Metabolomic reprogramming of the tumor microenvironment by dual arginase inhibitor OATD-02 boosts anticancer immunity

Metabolomic reprogramming of the tumor microenvironment by dual arginase inhibitor OATD-02 boosts anticancer immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Metabolomic reprogramming of the tumor microenvironment by dual arginase inhibitor OATD-02 boosts anticancer immunity Marcin Mikołaj Grzybowski, Yasemin Uçal, Angelika Muchowicz, Tomasz Rejczak, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6305179/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 May, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Metabolic reprogramming within the tumor microenvironment (TME) plays a central role in cancer progression and immune evasion, with L-arginine metabolism emerging as a key regulatory axis. Arginase overexpression depletes intratumoral L-arginine, thus suppressing T-cell proliferation while fuelling tumor growth through polyamine biosynthesis. OATD-02, a novel dual arginase (ARG1/ARG2) inhibitor, reprograms tumor metabolism by restoring L-arginine availability and reducing the levels of polyamines, thereby shifting the TME toward a more immunostimulatory state. Unlike ARG1-selective inhibitors with limited intracellular uptake, OATD-02 effectively inhibits both extracellular and intracellular arginases, thereby addressing a major limitation of first-generation arginase inhibitors. To visualize the pharmacodynamic effects of OATD-02 dosing in mice with spatial resolution, we employed MALDI mass spectrometry imaging (MALDI-MSI), thus enabling direct mapping of metabolic changes within tumor tissues. In preclinical models, OATD-02 treatment led to widespread accumulation of intratumoral L-arginine with concomitant depletion of polyamines and resulted in metabolic shifts that correlated with increased immune cell infiltration and an improved response to immune checkpoint blockade. These findings underscore the role of dual arginase inhibition in reshaping tumor metabolism and overcoming immune suppression by restoring the metabolic fitness of immune cells to fight cancer. The metabolic changes caused by OATD-02 treatment resulted in significantly enhanced antitumor immune responses, increased T-cell infiltration in tumors, expansion of CD8⁺ T cells in draining lymph nodes, and systemic upregulation of T-cell activation markers. These effects translated into a substantial survival benefit in the CT26 tumor model, particularly when combined with anti-PD-1 therapy, where OATD-02 improved checkpoint blockade efficacy by relieving metabolic constraints affecting tumor-infiltrating lymphocytes. By leveraging the unique capabilities of MALDI-MSI, this study provides high-resolution metabolic insights into the mechanism of action of OATD-02, reinforcing its potential as a next-generation metabolic-immunotherapeutic agent. The observed metabolic reprogramming, coupled with enhanced immune activation and prolonged survival, supports the clinical development of OATD-02 as a promising strategy for enhancing cancer immunotherapy efficacy. OATD-02 is currently undergoing clinical evaluation in a phase I/II trial (NCT05759923), which will further elucidate its safety and therapeutic impact. These findings highlight the potential of arginase-targeted therapies in cancer treatment and underscore the value of MALDI-MSI as a powerful tool for tracking metabolic responses to therapy. Biological sciences/Cancer/Cancer imaging Biological sciences/Drug discovery/Target validation Biological sciences/Drug discovery/Pharmaceutics Biological sciences/Immunology/Tumour immunology Biological sciences/Cancer/Cancer metabolism Biological sciences/Cancer/Cancer microenvironment Biological sciences/Cancer/Cancer models Biological sciences/Cancer/Cancer therapy/Drug development Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Health sciences/Oncology/Cancer/Cancer metabolism Health sciences/Oncology/Cancer/Cancer imaging Dual arginase inhibition OATD-02 MALDI imaging metabolic reprogramming arginine metabolism polyamines mitochondrial metabolism tumor metabolism anticancer therapy immune modulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Cancer cells undergo profound metabolic reprogramming to sustain proliferation, evade immune surveillance, and adapt to environmental stressors such as nutrient deprivation and hypoxia. Among the key metabolic pathways, L-arginine metabolism plays crucial roles in tumor growth and immune regulation ( 1 – 3 ). L-arginine is a conditionally essential amino acid involved in multiple biochemical processes, including polyamine biosynthesis ( 4 , 5 ), nitric oxide (NO) production ( 6 , 7 ), and proline metabolism ( 8 – 10 ), all of which influence tumor progression ( 8 , 11 – 13 ) and immune cell function ( 14 – 16 ). L-arginine degradation is regulated primarily by two enzymes: arginase (ARG1, ARG2) and nitric oxide synthase (NOS). As illustrated in Fig. 1 , ARG1, which is predominantly cytosolic, is a key component of the urea cycle in hepatocytes, where it catalyzes the hydrolysis of L-arginine to L-ornithine and urea, facilitating ammonia detoxification. ARG2, which is localized in mitochondria, performs a similar reaction but is more broadly involved in metabolic adaptation. L-ornithine serves as a precursor for polyamine biosynthesis (putrescine, spermidine, and spermine), which promotes tumor proliferation, and for proline synthesis, which plays a key role in extracellular matrix remodeling and redox homeostasis ( 8 , 17 ). NOS, on the other hand, converts L-arginine into NO, which can support tumor growth by promoting angiogenesis or inducing tumor cell death through immune-mediated cytotoxicity ( 7 ). Dysregulated L-arginine metabolism significantly influences the tumor microenvironment (TME), mediating interactions between tumor cells, stromal fibroblasts, and immune cells ( 18 , 19 ). Elevated arginase activity is detected in multiple malignancies, including lung, colorectal, breast, and prostate cancers, where it is correlated with poor prognosis, increased tumor invasiveness, and enhanced metastatic potential ( 20 , 21 ). Arginase-driven L-arginine depletion promotes tumor cell proliferation by increasing L-ornithine availability, enhancing polyamine synthesis, cell cycle progression, and resistance to apoptosis. Additionally, proline biosynthesis contributes to extracellular matrix remodeling, further facilitating tumor progression ( 8 ). Notably, L-arginine depletion impairs T-cell activation and proliferation by downregulating the expression of the CD3ζ chain, a key component of the T-cell receptor (TCR) complex, thereby suppressing antitumor immunity ( 22 ). Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) further increase ARG1 expression, exacerbating L-arginine depletion and creating an immune-privileged environment that facilitates tumor immune evasion ( 23 ). This metabolic alteration reduces the efficacy of immune checkpoint inhibitors, highlighting the need for strategies targeting arginase activity ( 14 , 15 ). In addition to its role in metabolic regulation, ARG2 is now recognized as a critical regulator of both tumor-intrinsic metabolism and immune responses. In contrast to ARG1, which primarily depletes extracellular L-arginine, ARG2 functions within mitochondria, where it directly influences tumor metabolic adaptation, nitrogen balance, and immune regulation (Fig. 1 ). In tumors, ARG2 expression is linked to glutaminolysis, polyamine biosynthesis, and mitochondrial metabolism, allowing cancer cells to survive in nutrient-deprived environments ( 24 , 25 ). The loss of ARG2 in pancreatic ductal adenocarcinoma (PDA) models results in nitrogen accumulation and impaired tumor growth, highlighting its role as a metabolic vulnerability factor in certain cancers ( 24 ). In addition to its metabolic functions, ARG2 acts as a cell-intrinsic regulator of CD8⁺ T cell activation, persistence, and antitumor efficacy ( 26 ). Unlike ARG1, which primarily affects T cells by depleting extracellular arginine, mitochondrial ARG2 regulates intracellular arginine metabolism, shaping T-cell fate independently of extracellular arginine availability. Deletion of ARG2 in CD8⁺ T cells enhances their expansion, effector function, and persistence, leading to improved tumor control in preclinical cancer models. Moreover, ARG2-deficient CD8⁺ T cells exhibit strong synergy with PD-1 blockade, suggesting that targeting ARG2 could enhance the efficacy of immune checkpoint therapies ( 26 ). Arginase inhibitors have been studied for over a century, with early studies identifying α-amino acids as weak inhibitors. A major breakthrough came with N-hydroxy-nor-arginine (nor-NOHA), a micromolar inhibitor that provided structural insights into arginase inhibition ( 27 ). This led to the development of boronic acid-based inhibitors such as 2(S)-amino-6-boronohexanoic acid (ABH) and ( S )-(2-boronoethyl)-L-cysteine (BEC), which are known for their potency and selectivity ( 17 ). These compounds laid the groundwork for more advanced inhibitors, such as OATD-02 and numidargistat. Preclinical studies have demonstrated that numidargistat (INCB001158) reversed T-cell immunosuppression and reduced tumor growth in multiple syngeneic mouse models, particularly when combined with anti-PD-L1 therapy, highlighting its potential to modulate the tumor immune microenvironment ( 28 ). However, despite pharmacodynamic evidence of arginase inhibition and increased plasma arginine levels in a phase I/II clinical trial, its antitumor efficacy has remained limited, suggesting the complexity of arginine metabolism in cancer ( 29 ). A key distinction between numidargistat and OATD-02 lies in their ability to inhibit intracellular ARG2. While numidargistat primarily targets extracellular ARG1, its limited intracellular penetration restricts its effect on ARG2-dependent metabolism in tumor and immune cells ( 30 ). In contrast, OATD-02 is designed to effectively inhibit both intracellular and extracellular arginases, granting it a broader and potentially stronger pharmacodynamic profile ( 31 , 32 ). The ongoing phase I/II clinical trial of OATD-02 aims to evaluate these multidimensional antitumor properties in patients with advanced solid tumors, providing a more comprehensive understanding of its therapeutic potential ( 33 ). To evaluate the intracellular effects of OATD-02 on tumor metabolism directly in tissue, the advanced imaging technique MALDI-MSI was used to assess spatially resolved metabolic alterations induced by arginase inhibition ( 34 , 35 ). This approach enabled the direct visualization of changes in key metabolites, including L-arginine and polyamines, within tumor tissue, complementing bulk quantification methods such as HPLC and LC-MS. By integrating MALDI-MSI with systemic pharmacokinetic and metabolic analyses, this study examines how OATD-02 modulates the tumor microenvironment. The spatial distribution of metabolic changes offers insights into the extent and localization of OATD-02-induced effects, contributing to a broader understanding of its potential as a metabolic modulator in cancer therapy and its role in enhancing immune responses in combination with immune checkpoint inhibitors. Methods Chemical compounds OATD-02 was synthesized at Molecure SA. For in vitro assays, OATD-02 was dissolved in Milli-Q water (Millipore) at a stock concentration of 20 mM and stored at -20°C until use. For in vivo studies, OATD-02 was dissolved in sterile saline at a final concentration of 10 mg/mL and stored at -20°C until use. Prior to administration, the solution was prewarmed to RT and used within its validated minimal stability period. Unless otherwise specified, all additional chemical reagents, including analytical standards, were obtained from Merck (Germany). Cell culture The CT26.WT (CRL-2638, mouse colon carcinoma) and K562 (CCL-243, human chronic myelogenous leukemia) cell lines were purchased from ATCC®. Both cell lines were maintained in RPMI-1640 medium (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher/Gibco™), 100 U/mL penicillin G and 100 µg/mL streptomycin (Antibiotic-Antimycotic, Gibco®) at 37°C in a humidified atmosphere containing 5% CO₂. CT26 cells were cultured as adherent monolayers and passaged upon 70–80% confluence using 0.25% trypsin-EDTA (Gibco®). Nonadherent K562 cells were maintained in suspension culture and passaged every 2–3 days by dilution with fresh medium to sustain logarithmic growth. The absence of mycoplasma contamination in both cell lines was confirmed via the MycoAlert™ Mycoplasma Detection Kit (Lonza). Cellular metabolite detection via HPLC The detection of amino acids and polyamines in CT26 and K562 cell extracts was performed via high-performance liquid chromatography (HPLC) with dabsyl derivatives, following a modified protocol of Krause et al. (36). CT26 adherent cells were seeded at a density of 750,000 cells per 10 mL in 75 cm² flasks and treated with 10 µM or 30 µM OATD-02. K562 suspension cells were seeded at a density of 2×10⁶ cells per 10 mL in 75 cm² flasks and treated with OATD-02 at the same concentrations. Treatment was initiated either 2 h postseeding, resulting in a total incubation time of 96 h, or two days postseeding, resulting in a total incubation time of 48 h. At the experimental endpoint, the CT26 monolayers were washed with 5 mL of prewarmed (37°C) 0.3 M mannitol solution. For K562 nonadherent cells, an initial centrifugation step was performed to pellet the cells, followed by rinsing with warm 0.3 M mannitol. Metabolite extraction was carried out via the addition of 1.5 mL of a cold extraction mixture consisting of HPLC-grade methanol, acetonitrile, and water at a 2:2:1 ratio. The cells were incubated on a laboratory shaker at 4°C for 5 min and then detached via cell scrapers (for CT26) or resuspended (for K562) in the extraction mixture. The resulting suspensions were centrifuged (10,000 × g, 10 min, 4°C), and the supernatants were transferred to coded tubes to ensure blinded analysis before being stored at −80°C until further HPLC processing. Dabsyl derivatization was performed by mixing 50 µL of NaHCO₃ buffer (0.4 M, pH 9) with 50 µL of a deproteinized sample or standard. Two hundred microliters of freshly prepared dabsyl chloride reagent (4 mg/mL in acetone) was subsequently added. The mixture was incubated at 70°C for 21 min with intermittent mixing. The reaction was terminated by adding 200 µL of cold dilution buffer (acetonitrile, ethanol, and sodium acetate buffer at a 2:1:1 ratio), followed by incubation on ice for 5 min. The samples were then centrifuged, and the clear supernatants were immediately analyzed by HPLC. HPLC separation was performed via a Dionex 3000 ICS system equipped with an Agilent Zorbax SB-C18 column (4.6 × 250 mm) maintained at 40°C. The mobile phase consisted of 45 mM sodium acetate buffer (pH 3.5; mobile phase A) and 100% acetonitrile (mobile phase B). The compounds were eluted at a flow rate of 1 mL/min via a gradient starting at 20% phase B, increasing to 100% over 40 min, followed by re-equilibration to 20% over 20 min. The total run time was 60 min. Detection was performed via a UV–VIS detector set at 436 nm. Quantification was achieved by comparing peak areas to standard calibration curves. Standard calibration curves were prepared via serial dilutions of reference standards in the same solvent matrix as the samples to ensure accurate quantification. LC-MS/MS analysis The concentrations of OATD-02 and L-arginine in blood serum were quantified via liquid chromatography coupled with tandem mass spectrometry (LC‒MS/MS). Sample preparation involved protein precipitation with acetonitrile, followed by centrifugation. The resulting supernatants were analyzed via hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry, which employs optimized multiple reaction monitoring (MRM) transitions. The quantification of OATD-02 was performed via matrix-matched calibration and quality control samples. Owing to the endogenous nature of L-arginine, a surrogate matrix approach was applied to ensure accurate detection and quantification. MALDI-MSI imaging Frozen mouse tumors were sectioned at 12-μm thickness and mounted onto indium-tin oxide (ITO)-coated glass slides (Bruker Daltonics, Bremen, Germany). The slides were stored in slide boxes, vacuum-sealed in foil (CASO, Arnsberg, Germany), and kept at −80°C until further processing. Prior to matrix application, the slides were equilibrated to room temperature (RT), removed from vacuum packaging, and dried for 10 min in a vacuum desiccator (SP Bel-Art). A deuterated standard of the OATD-02 derivative (5 mg/mL in 50% methanol) was prepared, and 100 µL was mixed with 5 mL of DHB matrix solution (40 mg/mL in 70% methanol). The matrix was uniformly applied via an M5 pneumatic sprayer (HTX Technologies LLC, Chapel Hill, North Carolina, USA) with the following parameters: 11 layers, flow rate of 0.05 mL/min, velocity of 1200 mm/min, track spacing of 3 mm, and CC pattern at a nozzle temperature of 65°C. MALDI-MSI measurements were performed via a timsTOF flex mass spectrometer (Bruker Daltonics, Bremen, Germany). External mass calibration was conducted via sodium formate clusters in electrospray ionization (ESI) mode, and online calibration was performed with the deuterated standard. Imaging was carried out with a lateral step size of 40 µm in the mass range of m/z 90–1300 in positive ion mode. Spectra were acquired via 400 shots per pixel. The following MS parameters were applied: Funnel 1 RF: 250 Vpp, Funnel 2 RF: 250 Vpp, Multipole RF: 250 Vpp, MS1 collision energy: 2 eV, Collision cell RF: 650 Vpp, low mass: m/z 90, TOF transfer time: 70 µs, TOF prepulse storage: 4 µs. The MALDI–MS data were processed via DataAnalysis 6.1 and SCiLS Lab 2025 software (Bruker Daltonics). The spectral intensities were normalized to the deuterated standard to ensure quantitative reliability. Statistical and spatial analyses were performed to assess metabolite distribution in the tissue sections. Animal studies All in vivo experiments were conducted using 7–9-week-old female BALB/c (BALB/cAnNCrl) mice obtained from Charles River Laboratories (certified SPF status). All procedures complied with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guidelines for the Welfare and Use of Animals in Cancer Research. Ethical approval for the study was granted by the 1st Local Ethics Committee for Animal Experiments in Warsaw, Poland (approval no. 891/2019). CT26 cells were harvested during the exponential growth phase, with viability exceeding 90%, as confirmed by trypan blue exclusion. The mice were subcutaneously implanted in the right flank with 5 × 10⁵ CT26 cells suspended in 50 µL of PBS. The animals were then randomized into experimental groups. Tumor growth was monitored at least three times per week via caliper measurements and calculated according to the following formula: width × length × depth × π/6, assuming an ellipsoidal shape. Humane endpoints were defined as body weight loss exceeding 20%, a tumor volume surpassing 2000 mm³, or the presence of persistent signs of pain or distress. Animals meeting these criteria were euthanized. For LC–MS and MALDI–MSI analyses, tumor-bearing mice were orally administered OATD-02 at a dose of 100 mg/kg. Blood, tumors and selected organs were collected after either three doses (16 h time point) or four doses (2 h time point). The control animals received an equivalent volume of vehicle (saline). The collected tissues were snap-frozen and stored at −80°C until further analysis (see the MALDI-MSI Imaging section for a detailed description). In the efficacy study, the mice were treated orally with OATD-02 at 100 mg/kg twice daily, starting one day after tumor implantation. The anti-mouse CD279 (PD-1) IgG2a rat antibody (clone RMP1-14, cat. 114115, lot B306588, BioXCell) was administered intraperitoneally at 2.5 mg/kg on days 6, 10, 14, and 18 postimplantation. The control animals received saline via oral gavage and an anti-KLH isotype control IgG2b rat antibody (clone LTF-2, cat. BE0090, lot 629816D1, BioXCell) via intraperitoneal injection. On day 15, six randomly selected mice from each group (n = 18) were sacrificed, and the tumors, spleens, and tumor-draining lymph nodes were collected for cytometric analysis (see the LC–MS/MS analysis and flow cytometry sections for details on sample processing). To minimize animal discomfort, appropriate analgesic and anesthetic measures were applied, and humane endpoints were consistently enforced. At the end of the study, the animals were deeply anesthetized with an intraperitoneal injection of ketamine (150 mg/kg) and xylazine (15 mg/kg). Euthanasia was performed via cervical dislocation in accordance with ethical guidelines. Flow cytometry The collected tumors, spleens, and lymph nodes were cut into small fragments (2–3 mm). Tumors and lymph nodes were enzymatically digested in DMEM containing collagenase (0.8 mg/mL) and DNase I (15 U/mL) for 40 min at 37°C in a CO₂ incubator. Following digestion, tumor and lymph node suspensions, along with spleen fragments, were passed through cell strainers to obtain single-cell suspensions. The cells were then washed with PBS and treated with ACK erythrocyte lysis buffer for 5 min on ice. Lysis was stopped by the addition of PBS, followed by centrifugation. The resulting cell pellets were resuspended in PBS, and the cell density and viability were assessed. Prior to antibody staining, dead cells were labeled via the Zombie Aqua™ Fixable Viability Kit (BioLegend). The following anti-mouse antibodies were used for flow cytometry: CD8 (PerCP-Cy5.5, clone 53-6.7, BioLegend), CD3 (APC, clone 17A2, eBioscience), CD4 (BV605, clone GK1.5, BioLegend), CD69 (PE, clone H1.2F3, eBioscience), CD45.2 (V500, clone 104, BioLegend), CD3e (PE-Cy7, clone 145-2C11, eBioscience), and CD11b (PE, clone M1/70, eBioscience). The samples were analyzed via a CytoFLEX Analyzer (Beckman Coulter), and the data were processed via FlowJo software (BD Biosciences). Statistical analysis Statistical analyses were performed using GraphPad Prism (version 10.0). Data distribution was assessed via D’Agostino-Pearson and Shapiro-Wilk normality tests. The sample size for in vivo studies was determined on the basis of a retrospective analysis of previous optimization experiments, ensuring a statistical power of approximately 80% with an alpha level of 0.05. Given the relatively small sample sizes in the in vivo experiments, which were balanced to maintain statistical sensitivity while adhering to the 3R principles, and the nonnormal distribution observed for some variables, statistical comparisons were performed via nonparametric tests to ensure methodological consistency and robustness. The results are presented as the median, with individual data points shown as scatter dot plots. Multiple group comparisons were conducted via the Kruskal‒Wallis test followed by Dunn’s post hoc test, and nonsignificant differences were reported alongside the observed trends when applicable. Metabolite concentrations obtained by HPLC were normalized relative to untreated control values to facilitate cross-condition comparisons. Normalization was performed using the formula “Normalized value = Treated/Mean untreated”. The resulting normalized values were averaged across replicates and visualized as heatmaps, illustrating relative changes in metabolite levels in response to treatment. Survival analysis was conducted using Kaplan-Meier survival curves, with comparisons performed using the log-rank (Mantel‒Cox) test. Additionally, the Gehan-Breslow-Wilcoxon test was applied to assess early differences in survival dynamics. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated via the Mantel–Haenszel method to estimate the relative risk of death between treatment groups. Median survival times and statistical comparisons were reported for each treatment condition. Differences were considered statistically significant at p ≤ 0.05. Results OATD-02 remodels tumor cell metabolism To evaluate the metabolic impact of intracellular arginase inhibition by OATD-02, we analyzed the intracellular levels of key metabolites involved in L-arginine metabolism, including L-arginine, L-ornithine, polyamines (spermidine and spermine), glutamine, and proline, in CT26 and K562 cell extracts via HPLC. We selected CT26 (murine colorectal carcinoma) and K562 (human chronic myeloid leukemia) cells because of their distinct tumor biology and relevance to in vivo models, allowing us to assess the direct anticancer metabolism-related effects of OATD-02. CT26 cells represent a murine model, enabling in vivo studies in an immunocompetent tumor environment, whereas K562 cells are human lymphoblasts that exhibit elevated ARG2 expression, making them an optimal system for assessing the impact of dual ARG1/ARG2 inhibition (37). Furthermore, OATD-02 has been tested in vivo in both models (38), reinforcing its relevance in evaluating tumor metabolism and therapeutic efficacy. The selection of OATD-02 concentrations was guided by pharmacokinetic data from in vivo studies, where a 100 mg/kg dose of OATD-02 resulted in plasma and tumor concentrations of approximately 5 µM and 30 nmol/g, respectively, two hours postdosing (38). Given the relatively short exposure, 10 and 30 µM were selected for in vitro experiments as physiologically relevant concentrations, reflecting the levels observed in the animal model. Heatmap visualization ( Figure 2 ) illustrates the relative metabolite levels normalized to those of untreated controls across different treatment conditions. OATD-02 treatment resulted in a dose- and time-dependent increase in the intracellular L-arginine level. After 48 hours, compared with untreated control cells, CT26 ( Figure 2, left panel ) and K562 cells ( Figure 2, right panel ) treated with 30 µM OATD-02 presented 1.57-fold and 1.62-fold increases in L-arginine levels, respectively. This effect was further amplified at 96 hours, reaching a 1.80-fold increase in CT26 cells and a 1.87-fold increase in K562 cells at the highest concentration tested. These findings confirm that OATD-02 effectively enhances L-arginine accumulation within tumor cells, supporting its mechanistic role as an intracellular arginase inhibitor. As expected, OATD-02 treatment significantly suppressed L-ornithine levels, confirming effective arginase inhibition. A marked reduction in intracellular L-ornithine was observed in CT26 cells, where levels decreased to 30% of control values after 48 hours of treatment with 30 µM OATD-02 and further declined to 19% at 96 hours. A similar pattern was detected in K562 cells, where L-ornithine levels decreased to 9% of the control values under prolonged exposure to OATD-02 ( Figure 2 ). These results confirm the functional inhibition of arginase, preventing the conversion of L-arginine to L-ornithine and thereby limiting its availability for downstream metabolic pathways. Consistent with the reduction in L-ornithine, OATD-02 treatment led to a dose- and time-dependent depletion of intracellular polyamines, specifically spermidine and spermine, which depend on L-ornithine as a precursor for biosynthesis. The most pronounced effect was observed at 96 hours, when the spermine level was reduced to 37% of the control value in CT26 cells and 23% in K562 cells at 30 µM OATD-02 ( Figure 2 ). The spermidine levels exhibited a similar trend, with a gradual decrease over time. These findings suggest that OATD-02 limits polyamine biosynthesis by depleting the substrate pool, further reinforcing its role in the metabolic reprogramming of tumor cells. In contrast to the substantial changes observed in L-arginine metabolism, the levels of glutamine and proline remained largely unaffected by OATD-02 treatment. The fluctuations were minor, with variations remaining within a ±15% range relative to untreated controls, indicating that OATD-02 specifically modulates the L-arginine metabolic axis without broadly perturbing other amino acid pathways ( Figure 2 ). These findings demonstrate that OATD-02 effectively increases intracellular L-arginine while depleting L-ornithine and polyamines in both the CT26 and K562 tumor models. The observed metabolic shifts are consistent with potent arginase inhibition, providing a mechanistic basis for the potential antitumor effects of OATD-02. The inclusion of 10 µM OATD-02, on the basis of in vivo exposure data, further strengthens the clinical relevance of these findings. OATD-02 increases systemic L-arginine levels and reshapes tumor metabolism in vivo To assess the biological consequences of the metabolic changes observed in vitro, we evaluated the pharmacodynamic effects of OATD-02 in vivo in CT26 tumor-bearing mice. The selected dose of 100 mg/kg (PO, BID) was confirmed in prior toxicokinetic studies to be safe and within the therapeutic window for long-term administration in BALB/c mice (38). Mice were treated with OATD-02 via oral gavage twice daily, and blood and tumor samples were collected at two pharmacokinetically relevant time points: 2 hours after the last dose (peak exposure) and 16 hours after the last dose (trough levels before the next administration) ( Figure 3A ). LC–MS/MS analysis of the plasma samples confirmed that OATD-02 remained detectable at both time points, with mean plasma concentrations of 2.97 µg/mL at 2 h and 1.05 µg/mL at 16 h ( Figure 3B, left panel ). These pharmacokinetic data indicate that OATD-02 achieves substantial systemic exposure, supporting its bioavailability and stability in circulation. Consistent with its role as an intracellular arginase inhibitor, OATD-02 treatment led to a marked increase in systemic L-arginine levels ( Figure 3B, right panel ). At 2 h posttreatment, the serum L-arginine concentration was approximately 10-fold greater than that in untreated control mice, with the mean concentration increasing from ~140 µM in controls to ~1.20 mM in treated mice. Although L-arginine levels decreased by 16 h, they remained significantly elevated (~930 µM) relative to those of the untreated controls, indicating a sustained pharmacodynamic effect of OATD-02 on systemic L-arginine homeostasis ( Figure 3B, right panel ). Further correlation analysis revealed a strong positive correlation between the OATD-02 plasma concentration and the serum L-arginine level (Spearman r = 0.797, p = 0.0153), confirming the dose-dependent modulation of L-arginine metabolism. To investigate the spatial distribution of metabolic alterations in tumors, MALDI-MSI was employed to visualize L-arginine and related metabolites, including proline, spermine, and spermidine, in tumor sections at 2 h and 16 h posttreatment ( Figure 3C, Suppl. Figure 1 ). Consistent with the serum data, intratumoral L-arginine levels were substantially increased in the OATD-02-treated mice compared with the untreated controls. This effect was evident as early as 2 h posttreatment and persisted at 16 h, with widespread but heterogeneous L-arginine enrichment across tumor tissue ( Figure 3C, Suppl. Figure 1 ). Notably, regions with the highest L-arginine accumulation coincided with the tumor parenchyma rather than the stromal compartments, suggesting a preferential metabolic impact on tumor cells. However, spatial analysis also revealed localized regions with lower L-arginine enrichment, which may correspond to hypoxic or necrotic areas within the tumor core. In parallel with L-arginine accumulation, MALDI-MSI analysis revealed a time-dependent reduction trend in the levels of the polyamines spermine and spermidine in tumor tissue. At 2 h posttreatment, a moderate decrease in polyamine levels was observed, particularly in the central regions of larger tumors. By 16 h, spermine and spermidine depletion became more pronounced and spatially widespread, maintaining this trend over time, suggesting that OATD-02-driven inhibition of arginase limits L-ornithine availability for polyamine biosynthesis over time ( Figure 3C, Suppl. Figure 1 ). Interestingly, the strongest reduction in polyamines was observed in tumor regions where L-arginine accumulation was highest, suggesting a metabolic shift favoring L-arginine retention over its downstream utilization. Unlike the substantial changes observed in L-arginine and polyamines, tumor proline levels exhibited only minor fluctuations in response to OATD-02 treatment ( Figure 3C, Suppl. Figure 1 ). Proline distribution appeared relatively uniform across tumor sections, suggesting that its biosynthesis from L-arginine-derived intermediates remained largely unaffected. This further supports the selective metabolic action of OATD-02 on the L-arginine-polyamine axis. Collectively, these findings demonstrate that OATD-02 effectively increases systemic and intratumoral L-arginine levels while simultaneously reducing polyamine metabolites in tumors. Spatial metabolic analysis suggests that these changes are not uniform across the tumor microenvironment, with larger tumors displaying distinct metabolic heterogeneity, potentially influenced by variations in vascularization and hypoxia. The temporal dynamics of these changes suggest that OATD-02 exerts a sustained metabolic effect, reshaping the tumor microenvironment in a manner that may contribute to its antitumor efficacy. Notably, the moderate impact observed for some metabolites, such as proline and early-stage polyamine depletion, may reflect the relatively short duration of OATD-02 administration in this study (only 3–4 doses before sample collection at 2 h and 16 h, respectively). Longer treatment regimens or combination therapies targeting hypoxic tumor regions may further amplify these metabolic shifts, potentially leading to a more pronounced reprogramming of tumor metabolism over time. OATD-02 improves survival and enhances antitumor immune response in vivo Having established the strong in vivo metabolic effects of OATD-02, we next evaluated its therapeutic potential in combination with immune checkpoint blockade. CT26 tumor-bearing mice were treated with OATD-02 (100 mg/kg, PO, BID) alone or in combination with anti-PD-1 antibodies (2.5 mg/kg, IP) according to the schedule outlined in Figure 4A . Tumor growth and survival were monitored, and immune profiling of tumors, spleens, and lymph nodes was conducted on day 15 postimplantation. Kaplan-Meier survival analysis revealed that both the OATD-02 and anti-PD-1 monotherapies significantly extended median survival compared with the vehicle-treated controls ( Figure 4B ). Compared with control mice, animals receiving OATD-02 alone had a median survival of 29.5 days, whereas anti-PD-1 monotherapy extended survival to 32 days. The log-rank test confirmed significant differences between the vehicle group and the anti-PD-1 group (p = 0.0097) and between the vehicle group and the OATD-02 group (p = 0.0470). However, the greatest survival benefit was observed in the combination therapy group (OATD-02 + anti-PD-1), with a median survival of 41.5 days, which was significantly longer than that in the monotherapy group (p = 0.0479 vs. anti-PD-1 alone). Hazard ratio (Mantel–Haenszel) analysis indicated that, compared with anti-PD-1 therapy alone, combination therapy reduced the risk of death by approximately 62% (HR = 0.382, 95% CI: 0.147–0.991), supporting a synergistic effect. To assess the immune mechanisms underlying this survival benefit, we analyzed tumor-infiltrating immune cells via flow cytometry. OATD-02 treatment led to a significant increase in the proportion of CD45⁺ T cells within tumors ( Figure 4C, top left panel ). This effect was further enhanced in the combination therapy group (p = 0.0429, Dunn’s test), suggesting more robust immune infiltration. Given that L-arginine availability is crucial for T-cell activation and proliferation, these findings indicate that OATD-02-mediated arginase inhibition relieves metabolic constraints on intratumoral T cells, facilitating their recruitment and expansion. To determine whether these effects extend beyond the tumor microenvironment, we analyzed tumor-draining lymph nodes (TDLNs), which are critical sites for priming antitumor immune responses. Compared with those in the vehicle group, the numbers of CD4⁺ and CD8⁺ T cells were significantly greater in the mice that received OATD-02 + anti-PD-1 (p = 0.0058 for CD4⁺ and p = 0.0099 for CD8⁺; Dunn’s test; Figure 4C, middle panels ). Neither monotherapy alone induced significant changes relative to the controls, suggesting that increased systemic L-arginine availability in the combination treatment group supports the expansion of tumor-reactive T cells in lymphoid organs, potentially enhancing long-term antitumor immunity. To further assess systemic immune activation, we measured the expression of CD69, a marker of early T-cell activation, on splenic CD4⁺ and CD8⁺ T cells ( Figure 4C, right panels ). While both monotherapies induced a modest increase in CD69 expression, the combination treatment led to highly significant upregulation compared with the vehicle (p < 0.0001 for CD4⁺, p = 0.0005 for CD8⁺, Dunn’s test). This finding suggests that systemic T-cell activation extends beyond the tumor site, potentially priming circulating T cells for enhanced antitumor responses. In contrast, CD11b⁺ myeloid cell populations, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), did not significantly change following treatment (p > 0.05 for all comparisons, Dunn’s test; Figure 4C, bottom left panel ). However, a visible trend toward reduced myeloid cell infiltration was observed in the combination therapy group, suggesting a potential shift in the tumor immune landscape. These results imply that while OATD-02 effectively promotes T-cell activation, its effects on immunosuppressive myeloid populations may require longer treatment durations or additional combinatorial approaches for full efficacy. These findings demonstrate that OATD-02 enhances antitumor immunity by increasing intratumoral T-cell infiltration, expanding tumor-reactive T cells in the lymph nodes, and increasing systemic T-cell activation. When combined with anti-PD-1 therapy, these effects translate into a significant survival benefit, with a 62% reduction in mortality risk compared with anti-PD-1 therapy alone ( Figure 4B ). Notably, the most pronounced immunological effects, including enhanced T-cell activation in the spleen and lymph nodes, were exclusive to the combination therapy group, underscoring the potential of OATD-02 as a metabolic adjuvant for immune checkpoint blockade. Discussion Metabolic reprogramming in the tumor microenvironment (TME) plays a pivotal role in cancer progression and immune evasion. Our study demonstrated that the dual ARG1/ARG2 arginase inhibitor OATD-02 effectively modulates the L-arginine metabolic axis, enhancing antitumor immune responses. Using a combination of in vitro, in vivo, and spatial metabolomics approaches, we demonstrated that OATD-02 increases L-arginine availability within tumors, depletes immunosuppressive polyamines, and enhances the efficacy of immune checkpoint inhibitors (ICIs). Notably, OATD-02 significantly elevated both intracellular and systemic L-arginine levels while concurrently reducing L-ornithine and polyamine concentrations. This effect was observed in CT26 colon carcinoma and K562 leukemia cell models, as well as in CT26 tumor-bearing mice. Our findings are consistent with those of previous studies demonstrating that arginase-mediated L-arginine depletion suppresses T-cell activation and facilitates tumor immune evasion ( 39 , 40 ). Unlike numidargistat (CB-1158), an arginase inhibitor with predominantly extracellular activity and limited efficacy against ARG2-driven pathways ( 30 , 41 ), OATD-02 effectively inhibits both extracellular and intracellular arginase isoforms. This distinction is critical, as ARG2 plays a tumor-intrinsic role in metabolic adaptation, regulating nitrogen balance and polyamine biosynthesis ( 42 – 44 ). The ability of OATD-02 to penetrate tumor cells and inhibit intracellular ARG2 represents a unique mechanistic advantage over other arginase-targeting therapies. While systemic metabolic alterations are critical for therapeutic efficacy, spatial heterogeneity within the tumor microenvironment may further shape treatment outcomes. To characterize the metabolic landscape of tumors, we employed MALDI mass spectrometry imaging (MALDI-MSI) to map metabolite distributions with high spatial resolution. Our results revealed heterogeneous L-arginine accumulation across tumor sections. Given that larger tumors often develop hypoxic cores with impaired circulation and tissue necrosis ( 45 ), these spatial differences may reflect microenvironmental constraints on metabolic fluxes. A similar approach was reported by Andersen et al. ( 46 ), who demonstrated that MALDI-MSI enables the spatially resolved detection of metabolic alterations in prostate cancer tissues, revealing distinct metabolic differences between tumor and normal regions. MALDI-MSI has also demonstrated that anti-cancer drugs frequently fail to penetrate tumors in both patients and mouse models ( 47 , 48 ). These findings underscore the power of MALDI-MSI in identifying spatial metabolic heterogeneity and highlight its value in assessing the metabolic reprogramming induced by OATD-02 in our study. Beyond the heterogeneous distribution of L-arginine, we observed a trend towards preferential depletion of polyamines, particularly in central tumor regions. This suggests that metabolic adaptation to OATD-02 treatment is influenced by tumor perfusion and oxygen availability. Hypoxic tumor regions are known to favor metabolic pathways that sustain proliferation under stress, including increased glutaminolysis and alternative nitrogen metabolism ( 49 ). The differential impact of OATD-02 across spatially distinct tumor compartments suggests that L-arginine-polyamine axis modulation may be influenced by oxygen and nutrient gradients within the tumor microenvironment. Since hypoxic regions often exhibit increased polyamine turnover to support tumor survival ( 13 , 45 , 50 ), this adaptation may shape the response to OATD-02. Expanding on these insights, our results show that metabolic modulation by OATD-02 can significantly enhance the efficacy of ICIs. By increasing L-arginine availability and reducing polyamines, OATD-02 creates a microenvironment more permissive for T-cell activation, thereby potentiating the effects of PD-1 blockade. OATD-02 combined with anti-PD-1 therapy led to increased T-cell infiltration in tumors, enhanced T-cell activation in tumor-draining lymph nodes, and improved systemic immune responses. These findings are consistent with those of Sosnowska et al. ( 39 ), who reported that arginase inhibition relieves metabolic constraints on T cells, thereby enhancing PD-1 blockade. Similarly, Grzywa et al. ( 51 ) and our previous studies ( 52 ) demonstrated that OATD-02 boosts antitumor immunity in multiple preclinical models, whereas Pilanc et al. ( 53 ) reported increased immune cell infiltration and potentiation of anti-PD-1 therapy by OATD-02 in glioblastoma models, underscoring the broad applicability of this cancer-agnostic mechanism across multiple tumor types. Furthermore, whereas CB-1158, an ARG1-restricted inhibitor, showed only modest increases in plasma L-arginine in clinical trials – likely owing to its limited intracellular activity ( 29 ) – our findings indicate that the dual targeting of both the intracellular and the extracellular arginases by OATD-02 results in superior metabolic and immunomodulatory effects. Indeed, our results align with those of previous experiments ( 38 ), in which we directly compared OATD-02 to CB-1158 and found that OATD-02 produced greater tumor growth inhibition in an ARG2-dependent model. The above observations underscore the crucial role of L-arginine metabolism in shaping the tumor microenvironment (TME) and highlight the importance of considering spatial metabolic heterogeneity when evaluating arginase-targeting therapies. The uneven metabolic reprogramming observed in OATD-02-treated tumors suggests the need to address regional metabolic constraints, particularly in hypoxic tumor cores. Arginases, particularly ARG2, regulate tumor metabolism and immune suppression ( 1 , 17 ). Their inhibition restores L-arginine availability, crucial for effective T-cell activation and antitumor immunity. Given its role in mitochondrial metabolism and nitrogen balance, intracellular ARG2 is a particularly relevant therapeutic target ( 1 ). By inhibiting both isoforms, OATD-02 may provide broader therapeutic benefits than agents that selectively target ARG1 ( 54 , 55 ). Tumor cells adapt metabolically to sustain growth and evade immune surveillance. L-arginine, polyamine, and proline metabolism play key roles in these processes, and OATD-02 disrupts their balance, reshaping the TME. L-arginine fuels tumor cell proliferation and supports processes such as extracellular matrix production, but it is also a critical resource for immune cell function (e.g., as a precursor for nitric oxide and polyamines). Tumors frequently exploit L-arginine metabolism by upregulating arginases, which deplete local L-arginine and redirect its metabolic flux toward polyamine and proline biosynthesis to support tumor growth and adaptation ( 17 ). Conversely, depriving immune cells of L-arginine has profound immunosuppressive effects. L-arginine deficiency impairs T-cell proliferation and activation by downregulating the CD3ζ chain of the T-cell receptor complex, thereby weakening TCR signaling ( 23 , 56 ). It also skews T-cell differentiation, leading to diminished IFN-γ and IL-2 production and a shift away from effective Th1 immune responses ( 1 ). Collectively, these effects create an immunosuppressive environment, highlighting why L-arginine availability is critical for antitumor immunity. L-arginine depletion promotes tumor growth and immune evasion. OATD-02 restores L-arginine levels, reinvigorating T-cell activity and enhancing antitumor immunity ( 39 , 57 ). Concurrent targeting of ARG1 and ARG2 may further enhance these effects. In addition to L-arginine, polyamines play a significant role in tumor progression and immune evasion through complementary metabolic mechanisms. Polyamines, such as spermidine and spermine, are essential for tumor cell proliferation and survival. They stabilize DNA, modify chromatin, and promote ribosome biogenesis, thereby accelerating tumor growth ( 58 ). These oncometabolites also contribute to immunosuppression. The spermidine secreted by tumor cells can directly inhibit CD8⁺ T-cell activation by preventing proper TCR clustering, effectively acting as a metabolic immune checkpoint ( 59 , 60 ) and highlighting how tumors exploit polyamine pathways to evade immune surveillance. Consistent with this notion, our study confirms the critical role of polyamine metabolism in the TME. OATD-02 treatment led to a significant reduction in intracellular spermidine and spermine levels in both CT26 and K562 cells. The depletion of polyamines observed in vitro was mirrored in tumor tissues – MALDI-MSI analysis of tumors from OATD-02-treated mice revealed a progressive decline in polyamine levels over time. Arginase inhibition by OATD-02 alters the metabolic balance in tumors, limiting the production of immunosuppressive polyamines and disrupting a key mechanism of tumor-mediated immune evasion. Given the dual role of polyamines in promoting tumor growth and suppressing immune responses, targeting polyamine synthesis has been explored as a therapeutic strategy to increase the efficacy of immunotherapy ( 58 ). In this context, the polyamine-lowering effect of OATD-02 may contribute to creating a tumor microenvironment that is less conducive to immune escape, thereby improving the effectiveness of anti-PD-1 therapy. Notably, the sustained reduction in tumor polyamine levels observed in our study further supports the combination of OATD-02 with immune checkpoint inhibitors, particularly in tumors characterized by high polyamine levels and an immunosuppressive microenvironment ( 59 ). Proline metabolism represents another aspect of tumor metabolic adaptation, influencing extracellular matrix (ECM) remodeling, redox homeostasis, and survival under stress. Proline biosynthesis contributes to oxidative stress resistance by maintaining the NADP⁺/NADPH balance, buffering redox status, and supporting tumor cell survival, particularly under hypoxic conditions ( 9 , 61 ). Additionally, proline plays a crucial role in collagen production, as its derivative, hydroxyproline, is a major structural component of collagen. Increased proline utilization promotes collagen-rich ECM deposition and stiffness, facilitating tumor invasion and metastasis. Notably, high collagen content in tumors often correlates with poor prognosis ( 62 , 63 ). Interestingly, despite the metabolic significance of proline, OATD-02 treatment did not significantly alter the intracellular proline levels in our models. In both CT26 and K562 cells, proline concentrations remained relatively stable across all conditions, even as L-arginine and L-ornithine underwent substantial changes. These findings suggest that tumor cells may preserve proline homeostasis through alternative pathways that are unaffected by arginase inhibition. For instance, cancer cells can recycle proline from collagen-derived hydroxyproline or upregulate glutamine metabolism to sustain proline synthesis ( 64 , 65 ). Indeed, the high glutamine content in our cell culture medium likely provided an alternative substrate for proline production, potentially masking any secondary effects of arginase inhibition on the L-ornithine–proline axis. The immunomodulatory and protumorigenic impact of arginases has been well documented in solid tumors, including lung, breast, colorectal, pancreatic, prostate, melanoma, renal, ovarian, and esophageal cancers, where elevated arginase activity frequently correlates with enhanced tumor growth, metastasis, or immune evasion ( 24 , 57 , 66 – 70 ). These findings underscore the broad relevance of L-arginine metabolism as a therapeutic target in oncology. However, the dependency on arginase activity varies among cancer types. For example, thymic epithelial tumors lack ARG1 expression, whereas in some lung cancers, ARG2 activity does not induce immunosuppression unless it is accompanied by additional factors such as nitric oxide production, highlighting the context-dependent role of arginases ( 71 , 72 ). Nevertheless, accumulating evidence suggests that dysregulated L-arginine metabolism also plays a crucial role in hematologic malignancies. Notably, acute myeloid leukemia (AML) cells secrete arginase to deplete L-arginine, leading to T-cell suppression; in AML models, arginase inhibition restores T-cell proliferation and induces leukemia cell apoptosis ( 37 , 73 ). Furthermore, multiple myeloma exhibits increased arginase activity in myeloid cells, contributing to systemic L-arginine depletion and T-cell dysfunction ( 74 ). Chronic myeloid leukemia (CML) provides another example, as CML cells rely on ARG2 to adapt to hypoxic conditions and resist therapy. Inhibiting arginase in CML has been shown to counteract these resistance mechanisms ( 75 ). Collectively, these findings extend the therapeutic rationale for arginase inhibition to a broad spectrum of malignancies. Given the broad immunosuppressive role of arginase in the TME, its inhibition represents a promising strategy to enhance cancer immunotherapy. Owing to its dual ARG1/ARG2 targeting ability and potent immunomodulatory effects, OATD-02 is a strong candidate for combination therapies designed to overcome resistance to ICIs. Multiple studies have demonstrated that increased arginase activity in tumors suppresses antitumor immunity by impairing CD8⁺ T-cell function and promoting immunosuppressive myeloid populations ( 39 ). Early trials with arginase inhibitors, such as CB-1158 (numidargistat), validated arginase as a therapeutic target by showing that its inhibition restores T-cell proliferation and improves checkpoint blockade efficacy in preclinical models ( 76 ). However, CB-1158 primarily targets extracellular ARG1 and has limited cell permeability, which may explain its modest clinical efficacy ( 55 ). In contrast, the ability of OATD-02 to effectively inhibit both intracellular and extracellular arginases provides broader immunomodulatory effects ( 52 ), potentially translating into enhanced therapeutic benefits when it is combined with ICIs. On the basis of preclinical data, combining arginase blockade with ICIs appears to be one of the most promising combinatorial cancer therapies. AZD0011 significantly increased intratumoral L-arginine levels while reducing ornithine, and its combination with anti–PD-L1 therapy resulted in markedly greater tumor growth inhibition than either treatment alone ( 77 , 78 ). In addition to checkpoint inhibitors, OATD-02–mediated metabolic reprogramming may enhance other immunotherapies. For example, Ye et al. ( 79 ) demonstrated that cotargeting arginase and L-arginine-depleting enzymes potentiated T-cell activation and tumor regression in a melanoma model, suggesting that increasing L-arginine availability could improve the outcomes of adoptive T-cell transfer or cancer vaccines. A promising avenue for enhancing antitumor immunity is combining arginase inhibition with other metabolic interventions. In our previous studies ( 38 ), we reported that adding OATD-02 to an IDO1 (indoleamine-2,3-dioxygenase) inhibitor (epacadostat) alongside anti–PD-L1 therapy resulted in significantly improved tumor control in a murine CT26 colorectal carcinoma model compared with dual or single agent treatments. This finding aligns with earlier studies indicating that simultaneous blockade of L-arginine and tryptophan catabolism can synergistically enhance antitumor immunity ( 28 ). Given the capacity of OATD-02 to modulate both L-arginine and polyamine pathways, pairing it with IDO1 inhibitors may be particularly effective in counteracting metabolic immunosuppression. Another promising approach for combination therapy involves the integration of arginase inhibitors with targeted anticancer agents. Preclinical studies suggest that arginase blockade can increase the efficacy of certain tyrosine kinase inhibitors. For example, combining an arginase inhibitor with cabozantinib or lenvatinib has been shown to remodel the immune microenvironment and improve tumor control ( 80 ). These findings suggest that OATD-02 may similarly enhance the effects of targeted therapies by mitigating tumor metabolic resistance mechanisms. In addition to targeted therapies, arginase inhibition may also act synergistically with other treatment modalities, including radiotherapy and NK cell–based immunotherapies. Notably, the combination of arginase inhibition with blockade of the inhibitory NK cell receptor NKG2A has been reported to improve tumor control and enhance immune activation ( 78 ), highlighting additional therapeutic opportunities for OATD-02 in multimodal treatment regimens. Our findings provide strong justification for the continued clinical development of OATD-02 as a metabolic-immunotherapeutic agent. While our studies demonstrated that dual ARG1/ARG2 inhibition enhances immune responses by increasing intratumoral L-arginine ( 38 ) and depleting polyamines, the full extent of tumor adaptation to prolonged L-arginine repletion remains to be explored. Future studies using patient-derived models will be essential to determine how metabolic compensation, such as glutamine or tryptophan dependence, may influence therapeutic outcomes ( 30 ). Moreover, integrating MSI with functional immune profiling may provide deeper insights into how spatial metabolic heterogeneity impacts therapeutic responses, helping to refine treatment strategies and optimize combination approaches. Encouragingly, the ongoing phase I clinical trial (NCT05759923) will provide critical insights into the safety, pharmacokinetics, and pharmacodynamics of OATD-02 in patients with advanced solid tumors. These results will guide the design of future combination strategies, reinforcing the potential of OATD-02 as a novel metabolic-immunotherapeutic approach. Conclusions Over the past years, studies have demonstrated the therapeutic potential of ARG1 inhibition in restoring L-arginine availability and enhancing antitumor immunity ( 30 , 77 , 78 , 81 ). However, recent studies have revealed a previously underappreciated role of ARG2, the mitochondrial isoform, in tumor metabolism and immune evasion, revealing its critical function in nitrogen balance, mitochondrial adaptation, and metabolic reprogramming ( 50 , 82 – 84 ). To our knowledge, this is the first study providing direct metabolic evidence of pharmacological ARG2 inhibition, demonstrating that dual ARG1/ARG2 blockade reshaped the tumor microenvironment by increasing L-arginine availability, depleting polyamines, and enhancing immune responses. This mechanistic advantage over ARG1-restricted inhibitors such as numidargistat ( 81 ) underscores the importance of targeting both arginase isoforms in metabolic therapy. Using MALDI-MSI spatial metabolomics, we mapped the metabolic consequences of OATD-02 at high spatial resolution, revealing widespread L-arginine accumulation in tumor tissues, a shift that aligns with enhanced T-cell activation. While metabolic adaptations to sustain L-arginine repletion remain a consideration, our findings reinforce the therapeutic relevance of ARG2 inhibition as a metabolic-immunotherapeutic strategy. Future research should focus on identifying tumor types that would benefit the most from dual ARG1/ARG2 inhibition. Dual arginase blockade represents a promising approach for overcoming immune suppression in tumors, offering a novel avenue for metabolic intervention in cancer treatment. Abbreviations 3R – Replacement, reduction, refinement (ethical principles for animal research) ABH – 2(S)-amino-6-boronohexanoic acid ARG1 – Arginase 1 ARG2 – Arginase 2 BEC – ( S )-(2-boronoethyl)-L-cysteine BID – twice a day CAFs – Cancer-Associated Fibroblasts CD – Cluster of Differentiation (e.g., CD8, CD4; surface markers on immune cells) CI – confidence interval CT26 – Murine Colon Carcinoma Cell Line FACS – Fluorescence-Activated Cell Sorting FBS - Fetal bovine serum HPLC – high-performance liquid chromatography HR – hazard ratio ICIs – Immune checkpoint inhibitors IgG2a – Immunoglobulin G, subclass 2a IP – intraperitoneal administration K562 – Human Chronic Myelogenous Leukemia Cell Line L-Arg – L-arginine LC‒MS/MS – Liquid chromatography‒tandem mass spectrometry MALDI-MSI – Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Imaging MDSCs – Myeloid-Derived Suppressor Cells NO – Nitric oxide NOS – nitric oxide synthase PBS – Phosphate-buffered saline PD-1 – Programmed Cell Death Protein 1 PO – per os (oral administration) RPMI-1640 – Roswell Park Memorial Institute Medium RT – room temperature SPF – specific pathogen-free TAMs – Tumor-associated macrophages TCR – T-cell receptor TME – Tumor microenvironment Declarations Ethical approval All procedures complied with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guidelines for the Welfare and Use of Animals in Cancer Research. Ethical approval for the study was granted by the 1st Local Ethics Committee for Animal Experiments in Warsaw, Poland (approval no. 891/2019). All experiments were performed in accordance with relevant institutional and national guidelines and regulations. The study is reported in accordance with the ARRIVE guidelines ( https://arriveguidelines.org ). Procedures involving animals, including anesthesia and euthanasia, were conducted in accordance with veterinary best practice and followed the recommendations outlined in the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). Competing Interests Several authors (MMG, AM, AK, KG, MS-R, MK, AZ, PP, MM, AT, TR, RB, ZZ) are employees of Molecure SA, which holds proprietary rights to OATD-02. The remaining authors declare that they have no competing interests. Author Contribution MMG, YU, AM, TR, RB, CH, and ZZ designed the study. MMG, MM, AK, and AZ performed the in vivo experiments. MMG, AZ, and PP conducted the in vitro experiments. AK, KG, MK, and MS-R carried out the flow cytometry analyses. MALDI–MSI analyses were performed by YU and TB. AT and TR conducted the LC–MS analyses, whereas AKJ performed the HPLC analyses. MMG, AM, AK, YU, and TB analyzed the data. MMG wrote the initial version of the manuscript, while ZZ, CH, and YU revised and edited it. MG, ZZ, and CH supervised the project and coordinated the study. All authors read and approved the final manuscript. Acknowledgement The authors would like to thank EU-OPENSCREEN for providing access to advanced research infrastructure, which enabled the MALDI‒MSI analyses conducted at CeMOS, Mannheim University of Applied Sciences (Mannheim, Germany). Their support was instrumental in visualizing the metabolic alterations induced by OATD-02. 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First-in-human phase 1 study of the arginase inhibitor INCB001158 alone or combined with pembrolizumab in patients with advanced or metastatic solid tumours. BMJ Oncology. 2024 May 9;3(1). Ochocki JD, Khare S, Hess M, Ackerman D, Qiu B, Daisak JI, et al. Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity. Cell Metab. 2018 Jun 5;27(6):1263-1280.e6. Zaytouni T, Tsai PY, Hitchcock DS, Dubois CD, Freinkman E, Lin L, et al. Critical role for arginase 2 in obesity-Associated pancreatic cancer. Nat Commun. 2017 Dec 1;8(1). Ino Y, Yamazaki-Itoh R, Oguro S, Shimada K, Kosuge T, Zavada J, et al. Arginase II Expressed in Cancer-Associated Fibroblasts Indicates Tissue Hypoxia and Predicts Poor Outcome in Patients with Pancreatic Cancer. PLoS One. 2013 Feb 12;8(2). Additional Declarations Competing interest reported. Several authors (MMG, AM, AK, KG, MS-R, MK, AZ, PP, MM, AT, TR, RB, ZZ) are employees of Molecure SA, which holds proprietary rights to OATD-02. The remaining authors declare that they have no competing interests. Supplementary Files OATD02supplementaryinformation26.03.25.docx Cite Share Download PDF Status: Published Journal Publication published 28 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Apr, 2025 Reviews received at journal 04 Apr, 2025 Reviews received at journal 03 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers invited by journal 31 Mar, 2025 Editor assigned by journal 31 Mar, 2025 Editor invited by journal 30 Mar, 2025 Submission checks completed at journal 27 Mar, 2025 First submitted to journal 25 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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15:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6305179/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6305179/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-03446-1","type":"published","date":"2025-05-28T15:57:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79674030,"identity":"cdceac99-80d8-458c-ace6-f983109f06ec","added_by":"auto","created_at":"2025-04-01 11:51:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":370271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of L-arginine metabolism by the arginase inhibitor OATD-02 in cytosolic and mitochondrial compartments.\u003c/strong\u003e OATD-02 inhibits arginase 1 (ARG1) in the cytosol and arginase 2 (ARG2) in mitochondria, altering key metabolic pathways. ARG1 inhibition prevents L-arginine catabolism into L-ornithine and urea, thereby reducing polyamine synthesis (putrescine, spermidine, and spermine) catalyzed by L-ornithine decarboxylase (ODC), spermidine synthase (SRM), and spermine synthase (SMS), which may limit tumor cell proliferation. ARG2 inhibition restricts L-ornithine availability in mitochondria, reducing proline biosynthesis via L-ornithine aminotransferase (OAT) and the pyrroline-5-carboxylate (P5C) pathway, which involves P5C synthase (P5CS), P5C dehydrogenase (PDG), and P5C reductase (P5CR). This metabolic shift may impair tumor adaptation to hypoxia and metabolic stress. The dual inhibition of ARG1 and ARG2 by OATD-02 may enhance antitumor effects by modulating polyamine metabolism, amino acid availability, and redox homeostasis.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6305179/v1/f1d78b7552817a77b938103c.png"},{"id":79674031,"identity":"76b2e2f8-8967-4480-bb26-f0ff9f8efd8d","added_by":"auto","created_at":"2025-04-01 11:51:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":283485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in the intracellular metabolism of CT26 and K562 cells\u003c/strong\u003e upon OATD-02 treatment. The heatmap represents the mean relative levels (n = 3) of L-arginine, L-ornithine, spermidine, spermine, glutamine, and proline in CT26 and K562 tumor cells treated with 10 µM or 30 µM OATD-02 for 48 h and 96 h. Metabolite levels are expressed as a fraction of those in the untreated control.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6305179/v1/3748efea1733da9d640413f5.png"},{"id":79674036,"identity":"a77147ac-cc36-4958-8213-4e38e0dd6801","added_by":"auto","created_at":"2025-04-01 11:51:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4325411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacodynamic effects of OATD-02 in CT26 tumor-bearing mice. \u003c/strong\u003e(A) Experimental design: Mice were treated with OATD-02 (100 mg/kg PO, BID), and blood and tumor samples were collected at 2 h and 16 h after the last dose. (B) LC-MS/MS analysis of the serum concentrations of OATD-02 (left) and L-arginine (right) at the indicated time points. (C) MALDI-MSI ion images of L-arginine, proline, spermine, and spermidine levels in tumors of untreated and OATD-02-treated mice at 2 h and 16 h after the last dose.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6305179/v1/957dd7f0914990b6334342ad.png"},{"id":79674034,"identity":"32591e89-f374-40a5-93c7-26d0a0d95603","added_by":"auto","created_at":"2025-04-01 11:51:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1541908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination therapy with OATD-02 and anti-PD-1 enhances antitumor immunity and prolongs survival in CT26 tumor-bearing mice. \u003c/strong\u003e(A) Experimental design: CT26 tumor-bearing mice were treated with OATD-02 (100 mg/kg, PO, BID) from day 1 until the end of the study. Anti-PD-1 antibodies (2.5 mg/kg, IP) were administered on days 6, 10, 14, and 18. On day 15, a subset of animals was sacrificed for flow cytometry analysis, while the remaining mice were monitored for survival. (B) Kaplan–Meier survival curves comparing treatment groups (Vehicle, Anti-PD-1, OATD-02, and combination therapy). Log-rank test significance: *p \u0026lt; 0.05. (C) Flow cytometry analysis of immune cell populations in tumors, lymph nodes, and spleens on day 15. Statistical analysis: Kruskal–Wallis test followed by Dunn’s post hoc test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6305179/v1/717356723e91bb4070f3b421.png"},{"id":83783509,"identity":"d6936c1c-1106-4e90-8bd2-dc23157ce47c","added_by":"auto","created_at":"2025-06-02 16:11:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7271662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6305179/v1/bd184e49-1f7a-45cf-a35e-2f3cb4e3d238.pdf"},{"id":79675007,"identity":"c48e644a-b4c5-4deb-80f3-aa8aed99e908","added_by":"auto","created_at":"2025-04-01 11:59:58","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":712659,"visible":true,"origin":"","legend":"","description":"","filename":"OATD02supplementaryinformation26.03.25.docx","url":"https://assets-eu.researchsquare.com/files/rs-6305179/v1/a7faaed2848536b897675f74.docx"}],"financialInterests":"Competing interest reported. Several authors (MMG, AM, AK, KG, MS-R, MK, AZ, PP, MM, AT, TR, RB, ZZ) are employees of Molecure SA, which holds proprietary rights to OATD-02. The remaining authors declare that they have no competing interests.","formattedTitle":"Metabolomic reprogramming of the tumor microenvironment by dual arginase inhibitor OATD-02 boosts anticancer immunity","fulltext":[{"header":"Background","content":"\u003cp\u003eCancer cells undergo profound metabolic reprogramming to sustain proliferation, evade immune surveillance, and adapt to environmental stressors such as nutrient deprivation and hypoxia. Among the key metabolic pathways, L-arginine metabolism plays crucial roles in tumor growth and immune regulation (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). L-arginine is a conditionally essential amino acid involved in multiple biochemical processes, including polyamine biosynthesis (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), nitric oxide (NO) production (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and proline metabolism (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), all of which influence tumor progression (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and immune cell function (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eL-arginine degradation is regulated primarily by two enzymes: arginase (ARG1, ARG2) and nitric oxide synthase (NOS). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, ARG1, which is predominantly cytosolic, is a key component of the urea cycle in hepatocytes, where it catalyzes the hydrolysis of L-arginine to L-ornithine and urea, facilitating ammonia detoxification. ARG2, which is localized in mitochondria, performs a similar reaction but is more broadly involved in metabolic adaptation. L-ornithine serves as a precursor for polyamine biosynthesis (putrescine, spermidine, and spermine), which promotes tumor proliferation, and for proline synthesis, which plays a key role in extracellular matrix remodeling and redox homeostasis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). NOS, on the other hand, converts L-arginine into NO, which can support tumor growth by promoting angiogenesis or inducing tumor cell death through immune-mediated cytotoxicity (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Dysregulated L-arginine metabolism significantly influences the tumor microenvironment (TME), mediating interactions between tumor cells, stromal fibroblasts, and immune cells (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Elevated arginase activity is detected in multiple malignancies, including lung, colorectal, breast, and prostate cancers, where it is correlated with poor prognosis, increased tumor invasiveness, and enhanced metastatic potential (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Arginase-driven L-arginine depletion promotes tumor cell proliferation by increasing L-ornithine availability, enhancing polyamine synthesis, cell cycle progression, and resistance to apoptosis. Additionally, proline biosynthesis contributes to extracellular matrix remodeling, further facilitating tumor progression (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Notably, L-arginine depletion impairs T-cell activation and proliferation by downregulating the expression of the CD3ζ chain, a key component of the T-cell receptor (TCR) complex, thereby suppressing antitumor immunity (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) further increase ARG1 expression, exacerbating L-arginine depletion and creating an immune-privileged environment that facilitates tumor immune evasion (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This metabolic alteration reduces the efficacy of immune checkpoint inhibitors, highlighting the need for strategies targeting arginase activity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to its role in metabolic regulation, ARG2 is now recognized as a critical regulator of both tumor-intrinsic metabolism and immune responses. In contrast to ARG1, which primarily depletes extracellular L-arginine, ARG2 functions within mitochondria, where it directly influences tumor metabolic adaptation, nitrogen balance, and immune regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In tumors, ARG2 expression is linked to glutaminolysis, polyamine biosynthesis, and mitochondrial metabolism, allowing cancer cells to survive in nutrient-deprived environments (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The loss of ARG2 in pancreatic ductal adenocarcinoma (PDA) models results in nitrogen accumulation and impaired tumor growth, highlighting its role as a metabolic vulnerability factor in certain cancers (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In addition to its metabolic functions, ARG2 acts as a cell-intrinsic regulator of CD8⁺ T cell activation, persistence, and antitumor efficacy (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Unlike ARG1, which primarily affects T cells by depleting extracellular arginine, mitochondrial ARG2 regulates intracellular arginine metabolism, shaping T-cell fate independently of extracellular arginine availability. Deletion of ARG2 in CD8⁺ T cells enhances their expansion, effector function, and persistence, leading to improved tumor control in preclinical cancer models. Moreover, ARG2-deficient CD8⁺ T cells exhibit strong synergy with PD-1 blockade, suggesting that targeting ARG2 could enhance the efficacy of immune checkpoint therapies (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eArginase inhibitors have been studied for over a century, with early studies identifying α-amino acids as weak inhibitors. A major breakthrough came with N-hydroxy-nor-arginine (nor-NOHA), a micromolar inhibitor that provided structural insights into arginase inhibition (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). This led to the development of boronic acid-based inhibitors such as 2(S)-amino-6-boronohexanoic acid (ABH) and (\u003cem\u003eS\u003c/em\u003e)-(2-boronoethyl)-L-cysteine (BEC), which are known for their potency and selectivity (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These compounds laid the groundwork for more advanced inhibitors, such as OATD-02 and numidargistat. Preclinical studies have demonstrated that numidargistat (INCB001158) reversed T-cell immunosuppression and reduced tumor growth in multiple syngeneic mouse models, particularly when combined with anti-PD-L1 therapy, highlighting its potential to modulate the tumor immune microenvironment (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, despite pharmacodynamic evidence of arginase inhibition and increased plasma arginine levels in a phase I/II clinical trial, its antitumor efficacy has remained limited, suggesting the complexity of arginine metabolism in cancer (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). A key distinction between numidargistat and OATD-02 lies in their ability to inhibit intracellular ARG2. While numidargistat primarily targets extracellular ARG1, its limited intracellular penetration restricts its effect on ARG2-dependent metabolism in tumor and immune cells (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In contrast, OATD-02 is designed to effectively inhibit both intracellular and extracellular arginases, granting it a broader and potentially stronger pharmacodynamic profile (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The ongoing phase I/II clinical trial of OATD-02 aims to evaluate these multidimensional antitumor properties in patients with advanced solid tumors, providing a more comprehensive understanding of its therapeutic potential (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo evaluate the intracellular effects of OATD-02 on tumor metabolism directly in tissue, the advanced imaging technique MALDI-MSI was used to assess spatially resolved metabolic alterations induced by arginase inhibition (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). This approach enabled the direct visualization of changes in key metabolites, including L-arginine and polyamines, within tumor tissue, complementing bulk quantification methods such as HPLC and LC-MS. By integrating MALDI-MSI with systemic pharmacokinetic and metabolic analyses, this study examines how OATD-02 modulates the tumor microenvironment. The spatial distribution of metabolic changes offers insights into the extent and localization of OATD-02-induced effects, contributing to a broader understanding of its potential as a metabolic modulator in cancer therapy and its role in enhancing immune responses in combination with immune checkpoint inhibitors.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eChemical compounds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOATD-02 was synthesized at Molecure SA. For in vitro assays, OATD-02 was dissolved in Milli-Q water (Millipore) at a stock concentration of 20 mM and stored at -20\u0026deg;C until use. For in vivo studies, OATD-02 was dissolved in sterile saline at a final concentration of 10 mg/mL and stored at -20\u0026deg;C until use. Prior to administration, the solution was prewarmed to RT and used within its validated minimal stability period. Unless otherwise specified, all additional chemical reagents, including analytical standards, were obtained from Merck (Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CT26.WT (CRL-2638, mouse colon carcinoma) and K562 (CCL-243, human chronic myelogenous leukemia) cell lines were purchased from ATCC\u0026reg;. Both cell lines were maintained in RPMI-1640 medium (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher/Gibco\u0026trade;), 100 U/mL penicillin G and 100 \u0026micro;g/mL streptomycin (Antibiotic-Antimycotic, Gibco\u0026reg;) at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. CT26 cells were cultured as adherent monolayers and passaged upon 70\u0026ndash;80% confluence using 0.25% trypsin-EDTA (Gibco\u0026reg;). Nonadherent K562 cells were maintained in suspension culture and passaged every 2\u0026ndash;3 days by dilution with fresh medium to sustain logarithmic growth. The absence of mycoplasma contamination in both cell lines was confirmed via the MycoAlert\u0026trade; Mycoplasma Detection Kit (Lonza).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular metabolite detection via HPLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe detection of amino acids and polyamines in CT26 and K562 cell extracts was performed via high-performance liquid chromatography (HPLC) with dabsyl derivatives, following a modified protocol of Krause et al. (36). CT26 adherent cells were seeded at a density of 750,000 cells per 10 mL in 75 cm\u0026sup2; flasks and treated with 10 \u0026micro;M or 30 \u0026micro;M OATD-02. K562 suspension cells were seeded at a density of 2\u0026times;10⁶ cells per 10 mL in 75 cm\u0026sup2; flasks and treated with OATD-02 at the same concentrations. Treatment was initiated either 2 h postseeding, resulting in a total incubation time of 96 h, or two days postseeding, resulting in a total incubation time of 48 h. At the experimental endpoint, the CT26 monolayers were washed with 5 mL of prewarmed (37\u0026deg;C) 0.3 M mannitol solution. For K562 nonadherent cells, an initial centrifugation step was performed to pellet the cells, followed by rinsing with warm 0.3 M mannitol. Metabolite extraction was carried out via the addition of 1.5 mL of a cold extraction mixture consisting of HPLC-grade methanol, acetonitrile, and water at a 2:2:1 ratio. The cells were incubated on a laboratory shaker at 4\u0026deg;C for 5\u0026nbsp;min and then detached via cell scrapers (for CT26) or resuspended (for K562) in the extraction mixture. The resulting suspensions were centrifuged (10,000 \u0026times; g, 10 min, 4\u0026deg;C), and the supernatants were transferred to coded tubes to ensure blinded analysis before being stored at \u0026minus;80\u0026deg;C until further HPLC processing. Dabsyl derivatization was performed by mixing 50 \u0026micro;L of NaHCO₃ buffer (0.4 M, pH 9) with 50 \u0026micro;L of a deproteinized sample or standard. Two hundred microliters of freshly prepared dabsyl chloride reagent (4 mg/mL in acetone) was subsequently added. The mixture was incubated at 70\u0026deg;C for 21 min with intermittent mixing. The reaction was terminated by adding 200\u0026nbsp;\u0026micro;L of cold dilution buffer (acetonitrile, ethanol, and sodium acetate buffer at a 2:1:1 ratio), followed by incubation on ice for 5 min. The samples were then centrifuged, and the clear supernatants were immediately analyzed by HPLC. HPLC separation was performed via a\u0026nbsp;Dionex 3000 ICS system equipped with an Agilent Zorbax SB-C18 column (4.6 \u0026times; 250 mm) maintained at 40\u0026deg;C. The mobile phase consisted of 45 mM sodium acetate buffer (pH 3.5; mobile phase A) and 100% acetonitrile (mobile phase B). The compounds were eluted at a flow rate of 1\u0026nbsp;mL/min via a gradient starting at 20% phase B, increasing to 100% over 40 min, followed by re-equilibration to 20% over 20 min. The total run time was 60 min. Detection was performed via a UV\u0026ndash;VIS detector set at 436 nm. Quantification was achieved by comparing peak areas to standard calibration curves. Standard calibration curves were prepared via serial dilutions of reference standards in the same solvent matrix as the samples to ensure accurate quantification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS/MS analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrations of OATD-02 and L-arginine in blood serum were quantified via liquid chromatography coupled with tandem mass spectrometry (LC‒MS/MS). Sample preparation involved protein precipitation with acetonitrile, followed by centrifugation. The resulting supernatants were analyzed via hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry, which employs optimized multiple reaction monitoring (MRM) transitions. The quantification of OATD-02 was performed via matrix-matched calibration and quality control samples. Owing to the endogenous nature of L-arginine, a surrogate matrix approach was applied to ensure accurate detection and quantification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMALDI-MSI imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen mouse tumors were sectioned at 12-\u0026mu;m thickness and mounted onto indium-tin oxide (ITO)-coated glass slides (Bruker Daltonics, Bremen, Germany). The slides were stored in slide boxes, vacuum-sealed in foil (CASO, Arnsberg, Germany), and kept at \u0026minus;80\u0026deg;C until further processing. Prior to matrix application, the slides were equilibrated to room temperature (RT), removed from vacuum packaging, and dried for 10 min in a vacuum desiccator (SP Bel-Art). A\u0026nbsp;deuterated standard of the OATD-02 derivative (5 mg/mL in 50% methanol) was prepared, and 100\u0026nbsp;\u0026micro;L was mixed with 5 mL of DHB matrix solution (40 mg/mL in 70% methanol). The matrix was uniformly applied via an M5 pneumatic sprayer (HTX Technologies LLC, Chapel Hill, North Carolina, USA) with the following parameters: 11 layers, flow rate of 0.05 mL/min, velocity of 1200 mm/min, track spacing of 3 mm, and CC pattern at a nozzle temperature of 65\u0026deg;C. MALDI-MSI measurements were performed via a timsTOF flex mass spectrometer (Bruker Daltonics, Bremen, Germany). External mass calibration was conducted via sodium formate clusters in electrospray ionization (ESI) mode, and online calibration was performed with the deuterated standard. Imaging was carried out with a lateral step size of 40 \u0026micro;m in the mass range of m/z 90\u0026ndash;1300 in positive ion mode. Spectra were acquired via 400 shots per pixel. The following MS parameters were applied: Funnel 1 RF: 250 Vpp, Funnel 2 RF: 250 Vpp, Multipole RF: 250 Vpp, MS1 collision energy: 2 eV, Collision cell RF: 650 Vpp, low mass: m/z 90, TOF transfer time: 70 \u0026micro;s, TOF prepulse storage: 4\u0026nbsp;\u0026micro;s. The MALDI\u0026ndash;MS data were processed via DataAnalysis 6.1 and SCiLS Lab 2025 software (Bruker Daltonics). The spectral intensities were normalized to the deuterated standard to ensure quantitative reliability. Statistical and spatial analyses were performed to assess metabolite distribution in the tissue sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll in vivo experiments were conducted using 7\u0026ndash;9-week-old female BALB/c (BALB/cAnNCrl) mice obtained from Charles River Laboratories (certified SPF status). All procedures complied with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guidelines for the Welfare and Use of Animals in Cancer Research. Ethical approval for the study was granted by the 1st Local Ethics Committee for Animal Experiments in Warsaw, Poland (approval no. 891/2019). CT26 cells were harvested during the exponential growth phase, with viability exceeding 90%, as confirmed by trypan blue exclusion. The mice were subcutaneously implanted in the right flank with 5 \u0026times; 10⁵ CT26 cells suspended in 50 \u0026micro;L of PBS. The animals were then randomized into experimental groups. Tumor growth was monitored at least three times per week via caliper measurements and calculated according to the following formula: width \u0026times; length \u0026times; depth \u0026times; \u0026pi;/6, assuming an ellipsoidal shape. Humane endpoints were defined as body weight loss exceeding 20%, a tumor volume surpassing 2000 mm\u0026sup3;, or the presence of persistent signs of pain or distress. Animals meeting these criteria were euthanized. For LC\u0026ndash;MS and MALDI\u0026ndash;MSI analyses, tumor-bearing mice were orally administered OATD-02 at a dose of 100 mg/kg. Blood, tumors and selected organs were collected after either three doses (16 h time point) or four doses (2 h time point). The control animals received an equivalent volume of vehicle (saline). The collected tissues were snap-frozen and stored at \u0026minus;80\u0026deg;C until further analysis (see the \u003cem\u003eMALDI-MSI Imaging\u003c/em\u003e section for a detailed description). In the efficacy study, the mice were treated orally with OATD-02 at 100 mg/kg twice daily, starting one day after tumor implantation. The anti-mouse CD279 (PD-1) IgG2a rat antibody (clone RMP1-14, cat. 114115, lot B306588, BioXCell) was administered intraperitoneally at 2.5 mg/kg on days 6, 10, 14, and 18 postimplantation. The control animals received saline via oral gavage and an anti-KLH isotype control IgG2b rat antibody (clone LTF-2, cat. BE0090, lot 629816D1, BioXCell) via intraperitoneal injection. On day 15, six randomly selected mice from each group (n = 18) were sacrificed, and the tumors, spleens, and tumor-draining lymph nodes were collected for cytometric analysis (see the \u003cem\u003eLC\u0026ndash;MS/MS analysis\u003c/em\u003e and \u003cem\u003eflow cytometry\u003c/em\u003e sections for details on sample processing). To minimize animal discomfort, appropriate analgesic and anesthetic measures were applied, and humane endpoints were consistently enforced. At the end of the study, the animals were deeply anesthetized with an intraperitoneal injection of ketamine (150 mg/kg) and xylazine (15 mg/kg). Euthanasia was performed via cervical dislocation in accordance with ethical guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collected tumors, spleens, and lymph nodes were cut into small fragments (2\u0026ndash;3 mm). Tumors and lymph nodes were enzymatically digested in DMEM containing collagenase (0.8\u0026nbsp;mg/mL) and DNase I (15 U/mL) for 40 min at 37\u0026deg;C in a CO₂ incubator. Following digestion, tumor and lymph node suspensions, along with spleen fragments, were passed through cell strainers to obtain single-cell suspensions. The cells were then washed with PBS and treated with ACK erythrocyte lysis buffer for 5 min on ice. Lysis was stopped by the addition of PBS, followed by centrifugation. The resulting cell pellets were resuspended in PBS, and the cell density and viability were assessed. Prior to antibody staining, dead cells were labeled via the Zombie Aqua\u0026trade; Fixable Viability Kit (BioLegend). The following anti-mouse antibodies were used for flow cytometry: CD8 (PerCP-Cy5.5, clone 53-6.7, BioLegend), CD3 (APC, clone 17A2, eBioscience), CD4 (BV605, clone GK1.5, BioLegend), CD69 (PE, clone H1.2F3, eBioscience), CD45.2 (V500, clone 104, BioLegend), CD3e\u0026nbsp;(PE-Cy7, clone 145-2C11, eBioscience), and CD11b (PE, clone M1/70, eBioscience). The samples were analyzed via a CytoFLEX Analyzer (Beckman Coulter), and the data were processed via FlowJo software (BD Biosciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism (version 10.0). Data distribution was assessed via D\u0026rsquo;Agostino-Pearson and Shapiro-Wilk normality tests. The sample size for in vivo studies was determined on the basis of a retrospective analysis of previous optimization experiments, ensuring a statistical power of approximately 80% with an alpha level of 0.05. Given the relatively small sample sizes in the in vivo experiments, which were balanced to maintain statistical sensitivity while adhering to the 3R principles, and the nonnormal distribution observed for some variables, statistical comparisons were performed via nonparametric tests to ensure methodological consistency and robustness. The results are presented as the median, with individual data points shown as scatter dot plots. Multiple group comparisons were conducted via the Kruskal‒Wallis test followed by Dunn\u0026rsquo;s post hoc test, and nonsignificant differences were reported alongside the observed trends when applicable. Metabolite concentrations obtained by HPLC were normalized relative to untreated control values to facilitate cross-condition comparisons. Normalization was performed using the formula \u0026ldquo;Normalized value = Treated/Mean untreated\u0026rdquo;. The resulting normalized values were averaged across replicates and visualized as heatmaps, illustrating relative changes in metabolite levels in response to treatment. Survival analysis was conducted using Kaplan-Meier survival curves, with comparisons performed using the log-rank (Mantel‒Cox) test. Additionally, the Gehan-Breslow-Wilcoxon test was applied to assess early differences in survival dynamics. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated via the Mantel\u0026ndash;Haenszel method to estimate the relative risk of death between treatment groups. Median survival times and statistical comparisons were reported for each treatment condition. Differences were considered statistically significant at p \u0026le; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOATD-02 remodels tumor cell metabolism\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the metabolic impact of intracellular arginase inhibition by OATD-02, we analyzed the intracellular levels of key metabolites involved in L-arginine metabolism, including L-arginine, L-ornithine, polyamines (spermidine and spermine), glutamine, and proline, in CT26 and K562 cell extracts via HPLC.\u003c/p\u003e\n\u003cp\u003eWe selected CT26 (murine colorectal carcinoma) and K562 (human chronic myeloid leukemia) cells because of their distinct tumor biology and relevance to in vivo models, allowing us to assess the direct anticancer metabolism-related effects of OATD-02. CT26 cells represent a murine model, enabling in vivo studies in an immunocompetent tumor environment, whereas K562 cells are human lymphoblasts that exhibit elevated ARG2 expression, making them an optimal system for assessing the impact of dual ARG1/ARG2 inhibition (37). Furthermore, OATD-02 has been tested in vivo in both models (38), reinforcing its relevance in evaluating tumor metabolism and therapeutic efficacy.\u003c/p\u003e\n\u003cp\u003eThe selection of OATD-02 concentrations was guided by pharmacokinetic data from in vivo studies, where a 100 mg/kg dose of OATD-02 resulted in plasma and tumor concentrations of approximately 5 \u0026micro;M and 30 nmol/g, respectively, two hours postdosing (38). Given the relatively short exposure, 10 and 30 \u0026micro;M were selected for in vitro experiments as physiologically relevant concentrations, reflecting the levels observed in the animal model.\u003c/p\u003e\n\u003cp\u003eHeatmap visualization (\u003cstrong\u003eFigure 2\u003c/strong\u003e) illustrates the relative metabolite levels normalized to those of untreated controls across different treatment conditions. OATD-02 treatment resulted in a dose- and time-dependent increase in the intracellular L-arginine level. After 48 hours, compared with untreated control cells, CT26 (\u003cstrong\u003eFigure 2, left panel\u003c/strong\u003e) and K562 cells (\u003cstrong\u003eFigure 2, right panel\u003c/strong\u003e) treated with 30 \u0026micro;M OATD-02 presented 1.57-fold and 1.62-fold increases in L-arginine levels, respectively. This effect was further amplified at 96 hours, reaching a 1.80-fold increase in CT26 cells and a 1.87-fold increase in K562 cells at the highest concentration tested. These findings confirm that OATD-02 effectively enhances L-arginine accumulation within tumor cells, supporting its mechanistic role as an intracellular arginase inhibitor.\u003c/p\u003e\n\u003cp\u003eAs expected, OATD-02 treatment significantly suppressed L-ornithine levels, confirming effective arginase inhibition. A marked reduction in intracellular L-ornithine was observed in CT26 cells, where levels decreased to 30% of control values after 48 hours of treatment with 30 \u0026micro;M OATD-02 and further declined to 19% at 96 hours. A similar pattern was detected in K562 cells, where L-ornithine levels decreased to 9% of the control values under prolonged exposure to OATD-02 (\u003cstrong\u003eFigure 2\u003c/strong\u003e). These results confirm the functional inhibition of arginase, preventing the conversion of L-arginine to L-ornithine and thereby limiting its availability for downstream metabolic pathways.\u003c/p\u003e\n\u003cp\u003eConsistent with the reduction in L-ornithine, OATD-02 treatment led to a dose- and time-dependent depletion of intracellular polyamines, specifically spermidine and spermine, which depend on L-ornithine as a precursor for biosynthesis. The most pronounced effect was observed at 96 hours, when the spermine level was reduced to 37% of the control value in CT26 cells and 23% in K562 cells at 30 \u0026micro;M OATD-02 (\u003cstrong\u003eFigure 2\u003c/strong\u003e). The spermidine levels exhibited a similar trend, with a gradual decrease over time. These findings suggest that OATD-02 limits polyamine biosynthesis by depleting the substrate pool, further reinforcing its role in the metabolic reprogramming of tumor cells.\u003c/p\u003e\n\u003cp\u003eIn contrast to the substantial changes observed in L-arginine metabolism, the levels of glutamine and proline remained largely unaffected by OATD-02 treatment. The fluctuations were minor, with variations remaining within a \u0026plusmn;15% range relative to untreated controls, indicating that OATD-02 specifically modulates the L-arginine metabolic axis without broadly perturbing other amino acid pathways (\u003cstrong\u003eFigure 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThese findings demonstrate that OATD-02 effectively increases intracellular L-arginine while depleting L-ornithine and polyamines in both the CT26 and K562 tumor models. The observed metabolic shifts are consistent with potent arginase inhibition, providing a mechanistic basis for the potential antitumor effects of OATD-02. The inclusion of 10 \u0026micro;M OATD-02, on the basis of in vivo exposure data, further strengthens the clinical relevance of these findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOATD-02 increases systemic L-arginine levels and reshapes tumor metabolism in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the biological consequences of the metabolic changes observed in vitro, we evaluated the pharmacodynamic effects of OATD-02 in vivo in CT26 tumor-bearing mice. The selected dose of 100 mg/kg (PO, BID) was confirmed in prior toxicokinetic studies to be safe and within the therapeutic window for long-term administration in BALB/c mice (38).\u003c/p\u003e\n\u003cp\u003eMice were treated with OATD-02 via oral gavage twice daily, and blood and tumor samples were collected at two pharmacokinetically relevant time points: 2 hours after the last dose (peak exposure) and 16 hours after the last dose (trough levels before the next administration) (\u003cstrong\u003eFigure 3A\u003c/strong\u003e). LC\u0026ndash;MS/MS analysis of the plasma samples confirmed that OATD-02 remained detectable at both time points, with mean plasma concentrations of 2.97 \u0026micro;g/mL at 2 h and 1.05 \u0026micro;g/mL at 16 h (\u003cstrong\u003eFigure 3B, left panel\u003c/strong\u003e). These pharmacokinetic data indicate that OATD-02 achieves substantial systemic exposure, supporting its bioavailability and stability in circulation.\u003c/p\u003e\n\u003cp\u003eConsistent with its role as an intracellular arginase inhibitor, OATD-02 treatment led to a marked increase in systemic L-arginine levels (\u003cstrong\u003eFigure 3B, right panel\u003c/strong\u003e). At 2 h posttreatment, the serum L-arginine concentration was approximately 10-fold greater than that in untreated control mice, with the mean concentration increasing from ~140 \u0026micro;M in controls to ~1.20 mM in treated mice. Although L-arginine levels decreased by 16 h, they remained significantly elevated (~930 \u0026micro;M) relative to those of the untreated controls, indicating a sustained pharmacodynamic effect of OATD-02 on systemic L-arginine homeostasis (\u003cstrong\u003eFigure 3B, right panel\u003c/strong\u003e). Further correlation analysis revealed a strong positive correlation between the OATD-02 plasma concentration and the serum L-arginine level (Spearman r\u0026nbsp;=\u0026nbsp;0.797, p = 0.0153), confirming the dose-dependent modulation of L-arginine metabolism.\u003c/p\u003e\n\u003cp\u003eTo investigate the spatial distribution of metabolic alterations in tumors, MALDI-MSI was employed to visualize L-arginine and related metabolites, including proline, spermine, and spermidine, in tumor sections at 2 h and 16 h posttreatment (\u003cstrong\u003eFigure 3C, Suppl. Figure 1\u003c/strong\u003e). Consistent with the serum data, intratumoral L-arginine levels were substantially increased in the OATD-02-treated mice compared with the untreated controls. This effect was evident as early as 2 h posttreatment and persisted at 16 h, with widespread but heterogeneous L-arginine enrichment across tumor tissue (\u003cstrong\u003eFigure 3C, Suppl. Figure 1\u003c/strong\u003e). Notably, regions with the highest L-arginine accumulation coincided with the tumor parenchyma rather than the stromal compartments, suggesting a preferential metabolic impact on tumor cells. However, spatial analysis also revealed localized regions with lower L-arginine enrichment, which may correspond to hypoxic or necrotic areas within the tumor core.\u003c/p\u003e\n\u003cp\u003eIn parallel with L-arginine accumulation, MALDI-MSI analysis revealed a time-dependent reduction trend in the levels of the polyamines spermine and spermidine in tumor tissue. At 2 h posttreatment, a moderate decrease in polyamine levels was observed, particularly in the central regions of larger tumors. By 16 h, spermine and spermidine depletion became more pronounced and spatially widespread, maintaining this trend over time, suggesting that OATD-02-driven inhibition of arginase limits L-ornithine availability for polyamine biosynthesis over time (\u003cstrong\u003eFigure 3C, Suppl. Figure 1\u003c/strong\u003e). Interestingly, the strongest reduction in polyamines was observed in tumor regions where L-arginine accumulation was highest, suggesting a metabolic shift favoring L-arginine retention over its downstream utilization.\u003c/p\u003e\n\u003cp\u003eUnlike the substantial changes observed in L-arginine and polyamines, tumor proline levels exhibited only minor fluctuations in response to OATD-02 treatment (\u003cstrong\u003eFigure 3C, Suppl. Figure 1\u003c/strong\u003e). Proline distribution appeared relatively uniform across tumor sections, suggesting that its biosynthesis from L-arginine-derived intermediates remained largely unaffected. This further supports the selective metabolic action of OATD-02 on the L-arginine-polyamine axis.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that OATD-02 effectively increases systemic and intratumoral L-arginine levels while simultaneously reducing polyamine metabolites in tumors. Spatial metabolic analysis suggests that these changes are not uniform across the tumor microenvironment, with larger tumors displaying distinct metabolic heterogeneity, potentially influenced by variations in vascularization and hypoxia.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe temporal dynamics of these changes suggest that OATD-02 exerts a sustained metabolic effect, reshaping the tumor microenvironment in a manner that may contribute to its antitumor efficacy. Notably, the moderate impact observed for some metabolites, such as proline and early-stage polyamine depletion, may reflect the relatively short duration of OATD-02 administration in this study (only 3\u0026ndash;4 doses before sample collection at 2 h and 16 h, respectively). Longer treatment regimens or combination therapies targeting hypoxic tumor regions may further amplify these metabolic shifts, potentially leading to a more pronounced reprogramming of tumor metabolism over time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOATD-02 improves survival and enhances antitumor immune response in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaving established the strong in vivo metabolic effects of OATD-02, we next evaluated its therapeutic potential in combination with immune checkpoint blockade. CT26 tumor-bearing mice were treated with OATD-02 (100 mg/kg, PO, BID) alone or in combination with anti-PD-1 antibodies (2.5 mg/kg, IP) according to the schedule outlined in \u003cstrong\u003eFigure 4A\u003c/strong\u003e. Tumor growth and survival were monitored, and immune profiling of tumors, spleens, and lymph nodes was conducted on day 15 postimplantation.\u003c/p\u003e\n\u003cp\u003eKaplan-Meier survival analysis revealed that both the OATD-02 and anti-PD-1 monotherapies significantly extended median survival compared with the vehicle-treated controls (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). Compared with control mice, animals receiving OATD-02 alone had a median survival of 29.5 days, whereas anti-PD-1 monotherapy extended survival to 32 days. The log-rank test confirmed significant differences between the vehicle group and the anti-PD-1 group (p = 0.0097) and between the vehicle group and the OATD-02 group (p = 0.0470). However, the greatest survival benefit was observed in the combination therapy group (OATD-02 + anti-PD-1), with a median survival of 41.5 days, which was significantly longer than that in the monotherapy group (p = 0.0479 vs. anti-PD-1 alone). Hazard ratio (Mantel\u0026ndash;Haenszel) analysis indicated that, compared with anti-PD-1 therapy alone, combination therapy reduced the risk of death by approximately 62% (HR = 0.382, 95% CI: 0.147\u0026ndash;0.991), supporting a synergistic effect.\u003c/p\u003e\n\u003cp\u003eTo assess the immune mechanisms underlying this survival benefit, we analyzed tumor-infiltrating immune cells via flow cytometry. OATD-02 treatment led to a significant increase in the proportion of CD45⁺ T cells within tumors (\u003cstrong\u003eFigure 4C, top left panel\u003c/strong\u003e). This effect was further enhanced in the combination therapy group (p = 0.0429, Dunn\u0026rsquo;s test), suggesting more robust immune infiltration. Given that L-arginine availability is crucial for T-cell activation and proliferation, these findings indicate that OATD-02-mediated arginase inhibition relieves metabolic constraints on intratumoral T cells, facilitating their recruitment and expansion.\u003c/p\u003e\n\u003cp\u003eTo determine whether these effects extend beyond the tumor microenvironment, we analyzed tumor-draining lymph nodes (TDLNs), which are critical sites for priming antitumor immune responses. Compared with those in the vehicle group, the numbers of CD4⁺ and CD8⁺ T cells were significantly greater in the mice that received OATD-02 + anti-PD-1 (p = 0.0058 for CD4⁺ and p = 0.0099 for CD8⁺; Dunn\u0026rsquo;s test; \u003cstrong\u003eFigure 4C, middle panels\u003c/strong\u003e). Neither monotherapy alone induced significant changes relative to the controls, suggesting that increased systemic L-arginine availability in the combination treatment group supports the expansion of tumor-reactive T cells in lymphoid organs, potentially enhancing long-term antitumor immunity.\u003c/p\u003e\n\u003cp\u003eTo further assess systemic immune activation, we measured the expression of CD69, a marker of early T-cell activation, on splenic CD4⁺ and CD8⁺ T cells (\u003cstrong\u003eFigure 4C, right panels\u003c/strong\u003e). While both monotherapies induced a modest increase in CD69 expression, the combination treatment led to highly significant upregulation compared with the vehicle (p \u0026lt; 0.0001 for CD4⁺, p = 0.0005 for CD8⁺, Dunn\u0026rsquo;s test). This finding suggests that systemic T-cell activation extends beyond the tumor site, potentially priming circulating T cells for enhanced antitumor responses.\u003c/p\u003e\n\u003cp\u003eIn contrast, CD11b⁺ myeloid cell populations, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), did not significantly change following treatment (p \u0026gt; 0.05 for all comparisons, Dunn\u0026rsquo;s test; \u003cstrong\u003eFigure 4C, bottom left panel\u003c/strong\u003e). However, a visible trend toward reduced myeloid cell infiltration was observed in the combination therapy group, suggesting a potential shift in the tumor immune landscape. These results imply that while OATD-02 effectively promotes T-cell activation, its effects on immunosuppressive myeloid populations may require longer treatment durations or additional combinatorial approaches for full efficacy.\u003c/p\u003e\n\u003cp\u003eThese findings demonstrate that OATD-02 enhances antitumor immunity by increasing intratumoral T-cell infiltration, expanding tumor-reactive T cells in the lymph nodes, and increasing systemic T-cell activation. When combined with anti-PD-1 therapy, these effects translate into a significant survival benefit, with a 62% reduction in mortality risk compared with anti-PD-1 therapy alone (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). Notably, the most pronounced immunological effects, including enhanced T-cell activation in the spleen and lymph nodes, were exclusive to the combination therapy group, underscoring the potential of OATD-02 as a metabolic adjuvant for immune checkpoint blockade.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMetabolic reprogramming in the tumor microenvironment (TME) plays a pivotal role in cancer progression and immune evasion. Our study demonstrated that the dual ARG1/ARG2 arginase inhibitor OATD-02 effectively modulates the L-arginine metabolic axis, enhancing antitumor immune responses. Using a combination of in vitro, in vivo, and spatial metabolomics approaches, we demonstrated that OATD-02 increases L-arginine availability within tumors, depletes immunosuppressive polyamines, and enhances the efficacy of immune checkpoint inhibitors (ICIs). Notably, OATD-02 significantly elevated both intracellular and systemic L-arginine levels while concurrently reducing L-ornithine and polyamine concentrations. This effect was observed in CT26 colon carcinoma and K562 leukemia cell models, as well as in CT26 tumor-bearing mice. Our findings are consistent with those of previous studies demonstrating that arginase-mediated L-arginine depletion suppresses T-cell activation and facilitates tumor immune evasion (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Unlike numidargistat (CB-1158), an arginase inhibitor with predominantly extracellular activity and limited efficacy against ARG2-driven pathways (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), OATD-02 effectively inhibits both extracellular and intracellular arginase isoforms. This distinction is critical, as ARG2 plays a tumor-intrinsic role in metabolic adaptation, regulating nitrogen balance and polyamine biosynthesis (\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). The ability of OATD-02 to penetrate tumor cells and inhibit intracellular ARG2 represents a unique mechanistic advantage over other arginase-targeting therapies.\u003c/p\u003e \u003cp\u003eWhile systemic metabolic alterations are critical for therapeutic efficacy, spatial heterogeneity within the tumor microenvironment may further shape treatment outcomes. To characterize the metabolic landscape of tumors, we employed MALDI mass spectrometry imaging (MALDI-MSI) to map metabolite distributions with high spatial resolution. Our results revealed heterogeneous L-arginine accumulation across tumor sections. Given that larger tumors often develop hypoxic cores with impaired circulation and tissue necrosis (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), these spatial differences may reflect microenvironmental constraints on metabolic fluxes. A similar approach was reported by Andersen et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), who demonstrated that MALDI-MSI enables the spatially resolved detection of metabolic alterations in prostate cancer tissues, revealing distinct metabolic differences between tumor and normal regions. MALDI-MSI has also demonstrated that anti-cancer drugs frequently fail to penetrate tumors in both patients and mouse models (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). These findings underscore the power of MALDI-MSI in identifying spatial metabolic heterogeneity and highlight its value in assessing the metabolic reprogramming induced by OATD-02 in our study. Beyond the heterogeneous distribution of L-arginine, we observed a trend towards preferential depletion of polyamines, particularly in central tumor regions. This suggests that metabolic adaptation to OATD-02 treatment is influenced by tumor perfusion and oxygen availability. Hypoxic tumor regions are known to favor metabolic pathways that sustain proliferation under stress, including increased glutaminolysis and alternative nitrogen metabolism (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). The differential impact of OATD-02 across spatially distinct tumor compartments suggests that L-arginine-polyamine axis modulation may be influenced by oxygen and nutrient gradients within the tumor microenvironment. Since hypoxic regions often exhibit increased polyamine turnover to support tumor survival (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e), this adaptation may shape the response to OATD-02.\u003c/p\u003e \u003cp\u003eExpanding on these insights, our results show that metabolic modulation by OATD-02 can significantly enhance the efficacy of ICIs. By increasing L-arginine availability and reducing polyamines, OATD-02 creates a microenvironment more permissive for T-cell activation, thereby potentiating the effects of PD-1 blockade. OATD-02 combined with anti-PD-1 therapy led to increased T-cell infiltration in tumors, enhanced T-cell activation in tumor-draining lymph nodes, and improved systemic immune responses. These findings are consistent with those of Sosnowska et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), who reported that arginase inhibition relieves metabolic constraints on T cells, thereby enhancing PD-1 blockade. Similarly, Grzywa et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) and our previous studies (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) demonstrated that OATD-02 boosts antitumor immunity in multiple preclinical models, whereas Pilanc et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) reported increased immune cell infiltration and potentiation of anti-PD-1 therapy by OATD-02 in glioblastoma models, underscoring the broad applicability of this cancer-agnostic mechanism across multiple tumor types. Furthermore, whereas CB-1158, an ARG1-restricted inhibitor, showed only modest increases in plasma L-arginine in clinical trials \u0026ndash; likely owing to its limited intracellular activity (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) \u0026ndash; our findings indicate that the dual targeting of both the intracellular and the extracellular arginases by OATD-02 results in superior metabolic and immunomodulatory effects. Indeed, our results align with those of previous experiments (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), in which we directly compared OATD-02 to CB-1158 and found that OATD-02 produced greater tumor growth inhibition in an ARG2-dependent model.\u003c/p\u003e \u003cp\u003eThe above observations underscore the crucial role of L-arginine metabolism in shaping the tumor microenvironment (TME) and highlight the importance of considering spatial metabolic heterogeneity when evaluating arginase-targeting therapies. The uneven metabolic reprogramming observed in OATD-02-treated tumors suggests the need to address regional metabolic constraints, particularly in hypoxic tumor cores. Arginases, particularly ARG2, regulate tumor metabolism and immune suppression (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Their inhibition restores L-arginine availability, crucial for effective T-cell activation and antitumor immunity. Given its role in mitochondrial metabolism and nitrogen balance, intracellular ARG2 is a particularly relevant therapeutic target (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). By inhibiting both isoforms, OATD-02 may provide broader therapeutic benefits than agents that selectively target ARG1 (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Tumor cells adapt metabolically to sustain growth and evade immune surveillance. L-arginine, polyamine, and proline metabolism play key roles in these processes, and OATD-02 disrupts their balance, reshaping the TME.\u003c/p\u003e \u003cp\u003eL-arginine fuels tumor cell proliferation and supports processes such as extracellular matrix production, but it is also a critical resource for immune cell function (e.g., as a precursor for nitric oxide and polyamines). Tumors frequently exploit L-arginine metabolism by upregulating arginases, which deplete local L-arginine and redirect its metabolic flux toward polyamine and proline biosynthesis to support tumor growth and adaptation (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Conversely, depriving immune cells of L-arginine has profound immunosuppressive effects. L-arginine deficiency impairs T-cell proliferation and activation by downregulating the CD3ζ chain of the T-cell receptor complex, thereby weakening TCR signaling (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). It also skews T-cell differentiation, leading to diminished IFN-γ and IL-2 production and a shift away from effective Th1 immune responses (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Collectively, these effects create an immunosuppressive environment, highlighting why L-arginine availability is critical for antitumor immunity. L-arginine depletion promotes tumor growth and immune evasion. OATD-02 restores L-arginine levels, reinvigorating T-cell activity and enhancing antitumor immunity (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Concurrent targeting of ARG1 and ARG2 may further enhance these effects.\u003c/p\u003e \u003cp\u003eIn addition to L-arginine, polyamines play a significant role in tumor progression and immune evasion through complementary metabolic mechanisms. Polyamines, such as spermidine and spermine, are essential for tumor cell proliferation and survival. They stabilize DNA, modify chromatin, and promote ribosome biogenesis, thereby accelerating tumor growth (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). These oncometabolites also contribute to immunosuppression. The spermidine secreted by tumor cells can directly inhibit CD8⁺ T-cell activation by preventing proper TCR clustering, effectively acting as a metabolic immune checkpoint (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) and highlighting how tumors exploit polyamine pathways to evade immune surveillance. Consistent with this notion, our study confirms the critical role of polyamine metabolism in the TME. OATD-02 treatment led to a significant reduction in intracellular spermidine and spermine levels in both CT26 and K562 cells. The depletion of polyamines observed in vitro was mirrored in tumor tissues \u0026ndash; MALDI-MSI analysis of tumors from OATD-02-treated mice revealed a progressive decline in polyamine levels over time. Arginase inhibition by OATD-02 alters the metabolic balance in tumors, limiting the production of immunosuppressive polyamines and disrupting a key mechanism of tumor-mediated immune evasion. Given the dual role of polyamines in promoting tumor growth and suppressing immune responses, targeting polyamine synthesis has been explored as a therapeutic strategy to increase the efficacy of immunotherapy (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In this context, the polyamine-lowering effect of OATD-02 may contribute to creating a tumor microenvironment that is less conducive to immune escape, thereby improving the effectiveness of anti-PD-1 therapy. Notably, the sustained reduction in tumor polyamine levels observed in our study further supports the combination of OATD-02 with immune checkpoint inhibitors, particularly in tumors characterized by high polyamine levels and an immunosuppressive microenvironment (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProline metabolism represents another aspect of tumor metabolic adaptation, influencing extracellular matrix (ECM) remodeling, redox homeostasis, and survival under stress. Proline biosynthesis contributes to oxidative stress resistance by maintaining the NADP⁺/NADPH balance, buffering redox status, and supporting tumor cell survival, particularly under hypoxic conditions (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Additionally, proline plays a crucial role in collagen production, as its derivative, hydroxyproline, is a major structural component of collagen. Increased proline utilization promotes collagen-rich ECM deposition and stiffness, facilitating tumor invasion and metastasis. Notably, high collagen content in tumors often correlates with poor prognosis (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Interestingly, despite the metabolic significance of proline, OATD-02 treatment did not significantly alter the intracellular proline levels in our models. In both CT26 and K562 cells, proline concentrations remained relatively stable across all conditions, even as L-arginine and L-ornithine underwent substantial changes. These findings suggest that tumor cells may preserve proline homeostasis through alternative pathways that are unaffected by arginase inhibition. For instance, cancer cells can recycle proline from collagen-derived hydroxyproline or upregulate glutamine metabolism to sustain proline synthesis (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Indeed, the high glutamine content in our cell culture medium likely provided an alternative substrate for proline production, potentially masking any secondary effects of arginase inhibition on the L-ornithine\u0026ndash;proline axis.\u003c/p\u003e \u003cp\u003eThe immunomodulatory and protumorigenic impact of arginases has been well documented in solid tumors, including lung, breast, colorectal, pancreatic, prostate, melanoma, renal, ovarian, and esophageal cancers, where elevated arginase activity frequently correlates with enhanced tumor growth, metastasis, or immune evasion (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan additionalcitationids=\"CR67 CR68 CR69\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). These findings underscore the broad relevance of L-arginine metabolism as a therapeutic target in oncology. However, the dependency on arginase activity varies among cancer types. For example, thymic epithelial tumors lack ARG1 expression, whereas in some lung cancers, ARG2 activity does not induce immunosuppression unless it is accompanied by additional factors such as nitric oxide production, highlighting the context-dependent role of arginases (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). Nevertheless, accumulating evidence suggests that dysregulated L-arginine metabolism also plays a crucial role in hematologic malignancies. Notably, acute myeloid leukemia (AML) cells secrete arginase to deplete L-arginine, leading to T-cell suppression; in AML models, arginase inhibition restores T-cell proliferation and induces leukemia cell apoptosis (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). Furthermore, multiple myeloma exhibits increased arginase activity in myeloid cells, contributing to systemic L-arginine depletion and T-cell dysfunction (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e). Chronic myeloid leukemia (CML) provides another example, as CML cells rely on ARG2 to adapt to hypoxic conditions and resist therapy. Inhibiting arginase in CML has been shown to counteract these resistance mechanisms (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). Collectively, these findings extend the therapeutic rationale for arginase inhibition to a broad spectrum of malignancies.\u003c/p\u003e \u003cp\u003eGiven the broad immunosuppressive role of arginase in the TME, its inhibition represents a promising strategy to enhance cancer immunotherapy. Owing to its dual ARG1/ARG2 targeting ability and potent immunomodulatory effects, OATD-02 is a strong candidate for combination therapies designed to overcome resistance to ICIs. Multiple studies have demonstrated that increased arginase activity in tumors suppresses antitumor immunity by impairing CD8⁺ T-cell function and promoting immunosuppressive myeloid populations (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Early trials with arginase inhibitors, such as CB-1158 (numidargistat), validated arginase as a therapeutic target by showing that its inhibition restores T-cell proliferation and improves checkpoint blockade efficacy in preclinical models (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). However, CB-1158 primarily targets extracellular ARG1 and has limited cell permeability, which may explain its modest clinical efficacy (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). In contrast, the ability of OATD-02 to effectively inhibit both intracellular and extracellular arginases provides broader immunomodulatory effects (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), potentially translating into enhanced therapeutic benefits when it is combined with ICIs.\u003c/p\u003e \u003cp\u003eOn the basis of preclinical data, combining arginase blockade with ICIs appears to be one of the most promising combinatorial cancer therapies. AZD0011 significantly increased intratumoral L-arginine levels while reducing ornithine, and its combination with anti\u0026ndash;PD-L1 therapy resulted in markedly greater tumor growth inhibition than either treatment alone (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). In addition to checkpoint inhibitors, OATD-02\u0026ndash;mediated metabolic reprogramming may enhance other immunotherapies. For example, Ye et al. (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e) demonstrated that cotargeting arginase and L-arginine-depleting enzymes potentiated T-cell activation and tumor regression in a melanoma model, suggesting that increasing L-arginine availability could improve the outcomes of adoptive T-cell transfer or cancer vaccines. A promising avenue for enhancing antitumor immunity is combining arginase inhibition with other metabolic interventions. In our previous studies (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), we reported that adding OATD-02 to an IDO1 (indoleamine-2,3-dioxygenase) inhibitor (epacadostat) alongside anti\u0026ndash;PD-L1 therapy resulted in significantly improved tumor control in a murine CT26 colorectal carcinoma model compared with dual or single agent treatments. This finding aligns with earlier studies indicating that simultaneous blockade of L-arginine and tryptophan catabolism can synergistically enhance antitumor immunity (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Given the capacity of OATD-02 to modulate both L-arginine and polyamine pathways, pairing it with IDO1 inhibitors may be particularly effective in counteracting metabolic immunosuppression. Another promising approach for combination therapy involves the integration of arginase inhibitors with targeted anticancer agents. Preclinical studies suggest that arginase blockade can increase the efficacy of certain tyrosine kinase inhibitors. For example, combining an arginase inhibitor with cabozantinib or lenvatinib has been shown to remodel the immune microenvironment and improve tumor control (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). These findings suggest that OATD-02 may similarly enhance the effects of targeted therapies by mitigating tumor metabolic resistance mechanisms. In addition to targeted therapies, arginase inhibition may also act synergistically with other treatment modalities, including radiotherapy and NK cell\u0026ndash;based immunotherapies. Notably, the combination of arginase inhibition with blockade of the inhibitory NK cell receptor NKG2A has been reported to improve tumor control and enhance immune activation (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e), highlighting additional therapeutic opportunities for OATD-02 in multimodal treatment regimens.\u003c/p\u003e \u003cp\u003eOur findings provide strong justification for the continued clinical development of OATD-02 as a metabolic-immunotherapeutic agent. While our studies demonstrated that dual ARG1/ARG2 inhibition enhances immune responses by increasing intratumoral L-arginine (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) and depleting polyamines, the full extent of tumor adaptation to prolonged L-arginine repletion remains to be explored. Future studies using patient-derived models will be essential to determine how metabolic compensation, such as glutamine or tryptophan dependence, may influence therapeutic outcomes (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Moreover, integrating MSI with functional immune profiling may provide deeper insights into how spatial metabolic heterogeneity impacts therapeutic responses, helping to refine treatment strategies and optimize combination approaches. Encouragingly, the ongoing phase I clinical trial (NCT05759923) will provide critical insights into the safety, pharmacokinetics, and pharmacodynamics of OATD-02 in patients with advanced solid tumors. These results will guide the design of future combination strategies, reinforcing the potential of OATD-02 as a novel metabolic-immunotherapeutic approach.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOver the past years, studies have demonstrated the therapeutic potential of ARG1 inhibition in restoring L-arginine availability and enhancing antitumor immunity (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e). However, recent studies have revealed a previously underappreciated role of ARG2, the mitochondrial isoform, in tumor metabolism and immune evasion, revealing its critical function in nitrogen balance, mitochondrial adaptation, and metabolic reprogramming (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan additionalcitationids=\"CR83\" citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e). To our knowledge, this is the first study providing direct metabolic evidence of pharmacological ARG2 inhibition, demonstrating that dual ARG1/ARG2 blockade reshaped the tumor microenvironment by increasing L-arginine availability, depleting polyamines, and enhancing immune responses. This mechanistic advantage over ARG1-restricted inhibitors such as numidargistat (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e) underscores the importance of targeting both arginase isoforms in metabolic therapy. Using MALDI-MSI spatial metabolomics, we mapped the metabolic consequences of OATD-02 at high spatial resolution, revealing widespread L-arginine accumulation in tumor tissues, a shift that aligns with enhanced T-cell activation. While metabolic adaptations to sustain L-arginine repletion remain a consideration, our findings reinforce the therapeutic relevance of ARG2 inhibition as a metabolic-immunotherapeutic strategy. Future research should focus on identifying tumor types that would benefit the most from dual ARG1/ARG2 inhibition. Dual arginase blockade represents a promising approach for overcoming immune suppression in tumors, offering a novel avenue for metabolic intervention in cancer treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e3R \u0026ndash; Replacement, reduction, refinement (ethical principles for animal research)\u003c/p\u003e\n\u003cp\u003eABH \u0026ndash; 2(S)-amino-6-boronohexanoic acid\u003c/p\u003e\n\u003cp\u003eARG1 \u0026ndash; Arginase 1\u003c/p\u003e\n\u003cp\u003eARG2 \u0026ndash; Arginase 2\u003c/p\u003e\n\u003cp\u003eBEC \u0026ndash; (\u003cem\u003eS\u003c/em\u003e)-(2-boronoethyl)-L-cysteine\u003c/p\u003e\n\u003cp\u003eBID \u0026ndash; twice a day\u003c/p\u003e\n\u003cp\u003eCAFs \u0026ndash; Cancer-Associated Fibroblasts\u003c/p\u003e\n\u003cp\u003eCD \u0026ndash; Cluster of Differentiation (e.g., CD8, CD4; surface markers on immune cells)\u003c/p\u003e\n\u003cp\u003eCI \u0026ndash; confidence interval\u003c/p\u003e\n\u003cp\u003eCT26 \u0026ndash; Murine Colon Carcinoma Cell Line\u003c/p\u003e\n\u003cp\u003eFACS \u0026ndash; Fluorescence-Activated Cell Sorting\u003c/p\u003e\n\u003cp\u003eFBS - Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eHPLC \u0026ndash; high-performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eHR \u0026ndash; hazard ratio\u003c/p\u003e\n\u003cp\u003eICIs \u0026ndash; Immune checkpoint inhibitors\u003c/p\u003e\n\u003cp\u003eIgG2a \u0026ndash; Immunoglobulin G, subclass 2a\u003c/p\u003e\n\u003cp\u003eIP \u0026ndash; intraperitoneal administration\u003c/p\u003e\n\u003cp\u003eK562 \u0026ndash; Human Chronic Myelogenous Leukemia Cell Line\u003c/p\u003e\n\u003cp\u003eL-Arg \u0026ndash; L-arginine\u003c/p\u003e\n\u003cp\u003eLC‒MS/MS \u0026ndash; Liquid chromatography‒tandem mass spectrometry\u003c/p\u003e\n\u003cp\u003eMALDI-MSI \u0026ndash; Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Imaging\u003c/p\u003e\n\u003cp\u003eMDSCs \u0026ndash; Myeloid-Derived Suppressor Cells\u003c/p\u003e\n\u003cp\u003eNO \u0026ndash; Nitric oxide\u003c/p\u003e\n\u003cp\u003eNOS \u0026ndash; nitric oxide synthase\u003c/p\u003e\n\u003cp\u003ePBS \u0026ndash; Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003ePD-1 \u0026ndash; Programmed Cell Death Protein 1\u003c/p\u003e\n\u003cp\u003ePO \u0026ndash; \u003cem\u003eper os\u003c/em\u003e (oral administration)\u003c/p\u003e\n\u003cp\u003eRPMI-1640 \u0026ndash; Roswell Park Memorial Institute Medium\u003c/p\u003e\n\u003cp\u003eRT \u0026ndash; room temperature\u003c/p\u003e\n\u003cp\u003eSPF \u0026ndash; specific pathogen-free\u003c/p\u003e\n\u003cp\u003eTAMs \u0026ndash; Tumor-associated macrophages\u003c/p\u003e\n\u003cp\u003eTCR \u0026ndash; T-cell receptor\u003c/p\u003e\n\u003cp\u003eTME \u0026ndash; Tumor microenvironment\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll procedures complied with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guidelines for the Welfare and Use of Animals in Cancer Research. Ethical approval for the study was granted by the 1st Local Ethics Committee for Animal Experiments in Warsaw, Poland (approval no. 891/2019). All experiments were performed in accordance with relevant institutional and national guidelines and regulations. The study is reported in accordance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003c/span\u003e). Procedures involving animals, including anesthesia and euthanasia, were conducted in accordance with veterinary best practice and followed the recommendations outlined in the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eSeveral authors (MMG, AM, AK, KG, MS-R, MK, AZ, PP, MM, AT, TR, RB, ZZ) are employees of Molecure SA, which holds proprietary rights to OATD-02. The remaining authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eMMG, YU, AM, TR, RB, CH, and ZZ designed the study. MMG, MM, AK, and AZ performed the in vivo experiments. MMG, AZ, and PP conducted the in vitro experiments. AK, KG, MK, and MS-R carried out the flow cytometry analyses. MALDI\u0026ndash;MSI analyses were performed by YU and TB. AT and TR conducted the LC\u0026ndash;MS analyses, whereas AKJ performed the HPLC analyses. MMG, AM, AK, YU, and TB analyzed the data. MMG wrote the initial version of the manuscript, while ZZ, CH, and YU revised and edited it. MG, ZZ, and CH supervised the project and coordinated the study. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank EU-OPENSCREEN for providing access to advanced research infrastructure, which enabled the MALDI‒MSI analyses conducted at CeMOS, Mannheim University of Applied Sciences (Mannheim, Germany). Their support was instrumental in visualizing the metabolic alterations induced by OATD-02.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are not publicly available due to the data policy of Molecure SA but can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCan\u0026egrave; S, Geiger R, Bronte V. The roles of arginases and arginine in immunity. Nature Reviews Immunology. Nature Research; 2024. \u003c/li\u003e\n\u003cli\u003eChen C, Han P, Qing Y. 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Arginase II Expressed in Cancer-Associated Fibroblasts Indicates Tissue Hypoxia and Predicts Poor Outcome in Patients with Pancreatic Cancer. PLoS One. 2013 Feb 12;8(2). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dual arginase inhibition, OATD-02, MALDI imaging, metabolic reprogramming, arginine metabolism, polyamines, mitochondrial metabolism, tumor metabolism, anticancer therapy, immune modulation","lastPublishedDoi":"10.21203/rs.3.rs-6305179/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6305179/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetabolic reprogramming within the tumor microenvironment (TME) plays a central role in cancer progression and immune evasion, with L-arginine metabolism emerging as a key regulatory axis. Arginase overexpression depletes intratumoral L-arginine, thus suppressing T-cell proliferation while fuelling tumor growth through polyamine biosynthesis. OATD-02, a novel dual arginase (ARG1/ARG2) inhibitor, reprograms tumor metabolism by restoring L-arginine availability and reducing the levels of polyamines, thereby shifting the TME toward a more immunostimulatory state. Unlike ARG1-selective inhibitors with limited intracellular uptake, OATD-02 effectively inhibits both extracellular and intracellular arginases, thereby addressing a major limitation of first-generation arginase inhibitors.\u003c/p\u003e \u003cp\u003eTo visualize the pharmacodynamic effects of OATD-02 dosing in mice with spatial resolution, we employed MALDI mass spectrometry imaging (MALDI-MSI), thus enabling direct mapping of metabolic changes within tumor tissues. In preclinical models, OATD-02 treatment led to widespread accumulation of intratumoral L-arginine with concomitant depletion of polyamines and resulted in metabolic shifts that correlated with increased immune cell infiltration and an improved response to immune checkpoint blockade. These findings underscore the role of dual arginase inhibition in reshaping tumor metabolism and overcoming immune suppression by restoring the metabolic fitness of immune cells to fight cancer.\u003c/p\u003e \u003cp\u003eThe metabolic changes caused by OATD-02 treatment resulted in significantly enhanced antitumor immune responses, increased T-cell infiltration in tumors, expansion of CD8⁺ T cells in draining lymph nodes, and systemic upregulation of T-cell activation markers. These effects translated into a substantial survival benefit in the CT26 tumor model, particularly when combined with anti-PD-1 therapy, where OATD-02 improved checkpoint blockade efficacy by relieving metabolic constraints affecting tumor-infiltrating lymphocytes.\u003c/p\u003e \u003cp\u003eBy leveraging the unique capabilities of MALDI-MSI, this study provides high-resolution metabolic insights into the mechanism of action of OATD-02, reinforcing its potential as a next-generation metabolic-immunotherapeutic agent. The observed metabolic reprogramming, coupled with enhanced immune activation and prolonged survival, supports the clinical development of OATD-02 as a promising strategy for enhancing cancer immunotherapy efficacy. OATD-02 is currently undergoing clinical evaluation in a phase I/II trial (NCT05759923), which will further elucidate its safety and therapeutic impact. These findings highlight the potential of arginase-targeted therapies in cancer treatment and underscore the value of MALDI-MSI as a powerful tool for tracking metabolic responses to therapy.\u003c/p\u003e","manuscriptTitle":"Metabolomic reprogramming of the tumor microenvironment by dual arginase inhibitor OATD-02 boosts anticancer immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 11:51:53","doi":"10.21203/rs.3.rs-6305179/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-04T12:14:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-04T07:36:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-03T08:48:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243084554346865384067236536263927598251","date":"2025-04-01T02:42:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251565396487156116512986199335169495413","date":"2025-03-31T21:36:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43987698792894930128995942896808380925","date":"2025-03-31T12:05:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T11:52:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-31T06:55:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-31T02:43:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-27T09:29:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-25T15:08:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"07cbdc5b-e539-49d8-862a-f3a15c814b31","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46482003,"name":"Biological sciences/Cancer/Cancer imaging"},{"id":46482004,"name":"Biological sciences/Drug discovery/Target validation"},{"id":46482005,"name":"Biological sciences/Drug discovery/Pharmaceutics"},{"id":46482006,"name":"Biological sciences/Immunology/Tumour immunology"},{"id":46482007,"name":"Biological sciences/Cancer/Cancer metabolism"},{"id":46482008,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":46482009,"name":"Biological sciences/Cancer/Cancer models"},{"id":46482010,"name":"Biological sciences/Cancer/Cancer therapy/Drug development"},{"id":46482011,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":46482012,"name":"Health sciences/Oncology/Cancer/Cancer metabolism"},{"id":46482013,"name":"Health sciences/Oncology/Cancer/Cancer imaging"}],"tags":[],"updatedAt":"2025-06-02T16:09:16+00:00","versionOfRecord":{"articleIdentity":"rs-6305179","link":"https://doi.org/10.1038/s41598-025-03446-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-28 15:57:51","publishedOnDateReadable":"May 28th, 2025"},"versionCreatedAt":"2025-04-01 11:51:53","video":"","vorDoi":"10.1038/s41598-025-03446-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-03446-1","workflowStages":[]},"version":"v1","identity":"rs-6305179","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6305179","identity":"rs-6305179","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}