Establishment of Zebrafish Patient-derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Zebrafish Patient-Derived Xenograft Model to Predict Treatment Outcomes of Colorectal Cancer Patients.

The use of zebrafish embryos for personalized medicine has become increasingly popular. We present a co-clinical trial aiming to evaluate the use of zPDX (zebrafish Patient-Derived Xenografts) in predicting the response to chemotherapy regimens used for colorectal cancer patients. zPDXs are generated by xenografting tumor tissues in two days post-fertilization zebrafish embryos. zPDXs were exposed to chemotherapy regimens (5-FU, FOLFIRI, FOLFOX, FOLFOXIRI) for 48 h. We used a linear mixed effect model to evaluate the zPDX-specific response to treatments showing for 4/36 zPDXs (11%), a statistically significant reduction of tumor size compared to controls. We used the RECIST criteria to compare the outcome of each patient after chemotherapy with the objective response of its own zPDX model. Of the 36 patients enrolled, 8 metastatic colorectal cancer (mCRC), response rate after first-line therapy, and the zPDX chemosensitivity profile were available. Of eight mCRC patients, five achieved a partial response and three had a stable disease. In 6/8 (75%) we registered a concordance between the response of the patient and the outcomes reported in the corresponding zPDX. Our results provide evidence that the zPDX model can reflect the outcome in mCRC patients, opening a new frontier to personalized medicine.

Precision Medicine in Head and Neck Cancers: Genomic and Preclinical Approaches.

Head and neck cancers (HNCs) represent the sixth most widespread malignancy worldwide. Surgery, radiotherapy, chemotherapeutic and immunotherapeutic drugs represent the main clinical approaches for HNC patients. Moreover, HNCs are characterised by an elevated mutational load; however, specific genetic mutations or biomarkers have not yet been found. In this scenario, personalised medicine is showing its efficacy. To study the reliability and the effects of personalised treatments, preclinical research can take advantage of next-generation sequencing and innovative technologies that have been developed to obtain genomic and multi-omic profiles to drive personalised treatments. The crosstalk between malignant and healthy components, as well as interactions with extracellular matrices, are important features which are responsible for treatment failure. Preclinical research has constantly implemented in vitro and in vivo models to mimic the natural tumour microenvironment. Among them, 3D systems have been developed to reproduce the tumour mass architecture, such as biomimetic scaffolds and organoids. In addition, in vivo models have been changed over the last decades to overcome problems such as animal management complexity and time-consuming experiments. In this review, we will explore the new approaches aimed to improve preclinical tools to study and apply precision medicine as a therapeutic option for patients affected by HNCs.

Zebrafish Avatar to Develop Precision Breast Cancer Therapies.

BACKGROUND: Zebrafish (Danio rerio) is a vertebrate that has become a popular alternative model for the cellular and molecular study of human tumors and for drug testing and validating approaches. Notably, zebrafish embryos, thanks to their accessibility, allow rapid collection of in vivo results prodromal to validation in the murine models in respect to the 3R principles. The generation of tumor xenograft in zebrafish embryos and larvae, or zebrafish avatar, represents a unique opportunity to study tumor growth, angiogenesis, cell invasion and metastatic dissemination, interaction between tumor and host in vivo avoiding immunogenic rejection, representing a promising platform for the translational research and personalized therapies. OBJECTIVE: In this mini-review, we report recent advances in breast cancer research and drug testing that took advantage of the zebrafish xenograft model using both breast cancer cell lines and patient's biopsy. CONCLUSION: Patient derived xenograft, together with the gene editing, the omics biotechnology, the in vivo time lapse imaging and the high-throughput screening that are already set up and largely used in zebrafish, could represent a step forward towards precision and personalized medicine in the breast cancer research field.

Zebrafish Patient-Derived Xenografts Identify Chemo-Response in Pancreatic Ductal Adenocarcinoma Patients.

It is increasingly evident the necessity of new predictive tools for the treatment of pancreatic ductal adenocarcinoma in a personalized manner. We present a co-clinical trial testing the predictiveness of zPDX (zebrafish patient-derived xenograft) for assessing if patients could benefit from a therapeutic strategy (ClinicalTrials.gov: XenoZ, NCT03668418). zPDX are generated xenografting tumor tissues in zebrafish embryos. zPDX were exposed to chemotherapy regimens commonly used. We considered a zPDX a responder (R) when a decrease ≥50% in the relative tumor area was reported; otherwise, we considered them a non-responder (NR). Patients were classified as Responder if their own zPDX was classified as an R for the chemotherapy scheme she/he received an adjuvant treatment; otherwise, we considered them a Non-Responder. We compared the cancer recurrence rate at 1 year after surgery and the disease-free survival (DFS) of patients of both groups. We reported a statistically significant higher recurrence rate in the Non-Responder group: 66.7% vs. 14.3% (p = 0.036), anticipating relapse/no relapse within 1 year after surgery in 12/16 patients. The mean DFS was longer in the R-group than the NR-group, even if not statistically significant: 19.2 months vs. 12.7 months, (p = 0.123). The proposed strategy could potentially improve preclinical evaluation of treatment modalities and may enable prospective therapeutic selection in everyday clinical practice.

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

BACKGROUND: The response to chemotherapy treatment of patients with pancreatic ductal adenocarcinoma (PDAC) 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. The need for in-vivo xenotransplantation models for cancer patients has increased exponentially, and for this reason zebrafish avatars have gained popularity. Preliminary studies were conducted also with PDAC tissue. AIM: To develop a simple, not expensive, diffusible zebrafish embryo model as avatar for patients affected by PDAC. METHODS: 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 (Gemcitabine; Gemcitabine and Oxaliplatin; Gemcitabine and nab-Paclitaxel; 5-Fluorouracil and Folinic acid and Oxaliplatin and Irinotecan). 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). We evaluated the effect of the chemotherapy exposition comparing the mean RTA of each treated subgroup and the control group and evaluating the percentage reduction of the mean RTA by comparing each treated subgroup with the control group. RESULTS: Between July 2018 and October 2019, a total of 15 patients with pancreatic 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. The histological examination confirmed the presence of a PDAC in all cases. 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 6/15 (40%) cases. 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. CONCLUSION: Our model seems to be effective for the xenotransplantation of PDAC tissue and evaluation of the effect of each chemotherapy scheme on the xenotransplanted tumor tissue.

A Model of a Zebrafish Avatar for Co-Clinical Trials.

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).

Neurotrophin-conjugated nanoparticles prevent retina damage induced by oxidative stress.

Glaucoma and other optic neuropathies are characterized by a loss of retinal ganglion cells (RGCs), a cell layer located in the posterior eye segment. Several preclinical studies demonstrate that neurotrophins (NTs) prevent RGC loss. However, NTs are rarely investigated in the clinic due to various issues, such as difficulties in reaching the retina, the very short half-life of NTs, and the need for multiple injections. We demonstrate that NTs can be conjugated to magnetic nanoparticles (MNPs), which act as smart drug carriers. This combines the advantages of the self-localization of the drug in the retina and drug protection from fast degradation. We tested the nerve growth factor and brain-derived neurotrophic factor by comparing the neuroprotection of free versus conjugated proteins in a model of RGC loss induced by oxidative stress. Histological data demonstrated that the conjugated proteins totally prevented RGC loss, in sharp contrast to the equivalent dose of free proteins, which had no effect. The overall data suggest that the nanoscale MNP-protein hybrid is an excellent tool in implementing ocular drug delivery strategies for neuroprotection and therapy.