Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model

Introduction: The transforming growth factor beta (TGF-beta) 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-beta signalling in human breast tumour cells. Methods: We 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-beta signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy. Results: Breast 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-beta 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-beta in breast cancer cells, blocked invasion and metastasis of breast cancer cells. Conclusions: The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-beta drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.