Background/Objectives: Breast cancer is a prevalent and heterogeneous disease with multiple subtypes, which are defined by characteristics such as molecular biomarkers and metastatic status. This study aimed to profile the metabolic activity of various breast cancer subtypes, both with and without chemotherapy (doxorubicin) application. Methods: Six human breast cell lines were evaluated, two non-tumorigenic controls and four cancerous lines. The cancer lines were clustered as primary-derived, metastasis-derived, triple-negative (TNBC), and strong hormone receptor-positive (ER+/PR+) and analyzed using the Biolog phenotype mammalian microarrays (PM-M1 to PM-M8) to assess metabolic activity via NADH production under a wide array of substrate parameters. Results: Unique metabolic profiles emerged across the subtypes and clusters; the TNBC and metastatic cells demonstrated enhanced utilization of glycolytic and anaerobic substrates consistent with the Warburg effect. The ER+/PR+ cells showed heightened glucose utilization and unique sensitivity to metabolic effectors and doxorubicin. Additionally, significant metabolic differences were observed in nucleoside and amino acid utilization between cancer and control cells, particularly in metastatic and TNBC lines. Conclusions: Our findings reveal the profound metabolic diversity among breast cancer subtypes and highlight distinct substrate dependencies for proliferation. The results additionally provide a framework for developing metabolic biomarkers and targeted therapies for chemotherapy resistance in breast cancer subtypes.

Thanks to incalculable efforts by teams of scientists and physicians over the past half‐century, many cancer diagnoses that would have previously seemed terminal are now considered treatable or even curable with optimization of existing treatment strategies and the introduction of novel treatment methodologies. One rising treatment paradigm with the potential to improve patient outcomes and experiences is photomedicine, a group of light‐activated treatments that can be applied systemically and activated locally. As the work of developing new phototherapeutic treatments and agents for use in cancer patients progresses, in vivo experimentation is a necessary hurdle, costing both time and resources. The use of zebrafish as a vertebrate tumor model for early‐stage animal testing has the potential to increase the overall efficiency of the research process. Zebrafish are a model organism that allow for numerous tumor presentations and drug‐delivery options to be tested at scale with a low turnaround time relative to other in vivo vertebrate models. The transparency of an embryonic or larval zebrafish's body allows for real‐time observation of drug dispersal and effects of treatment. These qualities allow zebrafish to serve as a useful screening tool for identifying promising candidate photosensitizers before embarking on mammalian studies.