• oncological patients
• personalized medicine
• zebrafish avatars
• zebrafish embryos

Background
The response to chemotherapy treatment of oncological patients is difficult to predict and the identification of patients who most likely will benefit from aggressive chemotherapy approaches is crucial. The concept of personalized medicine has emerged in the last years with the objective to tailor the medical treatment to the individual characteristics of each patient, and particularly to the tumor biology of each patient. 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. The need for in- vivo xenotransplantation models for cancer patients has increased exponentially, and for this reason zebrafish avatars have gained popularity. The aim of the research project is to develop a patient- derived xenograft (PDX) model using zebrafish embryos Avatars and evaluate its usability to predict the efficacy of the different chemotherapy schemes used for the treatment of oncological patients after the xenotransplantation of tumor tissue taken from the surgical specimen of each patient enrolled underwent surgical operation for hepato-biliary-pancreatic cancers and gastro-intestinal cancers
Methods and materials
To obtain the goal of the research project, the activities were divided in three principal phase. The “Part I: development of the xenotransplantation protocol of cancer cells in the zebrafish embryo and tests with chemotherapy drugs” comprised all the activities needed to define the protocol for the xenotransplantation and the tests with chemotherapy drugs. The key issue is related to the lack of an “equivalent dose” for translating the chemotherapy dosage used in humans to zebrafish embryos because the interspecies allometric approach for dose conversion from human to animal cannot be applied. Therefore, drug safety and efficacy assessments were performed to estimate the equivalent dose to administer. The “Part II: validation of the protocol with xenotransplantation of patients’ derived tumor tissue in the zebrafish embryo” comprised all the activities needed to validate the protocol for the xenotransplantation. At the end, the “Part III: Human Trial: Patient enrollment, surgery and xenotransplantation of cancer cells in Zebrafish embryos” comprised the enrolment of oncological patients and evaluation of the ability of the model to evaluate and theoretically predict the response to chemotherapy drugs tested after xenotransplantation of tumor tissue in zebrafish embryos.
For the xenotransplantation, tumor tissue was taken from the surgical specimen by the histopathologist. After its fragmentation into small pieces, they are stained with CM-Dil. Small pieces of stained tissue were transplanted into the yolk of wt AB zebrafish embryos with a glass capillary needle. Embryos were incubated at 35 °C in E3 medium supplemented with 1% Pen/Strep in the presence or absence of drugs for the following days in respect of the treatment plan. The response of zebrafish xenografts to the chemotherapy options has been analyzed by monitoring the fluorescent stained area at 2 h post injection (hpi), 1 d and 2 d post injection (dpi). In each time point, the mean size of the stained area was measured by ImageJ and it was normalized with respect to the 1 dpi time point mean relative tumor area (RTA). The effect of the chemotherapy exposition were evaluated comparing the mean RTA of each treated subgroup and the control group and evaluating the
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percentage reduction of the mean RTA by comparing each treated subgroup with the control group. Moreover, a generalized linear mixed effect model was used to analyze treatments effect on tumor size in zebrafishes and investigate patient specific response. A cluster analysis with K-means algorithm was performed to evaluate if there was a similar response among patients to treatments.
Results
First of all, by crossing data from safety and efficacy studies, a basic formula for estimating the equivalent dose for use in co-clinical trials was found and was validated in a clinical study enrolling 24 adult patients with solid cancers. Therefore, between July 2018 and July 2020, a total of 22 patients with pancreatic ductal adenocarcinoma (PDAC) and 35 patients with colorectal cancer were prospectively enrolled. In all cases, it was possible to take a fragment of the tumor from the surgical specimen for the xenotransplantation in the zebrafish embryos. In absence of chemotherapy (control group), over time the Dil-stained area showed a statistically significant increase in all cases. A statistically significant reduction of the mean RTA in the treated subgroups for at least one chemotherapy scheme was reported in 10/22 (45.5%) cases for PDAC and in 10/35 (28.6%) cases for colorectal cancer. The analysis of the percentage reduction of the RTA in treated subgroups in comparison to the control group revealed the presence of a linear relationship in each subgroup between the percentage reduction of the RTA and the number of cases reporting each percentage threshold considered for the analysis both for PDAC and colorectal cancer. The generalized linear mixed effect model showed that treatments were statistically significant in reducing the percent change tumor area, thus having a certain effect in hindering the growth of tumor cells compared to the absence of chemotherapy drugs in the control group. Moreover, comparing the treatment effect between patients and the population mean, a statistically significant reduction of tumor area was observed for the different chemotherapy schemes both for PDAC and for colorectal cancer in a percentage of cases, documenting an efficacy of the treatments to impair the tumor growth after the xenotransplantation in zebrafish embryos. The cluster analysis found out that for PDAC cases patients grouped together for most of the provided treatments with a group of patients that showed a trend of significantly decrease percent change area for every treatment proposed while for colorectal cancer found out that patients differed from each other in a treatment group, suggesting a patient specific response in respect to population mean.
Conclusion
The developed model with the use of zebrafish embryos appears to be an effective, usable and not expensive model for the xenotransplantation of pancreatic tumor or colorectal tumor tissue and for the evaluation of the efficacy of the different chemotherapy schemes available for the treatment of patients with PDAC or colorectal cancer. This could open a new frontier to personalized medicine because the results of the tests obtained in our model in the xenotransplanted zebrafish embryos could reflect the clinical course of the patients’ medical history, and such an approach might improve the evaluation of the patient’s prognosis and the identification of the most appropriate individualized therapy.