A Model of a Zebrafish Avatar for Co-Clinical Trials

Alice Usai, Gregorio Di Franco, Patrizia Colucci, Luca Emanuele Pollina, Enrico Vasile, Niccola Funel, Matteo Palmeri, Luciana Dente, Alfredo Falcone, Luca Morelli, Vittoria Raffa, Alice Usai, Gregorio Di Franco, Patrizia Colucci, Luca Emanuele Pollina, Enrico Vasile, Niccola Funel, Matteo Palmeri, Luciana Dente, Alfredo Falcone, Luca Morelli, Vittoria Raffa

Abstract

Animal "avatars" and co-clinical trials are being developed for possible use in personalized medicine in oncology. In a co-clinical trial, the cancer cells of the patient's tumor are xenotransplanted into the animal avatar for drug efficacy studies, and the data collected in the animal trial are used to plan the best drug treatment in the patient trial. Zebrafish have recently been proposed for implementing avatar models, however the lack of a general criterion for the chemotherapy dose conversion from humans to fish is a limitation in terms of conducting co-clinical trials. Here, we validate a simple, reliant and cost-effective avatar model based on the use of zebrafish embryos. By crossing data from safety and efficacy studies, we found a basic formula for estimating the equivalent dose for use in co-clinical trials which we validated in a clinical study enrolling 24 adult patients with solid cancers (XenoZ, NCT03668418).

Keywords: chemosensitivity; equivalent dose; patient-derived xenograft; translational research; zebrafish avatar.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemotherapy safety study. Zebrafish 2 days post fertilization (dpf) embryos were incubated with media (E3 supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin) modified with chemotherapy drugs or not modified at 35 °C for 3 days. At the end of the treatment, the percentages of dead embryos (red), aberrant (blue) and normal phenotype (green) were evaluated after fixation and stereomicroscope observation. In GEM, GEMOX, GEM/nab-P, GEMCIS, we report the Gemcitabine concentration in the x-axis (the concentrations of the other drugs are omitted but are provided in Table S1). In 5-FU, FOLFOX, FOLFIRI, FLOT, FOLFOXIRI, ECF, we report the 5-Fluorouracil concentration in the x-axis (the concentrations of the other drugs are omitted but are provided in Table S1). The control group showed a 10% alteration from the normal phenotype. For each chemotherapy regimen, a dose-response and the relative linear regression analysis of the normal phenotype (green line) and the dead embryos (red line) are shown. The resulting R square is reported. The results presented are a pool from three independent biological replicates (n = 90).
Figure 2
Figure 2
Estimation of the maximum tolerated dose (MTD). Box plot displaying equivalent plasma concentration EPC/IC50 and EPC/LD25 ratios for all chemotherapy protocols.
Figure 3
Figure 3
Efficacy analysis. Evaluation of the effects of chemotherapy on cancer cell lines (HCT 116, MIA PaCa-2) xenotransplanted in 2 dpf zebrafish embryos. Each embryo was imaged at 2 hpi, 1 dpi, 2 dpi and the relative area is the Dil-stained area normalized with respect to the 2 hpi time point. (A) Chemosensitivity of HCT 116 xenografts, d.f. = 8. A statistically significant increase in relative area was observed in all groups. (B) Chemosensitivity of HCT 116 xenografts, d.f. = 5. A statistically significant increase in the relative area was observed in the control, 5-FU, FOLFOX and FOLFIRI but not in FOLFOXIRI. (C) Chemosensitivity of MIA PaCa-2 xenografts, d.f. = 5. A statistically significant increase in relative area was observed in the control, GEMOX and FOLFOXIRI treatments but not in GEM and GEM/nab-P. Data are mean ± SEM and are representative of three independent assays. N = 15 (embryos), 2-way ANOVA followed by Bonferroni correction (all groups compared against control group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 4
Figure 4
Cancer cell engraftment into zebrafish embryos 2 days after injection. (A1,A2) HCT 116 cancer cell line DiI labeled (red) injected into the yolk sac (circled) of zebrafish embryo 2 dpf. (A3) Quantification of pyknotic nuclei by HCT 116 cancer cells xenotransplanted after two days of FOLFOXIRI treatment vs. control. Data are mean ± SEM, t-test, n > 15 * p < 0.05. Sections from HCT 116 cancer cells xenotransplanted in a zebrafish embryo treated with FOLFOXIRI (B1) vs. control (B2). Stars: cells with pyknotic nuclei. Scale bar: 50 µm.
Figure 5
Figure 5
Patient-derived tumor xenografts. (A1) The fragment of a pancreatic cancer tissue (patient P053) injected into the yolk sac survives and invades the yolk sac. DiI (red) cell membrane staining, Hoechst (blue) nuclear counterstain at 2 dpi. (A2) Higher magnification of the dashed rectangle of panel B1 (Hoechst nuclear staining). Stars show pyknotic nuclei. Scale bar 20 µm.
Figure 6
Figure 6
Anti-Human IgG immunohistochemistry (green). (A) HCT 116 cancer cell lines two days post injection into the zebrafish yolk. Patient’s pancreatic tumor before (B) and after xenotransplantation, 2 dpi (C). (D1,D2) Patient’s colon cancer cells spread throughout the vasculature reaching the zebrafish tail. Human cells are labeled with DiI (red) and anti-IgG (green). D1 is a magnification (90° rotation) of D2. Scale bar 50 μm.
Figure 7
Figure 7
A representative embryo xenotransplanted with a fresh tumor specimen of gastric cancer (patient S013). Bright-field images of the grafted embryo (A1–C1), epi-fluorescence images (A2–C2) and overlay (A3–C3), showing the region of interest (ROI; yellow line). All images are oriented so that the rostral end is on the left and dorsal end is on the top.
Figure 8
Figure 8
Quantitative analysis of six cases of patient-derived tumor xenografts. Dil-stained area at time points 2 hpi, 1 dpi and 2 dpi were normalized with respect to the time point 2 hpi. Patient enrollment codes are reported (C=Colon, P=Pancreas, S=Stomach), and the number of embryos analyzed for each case study is indicated in the image. Results are expressed as mean ± SEM. C020 (p = 0.04), C022 (p = 0.05), P023 (p = 0.003), S027 (p = 0.02), S042 (p = 0.02), P085 (p = 0.06) by 1-way repeated measures ANOVA.
Figure 9
Figure 9
Percentage of partial response (PR) and complete response (CR). FOLFOXIRI, FOLFIRI, FOLFOX and 5-FU treatments in zebrafish avatars xenotransplanted with colon tumor (n = 8 patient samples analyzed) (A); GEMOX, GEM/nab-P, GEM, FOLFOXIRI treatments in zebrafish avatars xenotransplanted with pancreas tumor (n = 12 patient samples analyzed) (B); ECF, FLOT, FOLFIRI and FOLFOX treatments in zebrafish avatars xenotransplanted with gastric tumor (n = 4 patient samples analyzed) (C).
Figure 10
Figure 10
Chemosensitivity assay. Two dpf embryos were injected with fragments of patient tumor tissue and incubated for 48 h with chemotherapy compounds at the ED. Representative cases of colon cancer (C024 and C031 patient-derived xenograft (PDX)) and pancreatic cancer (P025 and P030 PDX) with quantitative analysis of the relative tumor area (2 dpi/2 hpi for colon and 2 dpi/1 dpi for pancreas). All graphs show an increase in the stained area over time in the control group. C024, P025, P030 show a statistically significant regression of the stained area size in the FOLFOXIRI treated group compared with control non-treated xenografts. C031 shows significant stained area reduction in 5-FU and FOLFIRI treated groups compared with control non-treated xenografts. Results are expressed as mean ± SEM and analyzed by 1-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05. C024: n = 5, 7, 8, 9, 5 and C031: n = 8, 7, 5, 5, 4, respectively for control, 5-FU, FOLFOX, FOLFIRI, FOLFOXIRI. P025: n = 9, 9, 8, 9, 7 and P030: n = 5, 5, 9, 3 respectively for control, FOLFOXIRI, GEM, GEMOX, GEM/nab-P.

References

    1. Millner L.M., Strotman L.N. The Future of Precision Medicine in Oncology. Clin. Lab. Med. 2016;36:557–573. doi: 10.1016/j.cll.2016.05.003.
    1. Hidalgo M., Amant F., Biankin A.V., Budinská E., Byrne A.T., Caldas C., Clarke R., De Jong S., Jonkers J., Mælandsmo G.M., et al. Patient-Derived Xenograft Models: An Emerging Platform for Translational Cancer Research. Cancer Discov. 2014;4:998–1013. doi: 10.1158/-14-0001.
    1. Clohessy J.G., Pandolfi P.P. Mouse hospital and co-clinical trial project—From bench to bedside. Nat. Rev. Clin. Oncol. 2015;12:491–498. doi: 10.1038/nrclinonc.2015.62.
    1. Malaney P., Nicosia S.V., Davé V. One mouse, one patient paradigm: New avatars of personalized cancer therapy. Cancer Lett. 2013;344:1–12. doi: 10.1016/j.canlet.2013.10.010.
    1. Schmidt C. New mouse models mimic biology of human cancer. J. Natl. Cancer Inst. 2015:107. doi: 10.1093/jnci/djv024.
    1. Tentler J.J., Tan A.-C., Weekes C.D., Jimeno A., Leong S., Pitts T.M., Arcaroli J.J., Messersmith W.A., Eckhardt S.G. Patient-derived tumour xenografts as models for oncology drug development. Nat. Rev. Clin. Oncol. 2012;9:338–350. doi: 10.1038/nrclinonc.2012.61.
    1. Teng Y., Xie X., Walker S., White D.T., Mumm J., Cowell J.K. Evaluating human cancer cell metastasis in zebrafish. BMC Cancer. 2013;13:453. doi: 10.1186/1471-2407-13-453.
    1. Giannaccini M., Cuschieri A., Dente L., Raffa V. Non-mammalian vertebrate embryos as models in nanomedicine. Nanomed. Nanotechnol. Boil. Med. 2014;10:703–719. doi: 10.1016/j.nano.2013.09.010.
    1. Lee L.M., Seftor E.A., Bonde G., Cornell R.A., Hendrix M.J. The fate of human malignant melanoma cells transplanted into zebrafish embryos: Assessment of migration and cell division in the absence of tumor formation. Dev. Dyn. 2005;233:1560–1570. doi: 10.1002/dvdy.20471.
    1. Jackson S.J., Thomas G.J. Human tissue models in cancer research: Looking beyond the mouse. Dis. Model. Mech. 2017;10:939–942. doi: 10.1242/dmm.031260.
    1. Barriuso J., Nagaraju R., Hurlstone A. Zebrafish: A new companion for translational research in oncology. Clin. Cancer Res. 2015;21:969–975. doi: 10.1158/1078-0432.CCR-14-2921.
    1. Marques I.J., Weiss F.-U., Vlecken D.H., Nitsche C., Bakkers J., Lagendijk A.K., Partecke L.I., Heidecke C.-D., Lerch M.M., Bagowski C.P. Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model. BMC Cancer. 2009;9:128. doi: 10.1186/1471-2407-9-128.
    1. Haldi M., Ton C., Seng W.L., McGrath P. Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish. Angiogenesis. 2006;9:139–151. doi: 10.1007/s10456-006-9040-2.
    1. Fior R., Póvoa V., Mendes R.V., Carvalho T., Gomes A., Figueiredo N., Ferreira M.G. Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts. Proc. Natl. Acad. Sci. USA. 2017;114:E8234–E8243. doi: 10.1073/pnas.1618389114.
    1. Nair A.B., Jacob S. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm. 2016;7:27–31. doi: 10.4103/0976-0105.177703.
    1. Braekeveldt N., Bexell D. Patient-derived xenografts as preclinical neuroblastoma models. Cell Tissue Res. 2017;372:233–243. doi: 10.1007/s00441-017-2687-8.
    1. Méry B., Rancoule C., Guy J.-B., Espenel S., Wozny A.-S., Battiston-Montagne P., Ardail D., Beuve M., Alphonse G., Rodriguez-Lafrasse C., et al. Preclinical models in HNSCC: A comprehensive review. Oral Oncol. 2017;65:51–56. doi: 10.1016/j.oraloncology.2016.12.010.
    1. Krempley B.D., Yu K.H. Preclinical models of pancreatic ductal adenocarcinoma. Chin. Clin. Oncol. 2017;6:25. doi: 10.21037/cco.2017.06.15.
    1. Gengenbacher N., Singhal M., Augustin H.G. Preclinical mouse solid tumour models: Status quo, challenges and perspectives. Nat. Rev. Cancer. 2017;17:751–765. doi: 10.1038/nrc.2017.92.
    1. Day C.-P., Merlino G., Van Dyke T. Preclinical mouse cancer models: A maze of opportunities and challenges. Cell. 2015;163:39–53. doi: 10.1016/j.cell.2015.08.068.
    1. Taylor A.M., Zon L.I. Zebrafish Tumor Assays: The State of Transplantation. Zebrafish. 2009;6:339–346. doi: 10.1089/zeb.2009.0607.
    1. Marques R.P., Duarte G.S., Sterrantino C., Pais H.L., Quintela A., Martins A.P., Costa J. Triplet (FOLFOXIRI) versus doublet (FOLFOX or FOLFIRI) backbone chemotherapy as first-line treatment of metastatic colorectal cancer: A systematic review and meta-analysis. Crit. Rev. Oncol. 2017;118:54–62. doi: 10.1016/j.critrevonc.2017.08.006.
    1. Tehfe M., Dowden S., Kennecke H., El-Maraghi R., Lespérance B., Couture F., Létourneau R., Liu H., Romano A. nab-Paclitaxel Plus Gemcitabine Versus Gemcitabine in Patients with Metastatic Pancreatic Adenocarcinoma: Canadian Subgroup Analysis of the Phase 3 MPACT Trial. Adv. Ther. 2016;33:747–759. doi: 10.1007/s12325-016-0327-4.
    1. Mercatali L., La Manna F., Groenewoud A., Casadei R., Recine F., Miserocchi G., Pieri F., Liverani C., Bongiovanni A., Spadazzi C., et al. Development of a Patient-Derived Xenograft (PDX) of Breast Cancer Bone Metastasis in a Zebrafish Model. Int. J. Mol. Sci. 2016;17:1375. doi: 10.3390/ijms17081375.
    1. Progatzky F., Dallman M., Celso C.L. From seeing to believing: Labelling strategies for in vivo cell tracking experiments. Interface Focus. 2013;3:20130001. doi: 10.1098/rsfs.2013.0001.
    1. Giacchetti S., Perpoint B., Zidani R., Le Bail N., Faggiuolo R., Focan C., Chollet P., Llory J., Letourneau Y., Coudert B., et al. Phase III Multicenter Randomized Trial of Oxaliplatin Added to Chronomodulated Fluorouracil–Leucovorin as First-Line Treatment of Metastatic Colorectal Cancer. J. Clin. Oncol. 2000;18:136. doi: 10.1200/JCO.2000.18.1.136.
    1. Guimbaud R., Louvet C., Ries P., Ychou M., Maillard E., Andre T., Gornet J.-M., Aparicio T., Nguyen S., Azzedine A., et al. Prospective, Randomized, Multicenter, Phase III Study of Fluorouracil, Leucovorin, and Irinotecan Versus Epirubicin, Cisplatin, and Capecitabine in Advanced Gastric Adenocarcinoma: A French Intergroup (Fédération Francophone de Cancérologie Digestive, Fédération Nationale des Centres de Lutte Contre le Cancer, and Groupe Coopérateur Multidisciplinaire en Oncologie) Study. J. Clin. Oncol. 2014;32:3520–3526.
    1. Kimmel C.B., Ballard W.W., Kimmel S.R., Ullmann B., Schilling T.F. Stages of embryonic development of the zebrafish. Dev. Dyn. 1995;203:253–310. doi: 10.1002/aja.1002030302.

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