Abstract
IntroductionThe transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells.MethodsWe injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy.ResultsBreast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells.ConclusionsThe zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.
Highlights
The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis
Invasion and micrometastasis formation of human breast cancer cell lines in zebrafish Human cancer cell lines have provided a rich source of propagatable material for the molecular and cellular characterization of cancer pathogenesis
The MDA-MB-231 cell line is used extensively for the study of hormone-independent breast cancer. It is capable of forming tumours in immunedeficient mice, and has a high metastatic potential, thereby providing xenograft models for study cancer development in vivo [23]
Summary
The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. Several tumour transplantation assays with human and mammalian cells to study different aspects of tumour malignancies in embryo and adult zebrafish, such as tumour cell migration, proliferation, angiogenesis and tumour cell extravasation [6,12,14,15,16] have been developed Many of these assays are simplistic and are limited to one selected step of tumour development, and do not represent the full complexity of tumourigenesis in one model. A rapid and reproducible zebrafish embryonic xenograft model for simultaneous formation of a localized tumour and experimental micrometastasis, by intravascular injection of tumour cells into the blood circulation of zebrafish embryos, has been recently described by the group of Snaar-Jagalska [17] They have shown that with noninvasive high-resolution imaging, the critical steps of tumour progression, including tumour vascularisation and tissue invasion, can be characterized
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