatic ductal adenocarcinoma nuclei (orange arrows) in zebrafish patient-derived xenografts exposed to Gemcitabine + nab-Paclitaxel and 5-Fluorouracil + Folinic acid + Oxaliplatin + Irinotecan could be associated with a chemotherapy damage. Control group shows normal nuclei (black arrows). GEM/nab-P: Gemcitabine + nab-Paclitaxel; FOLFOXIRI: 5-Fluorouracil + Folinic acid + Oxaliplatin + Irinotecan.

Figure 4 Cases with a statistically significant reduction of the mean relative tumor area for at least one chemotherapy scheme (the code below each diagram corresponds to the case number). Results are expressed as average ± SEM, ^aP < 0.05, 1-way ANOVA followed by Dunnett’s multiple comparisons test. Control group shows normal nuclei (black arrows). GEM: Gemcitabine; GEMOX: Gemcitabine + Oxaliplatin; GEM/nab-P: Gemcitabine + nab-Paclitaxel; FOLFOXIRI: 5-Fluorouracil + Folinic acid + Oxaliplatin + Irinotecan.

Figure 5 Percentage of cases with a percentage reduction of mean relative tumor area equal or greater to each threshold value. RTA: relative tumor area; Control group shows normal nuclei (black arrows). k: Control group; GEM: Gemcitabine; GEMOX: Gemcitabine + Oxaliplatin; GEM/nab-P: Gemcitabine + nab-Paclitaxel; FOLFOXIRI: 5-Fluorouracil + Folinic acid + Oxaliplatin + Irinotecan.