Metabolic reprogramming is a hallmark of cancer. After infusion of ¹³C-enriched acetate or glucose, ¹³C-NMR analysis of biopsies from human primary or metastatic brain tumors has demonstrated oxidation of both substrates in the citric acid cycle [1, 2]. It is not known whether this capacity reflects growth in the microenvironment of the brain or a fundamental feature of cellular transformation. To investigate whether the capacity to oxidize both glucose and acetate in the citric acid cycle is a feature of an early cancer, we studied patients with early stage, non-metastatic breast cancer. After obtaining informed consent, eligible patients were enrolled in an Institutional Review Board (IRB) approved clinical protocol. All patients had standard histological analysis of the diagnostic biopsy prior to enrollment. Patients were infused intravenously with either [U-¹³C]glucose or [1,2-¹³C]acetate during tumor resection for 120 – 180 minutes prior to tissue sampling. Tumors were sampled by multiple biopsies or immediately after mastectomy to obtain 200-400 mg of tumor. After freeze-clamping and later aqueous extraction, ¹H-decoupled ¹³C-NMR spectra of the tumor extracts were acquired on an Agilent 14.1T system equipped with a cryogenically – cooled probe. Fractional enrichment in plasma glucose was measured by mass spectrometry. Fractional enrichment in plasma acetate was measured by ¹H NMR spectroscopy. Results are mean ± SEM.Eight patients were infused with ¹³C-glucose, achieving mean ¹³C-glucose enrichment in the blood of 56±4%. Figure 1A shows the ¹³C-NMR spectrum from a good prognosis, estrogen (ER) and progesterone receptor (ER) positive, HER2 negative tumor that has a low proliferation rate (Ki67 10%). The ¹³C-NMR spectra from the most aggressive and highly proliferative (Ki67 70-90%) triple negative breast cancers (ER-, PR- and HER2-) and HER2 positive cancers were remarkably similar (spectra not shown). Each spectrum showed dominant labeling in lactate with minimal to no labeling of glutamate and glutamine from infused ¹³C-glucose. In marked contrast, infusion of ¹³C-acetate led to significant labeling in glutamate and glutamine, with the generation of quartets reflecting multiple turns of the citric acid cycle (representative spectrum in Figure 1B from an ER+, PR+, HER2- tumor, Ki67 12%). This pattern was seen in all patients infused with ¹³C-acetate, including those with triple negative and HER2+ tumors. Among patients infused with [U-¹³C]glucose, the fraction of acetyl-CoA derived from glucose was < 5% except in two patients with HER2 + disease (17% and 10%). Among patients infused with [1,2-13C]acetate (n=8), the fraction of acetyl-CoA derived from acetate carbons was 48±4% (p < 0.001). No differences in acetate oxidation were detected among prognostic subgroups that were based on hormone receptor status. Interestingly, among patients infused with acetate, ¹³C-enriched beta-hydroxybutyrate (BHB) was also observed. The fractional ¹³C enrichment in BHB in plasma was 18±4%.Under these conditions infused ¹³C-enriched glucose contributed little to acetyl-CoA in most early stage breast cancers, but HER2+ disease was associated with a modest increase in glucose oxidation. However, ¹³C from infused ¹³C-enriched acetate contributed nearly 50% of acetyl-CoA, demonstrating an intact citric acid cycle in early stage breast cancer, regardless of prognostic subgroup or proliferative rate. The appearance of ¹³C-enriched BHB in both plasma and tumor is due to hepatic ketogenesis from the infused ¹³C-enriched acetate. Together, these data demonstrate that acetate and possibly ketones, but not glucose, may contribute significantly to energy production via the citric acid cycle in early stage breast cancer.