Systematic Roadmap for Cancer Drug Screening Using Zebrafish Embryo Xenograft Cancer Models: Melanoma Cell Line as a Case Study
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Image Analysis
2.2. RNA/DNA Genetic Extraction and Reverse Transcription
2.3. Primers Quality Test
2.4. qPCR Standard Curves
2.5. Zebrafish Maintenance and Egg Collection
2.6. Cell line and Culture Methods
2.7. Xenotransplantation and Injection Site Experiment
2.8. Engraftment Definition and Experimental Inclusion Criteria
2.8.1. Biodistribution and Dissemination Assay
2.8.2. Drug
2.8.3. In Vitro Cell Viability Assay (MTS)
2.8.4. Maximum Tolerated Dose (MTD) Assay
2.8.5. In Vivo Drug Efficacy Assays
2.8.6. Metrics Definition for Efficacy Assays
2.8.7. Statistical Methods
3. Results
3.1. Monitoring Approaches
3.2. Injection Site Assay
3.3. Compound Administration and Biodistribution Assay
3.4. In Vivo Efficacy Assays
4. Discussion
5. Conclusions
- (1)
- Experimental design. According to our results, twenty-four embryos per experimental group and three independent replicates were enough to see statistically significant results on the final day of the experiment and increased sensitivity to detect smaller antitumoral effects precluding false negatives. Our experimental setup was extended to 4 dpi, which had a clear added value and had a statistically significant impact on the efficacy assessment window and the DTG metric; therefore, ethical approval was required.
- (2)
- Cancer cell xenotransplantation. A total of 1000 cells was sufficient to detect tumor engraftment by imaging, and our results discourage the use of the yolk as the site of implantation. Conversely, the PCS was identified as the best site, as it yielded higher rates of cancer cell engraftment and was less harmful than the other sites, as determined by the engrafted embryos showing higher survival rates.
- (3)
- Sorting. At 1 dpi, sorting should be carefully performed using a fluorescence stereomicroscope to select properly microinjected embryos that exhibit cells at only the correct location and homogeneous tumor masses.
- (4)
- Tumor monitoring. As reported above, gDNA qPCR is a less time-consuming technique that provides some advantages for only ventral PVS implantation. However, due to its detrimental impact on statistical power (requires pools of embryos) and due to the PCS being selected as the site of tumor implantation, fluorescence imaging is the proposed tumor monitoring technique.
- (5)
- User-independent inclusion criteria. We aimed to propose a systematic screening roadmap, including a user-independent decision-making process, to minimize variability and maximize reproducibility. Therefore, we defined assay-based user-independent criteria, including the inclusion threshold (IT), to the efficacy assessment of only embryos that presented with a TA at 1 dpi that was higher than the IT, which was key for decision making.
- (6)
- Treatment. According to our results, compound administration by direct intratumoral inoculation is the best approach to treat engrafted embryos and for reliable efficacy assessments. As the experimental setup was extended to 4 dpi, compounds were administered for three consecutive days.
- (7)
- Final data analysis. Only embryos meeting the inclusion criteria (IT) were considered for efficacy assessment and decision making.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Letrado, P.; Mole, H.; Montoya, M.; Palacios, I.; Barriuso, J.; Hurlstone, A.; Díez-Martínez, R.; Oyarzabal, J. Systematic Roadmap for Cancer Drug Screening Using Zebrafish Embryo Xenograft Cancer Models: Melanoma Cell Line as a Case Study. Cancers 2021, 13, 3705. https://doi.org/10.3390/cancers13153705
Letrado P, Mole H, Montoya M, Palacios I, Barriuso J, Hurlstone A, Díez-Martínez R, Oyarzabal J. Systematic Roadmap for Cancer Drug Screening Using Zebrafish Embryo Xenograft Cancer Models: Melanoma Cell Line as a Case Study. Cancers. 2021; 13(15):3705. https://doi.org/10.3390/cancers13153705
Chicago/Turabian StyleLetrado, Patricia, Holly Mole, María Montoya, Irene Palacios, Jorge Barriuso, Adam Hurlstone, Roberto Díez-Martínez, and Julen Oyarzabal. 2021. "Systematic Roadmap for Cancer Drug Screening Using Zebrafish Embryo Xenograft Cancer Models: Melanoma Cell Line as a Case Study" Cancers 13, no. 15: 3705. https://doi.org/10.3390/cancers13153705