Construction And Application Of A Zebrafish Model For Tumor Brain Metastasis

Malignant tumor has become one of the top causes of death worldwide.The high metastasis rate of tumor is worst results of malignant tumors,in which brain metastasis is one of the most serious consequences.The prognosis of patients with brain metastasis is very poor and 5 year survival rate is very low.Melanoma,lung cancer and breast cancer are the top 3 cancer types with the highest ratio of brain metastases.Therefore,to study on the mechanism of brain metastatic of cancer cells has important significance for better therapeutic methods.The present project aims to establish an in vivo animal model for the study of brain metastasis taking advantage of the model organism zebrafish and to explore molecular mechanisms as well as the role of tumor cell-derived exosomes in brain metastasis.Our results are summarized as follows:1.Melanoma tfRFP-B16-F10 cells,which were genetically labelled with red fluorescent protein,were into the pericardial cavity of zebrafish embryos before(2 dpf)and after(4 dpf)the formation of the blood-brain barrier(BBB).Metastasis of tumor cells were examined: survival rate,the number of tumor colonies at the injection site and metastases at different sites(head,trunk,and tail),and the total metastases.We also did in vivo imaging on the transplanted larvae to investigate how tumor cells enter the brain.We found that tumor cells enter the brain by constantly deforming through the gaps between the brain's endothelial cells.The tight junctions between brain endothelial cells is one of the main components of the blood-brain barrier.Our results demonstrated that the brain metastasis rate of tumor cells is greatly reduced after the tight junctions appear,suggesting that the BBB plays an important role in inhibiting the brain metastasis of tumor cells.In addition,we did long-term live imaging after 2 days of brain metastases under confocal microscope.We found that brain metastasized tumor cells expanded along the blood vessel.We have successfully established a zebrafish xenograft model to study brain metastasis of cancer.2.3-D cultured tumorigenic melanoma tfRFP-B16-F10 cells,as well as those cultured on regular rigid surface,were separately injected into the pericardial cavity of different zebrafish embryos on 3dpf.Metastasis of tumor cells were examined: survival rate,the number of tumor colonies at the injection site and metastases at different sites(head,trunk,and tail),and the total metastases.We found that both the total metastasis and brain metastasis,the 3-D cultured tumorigenic tumor cells have higher metastatic rate than regular tumor cells,indicating that the tumorigenic tumor cells have higher potential to spread to other tissues including the brain.Furthermore,we use fluorescence microscope to observe the same zebrafish larvae within a time window and observed the growth of tumor cells at the metastatic site.3.Exosome is a 30-100 um vesicle structure,which plays an important role in the metastasis process of tumor cells.However,little is known on the molecular mechanism of the exosomal effects on brain metastasis.Therefore,we intend to use the brain metastasis zebrafish model established in this project to study the role of exosomes in of brain metastasis.We used DiI to label exosomes extracted in vitro with red fluorescence.The DiI-exosomes were injected into the pericardial cavity of 2dpf zebrafish embryo,and confocal live imaging was performed to observe the interaction between exosomes and endothelial cells of the brain.We found that exosomes can be absorbed by brain endothelial cells and red fluorescence stays co-localized with the green fluorescence of brain endothelial cells for up to 4 hours or even longer,indicating the existence of interactions between exosomes and brain endothelial cells which may play an important role in the tumor cells penetrating the blood-brain barrier.We have successively attempted 5 times injection of tfRFP-B16-F10 and its exosomes,and one times injection of NSCLC: mCherry-A549 and its exosomes,monitoring brain metastasis,and conducting statistical analysis.However,the experimental results are quite different from the expectation,so we need to explore new modeling methods to construct the zebrafish model to study the effect of exosomes on the brain metastasis of tumor cells.