Background: Antibiotic residues in food and environmental matrices have raised increasing concern because of their risks to human health, ecological safety, and the spread of antimicrobial resistance. Although conventional instrumental methods offer high sensitivity and selectivity, their dependence on expensive equipment, complex pretreatment, and skilled operators limits rapid on-site application. Molecularly imprinted polymers (MIPs)based sensors have therefore emerged as promising alternatives owing to their high selectivity, stability, low cost, and compatibility with portable platforms. Scope and approach: This review systematically summarizes recent advances in MIPs-based sensors for antibiotic detection. A total of 213 original studies published between 2021 and February 10, 2026 were surveyed, and 60 representative studies were selected for in-depth discussion. The review covers MIPs fabrication methods, optimization strategies, and sensing applications across electrochemical, electrochemiluminescence, photoelectrochemical, fluorescence, colorimetric, and surface-enhanced Raman scattering platforms. Key findings and conclusions: Recent studies showed that computation-guided formulation design, synergistic monomer selection, oriented and surface imprinting, antifouling architectures, stimuli-responsive systems, and green synthesis strategies markedly improved the selectivity, sensitivity, and applicability of MIPs-based sensors. Electrochemical and fluorescence platforms were the most extensively investigated. Despite substantial progress, challenges remain in site homogeneity, matrix interference, reproducibility, and multi-analyte discrimination. Overall, this review highlights current advances and provides guidance for the rational design of highperformance MIPs-based sensors for antibiotic analysis in food safety and environmental monitoring.

Pesticide residues in vegetables have raised an extensive concern. However, the residues and integrated risks of multiple pesticides in various vegetables from Shandong province, a leading vegetable producer in China, remain unclear thus far. Herein, we performed a 4-year investigation on residues of 33 commonly-used pesticides in 23 daily-consumed vegetables from Shandong province during 2018-2021, and comprehensively assessed their chronic health risks for different age groups using both deterministic and probabilistic approaches. Among the 1067 samples, procymidone, carbendazim, and thiamethoxam were the top three detected pesticides. Notably, 6 prohibited pesticides, including carbofuran, omethoate, phorate, methamidophos, chlorpyrifos, and fipronil, were also found in vegetables. Based on the maximum residue limits (MRLs) stipulated in GB 2763, carbofuran, omethoate, and avermectins exhibited the highest over-MRL rates. Moreover, 34.7% of the samples contained pesticide residues. Multiple pesticides were simultaneously detected in 10.2% of the samples. Among multi-residue samples, the percentage of samples containing 2 pesticides was the highest, and the most common combination of residues was procymidone + carbendazim. Additionally, 2.3% of the samples were found with pesticides above the MRLs. More attention should be paid to leaf lettuce, kidney bean, leek, and celery, given the relatively higher over-MRL rates. Although we found that both the individual and cumulative exposure risks of these pesticides for different age groups were within established safety limits (below 1), the dietary exposure risks for children were higher than that for the other three age groups. Omethoate, carbofuran, phorate, and avermectins were the key contributors to the potential health risk for inhabitants. Given the ubiquity of pesticide residues as well as the detection and exceeding MRLs of banned pesticides, the potential health hazards of pesticides in vegetables should be continuously assessed. These findings provide a valuable reference for future pesticide management and risk assessment programs.

Abstract Liver fibrosis is a dynamic pathological consequence of chronic liver injury, in which persistent oxidative stress and inflammation drive progressive extracellular matrix deposition. Cyclic nitroxide radicals exhibit diverse biological activities, but their effects on liver fibrosis remain unclear. This study systematically evaluates the therapeutic potential of 3-carbamoyl proxyl nitroxide (3-CP) against carbon tetrachloride (CCl₄)-induced liver fibrosis. In vitro, 3-CP inhibited hepatic stellate cell (HSC) activation, migration, and proliferation, and reduced α-smooth muscle actin (α-SMA) and collagen I (COL1) expression. In a BALB/c mouse model of CCl4-induced liver fibrosis, 20 and 40 mg/kg 3-CP reduced the fibrosis area from 13.6 ± 1.0% (model group) to 6.9 ± 0.9% and 5.7 ± 1.3%, respectively, accompanied by decreased serum transaminase levels, restored liver architecture, and diminished collagen deposition. Mechanistic studies revealed that 3-CP modulated the TLR4/NF-κB signaling pathway, downregulating phosphorylated NF-κB p65 (p-p65) and reducing hepatic mRNA levels of pro-inflammatory (IL-1β, IL-6, TNF-α) and pro-fibrotic (TGF-β) cytokines by approximately 35–55%. Supportive in silico analysis suggested potential interactions between 3-CP and key pathway proteins (TLR4, MyD88, IKKβ, p65, IκBα). These findings indicate that 3-CP represents a promising therapeutic candidate that concurrently addresses oxidative damage and inflammatory signaling during liver fibrogenesis.

The effects of L-arginine on the activity and structure of invertase as well as the influence of β-cyclodextrin (β-CD), sodium alginate (SA), fenugreek gum (FG), and inulin on the denatured invertase were investigated. The results demonstrated that L‑arginine inhibits the activity of invertase, as this guanidinium compound alters the secondary structure of enzyme and disrupts the hydrogen‑bond network of water. In the presence natural polysaccharides, the activity and secondary structure of invertase were restored. The crowding factor of polysaccharides, as well as their ability to promote or disrupt the hydrogen-bond network of water, is directly correlated with the recovery of enzyme activity and the refolding of enzyme structure. Such correlations suggest that quantitative characterization of the physicochemical parameters of polysaccharide media is of great importance for the scientific analysis and prediction of enzyme activity and structural dynamics in complex food system.

Digestive enzyme stability, which depends on its macromolecular environments, is essential for maintaining its function during food processing, storage, and digestion. Conformational transitions of porcine pancreatic lipase (PPL) induced by urea-containing macromolecules including polyethylene glycol, dextran, and Ficoll, followed the unfolding process. The Ficoll 70 system showed two intermediate states of PPL. Lipase in Pluronics maintained a high proportion of the native conformation, illustrating the role of macromolecules in protecting the structure of lipase, in PEG and dextran. Intermediate states of lipase occurred in high concentrations of denaturants. The heterogeneity observed among intermediate states highlights the need for an effective quantitative framework to characterize conformational transitions in macromolecular environments. To further quantify these transitions, analysis of the equilibrium constants (τ) and denaturant binding numbers (m) revealed an antagonistic relationship, reflecting kinetic and thermodynamic differences between the N→I and I→U transitions. The quantitative model employing the equilibrium constant and denaturant binding number describes the conformational transitions of proteins in macromolecular media. Herein, a theoretical framework and experimental approach aid in understanding the regulating effects induced by exposure to food macromolecules and the biophysics of digestive enzymes.

An aptamer sensor for the rapid and sensitive recognition of oxytetracycline (OTC) was developed by modifying a screen-printed carbon electrode with Cu-based metal organic framework (MOF), electrochemically reduced graphene oxide (ErGO), and gold nanoparticles (AuNPs). The Cu-MOF had a large surface area and accommodated AuNPs and ErGO. The electrodeposition of AuNPs and the immobilization of ErGO improved the conductivity to amplify the sensor response. The developed sensor had an extremely broad linear range (0.1-105 ng/ mL) and a very low limit of detection (0.03 ng/mL), and it was highly stable, specific, and reproducible. When used to detect OTC in milk and pork samples, the recovery ranged from 87.0 % to 110.2 %.

Sodium alginate enhanced invertase activity, whereas fenugreek gum suppressed it. Further investigation into the physicochemical properties of the medium revealed a bidirectional regulatory mechanism. Sodium alginate, characterized by a positive logarithm of the crowding factor (ln Γ), a smaller excluded volume (Vₑₓ), and improved hydration, interacted with invertase via enthalpy-driven hydrogen bonding. This interaction stabilized the α-helix and β-sheet structures of the enzyme, thereby increasing its activity. In contrast, fenugreek gum exhibited a negative ln Γ, a larger excluded volume, and lower hydration capacity. It perturbed the enzyme through entropy-driven hydrophobic interactions, leading to a reduction in β-sheet content and a consequent decrease in enzymatic activity. The overall influence of the medium-comprising viscosity, macromolecular crowding, and soft interactions-enables quantitative assessment of how polysaccharides affect invertase activity. This study offers valuable insights for the rational selection of polysaccharides in dietary formulations aimed at modulating digestive enzyme activity.

Colorectal cancer is the second leading cause of cancer-related deaths worldwide, and its development typically involves complex metabolic reprogramming. By mapping the spatial distributions of metabolites and N-glycans in heterogeneous colorectal cancer tissues, we can elucidate cancer-associated metabolic and N-glycan changes. Herein, we combine mass spectrometry imaging-based metabolomics and N-glycomics to characterize the spatially resolved reprogramming of metabolites and N-glycans in colorectal cancer tissues. The metabolic characteristics of different regions of colorectal cancer were evaluated through the utilization of orthogonal partial least squares discriminant analysis. In combination with metabolic pathway enrichment analysis, significant alterations were identified in the fatty acid metabolism, arginine and proline metabolism of colorectal cancer. Cancer cell regions exhibited a marked upregulation of saturated fatty acids, monounsaturated fatty acids, polyamines, and histidine. Additionally, we discovered that the high-mannose N-glycans were predominantly distributed in tumor tissue regions, whereas complex N-glycans were more commonly found in the normal tissue regions adjacent to the tumor. Such findings provide new insights into the spatial signatures of metabolites and N-glycans in colorectal cancer, thereby offering a crucial basis for the diagnosis of colorectal cancer and potential vulnerabilities that might be targeted for cancer therapy.

ETHNOPHARMACOLOGICAL RELEVANCE:Schisandra chinensis, a renowned medicinal plant in traditional Chinese, Korean, and Russian ethnomedicine, has been widely used for its hepatoprotective, adaptogenic, and tonic properties. In traditional Chinese medicine, it is classified as a superior herb known to astringe essence, nourish the kidneys, and calm the mind. Historically, it has been prescribed for fatigue, insomnia, and respiratory ailments, but its most notable application lies in liver protection. It has long been used to treat liver disorders, including hepatitis and toxin-induced liver injury, aligning with modern pharmacological evidence demonstrating its potent hepatoprotective effects. AIM OF THE STUDY:This study aimed to develop a novel comprehensive two-dimensional primary Hepatic stellate cell (HSC) membrane chromatographic system to screen anti-fibrotic components from S. chinensis and to investigate the anti-fibrotic effects and mechanisms of gomisin N, a key lignan compound. MATERIALS AND METHODS:A novel bioaffinity-based chromatographic screening system was designed and integrated with mass spectrometry to identify compounds with high HSC membrane affinity. The anti-fibrotic effects of the identified compound were then evaluated in vitro using primary HSCs and in vivo using a carbon tetrachloride-induced liver fibrosis mouse model. Primary HSC membrane chromatography coupled with high-resolution time-of-flight mass spectrometry was used to screen S. chinensis components. The affinity of the identified compounds was validated, and gomisin N was selected for further study. Cellular assays assessed its effects on HSC activation, while in vivo studies evaluated its impact on liver fibrosis. Mechanistic studies involved Western blotting, immunofluorescence, and real-time quantitative reverse transcription PCR to assess inflammation, oxidative stress, hepatic microvascular formation, and apoptosis-related pathways. RESULTS:Gomisin N exhibited the highest retention time on the HSC membrane chromatographic column, indicating strong HSC binding affinity. It significantly inhibited HSC activation and proliferation in vitro. In the CCl4-induced liver fibrosis mouse model, gomisin N ameliorated liver fibrosis, reduced inflammatory cytokine expression, decreased oxidative stress markers, and suppressed hepatic microvascular formation. Mechanistically, it inhibited the Nuclear factor kappa B pathway, reduced hepatocyte apoptosis via regulation of B-cell lymphoma 2/Bcl-2-associated X protein and Caspase-3, and demonstrated a favourable safety profile. CONCLUSION:This study presents a novel two-dimensional primary HSC membrane chromatographic system for screening anti-fibrotic compounds and is the first to report the anti-fibrotic effects of gomisin N. The findings provide experimental evidence supporting gomisin N as a promising lead compound for anti-fibrotic drug development.

Clematis L. is a genus with global distribution and significant usage in traditional Chinese medicine. Traditionally, species authentication for Clematis L. has relied on morphological characteristics, but such method is susceptible to errors and lacks reproducibility. In this study, an untargeted UPLC-MS/MS-based metabolomics approach was employed to comprehensively discriminate Clematis tangutica (Maxim.) Korsh, C. intricata and Clematidis Radix et Rhizoma (CRR) from eight provinces in China. 2331 differential metabolites were identified by principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA), which revealed distinct separations among the studied regions. The KEGG metabolic pathway analysis showed that flavone and flavonol biosynthesis and flavonoid biosynthesis were closely associated with geographical origin. This work established the metabolomics evidence that flavonoid biosynthesis serves as a biochemical signature of geographical adaptation in Clematis, providing a scientific foundation for precise origin traceability, resource conservation, and quality standardization of medicinal species in traditional Chinese medicine.

Wheat is a staple food for about 35 % of the world's population, but it is susceptible to contamination by various substances. Ensuring the quality and safety of wheat is vital for protecting public health and maintaining consumer confidence. In this work, we collected 350 wheat samples from Shandong, China in 2020 and 2021 and determined the occurrence of type B trichothecenes, pesticides, and heavy metals. A probabilistic analysis based on Monte Carlo simulation was then used to assess the potential health risks of the measured substances, quantified by the hazard quotient (HQ) and the hazard index (HI) values. Deoxynivalenol (DON) was detected in 50 % of the collected wheat samples. Carbendazim, tebuconazole, and imidachloprid were the most detected pesticides. More than 70 % of the wheat samples exhibited heavy metal contamination. For children, the mean HI of trichothecenes was 1.06, and the HQ of DON and its acetyl derivatives at the 95th percentile was 1.13, which indicated notable health risk. The mean HI of pesticides was 0.89, and the HI of pesticides at the 95th percentile was 1.02. For adults, the mean HI of trichothecenes was 0.49, but the HI trichothecenes reached 1.04 and 1.33 at the 90th and 95th percentile, respectively. The mean HI of pesticides was 0.43, and the HI of pesticides at the 95th percentile was 0.78. For both children and adults, the non-carcinogenic risks of heavy metals were trivial, but there was probably carcinogenic risk from Cr. When substances across categories were considered in aggregate, the mean HI reached 1.95 for children, and the HI at the 75th percentile was 1.03 for adults. In view of the cumulative risk, environmentally sustainable pest and disease prevention and control techniques are needed for wheat cultivation in Shandong.

Curcumin is widely recognized for its diverse antitumor properties, ranging from breast cancer to many other types of cancers. However, its role in the tumor microenvironment remains to be elucidated. In this study, we established a 3D tumor spheroids model that can simulate the growth environment of tumor cells and visualized the antitumor metabolic alteration caused by curcumin using mass spectrometry imaging technology. Our results showed that curcumin not only exerts a profound impact on the growth and proliferation of breast cancer cells but in situ multivariate statistical analysis also reveals the significant effect on the overall metabolic profile of tumor spheroids. Meanwhile, our visualization map characterized curcumin metabolic processes of reduction and glucuronidation in tumor spheroids. More importantly, abnormal metabolic pathways related to lipid metabolism and polyamine metabolism were also remodeled at the metabolite and gene levels after curcumin intervention. These insights deepen our comprehension of the regulatory mechanism of curcumin on the tumor metabolic network, furnishing powerful references for antitumor treatment.

Enhancing the sensitivity of targeted substance detection is crucial, yet prior research seldom incorporates more than two signal amplification methods. In this study, we introduced a novel triple signal amplification strategy for tetracycline detection using an aptasensor. This strategy integrates a graphene and multi-walled carbon nanotubes composite (GO-MWCNTs), Exonuclease I (Exo I), and a hybrid DNA-gold nanoparticle (AuNPs)horseradish peroxidase (HRP) system. The GO-MWCNTs serve as a conductive carrier, boosting electron transfer for initial signal amplification. Exo I, targeting single-stranded DNA, facilitates target recovery and secondary signal amplification. The gold nanoprobe, through specific base pairing, binds to the tetracycline aptamer's complementary chains on the electrode surface. Horseradish peroxidase's catalytic action then generates a robust electrochemical signal, culminating in three-stage signal amplification. This optimized approach achieved a low detection limit of 3.3 x 10-4 ng mL- 1, with a range from 1 x 10-3 ng mL- 1 to 1 x 103 ng mL- 1. Notably, the aptasensor demonstrated high selectivity, repeatability, stability, and reliability. These findings offer a promising reference for developing effective aptasensors in antibiotic detection.

Eclipta prostrata L. has been used in traditional medicine and known for its liver-protective properties for centuries. Wedelolactone (WEL) and demethylwedelolactone (DWEL) are the major coumarins found in E. prostrata L. However, the comprehensive characterization of these two compounds on non-alcoholic fatty liver disease (NAFLD) still remains to be explored. Utilizing a well-established zebrafish model of thioacetamide (TAA)-induced liver injury, the present study sought to investigate the impacts and mechanisms of WEL and DWEL on NAFLD through integrative spatial metabolomics with liver-specific transcriptomics analysis. Our results showed that WEL and DWEL significantly improved liver function and reduced the accumulation of fat in the liver. The biodistributions and metabolism of these two compounds in whole-body zebrafish were successfully mapped, and the discriminatory endogenous metabolites reversely regulated by WEL and DWEL treatments were also characterized. Based on spatial metabolomics and transcriptomics, we identified that steroid biosynthesis and fatty acid metabolism are mainly involved in the hepatoprotective effects of WEL instead of DWEL. Our study unveils the distinct mechanism of WEL and DWEL in ameliorating NAFLD, and presents a “multi-omics” platform of spatial metabolomics and liver-specific transcriptomics to develop highly effective compounds for further improved therapy.

Liver's distinctive function renders it highly susceptible to diverse damage sources. Characterizing the metabolic profiles and spatial signatures in different liver injuries is imperative for early diagnosis and etiology-oriented treatment. In this comparative study, we conducted whole-body spatial metabolomics on zebrafish with liver injury induced by ethanol (EtOH), acetaminophen (APAP), and thioacetamide (TAA). The two specific levels, the whole-body and liver-specific metabolic profiles, as well as their regional distributions, were systematically mapped in situ by mass spectrometry imaging, which is distinct from conventional LC-MS and GC-MS methods. We found that liver injury regions exhibited more pronounced metabolic reprogramming than the entire organism, leading to significant alterations in eight fatty acids, three phospholipids, and four low-molecular-weight metabolites. More importantly, fatty acids as well as small molecule metabolites including glutamine, glutamate, taurine and malic acid displayed contrasting changes between alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). In addition, phospholipids, including Lyso PC (16:0) and Lyso PE (18:0), demonstrated notable down-regulation in all damaged liver, whereas PC (34:1) underwent upregulation. This study not only deepens insights into distinct potential biomarkers for liver injuries, but also underscores spatial metabolomics as a powerful tool to elucidate possible pathogenic mechanisms in other metabolic diseases.

Berberine (BR), an alkaloid isolated from the Chinese traditional medicine Coptidis rhizoma, exhibits therapeutic effects on several diseases including bacterial infections, diabetes, and hyperlipidemia, but the oral availability is poor. In this work, we prepared the chitosan microneedle array–loaded BR (BR-CS MNAs) to transdermally deliver BR, and the spatial distribution of BR in heterogeneous skin tissues was analyzed and imaged by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Some endogenous phospholipids with specific spatial distribution were used to differentiate the epidermis and dermis regions of the skin. The results showed that BR was effectively delivered and could permeate to both epidermis and dermis regions of the skin. This demonstrated the feasibility of MALDI-MSI to evaluate the transdermal delivery efficiency of microneedle arrays and suggested BR could be transdermally delivered by CS MNAs. The chitosan microneedle array–loaded BR (BR-CS MNAs) was prepared and used to transdermal deliver BR. The spatial distribution of BR during the transdermal deliver process was analyzed and imaged by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Some endogenous phospholipids with specific spatial distribution were used to help differentiate the epidermis and dermis regions of skin

In the present study, we aimed to evaluate the concentration and health risk of aflatoxin B1 (AFB1), heavy metals (HMs), and phthalate acid esters (PAEs) in peanut samples in Shandong Province, China, and 255 peanut samples were collected from 20 counties in 2020. AFB1, cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), arsenic (As), Di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and diisobutyl phthalate (DIBP) were detected by the UPLC-MS method, GC-MS method, and ICP-MS method. We found that 4.7 % of samples were contaminated by AFB1. The margin of exposure (MOE) values of AFB1 ranged from 3549 to 3281 (LB to UB), which were lower than the threshold. At the mean and high percentile (P97.5), the liver cancer risk associated with AFB1 was lower than the annual incidence of liver cancer in China (17.7 cases/105 persons·per year), indicating that consumption of peanuts would not pose significant risks of cancer. Moreover,100 % of peanut samples were contaminated by Cd, Pb, and Cr, and 73.3 % and 61.5 % of samples were contaminated by Hg and As, respectively. In addition, 100 %, 98 %, and 99.3 % of peanut samples were contaminated by DEHP, DBP, and DIBP, respectively. The mean and P97.5 values for THQ and HI of HMs and PAEs were found to be less than 1, indicating that there was no significant health risk associated with peanut consumption among the local population in Shandong Province. The P97.5 HI values for the cumulative risk of HMs and PAEs were found to be less than 1, indicating that consuming peanuts may not pose a significant health risk from these substances. However, as peanut oil is the primary way in which peanuts are consumed, measures to mitigate and enrich AFB1, HMs, and PAEs should not be overlooked. Collectively, the effects of these chemicals on peanut products and the health risks to humans should be further studied.

Short-chain fatty acids (SCFAs), as the main metabolites of gut microbiota, are recognized as crucial players in the host's inflammatory response and metabolic disease. Imaging the spatial distributions and calculating the accurate contents of SCFAs in the heterogeneous intestinal tissue are critical to reveal their biological functions. Here, we develop an isotope-coded on-tissue derivatization method combined with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to map the spatial expressions of SCFAs in the colon tissue based on pair-labeled N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide (TMPA) and D3-TMPA. A noticeable increase in the MALDI-MSI sensitivity of SCFAs was achieved after on-tissue derivatization, which enables the visualization of acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, hydroxy acetic acid, and hydroxy propionic acid in the colon tissue. Moreover, the introduction of D3-TMPA-tagged SCFAs as internal standards can significantly reduce quantitation deviation from the matrix effects, ensuring the quantitative MALDI-MSI of SCFAs. We further used this method to characterize the spatial alterations of SCFAs in the colon tissues of mice with enterocolitis. The development of this strategy provides a reliable approach to image the spatial expressions of SCFAs in tissues and paves an insight way to study the roles of SCFAs in the gut microbiota and disease.

Carbohydrates play crucial regulatory roles in various physiological and pathological processes. However, the low ionization efficiency and the presence of linkage pattern, monosaccharide composition and anomeric configuration isomers make their in-depth analysis very challenging, especially for heterogeneous biological tissues. In this study, we propose a high-sensitive and isomer-specific imaging approach to visualize the spatial distributions of monosaccharide and disaccharide isomers by integrating chemical derivatization and matrix-assisted laser desorption/ionization tandem mass spectrometry imaging (MALDI-MS2I). 2-Pyridinecarbohydrazide (PYD) is developed as a novel derivatization reagent which can not only improves the MS sensitivity of carbohydrates, but also enables the identification and visualization of ketose and aldose monosaccharide isomers, as well as linkage pattern, monosaccharide composition and anomeric configuration disaccharide isomers by mass spectrometry imaging of isomer-specific MS/MS fragment ions. Moreover, we build quantitative MALDI-MS2 and MALDI-MS2I methods for disaccharide isomers based on the diagnostic fragment ions, and good linear relationships could be achieved both in solution and on glass slides. We expect that this study should provide new ideas for in-depth profiling of the spatial signatures of carbohydrates in biological tissues and lay the foundation for a deeper understanding of carbohydrates' structure.

The metabolic cross-talk between tumor and immune cells plays key roles in immune cell function and immune checkpoint blockade therapy. However, the characterization of tumor immunometabolism and its spatiotemporal alterations during immune response in a complex tumor microenvironment is challenging. Here, a 3D tumor-immune cell coculture spheroid model was developed to mimic tumor-immune interactions, combined with mass spectrometry imaging-based spatially resolved metabolomics to visualize tumor immunometabolic alterations during immune response. The inhibition of T cells was simulated by coculturing breast tumor spheroids with Jurkat T cells, and the reactivation of T cells can be monitored through diminishing cancer PD-L1 expressions by berberine. This system enables simultaneously screening and imaging discriminatory metabolites that are altered during T cell-mediated antitumor immune response and characterizing the distributions of berberine and its metabolites in tumor spheroids. We discovered that the transport and catabolism of glutamine were significantly reprogrammed during the antitumor immune response at both metabolite and enzyme levels, corresponding to its indispensable roles in energy metabolism and building new biomass. The combination of spatially resolved metabolomics with the 3D tumor-immune cell coculture spheroid visually reveals metabolic interactions between tumor and immune cells and possibly helps decipher the role of immunometabolic alterations in tumor immunotherapy.