Simple SummaryHeparanase (HPSE) has been demonstrated to enhance the progression and metastasis of solid tumours, leading to a poor clinical prognosis. However, robust genetic ablation animal models to investigate the role of HPSE in breast cancer have not been described. The aim of this study was to utilise an HPSE-deficient strain of the well-established MMTV-PyMT murine mammary carcinoma model (MMTV-PyMTxHPSE−/−mice) to investigate the role of HPSE in early establishment, progression, and metastasis of mammary tumours. Contrary to our current understanding, we observed that even though HPSE regulated tumour angiogenesis, the establishment, progression, and metastasis of mammary tumours in MMTV-PyMT animals were HPSE-independent. We further observed no compensation by matrix metalloproteinases in response to the lack of HPSE in MMTV-PyMTxHPSE−/−animals. These findings may have significant implications in the development and clinical utility of HPSE inhibitors.AbstractBreast cancer is the second most common human malignancy and is a major global health burden. Heparanase (HPSE) has been widely implicated in enhancing the development and progression of solid tumours, including breast cancer. In this study, the well-established spontaneous mammary tumour-developing MMTV-PyMT murine model was utilised to examine the role of HPSE in breast cancer establishment, progression, and metastasis. The use of HPSE-deficient MMTV-PyMT (MMTV-PyMTxHPSE−/−) mice addressed the lack of genetic ablation models to investigate the role of HPSE in mammary tumours. It was demonstrated that even though HPSE regulated mammary tumour angiogenesis, mammary tumour progression and metastasis were HPSE-independent. Furthermore, there was no evidence of compensatory action by matrix metalloproteinases (MMPs) in response to the lack of HPSE expression in the mammary tumours. These findings suggest that HPSE may not play a significant role in the mammary tumour development of MMTV-PyMT animals. Collectively, these observations may have implications in the clinical setting of breast cancer and therapy using HPSE inhibitors.