Metabolomic properties of the fluid from the surgical ligation after breast-conserving therapy and intraoperative radiotherapy.

Intraoperative radiotherapy with low-energy X-rays (IOXRT) is an increasingly utilized modality during breast conserving therapy (BCT). However, the molecular mechanisms by which it affects the postoperative microenvironment have not yet been fully elucidated. Surgical wound fluid (WF) has been shown to modulate cancer cell behavior; however, its metabolomic composition has not previously been characterized in the context of breast cancer. The objective of this study was to evaluate metabolic alterations in postoperative WF and to determine whether IOXRT induces different metabolic signatures compared to mastectomy (AMP). Postoperative WF was collected from 54 breast cancer patients (38 BCT IOXRT; 16 AMP) at two time points: day 1 (A) and day 5 (B) after surgery. The samples were then analyzed by1H NMR spectroscopy using NOESY, CPMG, and JRES techniques. A total of 114 spectral signals were quantified, and 42 metabolites were identified. Multivariate analyses, such as Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS DA), and Orthogonal Partial Least Squares Discriminant Analysis (OPLS DA), as well as the univariate Wilcoxon signed rank tests were applied to assess temporal and intergroup differences. A clear metabolic separation was observed between time points A and B in both treatment groups. However, statistical analysis did not reveal significant differences between BCT IOXRT and AMP. In BCT IOXRT, on the fifth day, WF exhibited a decrease in the branched chain amino acids, asparagine, lysine, methionine, and glutamate, concomitant with an increase in lactate and pyruvate. AMP-specific alterations included a decrease in 2-oxoglutarate and hypoxanthine on the first day, along with an increase in glucose and creatinine on the fifth day. A decline in ketone bodies (3-hydroxybutyrate, acetoacetate, acetone) was observed in both groups. Postoperative WF demonstrates dynamic metabolic changes reflecting early wound healing processes and treatment-related effects. IOXRT has been found to be associated with enhanced glycolytic signatures and reduced amino acid levels, suggesting altered metabolic activity in the irradiated tumor bed. The WF metabolomic profile has the potential to offer a novel source of biomarkers, which could facilitate the evaluation of treatment response and tumor microenvironment characteristics.