Cancer metabolic reprogramming plays a critical role in tumor progression and therapeutic resistance, underscoring the need for advanced analytical strategies. Metabolomics, leveraging mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, offers a comprehensive and functional readout of tumor biochemistry. By enabling both targeted metabolite quantification and untargeted profiling, metabolomics captures the dynamic metabolic alterations associated with cancer. The integration of metabolomics with machine learning (ML) approaches further enhances the interpretation of these complex, high-dimensional datasets, providing powerful insights into cancer biology from biomarker discovery to therapeutic targeting. This review systematically examines the transformative role of ML in cancer metabolomics. We discuss how various ML methodologies—including supervised algorithms (e.g., Support Vector Machine, Random Forest), unsupervised techniques (e.g., Principal Component Analysis, t-SNE), and deep learning frameworks—are advancing cancer research. Specifically, we highlight three major applications of ML–metabolomics integration: (1) cancer subtyping, exemplified by the use of Similarity Network Fusion (SNF) and LASSO regression to classify triple-negative breast cancer into subtypes with distinct survival outcomes; (2) biomarker discovery, where Random Forest and Partial Least Squares Discriminant Analysis (PLS-DA) models have achieved >90% accuracy in detecting breast and colorectal cancers through biofluid metabolomics; and (3) prognostic modeling, demonstrated by the identification of race-specific metabolic signatures in breast cancer and the prediction of clinical outcomes in lung and ovarian cancers. Beyond these areas, we explore applications across prostate, thyroid, and pancreatic cancers, where ML-driven metabolomics is contributing to earlier detection, improved risk stratification, and personalized treatment planning. We also address critical challenges, including issues of data quality (e.g., batch effects, missing values), model interpretability, and barriers to clinical translation. Emerging solutions, such as explainable artificial intelligence (XAI) approaches and standardized multi-omics integration pipelines, are discussed as pathways to overcome these hurdles. By synthesizing recent advances, this review illustrates how ML-enhanced metabolomics bridges the gap between fundamental cancer metabolism research and clinical application, offering new avenues for precision oncology through improved diagnosis, prognosis, and tailored therapeutic strategies.

Lactic acid bacteria (LAB) possess the ability to argument T cell activity through functional modification of antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages. Nevertheless, the precise mechanism underlying LAB-induced enhancement of antigen presentation in APCs remains incompletely understood. To address this question, we investigated the detailed mechanism underlying the enhancement of major histocompatibility complex (MHC) class I-restricted antigen presentation in DCs using a probiotic strain known as Lactococcus lactis subsp. Cremoris C60. We found that Heat-killed-C60 (HK-C60) facilitated the processing and presentation of ovalbumin (OVA) peptide antigen OVA_257-264 (SIINFEKL) via H-2K^b in bone marrow-derived dendritic cells (BMDCs), leading to increased generation of effector CD8+ T cells both in vitro and in vivo. We also revealed that HK-C60 stimulation augmented the activity of 20S immunoproteasome (20SI) in BMDCs, thereby enhancing the MHC class I-restricted antigen presentation machinery. Furthermore, we assessed the impact of HK-C60 on CD8+ T cell activation in an OVA-expressing B16-F10 murine melanoma model. Oral administration of HK-C60 significantly attenuated tumor growth compared to control treatment. Enhanced Ag processing and presentation machineries in DCs from both Peyer’s Patches (PPs) and lymph nodes (LNs) resulted in an increased tumor antigen specific CD8+ T cells. These findings shed new light on the role of LAB in MHC class-I restricted antigen presentation and activation of CD8+ T cells through functional modification of DCs.

Severe SARS-CoV-2 infection is associated with significant immune dysregulation involving different immune cell subsets. In this study, when analyzing critically ill COVID-19 patients versus those with mild disease, we observed a significant reduction in total and memory B cell subsets but an increase in naive B cells. Moreover, B cells from COVID-19 patients displayed impaired effector functions, evidenced by diminished proliferative capacity, reduced cytokine, and Ab production. This functional impairment was accompanied by an increased apoptotic potential upon stimulation in B cells from severely ill COVID-19 patients. Our further studies revealed the expansion of B cells expressing coinhibitory molecules (PD-1, PD-L1, TIM-1, VISTA, CTLA-4, and Gal-9) in intensive care unit (ICU)–admitted patients but not in those with mild disease. The coinhibitory receptor expression was linked to altered IgA and IgG expression and increased the apoptotic capacity of B cells. Also, we found a reduced frequency of CD24hiCD38hi regulatory B cells with impaired IL-10 production. Our mechanistic studies revealed that the upregulation of PD-L1 was linked to elevated plasma IL-6 levels in COVID-19 patients. This implies a connection between the cytokine storm and altered B cell phenotype and function. Finally, our metabolomic analysis showed a significant reduction in tryptophan but elevation of kynurenine in ICU-admitted COVID-19 patients. We found that kynurenine promotes PD-L1 expression in B cells, correlating with increased IL-6R expression and STAT1/STAT3 activation. Our observations provide novel insights into the complex interplay of B cell dysregulation, implicating coinhibitory receptors, IL-6, and kynurenine in impaired B cell effector functions, potentially contributing to the pathogenesis of COVID-19.

Creatine is an indispensable organic compound utilized in physiological environments; however, its role in immunity is still poorly understood. Here, we show that creatine supplementation enhances anti-tumor immunity through the functional upregulation of macrophages by increasing adenosine triphosphate (ATP) production. Creatine supplementation significantly suppressed B16-F10-originated tumor growth in mice compared with the control treatment. Under these conditions, intratumor macrophages polarized towards the M1 phenotype rather than the M2 phenotype, and there was an increase in tumor antigen-specific CD8+ T cells in the mice. The cytokine production and antigen-presenting activity in the macrophages were enhanced by creatine supplementation, resulting in a substantial increase in tumor antigen-specific CD8+ T cells. ATP upregulation was achieved through the cytosolic phosphocreatine (PCr) system via extracellular creatine uptake, rather than through glycolysis and mitochondrial oxidative phosphorylation in the macrophages. Blockade of the creatine transporter (CrT) failed to upregulate ATP and enhance the immunological activity of macrophages in creatine supplementation, which also impaired CD8+ T cell activity. Consequently, CrT blockade failed to suppress tumor growth in the creatine-supplemented mice. Thus, creatine is an important nutrient that promotes macrophage function by increasing ATP levels, ultimately contributing to enhanced anti-tumor immunity orchestrated by CD8+ T cells.

The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery.

Creatine is an organic compound which is utilized in biological activities, especially for ATP production in the phosphocreatine system. This is a well-known biochemical reaction that is generally recognized as being mainly driven in specific parts of the body, such as the skeletal muscle and brain. However, our report shows a novel aspect of creatine utilization and ATP synthesis in innate immune cells. Creatine supplementation enhanced immune responses in neutrophils, such as cytokine production, ROS production, phagocytosis, and NETosis, which were characterized as antibacterial activities. This creatine-induced functional upregulation of neutrophils provided a protective effect in a murine bacterial sepsis model. The mortality rate in mice challenged with E. coli K-12 was decreased by creatine supplementation compared with the control treatment. Corresponding to this decrease in mortality, we found that creatine supplementation decreased blood pro-inflammatory cytokine levels and bacterial colonization in organs. Creatine supplementation significantly increased the cellular ATP level in neutrophils compared with the control treatment. This ATP increase was due to the phosphocreatine system in the creatine-treated neutrophils. In addition, extracellular creatine was used in this ATP synthesis, as inhibition of creatine uptake abolished the increase in ATP in the creatine-treated neutrophils. Thus, creatine is an effective nutrient for modifying the immunological function of neutrophils, which contributes to enhancement of antibacterial immunity.