Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model

Abstract

BackgroundAberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour.MethodsWe fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish.ResultsIn the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature.ConclusionOur results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.

Up‐regulation of golgi α‐mannosidase IA and down‐regulation of golgi α‐mannosidase IC activates unfolded protein response during hepatocarcinogenesis

“…Recently, the xenotransplantation in vivo migration assay has been used in zebrafish embryos as a tool to investigate metastasis 23. Using the stable clones generated above, the effects of MAN1A1 and MAN1C1 on cell migration and proliferation were examined in vivo .…”

“…Zebrafish husbandry and xenotransplantation were performed as previously described 23, 24, 25. Detailed protocols and reagents are included in the Supporting Materials and Methods.…”

P2X7 receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness

“…In addition, we used the zebrafish model to study the in vivo invasive properties of MDA-MB-435s cancer cells (Marques et al, 2009). Pharmacologically targeting of P2X 7 R with selective inhibitor A-438079 decreased the metastases rate of MDA-MB-435s cells from the yolk sac of zebrafish embryos, but did not altered metastases rate of a non-expressing P2X 7 R cancer cell line, confirming the in vivo involvement of P2X 7 R in the invasiveness properties of MDA-MB-435s.…”

“…P2X 7 R activity is required for in vivo MDA-MB-435s invasiveness We finally analyzed the in vivo relevance of P2X 7 R function on MDA-MB-435s metastases, taking advantage of the zebrafish as a model to study the invasive and metastatic behaviour of human tumour cells (Marques et al, 2009). First, we validated this model by comparing the ability of highly invasive MDA-MB-435s cells with Same invasion experiments were performed on MDA-MB-435s cancer cells transfected with siRNA directed against P2RX7 mRNA (siP2X7) or scramble siRNA-A as a control (siCTL), in control condition (no ATP), or in presence of 3 mM ATP with or without 1 mM KN62 (n ¼ 3 independent experiments).…”

“…Labelled cells were incubated for 30 min with 10 mM of the specific P2X 7 R antagonist A-438079 (Tocris, Bristol, UK) or vehicle (DMSO), centrifuged, resuspended in 67% of DPBS, 5 fetal calf serum, 0.05% Phenol Red and injected into the yolk sac of dechorionated zebrafish embryos using the method described in Marques et al, 2009. Briefly 100-150 labelled cells in 4 nl were injected into the yolk sac of zebrafish embryos using a manual injector (Narishige, East Meadow (Long Island), NY, USA).…”