HCT116 colorectal and MCF7 brest cancer cell lines xenografted into zebrafish embryos gives insight into the importance of microenviroment in tumor growth and metastasis for a future use of the model in clinical research

Zebrafish has emerged as one of the best models to characterize different human diseases due to his genetic similarity (75% of orthologous genes), and more specific, cancer. For this reason, zebrafish has been used in cancer research by means of the xenograft technique (injection of human cancer cells in embryos or adults of this model organism). The objective of this technique is simulate a human-like microenvironment inside a model organism in order to study the disease development with a fastest and accurate approach. As long as the xenograft technique has been implemented in zebrafish, mentioned before, there has been improvements to this technique and the conditions surrounding it. Nevertheless, there are some bottle necks with the necessity of being addressed with the objective of getting a more robust and informative technique in order to establish it in a near future in the personalized medicine field

Modeling Cancer Using Zebrafish Xenografts: Drawbacks for Mimicking the Human Microenvironment

The first steps towards establishing xenografts in zebrafish embryos were performed by Lee et al., 2005 and Haldi et al., 2006, paving the way for studying human cancers using this animal species. Since then, the xenograft technique has been improved in different ways, ranging from optimizing the best temperature for xenografted embryo incubation, testing different sites for injection of human tumor cells, and even developing tools to study how the host interacts with the injected cells. Nonetheless, a standard protocol for performing xenografts has not been adopted across laboratories, and further research on the temperature, microenvironment of the tumor or the cell–host interactions inside of the embryo during xenografting is still needed. As a consequence, current non-uniform conditions could be affecting experimental results in terms of cell proliferation, invasion, or metastasis; or even overestimating the effects of some chemotherapeutic drugs on xenografted cells. In this review, we highlight and raise awareness regarding the different aspects of xenografting that need to be improved in order to mimic, in a more efficient way, the human tumor microenvironment, resulting in more robust and accurate in vivo resultsConsellería de Educación, Universidade e Formación Profesional (ED431C 2018/28)S

Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool

Background Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model. Methods This study improved the conditions of the xenotransplantation technique – quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a ‘proliferation index’. Results The analysis of tumor cell proliferation at different temperatures (34 °C and 36 °C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36°, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5- Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 °C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compared with the control group. This was not the case at 34 °C. Conclusions Our results demonstrate that the proliferation of the injected cells is better at 36 °C and that this temperature is the most suitable for testing chemotherapeutic drugs like the 5-FluorouracilThis research was funded by the Fondo de Investigación Sanitaria (Instituto Carlos III) - FIS project (PI13/01388). The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing of this manuscriptS

Circulating Tumor Cells Characterization Revealed TIMP1 as a Potential Therapeutic Target in Ovarian Cancer

Background: Recent studies showed a relevant role of hematogenous spread in ovarian cancer and the interest of circulating tumor cells (CTCs) monitoring as a prognosis marker. The aim of the present study was the characterization of CTCs from ovarian cancer patients, paying special attention to cell plasticity characteristics to better understand the biology of these cells. Methods: CTCs isolation was carried out in 38 patients with advanced high-grade serous ovarian cancer using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize these cells. Results: Epithelial CTCs were found in 21% of patients, being their presence higher in patients with extraperitoneal metastasis. Importantly, this population was characterized by the expression of epithelial markers as MUC1 and CK19, but also by genes associated with mesenchymal and more malignant features as TIMP1, CXCR4 and the stem markers CD24 and CD44. In addition, we evidenced the relevance of TIMP1 expression to promote tumor proliferation, suggesting its interest as a therapeutic target. Conclusions: Overall, we evidenced the utility of the molecular characterization of EpCAM+ CTCs from advanced ovarian cancer patients to identify biomarkers with potential applicability for disseminated disease detection and as therapeutic targets such as TIMP1Part of this research was supported by CIBERONC funds (CB16/12/00328)S

Morphological Abnormalities and Gene Expression Changes Caused by High Incubation Temperatures in Zebrafish Xenografts with Human Cancer Cells

Published studies show that most of the human cancer xenograft studies in zebrafish embryos have used incubation temperatures in the range of 32–34 °C for 3–6 days post-injection, trying to find a compromise temperature between the zebrafish embryos (28 °C) and the human injected cells (37 °C). While this experimental setup is widely used, a question remains: is possible to overcome the drawbacks caused by a suboptimal temperature for the injected cells? To clarify the effect of temperature and injected cells on the host, in this study, we analyzed the development and health of the last in response to different temperatures in the presence or absence of injected human cancer cells. Comparing different incubation temperatures (28, 34 and 36 °C), we determined morphological abnormalities and developmental effects in injected and non-injected embryos at different time points. Besides this, the expression of selected genes was determined by qPCR to determine temperature affected metabolic processes in the embryos. The results indicate that an incubation temperature of 36 °C during a period of 48 h is suitable for xenotransplantation without morphological or metabolic changes that could be affecting the host or the injected cells, allowing them to proliferate near their optimal temperatureThis research is funded by ‘Consolidación e estruturación de unidades de investigación competitivas do SUG. Grupos de referencia competitiva’ (ED431C 2018/29) and ‘Axuda á formación da etapa predoutoral’ of Xunta de GaliciaS

Severe neurometabolic phenotype in npc1−/− zebrafish with a C-terminal mutation

Niemann Pick disease type C (NPC) is an autosomal recessive neurodegenerative lysosomal disorder characterized by an accumulation of lipids in different organs. Clinical manifestations can start at any age and include hepatosplenomegaly, intellectual impairment, and cerebellar ataxia. NPC1 is the most common causal gene, with over 460 different mutations with heterogeneous pathological consequences. We generated a zebrafish NPC1 model by CRISPR/Cas9 carrying a homozygous mutation in exon 22, which encodes the end of the cysteine-rich luminal loop of the protein. This is the first zebrafish model with a mutation in this gene region, which is frequently involved in the human disease. We observed a high lethality in npc1 mutants, with all larvae dying before reaching the adult stage. Npc1 mutant larvae were smaller than wild type (wt) and their motor function was impaired. We observed vacuolar aggregations positive to cholesterol and sphingomyelin staining in the liver, intestine, renal tubules and cerebral gray matter of mutant larvae. RNAseq comparison between npc1 mutants and controls showed 284 differentially expressed genes, including genes with functions in neurodevelopment, lipid exchange and metabolism, muscle contraction, cytoskeleton, angiogenesis, and hematopoiesis. Lipidomic analysis revealed significant reduction of cholesteryl esters and increase of sphingomyelin in the mutants. Compared to previously available zebrafish models, our model seems to recapitulate better the early onset forms of the NPC disease. Thus, this new model of NPC will allow future research in the cellular and molecular causes/consequences of the disease and on the search for new treatments