Mai M. Hassanein ^1,2 Yousra A. Hagyousif ^1,2 Ruba A. Zenati ^1,2 Hamza M. Al-Hroub ² Farman Khan ³ Ahmad Y. Abuhelwa ^2,3 Karem H. Alzoubi ^2,3 Nelson C. Soares ^4,5 Waseem El-Huneidi ^2,6 Eman Abu-Gharbieh ^2,7,8 Hany Omar ^2,3 Dana M. Zaher ² Yasser Bustanji ^2,6,8 Mohammad H. Semreen ^1,2*

• ¹ Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates
• ² Research Institute of Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates
• ³ Department of Pharmacy Practice and Pharmacotherapeutics, College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates
• ⁴ National Health Institute Doutor Ricardo Jorge (INSA), Lisbon, Portugal
• ⁵ Center for Applied and Translational Genomics (CATG), Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates
• ⁶ Department of Basic Medical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
• ⁷ Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
• ⁸ School of Pharmacy, The University of Jordan, Amman, Amman, Jordan

Background: Breast cancer is one of the most prevalent malignancies and a leading cause of death among women worldwide. Among its subtypes, triple-negative breast cancer (TNBC) poses significant clinical challenges due to its aggressive behavior and limited treatment options. This study aimed to investigate the effects of doxorubicin (DOX) and 5-fluorouracil (5-FU) as monotherapies and in combination using an established MDA-MB-231 xenograft model in female BALB/C nude mice employing advanced metabolomics analysis to identify molecular alterations induced by these treatments. Methods: We conducted comprehensive plasma and tumor tissue sample profiling using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). Results: Each treatment group exhibited unique metabolic profiles in plasma and tumor analysis. Univariate and enrichment analyses identified alterations in metabolic pathways. The combination treatment of DOX + 5-FU induced the most extensive metabolic alterations disrupting key pathways including purine, pyrimidine, beta-alanine, and sphingolipid metabolism. It significantly reduced critical metabolites such as guanine, xanthine, inosine, L-fucose, and sphinganine, demonstrating enhanced cytotoxic effects compared to individual treatments. The DOX treatment uniquely increased ornithine levels, while 5-FU altered sphingolipid metabolism, promoting apoptosis. Significance: This in-vivoin vivo study highlights TNBC's metabolic alterations to chemotherapeutics, identifying potential biomarkers like L-fucose and beta-alanine, and provides insights for improving treatment strategies.