ZEBRAFISH EMBRYO AS AVATAR OF PATIENTS WITH PANCREATIC CANCER: PRELIMINARY EXPERIENCE TOWARD A PERSONALIZED MEDICINE

Background: In the last years, a new concept of personalized medicine called ‘Mouse Avatars’ or ‘co-clinical trials’ has emerged, with the purpose to develop models to study the response of tumor to therapy on an individual basis. A big limitation of xenograft experiments in murine hosts is the requirement of immune-permissive strains and the long duration of time before the detection of human engrafted cells. The aim of this study is to evaluate the usability of Zebrafish embryos (ZE) as avatar and to test the chemosensibility to the different chemotherapy schemes used for the treatment of patients affected by pancreatic cancer. Methods: For each enrolled patient, a fragment of the tumor, fluorescently labeled with Dil, was xenotransplanted inside the yolk of 2 day post-fertilization ZE. Two hours post-injection (hpi) transplanted ZE were incubated in only E3 media (control-ZE group) or with added standard combinations of chemotherapy (GEMOX, nab-Paclitaxel/Gemcitabine, Gemcitabine, FOLFOXIRI) (treated-ZE subgroups) for 48 hours. Each subgroup was composed by 10 ZE. For each protocol, we used a conversion factor human-to-fish calculated with a toxicity/efficacy study on zebrafish model. A ratio between the tumor mass area at 24 hpi and 48 hpi and the percentages of cell migration through the ZE caudal vein were calculated in the control-ZE and in each treated-ZE subgroup and compared to each other. A p<0.1 was considered statistically significant. Results: Ten patients with pancreatic cancer were enrolled between January 2018 and November 2018. The first four cases were used for the definition of the protocol for the xenotransplantation and the tests of chemosensibility were not performed. In all cases the tumor cells successfully grafted in the yolk sack of zebrafish embryos. In all cases of the control-ZE group the tumor mass increased at 48 hpi respect 24 hpi in all cases. The mass progression/regression analysis revealed a statistically significant difference of tumor mass progression in the treated-ZE subgroups respect the control-ZE subgroup in 3/6 cases (50%). One chemotherapy scheme was statistically more efficient in 2/6 cases (nab-Paclitaxel/Gemcitabine and FOLFOXIRI, respectively), while two chemotherapy schemes in 1/6 case (nab-Paclitaxel/Gemcitabine and FOLFOXIRI). Cell migration was detected in a percentage of grafted zebrafish embryos of the control-ZE subgroup in 3/6 cases (50%). A significant reduction of the cell migration percentage in the treated-ZE subgroups compared to the control-ZE subgroup was revealed for almost one chemotherapy scheme in 3/3 cases. The reduction of cell migration percentage was revealed for two in 1/3 case, for three chemotherapy schemes in 1/3 case and for all chemotherapy schemes in 1/3 cases. Conclusions: In our experience, zebrafish embryos could be a used as avatar for oncological patients because in all cases the tumor cells successfully grafted in the yolk sack of zebrafish embryos. Probably the protocol used for the tests of chemosensibility should be improved and the human-to-fish dose should be better defined. Since we have noticed a tendency to inhibit the tumor cells proliferation by the chemotherapy treatments without reaching statistically significance, we have already tried to increase the chemotherapy doses with the intent to reach it. Moreover, a prospective co-clinical trial is under way to evaluate the concordance between the results of tests in zebrafish and the response to chemotherapy in oncological patients