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Updated: Aug 16, 2025
Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis
Published on: June 19, 2021
A zebrafish HCT116 xenograft model to predict anandamide outcomes on colorectal cancer
Francesca Maradonna1,2, Camilla M Fontana1,3, Fiorenza Sella1
1Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy.
Abstract:
Colon cancer is one of the leading causes of death worldwide. In recent years, cannabinoids have been extensively studied for their potential anticancer effects and symptom management. Several in vitro studies reported anandamide's (AEA) ability to block cancer cell proliferation and migration, but evidence from in vivo studies is still lacking. Thus, in this study, the effects of AEA exposure in zebrafish embryos transplanted with HCT116 cells were evaluated. Totally, 48 hpf xenografts were exposed to 10 nM AEA, 10 nM AM251, one of the cannabinoid 1 receptor (CB1) antagonist/inverse agonists, and to AEA + AM251, to verify the specific effect of AEA treatment. AEA efficacy was evaluated by confocal microscopy, which demonstrated that these xenografts presented a smaller tumor size, reduced tumor angiogenesis, and lacked micrometastasis formation. To gain deeper evidence into AEA action, microscopic observations were completed by molecular analyses. RNA seq performed on zebrafish transcriptome reported the downregulation of genes involved in cell proliferation, angiogenesis, and the immune system. Conversely, HCT116 cell transcripts resulted not affected by AEA treatment. In vitro HCT116 culture, in fact, confirmed that AEA exposure did not affect cell proliferation and viability, thus suggesting that the reduced tumor size mainly depends on direct effects on the fish rather than on the transplanted cancer cells. AEA reduced cell proliferation and tumor angiogenesis, as suggested by socs3 and pcnp mRNAs and Vegfc protein levels, and exerted anti-inflammatory activity, as indicated by the reduction of il-11a, mhc1uba, and csf3b mRNA. Of note, are the results obtained in groups exposed to AM251, which presence nullifies AEA's beneficial effects. In conclusion, this study promotes the efficacy of AEA in personalized cancer therapy, as suggested by its ability to drive tumor growth and metastasis, and strongly supports the use of zebrafish xenograft as an emerging model platform for cancer studies.
Insights
Anandamide (AEA) significantly reduced colon cancer growth and metastasis in zebrafish xenografts, primarily by affecting the host fish rather than cancer cells. This suggests AEA
Area of Science:
- Oncology
- Pharmacology
- Developmental Biology
Background:
- Colon cancer is a leading global cause of mortality.
- Cannabinoids, including anandamide (AEA), show potential in cancer treatment and symptom management.
- In vivo evidence for AEA's anti-cancer effects is limited, necessitating further investigation.
Purpose of the Study:
- To evaluate the in vivo effects of anandamide (AEA) on colon cancer xenografts in zebrafish embryos.
- To investigate the specific role of the cannabinoid 1 receptor (CB1) in AEA's anti-cancer activity.
- To explore the molecular mechanisms underlying AEA's impact on tumor growth, angiogenesis, and metastasis.
Main Methods:
- Zebrafish embryos (48 hpf) were xenografted with HCT116 colon cancer cells.
- Xenografts were treated with AEA, the CB1 antagonist AM251, or a combination of both.
- Tumor size, angiogenesis, and metastasis were assessed using confocal microscopy and molecular analyses (RNA-seq).
Main Results:
- AEA treatment significantly reduced tumor size, inhibited angiogenesis, and prevented micrometastasis formation in zebrafish xenografts.
- Molecular analysis revealed AEA downregulates genes involved in proliferation, angiogenesis, and the immune system in the host fish.
- AEA did not affect HCT116 cell proliferation or viability in vitro, indicating AEA's effects are primarily host-mediated.
Conclusions:
- Anandamide (AEA) demonstrates significant anti-cancer efficacy in a zebrafish xenograft model, impacting tumor growth and metastasis.
- AEA's beneficial effects are mediated through host-dependent mechanisms, involving anti-proliferative, anti-angiogenic, and anti-inflammatory actions.
- The cannabinoid 1 receptor (CB1) is crucial for AEA's anti-tumor activity, and zebrafish xenografts represent a promising platform for cancer drug discovery.
