Zebrafish embryo as avatar of patients with colorectal cancer and hepatic colorectal metastasis: 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. We propose the use of rejection-free 0-5 days post fertilization (dpf) Zebrafish Avatars for patients affected by colorectal (CR) cancer or colorectal liver metastasis (CRLM), in alternative to the expensive, time consuming and high ethical impact models of nude mouse xenografts. The aim of the project is to evaluate the usability of Zebrafish embryos as avatar and to test the chemosensibility to the different chemotherapy schemes used for the treatment of patients affected by CR cancer or CRLM. Methods: Patients with CR cancer or CRLM were enrolled. For each enrolled patient, a fragment of the tumor was taken after the specimen removal. After its elaboration, the tissue, fluorescently labeled with Dil, is pushed inside the yolk of 2 dpf zebrafish. At 2 hours post-injection (hpi) transplanted embryos were distributed in 24-well plates and incubated in E3 media with the presence or absence of drugs. The transplanted embryos were exposed for 48 hours to the standard combinations of chemotherapy used for the treatment of colorectal cancer (5-FU, FOLFIRI, FOLFOX, FOLFOXIRI) added to E3 medium (treated grated zebrafish embryos subgroups – treated-ZE subgroups). The control grafted zebrafish embryos subgroup (control-ZE) was exposed only to E3 medium. Each subgroup (control-ZE vs treated-ZE) were composed by 10 zebrafish embryos. For each protocol, the human plasma equivalent concentration was used as reference and we used a conversion factor human-to-fish calculated with a toxicity/efficacy study on the zebrafish model. Every day the fluorescent cancer cells were imaged by microscopic observation to evaluate the effect of the drug of interest. We used mass progression/regression as primary measure and cell migration along the zebrafish embryos’ body and tail as secondary measure. A ratio between the tumor mass area at 2 hpi and 48 hpi was calculated for each ZE subgroup and compared to each other. The percentage of cell migration revealed in the grafted zebrafish of the control-ZE subgroup was compared with the percentage revealed in each treated-ZE subgroup. A p<0.1 was considered statistically significant. Results: Ten patients with colon cancer (CR group) and two patients with colorectal liver metastasis (CRLM group) were enrolled between January 2018 and November 2018. In all cases of both CR and CRLM group the tumor cells successful grafted in the yolk sack of zebrafish embryos and in the control-ZE subgroup the tumor mass increased at 48 hpi respect 2 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 4/10 cases (40%) of CR group and 2/2 (100%) cases of CRLM group. In the CR group, one chemotherapy scheme was statistically more efficient in 2/10 cases, two chemotherapy schemes in 1/10 case and three chemotherapy schemes in 1/10 case. In the CRLM group, one chemotherapy scheme was statistically more efficient in 1 case while in the other case two chemotherapy schemes resulted more efficient. Cell migration was detected in a percentage of grafted zebrafish embryos of the control-ZE subgroup in 7/10 cases (70%) of CR group and in 2/2 cases (100%) of CRLM group. 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 7/7 cases of CR group and in 2/2 cases of CRLM group. In the CR group, the reduction of cell migration percentage was revealed for one chemotherapy scheme in 3/7 cases, for two and three chemotherapy schemes in 1/7 case and for all chemotherapy schemes in 2/4 cases. In the CRLM group, the reduction of cell migration percentage was revealed for three chemotherapy schemes in one case and for all in the other case. Conclusions: In our experience, zebrafish embryos could be a model as avatar for oncological patients because in all cases the tumor cells successful grafted in the yolk sack of zebrafish embryos. Probably the protocol used for the tests of chemosensibility should be improved and better defined the human-to-fish dose. Since we have noticed a tendency to inhibit the tumor cells proliferation by the chemotherapy treatments without reaching statistically significant, 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.