Enhancement of anti-programmed cell death protein-1 immunotherapy in non-small cell lung cancer using arginine and citrulline supplementation

Enhancement of anti-programmed cell death protein-1 immunotherapy in non-small cell lung cancer using arginine and citrulline supplementation | 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 Research Article Enhancement of anti-programmed cell death protein-1 immunotherapy in non-small cell lung cancer using arginine and citrulline supplementation Naoki Miyamoto, Mitsuteru Yoshida, Shinichi Tsukumo, Hirohisa Ogawa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5294074/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study aimed to examine the role of citrulline in the lung cancer microenvironment and its potential synergistic effects with other therapies. Methods Murine lung cancer cells (CMT167) were subcutaneously implanted into mice to establish tumor models, followed by treatment with the anti-PD-1 antibody either alone or in combination with arginine or arginine and citrulline. Tumor growth, survival rate, cytokine levels, immune cell populations, and metabolic marker expression were assessed using histological, immunostaining, flow cytometry, and serum analyses. Results Mice in the treatment groups exhibited significantly lower tumor volumes than those in the control group (control, 1161.59 ± 294.73; anti-PD-1, 427.38 ± 355.34; anti-PD-1 plus arginine, 452.10 ± 332.04; anti-PD-1 plus arginine and citrulline, 198.45 ± 236.22 mm 3 ; P < 0.0001). Furthermore, the anti-PD-1 plus arginine and citrulline group exhibited significantly improved progression-free survival compared with that of the control group ( P = 0.00039). The anti-PD-1 plus arginine and citrulline group also showed a significantly higher number of tumor-infiltrating CD8 + lymphocytes per high-power field (hpf) than the control group (control, 24.22 ± 9.13; anti-PD-1, 29.20 ± 9.41; anti-PD-1 plus arginine, 34.33 ± 8.81; anti-PD-1 plus arginine and citrulline, 46.56 ± 10.01 cells/hpf). Conclusion Arginine and citrulline supplementation facilitated CD8 + lymphocyte infiltration into the tumor microenvironment, thereby augmenting the efficacy of lung cancer immunotherapy. anti-PD-1 immunotherapy CD8 T cells non-small cell lung cancer arginine citrulline supplementation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Lung cancer is the most prevalent cancer globally, accounting for approximately 2.2 million new cases and 1.8 million deaths in 2020 (Sung et al. 2021). Over the past decade, the treatment landscape for non-small cell lung cancer (NSCLC) has been revolutionized by the advent of molecular-targeted therapeutics and immune checkpoint inhibitors (ICIs) (Yang et al. 2016; Herbst et al. 2018). In cases lacking targetable molecular alterations, the assessment of programmed death ligand 1 (PD-L1) expression is crucial for directing treatment strategies for both squamous and non-squamous lung cancers (Alexander et al. 2020). Although ICIs have substantially improved outcomes in previously untreatable cancer cases, their effectiveness as monotherapy is limited, with response rates ranging from 14–20% (Xia and Liu 2019). Therefore, identifying the immunological mechanisms that confer resistance to these inhibitors is essential for enhancing therapeutic efficacy and overcoming tumor resistance. A mechanism by which tumors evade the host immune response involves arginine metabolism by arginases (Munder 2009). Arginine is crucial for long-term survival, immune memory generation, and tumor-killing efficiency of T cells. Consequently, its deficiency reduces T-cell activity and increases tumor size (Martí I Líndez and Reith 2021). Conversely, augmenting arginine levels in the tumor microenvironment activates T cells and enhances the efficacy of ICIs (He et al. 2017). Both arginase inhibitors and arginine itself can inhibit tumor growth, suggesting arginine metabolism as a viable therapeutic target (Miret et al. 2019; Sosnowska et al. 2021). Arginine also plays a vital role in the synthesis of nitric oxide (NO), polyamines, and proteins. However, after oral intake, arginine is metabolized and converted to various forms (Agarwal et al. 2017) through two primary metabolic pathways—conversion to NO and citrulline by nitric oxide synthase (NOS) and hydrolysis to ornithine and urea by arginase. Within the NOS pathway, the enzyme argininosuccinate synthetase, coupled with constitutively expressed argininosuccinate lyase, facilitates the recycling of citrulline for de novo arginine synthesis (Rath et al. 2014). Considering these roles of arginine, citrulline may serve as a promising alternative to enhance arginine availability. Citrulline supplementation is reportedly more effective than arginine alone in boosting arginine availability and NO synthesis (El-Hattab et al. 2012; Agarwal et al. 2017). We hypothesized that supplementing anti-PD-1 antibodies and arginine with citrulline can augment their antitumor effects. Accordingly, we investigated the role of citrulline in the tumor microenvironment of lung cancer and assessed whether citrulline supplementation contributed to the antitumor immune response. Materials and methods Mice All experiments were performed in wild-type (WT) C57BL/6 mice (10 − 12 weeks old, male) obtained from the Animal House of the Medical Research Center (CLEA Japan, Inc.) and maintained in our laboratory for animal experiments (Tokushima University, Tokushima, Japan) under controlled environmental conditions (22°C and 55% relative humidity under a fixed 12:12 h light/dark regime). All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Tokushima, School of Medicine, and were performed according to the relevant guidelines (Approval No.: T202422). All procedures performed in this study adhered to the ARRIVE Guidelines for reporting animal research (Percie du Sert et al. 2020). Cell lines The murine lung adenocarcinoma cell line CMT167 (Cat#: EC10032302-F0) was sourced from KAC (Kyoto, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (Bio-West, Bradenton, FL, USA) at 37°C under a 5% CO 2 atmosphere. Cultures were not maintained beyond 12 weeks of thawing. Reagents Anti-PD-1 (clone: RMP1-14, Cat#: BE0146) and rat IgG2a isotype controls (clone: 2A3, Cat#: BE0089) were purchased from BioXCell (Lebanon, NH, USA). L-Arginine (Cat#: 015-04615) and L-citrulline (Cat#: 036-21402) were acquired from FUJIFILM Wako Chemicals (Osaka, Japan). Tumor models and treatment To establish tumor models, CMT167 cells (1 × 10 4 cells) were resuspended in 20 µl of Cultrex (Cat#: 3433-005-01; R & D Systems, Minneapolis, MN, USA) and injected subcutaneously into the left lateral thigh under inhaled isoflurane anesthesia. The following day (day 1), the mice were randomized into the following four groups: 1) control, 2) anti-PD-1, 3) anti-PD-1 plus arginine, and 4) anti-PD-1 plus arginine and citrulline. Anti-PD-1 or isotype control antibodies were administered intraperitoneally at 200 µg/body on days 1, 4, 6, 8, 11, 13, 15, 18, 20, 22, 25, and 27 (Fig. 1 ). The control group mice were intraperitoneally administered IgG2a isotype controls. L-Arginine and L-citrulline were diluted in phosphate-buffered saline (PBS) and administered orally at 2 g/kg body weight daily. Tumor dimensions were measured with a caliper, and volume was calculated using the formula: tumor volume = (short diameter) 2 × (long diameter)/2. Efforts were made to minimize suffering. We defined progression-free survival as the period until tumor growth could be detected and conducted the experiments accordingly. Four weeks post-implantation, the mice were anesthetized and euthanized humanely via vertebral dislocation. Histology and immunostaining At the end of the experiment, tumors were harvested, and 3 µm-thick sections were prepared from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. All sections were stained with hematoxylin and eosin (H&E) by incubating them with hematoxylin for 5 min and subsequently with eosin for 4 min at 25°C. The stained sections were examined under a microscope. For immunostaining, primary antibodies against CD4 (Cat#: 25229), CD8α (Cat#: 98941), and F4/80 (Cat#: 70076) from Cell-Signaling Technologies (Danvers, MA, USA) were used. Tumor-infiltrating lymphocytes (TILs) were identified as lymphocytes within tumor nests. For group comparisons, three random fields (40× magnification) per tumor were independently quantified by two blinded observers (N. M. and H. T.), with the mean values used for analysis. In brief, for immunohistochemistry (IHC), the sections were boiled in citrate buffer (pH 6.0) for 10 min for antigen retrieval, and then blocked with 2.5% horse serum. The sections were then incubated overnight with primary antibodies at 4°C. IHC staining was performed using the ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Detection Kit (Vector Laboratory, Newark, CA, USA). The sections were incubated at room temperature for 10 min in PBS containing 3,3-diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratory, Burlingame, CA, USA) and were counterstained with hematoxylin. Flow cytometry analysis Tumors were dissected into small pieces and digested with Dri Tumor & Tissue Dissociation Reagent (Cat#: 661563; BD Biosciences, Franklin Lakes, NJ, USA) diluted in DMEM for 20 min at 37°C. The tissues were then washed with PBS and filtered through a 70 µm cell strainer. Two antibody panels were used for flow cytometry: Panel 1 included CD11b-PB (Cat#: 101223), MHCII-BV510 (Cat#: 107636), F4/80-PE (Cat#: 123110), Ly6c-PECy7 (Cat#: 128018), CD45-FITC (Cat#: 157214), CD163-APC (Cat#: 155305), and ZombieNIR-APCCy7 (Cat#: 423106), all from BioLegend (San Diego, CA, USA); Panel 2 included CD4-PECy7 (Cat#: 100422), TCRβ-APC (Cat#: 109212), CD45-FITC (Cat#: 157214), FOXP3-PE (Cat#: 320008), and ZombieAqua-BV510 (Cat#: 423101), also from BioLegend. The cells were stained using the Zombie Fixable Viability Kit according to the manufacturer’s instructions, followed by antibody staining at room temperature for 30 min. For intracellular staining, surface antigen-stained cells were fixed by incubating in a fixation buffer for 18 h in a refrigerator at 4°C, washed with permeabilization buffer, and stained with antibodies in permeabilization buffer for 60 min using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Cat#: 00-5523-00). Data were analyzed using the CytExpert software (Beckman Coulter, Brea, CA, USA). Flow cytometry was restricted to mice with tumors weighing ≥ 0.3 g. Detection of cytokine expression Blood samples were collected from the heart at the end of the experiment, and serum was separated by centrifugation at 3,000 rpm for 15 min and stored at − 80°C until use. The levels of serum cytokines, namely interleukin (IL)-1β, IL-2, IL-6, IL-10, IL-12p70, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), were quantified using the Luminex® Assay Mouse Premixed Multi-Analyte Kit (Cat#: F-RD-LuminexMM-07; R&D Systems, Minneapolis, MN, USA). F-Fluorodeoxyglucose - positron emission tomography/computed tomography (FDG-PET/CT)-based analysis of the maximum standardized uptake value (SUVmax) On day 22 post-CMT167 tumor cell transplantation, 1 − 2 mice were randomly selected from each group for FDG-PET/CT scanning. The mice were injected with 10 MBq/0.1 − 0.2 ml FDG via tail-vein catheter, and then anesthetized with isoflurane inhalation (3% for anesthesia induction and 2% for anesthesia maintenance). PET data were acquired for 20 min after a 40 min uptake period. SUVmax and SUVmean were measured, with SUVmax calculated from the maximum voxel value (Bq/ml) in the volume of interest (VOI) on fused PET images. The scans were performed using a Siemens Inveon small-animal CT scanner (Siemens Healthcare, Erlangen, Germany). Statistical analysis Data are presented as mean ± standard deviation (SD). For comparisons of three or more groups, a one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test was used. Kaplan–Meier plots and the log-rank test were used to compute and analyze the progression-free survival rates. Statistical significance was defined as P < 0.05. All analyses were conducted using EZR version 1.55 (Kanda 2013). Results Combination therapy with anti-PD-1, arginine, and citrulline significantly improved tumor growth suppression and survival We determined tumor volumes in mice subjected to different treatment regimens. The mice in the treatment groups exhibited significantly lower tumor volume than those in the control group (Fig. 2 a). On day 28, the tumor volume in the control group was 1161.59 ± 294.73 mm³, whereas those in the anti-PD-1, anti-PD-1 plus arginine, and anti-PD-1 plus arginine and citrulline groups were 427.38 ± 355.36, 452.10 ± 332.04, and 198.45 ± 236.22 mm 3 , respectively. Despite the reduction in the volume, the differences among the treatment groups were not significant. We also compared progression-free survival across the groups (Fig. 2 b). Median progression-free survival was 11.0, 11.0, 11.5, and 17.0 days for the control, anti-PD-1, anti-PD-1 plus arginine, and anti-PD-1 plus arginine and citrulline groups, respectively (P = 0.00039). Throughout the experimental period, the maximum body weight loss remained below 10% (Fig. 2 c). Thus, our findings suggest that the combination of arginine and citrulline with anti-PD-1 therapy effectively suppressed tumor growth and significantly improved progression-free survival. Citrulline combination therapy markedly reduced tumor metabolic activity Axial PET/CT images from day 22 post-implantation are presented in Fig. 3 . FDG uptake patterns corresponded to tumor presence and activity. Control group mice exhibited the largest tumors with the highest FDG uptake (Fig. 3 a), whereas those in the citrulline combination group showed minimal FDG uptake (Fig. 3 d). The SUVmax at the tumor site was 5.23 in the control group, 2.54 in the anti-PD-1 group, 4.23 in the anti-PD-1 plus arginine group, and 1.30 in the citrulline combination group. The mean SUVs were 2.34, 1.31, 1.70, and 0.86, respectively, for thsee groups. Enhanced infiltration of CD8 + lymphocytes with arginine and citrulline combination therapy FFPE sections from tumor-bearing mice were stained for CD8, CD4, and F4/80 markers (Fig. 4 a). Quantification of TILs revealed a higher number of CD8 + lymphocytes per high-power field (hpf) in the anti-PD-1 plus arginine and citrulline group than in the control group (control, 24.22 ± 9.13; anti-PD-1, 29.20 ± 9.41; anti-PD-1 plus arginine, 34.33 ± 8.81; anti-PD-1 plus arginine and citrulline, 46.56 ± 10.01 cells/hpf; Fig. 4 b). CD4 + lymphocyte counts did not differ significantly across the groups (control, 3.00 ± 1.94; anti-PD-1, 2.33 ± 1.63; anti-PD-1 plus arginine, 4.00 ± 3.05; anti-PD-1 plus arginine and citrulline, 4.44 ± 2.60 cells/hpf; Fig. 4 c). Tumor immune cell populations showed no significant differences Flow cytometry was used to analyze immune cell populations within tumors across treatment groups (Fig. 5 ). Owing to the limited number of mice developing sufficiently large tumor lesions, only tumors from three mice in the anti-PD-1 group, three in the anti-PD-1 plus arginine group, and one in the anti-PD-1 plus arginine and citrulline group were analyzed. No significant differences were observed in the numbers of CD8 + T cells (CD45 + CD4 − ; Fig. 5 a), CD4 + T cells (CD45 + CD4 + ; Fig. 5 b), regulatory T cells (CD45 + CD4 + FOXP + ; Fig. 5 c), M1 macrophages (F4/80 + CD11b + Ly6c − CD163 − MHCⅡ + ; Fig. 5 d), and M2 macrophages (F4/80 + CD11b + Ly6c − CD163 + MHCⅡ − ; Fig. 5 e). Serum cytokine levels remained unchanged across treatment groups Multiplex analysis was performed to measure the serum levels of immune-specific markers, namely IL-1β, IL-2, IL-6, IL-10, IL-12p70, TNF-α, and IFN-γ. The expression levels of these cytokines did not show significant differences among the treatment groups (Fig. 5 f-i). Discussion Anti-PD-1 immunotherapy inhibits tumor growth in mice with CMT167 lung cancer (Li et al. 2017). Furthermore, arginine enhances the anticancer effects of anti-PD-1 antibodies (He et al. 2017; Satoh et al. 2020). Conversely, citrulline exhibits toxicity against human cervical adenocarcinoma cells in vitro (Huerta 2015); however, its in vivo effects on cancer remain largely unexplored. In the present study, we found that tumor growth was inhibited in the treatment groups, with enhanced effects observed upon the addition of arginine and citrulline. To the best of our knowledge, this study is the first to propose citrulline as a supplemental agent in lung cancer immunotherapy. We observed that combination therapy with anti-PD-1 plus arginine and citrulline significantly improved progression-free survival and reduced tumor growth compared with the control. Moreover, this combination therapy resulted in a significantly higher number of tumor-infiltrating CD8 + lymphocytes in the tumor microenvironment. The state of the immune system in patients with cancer depends on the interaction between the tumor microenvironment and antitumor immune response. TILs, particularly CD8 + T cells, play crucial roles in antitumor immunity (Ahmadzadeh et al. 2009). Zeng et al (2016) reported that high levels of CD8 + TILs are associated with better prognosis and survival in patients with NSCLC. The metabolic balance within the tumor microenvironment regulates tumor immunity and contributes to resistance to immunotherapy. Lower levels of tumor-infiltrating T cells have been associated with poor prognosis and diminished response to ICIs (Gooden et al. 2011; Geng et al. 2015). In the present study, arginine supplementation increased the number of CD8 + lymphocytes more effectively than the anti-PD-1 treatment alone. Arginine enhances immune responses; the effects of arginine deprivation on human T lymphocytes were first reported in 1968, demonstrating a causal relationship between arginine deficiency and impaired in vitro activation of lymphocytes (Barile and Leventhal 1968). Arginine deprivation impairs T-cell function by downregulating the expression of the CD3 subunit of the T-cell receptor complex (Minami et al. 1987; Weissman et al. 1988; Rodriguez et al. 2002). Arginine level in the tumor microenvironment is lower than that in the plasma, suggesting a role of arginine supplementation in enhancing tumor immunity (Sullivan et al. 2019). Oral arginine is subject to first-pass metabolism in the gastrointestinal tract and liver, reducing its bioavailability. Marini et al. reported that the first-pass metabolism in wild-type mice was 75%, leading to plasma arginine level lower than that in arginase 2-deficient mice (Marini et al. 2011). Owing to these limitations, citrulline has been proposed as an alternative to increase arginine availability. The findings of the present study suggest that anti-PD-1 combined with arginine and citrulline suppresses tumor growth and increase CD8 + lymphocyte count in the tumor microenvironment. Citrulline bypasses the first-pass metabolism, entering systemic circulation where it is converted to arginine in the kidney by argininosuccinate synthetase and lyase (Wu and Morris, 1998; Levillain, 2012). In a pharmacokinetic study, Moinard et al (2008) showed that oral citrulline supplementation led to a dose-dependent increase in plasma level of arginine as well as that of citrulline and ornithine. Schwedhelm et al (2008) indicated that supplemental citrulline is more effective than arginine itself at increasing systemic arginine availability when administered at equivalent doses. Ouaknine Krief et al (2019) reported that patients with lung cancer with high plasma citrulline level showed longer progression-free survival and overall survival than those with low plasma citrulline level. Citrulline serves as a precursor to arginine and indirectly enhances NO biosynthesis. NO is an endogenous, water-soluble free radical with diverse biological functions, particularly in endothelial vasodilation. In recent decades, research has increasingly focused on the role of NO in inhibiting tumor growth. High concentration of NO has been demonstrated to have therapeutic potential against cancer across human and murine models (Wink et al. 1997; Huerta, 2015; Li et al. 2023). Sullivan and Graham (2008) reported that elevated NO level achieved by delivering NO donors induces apoptosis in tumor cells. Furthermore, NO sensitizes drug-resistant tumor cells to certain chemotherapeutic agents (Liu et al. 2004; Bonavida 2020). These findings suggest that highly active NO donors could be effective in cancer treatment when used in combination with other therapeutic modalities. For characterizing the tumor microenvironment, it might be useful to measure NO and other metabolites using tumor suspensions. Despite the induction of CD8 + lymphocytes, we observed no significant differences in the abundance of other immune cell populations, including CD4 + lymphocytes, regulatory T cells, and macrophages. Tumor-associated macrophages, which express arginases and catabolize arginine, contribute to tumor progression and pro-tumoral remodeling. Sangaletti et al (2008) reported that tumor-associated macrophages in mammary carcinoma express SPARC and facilitate cancer cell migration. Curiel et al (2004). indicated that these macrophages secrete CCL22 chemokines, promoting tumor growth. Further research is required to verify whether coadministration of arginine and citrulline can enhance the therapeutic efficacy of anti-PD-1 antibodies. Although high doses of oral arginine can induce adverse gastrointestinal events attributed to the first-pass effect (Heyland et al. 2003; Grimble, 2007), citrulline administration has no apparent adverse effects and may be more suitable for clinical use (Agarwal et al. 2017). Given their use in cardiovascular disease treatments (Figueroa et al. 2016; Pahlavani et al. 2017; Suzuki et al. 2019), arginine and citrulline could be safely tested in clinical trials. This study had several limitations. First, several mice in the treatment groups developed small lesions and were unsuitable for flow cytometry analysis. We could not efficiently evaluate the effect of the therapies on small lesion samples. For mice with lesions weighing less than 0.3 g at the time of sacrifice, we performed only IHC, which could account for discrepancies between immunostaining and flow cytometry results. Additionally, tumor evaluation was not feasible for tumor-free mice in the treatment groups. In such cases, evaluation of immune cells in other lymphoid organs could serve as a substitute for their evaluation in the tumors, regardless of the effect of the tumor microenvironment, and might be useful for assessing tumor-free mice. Second, the mechanism by which the combination therapy of anti-PD-1 plus arginine and citrulline induces more CD8 + lymphocytes remains unclear. In vitro experiments and bioinformatic analyses may be useful in elucidating the interactions between tumors and immune cells and should be performed in future. Finally, our study did not include an anti-PD-1 plus citrulline group. In future studies, this group should be compared with the anti-PD-1 plus arginine group or the anti-PD-1 plus arginine and citrulline combination group for more clarity on the effects of arginine and citrulline. In conclusion, we demonstrate that arginine and citrulline supplementation with anti-PD-1 therapy suppresses lung cancer growth in mice and improves progression-free survival. This combination therapy enhances CD8 + lymphocyte infiltration and the efficacy of anti-PD-1 drugs. Future studies should be conducted to investigate the mechanisms through which arginine and citrulline combination therapy induces CD8 + lymphocytes. Declarations Acknowledgments Not applicable. <Funding No funding was received. Competing interests The authors declare that there are no conflicts of interest Author contributions NM and HT designed the study. NM performed the experiments and wrote the manuscript. NM and MY conducted the animal experiments. HO performed histological analysis. ST and TO analyzed the data. HT, MY, and KY supervised the study. Data availability The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request. All data generated or analyzed in this study are included in this published article. Ethics approval All experiments were performed in accordance with the guidelines established by the Tokushima University Committee on Animal Care and Use. All experimental protocols were reviewed and approved by the Animal Research Committee of the University of Tokushima (Approval No.: T202422). Consent to participate Not applicable. Consent to publish Not applicable. References Agarwal U, Didelija IC, Yuan Y, Wang X, Marini JC (2017) Supplemental citrulline is more efficient than arginine in increasing systemic arginine availability in mice. 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J Biol Chem 277:21123–21129. doi:10.1074/jbc.M110675200 Sangaletti S, Di Carlo E, Gariboldi S, Miotti S, Cappetti B, Parenza M, Rumio C, Brekken RA, Chiodoni C, Colombo MP (2008) Macrophage-derived SPARC bridges tumor cell-extracellular matrix interactions toward metastasis. Cancer Res 68:9050–9059. doi:10.1158/0008-5472.CAN-08-1327 Satoh Y, Kotani H, Iida Y, Taniura T, Notsu Y, Harada M (2020) Supplementation of l-arginine boosts the therapeutic efficacy of anticancer chemoimmunotherapy. Cancer Sci 111:2248–2258. doi:10.1111/cas.14490 Schwedhelm E, Maas R, Freese R, Jung D, Lukacs Z, Jambrecina A, Spickler W, Schulze F, Böger RH (2008) Pharmacokinetic and pharmacodynamic properties of Oral L-citrulline and L-arginine: impact on nitric oxide metabolism. Br J Clin Pharmacol 65:51–59. doi:10.1111/j.1365-2125.2007.02990.x Sosnowska A, Chlebowska-Tuz J, Matryba P et al (2021) Inhibition of arginase modulates T-cell response in the tumor microenvironment of lung carcinoma. Oncoimmunology 10:1956143. doi:10.1080/2162402X.2021.1956143 Sullivan MR, Danai LV, Lewis CA, Chan SH, Gui DY, Kunchok T, Dennstedt EA, Vander Heiden MG, Muir A (2019) Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. eLife 8:e44235. doi:10.7554/eLife.44235 Sullivan R, Graham CH (2008) Chemosensitization of cancer by nitric oxide. Curr Pharm Des 14:1113–1123. doi:10.2174/138161208784246225 Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 71:209–249. doi:10.3322/caac.21660 Suzuki I, Sakuraba K, Horiike T, Kishi T, Yabe J, Suzuki T, Morita M, Nishimura A, Suzuki Y (2019) A combination of oral L-citrulline and L-arginine improved 10-min full-power cycling test performance in male collegiate soccer players: A randomized crossover trial. Eur J Appl Physiol 119:1075–1084. doi:10.1007/s00421-019-04097-7 Weissman AM, Ross P, Luong ET, Garcia-Morales PG, Jelachich ML, Biddison WE, Klausner RD, Samelson LE (1988) Tyrosine phosphorylation of the human T cell antigen receptor zeta-chain: activation via CD3 but not CD2. J Immunol 141:3532–3536. doi:10.4049/jimmunol.141.10.3532 Wink DA, Cook JA, Christodoulou D et al (1997) Nitric oxide and some nitric oxide donor compounds enhance the cytotoxicity of cisplatin. Nitric Oxide 1:88–94. doi:10.1006/niox.1996.0108 Wu G, Morris SM Jr (1998) Arginine metabolism: nitric oxide and beyond. Biochem J 336:1–17. doi:10.1042/bj3360001 Xia L, Liu Y, Wang Y (2019) PD-1/PD‐L1 blockade therapy in advanced non‐small‐cell lung cancer: current status and future directions. Oncologist 24:S31–S41. Yang J, Chen J, Wei J, Liu X, Cho WC (2016) Immune checkpoint blockade as a potential therapeutic target in non-small cell lung cancer. Expert Opin Biol Ther 16:1209–1223. doi:10.1080/14712598.2016.1214265 Zeng DQ, Yu YF, Ou QY, Li XY, Zhong RZ, Xie CM, Hu QG (2016) Prognostic and predictive value of tumor-infiltrating lymphocytes for clinical therapeutic research in patients with non-small cell lung cancer. Oncotarget 7:13765–13781. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5294074","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":369730120,"identity":"d09b0783-2516-406b-8b4d-6d7a27e3ae7a","order_by":0,"name":"Naoki Miyamoto","email":"","orcid":"","institution":"The University of Tokushima","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Miyamoto","suffix":""},{"id":369730121,"identity":"a919a776-1d8d-4dfc-a042-c67a6b0f1f12","order_by":1,"name":"Mitsuteru Yoshida","email":"","orcid":"","institution":"Kochi Red Cross Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mitsuteru","middleName":"","lastName":"Yoshida","suffix":""},{"id":369730122,"identity":"09642d90-3b66-4e5a-ab96-e2503ff99fc2","order_by":2,"name":"Shinichi Tsukumo","email":"","orcid":"","institution":"Tokushima University","correspondingAuthor":false,"prefix":"","firstName":"Shinichi","middleName":"","lastName":"Tsukumo","suffix":""},{"id":369730123,"identity":"609e4953-45ba-438e-831c-62a5e8b17c48","order_by":3,"name":"Hirohisa Ogawa","email":"","orcid":"","institution":"Tokushima University Graduate School of Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hirohisa","middleName":"","lastName":"Ogawa","suffix":""},{"id":369730124,"identity":"47fc2db4-0851-4b1d-8721-8885bdaa3e11","order_by":4,"name":"Tamaki Otani","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Tamaki","middleName":"","lastName":"Otani","suffix":""},{"id":369730125,"identity":"a50afc5b-ee5e-400d-a819-2e074d5aafbf","order_by":5,"name":"Koji Yasutomo","email":"","orcid":"","institution":"Tokushima University","correspondingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Yasutomo","suffix":""},{"id":369730126,"identity":"299872aa-1a74-4f5a-8416-919929dc85db","order_by":6,"name":"Hiromitsu Takizawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie3QMUvDQBTA8XcErssrXU8Q+wmEhoPgYvNVEg7i6BfoECmcW7sG1O8gCJ0vHNwkdA1EsFNdHHQJHUL1WtEpqWQTuf9yw/HjvTsAl+tPRkHtjgGAZw+FPxckbQb4TY5kF7JvZL7I72uFPRPnm4ke8uep5lg/HZ9eK8pgMgbvpnkMYqIUM9pfGCpEX64xeIwsMQLIbfNIhItUjWhJFga57qcaA3VZMaAKSBY1k8FLqqJtGT5IS7C2ZLmyU7YHCLOL5bKM7ylygdSSwi5G5AFSrKP8avYhMpMI/07uyMo7i2cC297Smyf8fVMl5/Op1uy11mGwjEjxVo1P/JYfa8muhH7WRewbss7E5XK5/mefMdNhxBLt7QwAAAAASUVORK5CYII=","orcid":"","institution":"The University of Tokushima","correspondingAuthor":true,"prefix":"","firstName":"Hiromitsu","middleName":"","lastName":"Takizawa","suffix":""}],"badges":[],"createdAt":"2024-10-19 10:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5294074/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5294074/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67857297,"identity":"cd7a76b6-263b-47fb-80ab-d8f94772a43d","added_by":"auto","created_at":"2024-10-30 11:46:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the experimental setup.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were inoculated with 1.0 × 10\u003csup\u003e5\u003c/sup\u003e CMT167 tumor cells. The control and anti-PD-1 group mice were intraperitoneally administered IgG2a isotype control and anti-PD-1, respectively. The anti-PD-1 plus arginine group mice were intraperitoneally administered anti-PD-1 and orally administered arginine every day. The anti-PD-1 plus arginine and citrulline group mice were intraperitoneally administered anti-PD-1 and orally administered both arginine and citrulline every day. The number of mice per group was as follows: control (n = 12), anti-PD-1 (n = 12), anti-PD-1 + arginine (n = 12), and anti-PD-1 + arginine + citrulline (n = 12).\u003c/p\u003e","description":"","filename":"OnlineFigure112.png","url":"https://assets-eu.researchsquare.com/files/rs-5294074/v1/0ab295703dc9f39de1fbfc16.png"},{"id":67858327,"identity":"f1cf5b20-3edc-41b0-9afa-f7ed6506810b","added_by":"auto","created_at":"2024-10-30 11:54:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of arginine and citrulline enhances the antitumor efficacy of the immunotherapies.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Tumor volume growth curves for mice treated with the IgG control antibody, anti-PD-1, anti-PD-1 plus arginine, and anti-PD-1 plus arginine and citrulline. Data are presented as mean ± SD; n = 12. Statistical significance was assessed using the one-way ANOVA (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). (b) Progression-free survival curve for each treatment group. Statistical significance was assessed using the log-rank test (*\u003cem\u003eP\u003c/em\u003e = 0.00039). (c) Changes in body weight during the treatment period for each group.\u003c/p\u003e","description":"","filename":"OnlineFigure29.png","url":"https://assets-eu.researchsquare.com/files/rs-5294074/v1/9d85433b26088d4d067e7166.png"},{"id":67858326,"identity":"539b0c33-9e74-438f-b533-fc4417e34635","added_by":"auto","created_at":"2024-10-30 11:54:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1038215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAxial PET/CT images of mice on day 22 post-transplantation of CMT167 tumor cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages represent each treatment group: (a) control, (b) anti-PD-1, (c) anti-PD-1 plus arginine, and (d) anti-PD-1 plus arginine and citrulline. Arrowheads indicate the tumor locations.\u003c/p\u003e","description":"","filename":"Figure330.png","url":"https://assets-eu.researchsquare.com/files/rs-5294074/v1/c58fe12733d0625e4af077a0.png"},{"id":67857300,"identity":"f8d05f4c-b454-497b-84d3-43f41b30c7c4","added_by":"auto","created_at":"2024-10-30 11:46:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1235716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of immune cells within tumors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Immunohistochemical staining for CD8a, CD4, and F4/80 in each treatment group. Scale bar represents 100 µm. (b) Quantification of tumor-infiltrating CD8a\u003csup\u003e+\u003c/sup\u003e T cells across treatment groups. Statistically significant differences, determined using ANOVA, are indicated (*\u003cem\u003eP\u003c/em\u003e ≤ 0.05, **\u003cem\u003eP\u003c/em\u003e ≤ 0.001). (c) Quantification of tumor-infiltrating CD4\u003csup\u003e+\u003c/sup\u003e T cells across treatment groups.\u003c/p\u003e","description":"","filename":"OnlineFigure47.png","url":"https://assets-eu.researchsquare.com/files/rs-5294074/v1/e41659b6706c9a352b758af5.png"},{"id":67857301,"identity":"2b75b2c7-fcb3-4267-b1c5-7b4287375af1","added_by":"auto","created_at":"2024-10-30 11:46:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometric analysis of immune cell populations in CMT167 tumors across treatment groups and serum multiplex assay results for each treatment group.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometric analysis of CMT167 tumors across different treatment groups for: (a) CD8\u003csup\u003e+\u003c/sup\u003e T cells, (b) CD4\u003csup\u003e+\u003c/sup\u003e T cells, (c) regulatory T cells (Treg, defined by Foxp3 expression in CD4\u003csup\u003e+\u003c/sup\u003e T cells), (d) M1 macrophages, and (e) M2 macrophages. (f-i) Serum multiplex assay results for each treatment group. Statistically significant differences were assessed using ANOVA.\u003c/p\u003e","description":"","filename":"OnlineFigure55.png","url":"https://assets-eu.researchsquare.com/files/rs-5294074/v1/d8796592bf39253cb88af881.png"},{"id":68371750,"identity":"3ad37d44-173f-4b6d-8a64-feec4ff9a0ae","added_by":"auto","created_at":"2024-11-06 14:23:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3208161,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5294074/v1/041f40bd-fd9f-4335-9e1d-ad7137a1ad4b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancement of anti-programmed cell death protein-1 immunotherapy in non-small cell lung cancer using arginine and citrulline supplementation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung cancer is the most prevalent cancer globally, accounting for approximately 2.2\u0026nbsp;million new cases and 1.8\u0026nbsp;million deaths in 2020 (Sung et al. 2021). Over the past decade, the treatment landscape for non-small cell lung cancer (NSCLC) has been revolutionized by the advent of molecular-targeted therapeutics and immune checkpoint inhibitors (ICIs) (Yang et al. 2016; Herbst et al. 2018). In cases lacking targetable molecular alterations, the assessment of programmed death ligand 1 (PD-L1) expression is crucial for directing treatment strategies for both squamous and non-squamous lung cancers (Alexander et al. 2020). Although ICIs have substantially improved outcomes in previously untreatable cancer cases, their effectiveness as monotherapy is limited, with response rates ranging from 14\u0026ndash;20% (Xia and Liu 2019). Therefore, identifying the immunological mechanisms that confer resistance to these inhibitors is essential for enhancing therapeutic efficacy and overcoming tumor resistance.\u003c/p\u003e \u003cp\u003eA mechanism by which tumors evade the host immune response involves arginine metabolism by arginases (Munder 2009). Arginine is crucial for long-term survival, immune memory generation, and tumor-killing efficiency of T cells. Consequently, its deficiency reduces T-cell activity and increases tumor size (Mart\u0026iacute; I L\u0026iacute;ndez and Reith 2021). Conversely, augmenting arginine levels in the tumor microenvironment activates T cells and enhances the efficacy of ICIs (He et al. 2017). Both arginase inhibitors and arginine itself can inhibit tumor growth, suggesting arginine metabolism as a viable therapeutic target (Miret et al. 2019; Sosnowska et al. 2021).\u003c/p\u003e \u003cp\u003eArginine also plays a vital role in the synthesis of nitric oxide (NO), polyamines, and proteins. However, after oral intake, arginine is metabolized and converted to various forms (Agarwal et al. 2017) through two primary metabolic pathways\u0026mdash;conversion to NO and citrulline by nitric oxide synthase (NOS) and hydrolysis to ornithine and urea by arginase. Within the NOS pathway, the enzyme argininosuccinate synthetase, coupled with constitutively expressed argininosuccinate lyase, facilitates the recycling of citrulline for de novo arginine synthesis (Rath et al. 2014). Considering these roles of arginine, citrulline may serve as a promising alternative to enhance arginine availability. Citrulline supplementation is reportedly more effective than arginine alone in boosting arginine availability and NO synthesis (El-Hattab et al. 2012; Agarwal et al. 2017).\u003c/p\u003e \u003cp\u003eWe hypothesized that supplementing anti-PD-1 antibodies and arginine with citrulline can augment their antitumor effects. Accordingly, we investigated the role of citrulline in the tumor microenvironment of lung cancer and assessed whether citrulline supplementation contributed to the antitumor immune response.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eAll experiments were performed in wild-type (WT) C57BL/6 mice (10\u0026thinsp;\u0026minus;\u0026thinsp;12 weeks old, male) obtained from the Animal House of the Medical Research Center (CLEA Japan, Inc.) and maintained in our laboratory for animal experiments (Tokushima University, Tokushima, Japan) under controlled environmental conditions (22\u0026deg;C and 55% relative humidity under a fixed 12:12 h light/dark regime). All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Tokushima, School of Medicine, and were performed according to the relevant guidelines (Approval No.: T202422). All procedures performed in this study adhered to the ARRIVE Guidelines for reporting animal research (Percie du Sert et al. 2020).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines\u003c/h3\u003e\n\u003cp\u003eThe murine lung adenocarcinoma cell line CMT167 (Cat#: EC10032302-F0) was sourced from KAC (Kyoto, Japan). The cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) with 10% fetal bovine serum (Bio-West, Bradenton, FL, USA) at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Cultures were not maintained beyond 12 weeks of thawing.\u003c/p\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eAnti-PD-1 (clone: RMP1-14, Cat#: BE0146) and rat IgG2a isotype controls (clone: 2A3, Cat#: BE0089) were purchased from BioXCell (Lebanon, NH, USA). L-Arginine (Cat#: 015-04615) and L-citrulline (Cat#: 036-21402) were acquired from FUJIFILM Wako Chemicals (Osaka, Japan).\u003c/p\u003e\n\u003ch3\u003eTumor models and treatment\u003c/h3\u003e\n\u003cp\u003eTo establish tumor models, CMT167 cells (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells) were resuspended in 20 \u0026micro;l of Cultrex (Cat#: 3433-005-01; R \u0026amp; D Systems, Minneapolis, MN, USA) and injected subcutaneously into the left lateral thigh under inhaled isoflurane anesthesia. The following day (day 1), the mice were randomized into the following four groups: 1) control, 2) anti-PD-1, 3) anti-PD-1 plus arginine, and 4) anti-PD-1 plus arginine and citrulline. Anti-PD-1 or isotype control antibodies were administered intraperitoneally at 200 \u0026micro;g/body on days 1, 4, 6, 8, 11, 13, 15, 18, 20, 22, 25, and 27 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The control group mice were intraperitoneally administered IgG2a isotype controls. L-Arginine and L-citrulline were diluted in phosphate-buffered saline (PBS) and administered orally at 2 g/kg body weight daily. Tumor dimensions were measured with a caliper, and volume was calculated using the formula: tumor volume = (short diameter)\u003csup\u003e2\u003c/sup\u003e \u0026times; (long diameter)/2. Efforts were made to minimize suffering. We defined progression-free survival as the period until tumor growth could be detected and conducted the experiments accordingly. Four weeks post-implantation, the mice were anesthetized and euthanized humanely via vertebral dislocation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHistology and immunostaining\u003c/h3\u003e\n\u003cp\u003eAt the end of the experiment, tumors were harvested, and 3 \u0026micro;m-thick sections were prepared from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. All sections were stained with hematoxylin and eosin (H\u0026amp;E) by incubating them with hematoxylin for 5 min and subsequently with eosin for 4 min at 25\u0026deg;C. The stained sections were examined under a microscope. For immunostaining, primary antibodies against CD4 (Cat#: 25229), CD8α (Cat#: 98941), and F4/80 (Cat#: 70076) from Cell-Signaling Technologies (Danvers, MA, USA) were used. Tumor-infiltrating lymphocytes (TILs) were identified as lymphocytes within tumor nests. For group comparisons, three random fields (40\u0026times; magnification) per tumor were independently quantified by two blinded observers (N. M. and H. T.), with the mean values used for analysis. In brief, for immunohistochemistry (IHC), the sections were boiled in citrate buffer (pH 6.0) for 10 min for antigen retrieval, and then blocked with 2.5% horse serum. The sections were then incubated overnight with primary antibodies at 4\u0026deg;C. IHC staining was performed using the ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Detection Kit (Vector Laboratory, Newark, CA, USA). The sections were incubated at room temperature for 10 min in PBS containing 3,3-diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratory, Burlingame, CA, USA) and were counterstained with hematoxylin.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eTumors were dissected into small pieces and digested with Dri Tumor \u0026amp; Tissue Dissociation Reagent (Cat#: 661563; BD Biosciences, Franklin Lakes, NJ, USA) diluted in DMEM for 20 min at 37\u0026deg;C. The tissues were then washed with PBS and filtered through a 70 \u0026micro;m cell strainer. Two antibody panels were used for flow cytometry: Panel 1 included CD11b-PB (Cat#: 101223), MHCII-BV510 (Cat#: 107636), F4/80-PE (Cat#: 123110), Ly6c-PECy7 (Cat#: 128018), CD45-FITC (Cat#: 157214), CD163-APC (Cat#: 155305), and ZombieNIR-APCCy7 (Cat#: 423106), all from BioLegend (San Diego, CA, USA); Panel 2 included CD4-PECy7 (Cat#: 100422), TCRβ-APC (Cat#: 109212), CD45-FITC (Cat#: 157214), FOXP3-PE (Cat#: 320008), and ZombieAqua-BV510 (Cat#: 423101), also from BioLegend. The cells were stained using the Zombie Fixable Viability Kit according to the manufacturer\u0026rsquo;s instructions, followed by antibody staining at room temperature for 30 min. For intracellular staining, surface antigen-stained cells were fixed by incubating in a fixation buffer for 18 h in a refrigerator at 4\u0026deg;C, washed with permeabilization buffer, and stained with antibodies in permeabilization buffer for 60 min using the eBioscience\u0026trade; Foxp3/Transcription Factor Staining Buffer Set (Cat#: 00-5523-00). Data were analyzed using the CytExpert software (Beckman Coulter, Brea, CA, USA). Flow cytometry was restricted to mice with tumors weighing\u0026thinsp;\u0026ge;\u0026thinsp;0.3 g.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetection of cytokine expression\u003c/h3\u003e\n\u003cp\u003eBlood samples were collected from the heart at the end of the experiment, and serum was separated by centrifugation at 3,000 rpm for 15 min and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use. The levels of serum cytokines, namely interleukin (IL)-1β, IL-2, IL-6, IL-10, IL-12p70, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), were quantified using the Luminex\u0026reg; Assay Mouse Premixed Multi-Analyte Kit (Cat#: F-RD-LuminexMM-07; R\u0026amp;D Systems, Minneapolis, MN, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eF-Fluorodeoxyglucose\u003c/b\u003e-\u003cb\u003epositron emission tomography/computed tomography (FDG-PET/CT)-based analysis of the maximum standardized uptake value (SUVmax)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOn day 22 post-CMT167 tumor cell transplantation, 1\u0026thinsp;\u0026minus;\u0026thinsp;2 mice were randomly selected from each group for FDG-PET/CT scanning. The mice were injected with 10 MBq/0.1\u0026thinsp;\u0026minus;\u0026thinsp;0.2 ml FDG via tail-vein catheter, and then anesthetized with isoflurane inhalation (3% for anesthesia induction and 2% for anesthesia maintenance). PET data were acquired for 20 min after a 40 min uptake period. SUVmax and SUVmean were measured, with SUVmax calculated from the maximum voxel value (Bq/ml) in the volume of interest (VOI) on fused PET images. The scans were performed using a Siemens Inveon small-animal CT scanner (Siemens Healthcare, Erlangen, Germany).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). For comparisons of three or more groups, a one-way analysis of variance (ANOVA) followed by Bonferroni\u0026rsquo;s post-hoc test was used. Kaplan\u0026ndash;Meier plots and the log-rank test were used to compute and analyze the progression-free survival rates. Statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All analyses were conducted using EZR version 1.55 (Kanda 2013).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCombination therapy with anti-PD-1, arginine, and citrulline significantly improved tumor growth suppression and survival\u003c/h2\u003e \u003cp\u003eWe determined tumor volumes in mice subjected to different treatment regimens. The mice in the treatment groups exhibited significantly lower tumor volume than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). On day 28, the tumor volume in the control group was 1161.59\u0026thinsp;\u0026plusmn;\u0026thinsp;294.73 mm\u0026sup3;, whereas those in the anti-PD-1, anti-PD-1 plus arginine, and anti-PD-1 plus arginine and citrulline groups were 427.38\u0026thinsp;\u0026plusmn;\u0026thinsp;355.36, 452.10\u0026thinsp;\u0026plusmn;\u0026thinsp;332.04, and 198.45\u0026thinsp;\u0026plusmn;\u0026thinsp;236.22 mm\u003csup\u003e3\u003c/sup\u003e, respectively. Despite the reduction in the volume, the differences among the treatment groups were not significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also compared progression-free survival across the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Median progression-free survival was 11.0, 11.0, 11.5, and 17.0 days for the control, anti-PD-1, anti-PD-1 plus arginine, and anti-PD-1 plus arginine and citrulline groups, respectively (P\u0026thinsp;=\u0026thinsp;0.00039). Throughout the experimental period, the maximum body weight loss remained below 10% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Thus, our findings suggest that the combination of arginine and citrulline with anti-PD-1 therapy effectively suppressed tumor growth and significantly improved progression-free survival.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCitrulline combination therapy markedly reduced tumor metabolic activity\u003c/h2\u003e \u003cp\u003eAxial PET/CT images from day 22 post-implantation are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. FDG uptake patterns corresponded to tumor presence and activity. Control group mice exhibited the largest tumors with the highest FDG uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), whereas those in the citrulline combination group showed minimal FDG uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The SUVmax at the tumor site was 5.23 in the control group, 2.54 in the anti-PD-1 group, 4.23 in the anti-PD-1 plus arginine group, and 1.30 in the citrulline combination group. The mean SUVs were 2.34, 1.31, 1.70, and 0.86, respectively, for thsee groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEnhanced infiltration of CD8\u0026thinsp;+\u0026thinsp;lymphocytes with arginine and citrulline combination therapy\u003c/h2\u003e \u003cp\u003eFFPE sections from tumor-bearing mice were stained for CD8, CD4, and F4/80 markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Quantification of TILs revealed a higher number of CD8\u003csup\u003e+\u003c/sup\u003e lymphocytes per high-power field (hpf) in the anti-PD-1 plus arginine and citrulline group than in the control group (control, 24.22\u0026thinsp;\u0026plusmn;\u0026thinsp;9.13; anti-PD-1, 29.20\u0026thinsp;\u0026plusmn;\u0026thinsp;9.41; anti-PD-1 plus arginine, 34.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.81; anti-PD-1 plus arginine and citrulline, 46.56\u0026thinsp;\u0026plusmn;\u0026thinsp;10.01 cells/hpf; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). CD4\u0026thinsp;+\u0026thinsp;lymphocyte counts did not differ significantly across the groups (control, 3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94; anti-PD-1, 2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63; anti-PD-1 plus arginine, 4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05; anti-PD-1 plus arginine and citrulline, 4.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60 cells/hpf; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTumor immune cell populations showed no significant differences\u003c/h2\u003e \u003cp\u003eFlow cytometry was used to analyze immune cell populations within tumors across treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Owing to the limited number of mice developing sufficiently large tumor lesions, only tumors from three mice in the anti-PD-1 group, three in the anti-PD-1 plus arginine group, and one in the anti-PD-1 plus arginine and citrulline group were analyzed. No significant differences were observed in the numbers of CD8\u003csup\u003e+\u003c/sup\u003e T cells (CD45\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e\u0026minus;\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD45\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), regulatory T cells (CD45\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e FOXP\u003csup\u003e+\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), M1 macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e Ly6c\u003csup\u003e\u0026minus;\u003c/sup\u003e CD163\u003csup\u003e\u0026minus;\u003c/sup\u003e MHCⅡ\u003csup\u003e+\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), and M2 macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e Ly6c\u003csup\u003e\u0026minus;\u003c/sup\u003e CD163\u003csup\u003e+\u003c/sup\u003e MHCⅡ\u003csup\u003e\u0026minus;\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSerum cytokine levels remained unchanged across treatment groups\u003c/h2\u003e \u003cp\u003eMultiplex analysis was performed to measure the serum levels of immune-specific markers, namely IL-1β, IL-2, IL-6, IL-10, IL-12p70, TNF-α, and IFN-γ. The expression levels of these cytokines did not show significant differences among the treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-i).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAnti-PD-1 immunotherapy inhibits tumor growth in mice with CMT167 lung cancer (Li et al. 2017). Furthermore, arginine enhances the anticancer effects of anti-PD-1 antibodies (He et al. 2017; Satoh et al. 2020). Conversely, citrulline exhibits toxicity against human cervical adenocarcinoma cells in vitro (Huerta 2015); however, its in vivo effects on cancer remain largely unexplored. In the present study, we found that tumor growth was inhibited in the treatment groups, with enhanced effects observed upon the addition of arginine and citrulline. To the best of our knowledge, this study is the first to propose citrulline as a supplemental agent in lung cancer immunotherapy.\u003c/p\u003e \u003cp\u003eWe observed that combination therapy with anti-PD-1 plus arginine and citrulline significantly improved progression-free survival and reduced tumor growth compared with the control. Moreover, this combination therapy resulted in a significantly higher number of tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e lymphocytes in the tumor microenvironment. The state of the immune system in patients with cancer depends on the interaction between the tumor microenvironment and antitumor immune response. TILs, particularly CD8\u003csup\u003e+\u003c/sup\u003e T cells, play crucial roles in antitumor immunity (Ahmadzadeh et al. 2009). Zeng et al (2016) reported that high levels of CD8\u003csup\u003e+\u003c/sup\u003e TILs are associated with better prognosis and survival in patients with NSCLC. The metabolic balance within the tumor microenvironment regulates tumor immunity and contributes to resistance to immunotherapy. Lower levels of tumor-infiltrating T cells have been associated with poor prognosis and diminished response to ICIs (Gooden et al. 2011; Geng et al. 2015). In the present study, arginine supplementation increased the number of CD8\u003csup\u003e+\u003c/sup\u003e lymphocytes more effectively than the anti-PD-1 treatment alone. Arginine enhances immune responses; the effects of arginine deprivation on human T lymphocytes were first reported in 1968, demonstrating a causal relationship between arginine deficiency and impaired in vitro activation of lymphocytes (Barile and Leventhal 1968). Arginine deprivation impairs T-cell function by downregulating the expression of the CD3 subunit of the T-cell receptor complex (Minami et al. 1987; Weissman et al. 1988; Rodriguez et al. 2002). Arginine level in the tumor microenvironment is lower than that in the plasma, suggesting a role of arginine supplementation in enhancing tumor immunity (Sullivan et al. 2019).\u003c/p\u003e \u003cp\u003eOral arginine is subject to first-pass metabolism in the gastrointestinal tract and liver, reducing its bioavailability. Marini et al. reported that the first-pass metabolism in wild-type mice was 75%, leading to plasma arginine level lower than that in arginase 2-deficient mice (Marini et al. 2011). Owing to these limitations, citrulline has been proposed as an alternative to increase arginine availability. The findings of the present study suggest that anti-PD-1 combined with arginine and citrulline suppresses tumor growth and increase CD8\u003csup\u003e+\u003c/sup\u003e lymphocyte count in the tumor microenvironment. Citrulline bypasses the first-pass metabolism, entering systemic circulation where it is converted to arginine in the kidney by argininosuccinate synthetase and lyase (Wu and Morris, 1998; Levillain, 2012). In a pharmacokinetic study, Moinard et al (2008) showed that oral citrulline supplementation led to a dose-dependent increase in plasma level of arginine as well as that of citrulline and ornithine. Schwedhelm et al (2008) indicated that supplemental citrulline is more effective than arginine itself at increasing systemic arginine availability when administered at equivalent doses. Ouaknine Krief et al (2019) reported that patients with lung cancer with high plasma citrulline level showed longer progression-free survival and overall survival than those with low plasma citrulline level. Citrulline serves as a precursor to arginine and indirectly enhances NO biosynthesis. NO is an endogenous, water-soluble free radical with diverse biological functions, particularly in endothelial vasodilation. In recent decades, research has increasingly focused on the role of NO in inhibiting tumor growth. High concentration of NO has been demonstrated to have therapeutic potential against cancer across human and murine models (Wink et al. 1997; Huerta, 2015; Li et al. 2023). Sullivan and Graham (2008) reported that elevated NO level achieved by delivering NO donors induces apoptosis in tumor cells. Furthermore, NO sensitizes drug-resistant tumor cells to certain chemotherapeutic agents (Liu et al. 2004; Bonavida 2020). These findings suggest that highly active NO donors could be effective in cancer treatment when used in combination with other therapeutic modalities. For characterizing the tumor microenvironment, it might be useful to measure NO and other metabolites using tumor suspensions.\u003c/p\u003e \u003cp\u003eDespite the induction of CD8\u003csup\u003e+\u003c/sup\u003e lymphocytes, we observed no significant differences in the abundance of other immune cell populations, including CD4\u003csup\u003e+\u003c/sup\u003e lymphocytes, regulatory T cells, and macrophages. Tumor-associated macrophages, which express arginases and catabolize arginine, contribute to tumor progression and pro-tumoral remodeling. Sangaletti et al (2008) reported that tumor-associated macrophages in mammary carcinoma express SPARC and facilitate cancer cell migration. Curiel et al (2004). indicated that these macrophages secrete CCL22 chemokines, promoting tumor growth. Further research is required to verify whether coadministration of arginine and citrulline can enhance the therapeutic efficacy of anti-PD-1 antibodies. Although high doses of oral arginine can induce adverse gastrointestinal events attributed to the first-pass effect (Heyland et al. 2003; Grimble, 2007), citrulline administration has no apparent adverse effects and may be more suitable for clinical use (Agarwal et al. 2017). Given their use in cardiovascular disease treatments (Figueroa et al. 2016; Pahlavani et al. 2017; Suzuki et al. 2019), arginine and citrulline could be safely tested in clinical trials.\u003c/p\u003e \u003cp\u003eThis study had several limitations. First, several mice in the treatment groups developed small lesions and were unsuitable for flow cytometry analysis. We could not efficiently evaluate the effect of the therapies on small lesion samples. For mice with lesions weighing less than 0.3 g at the time of sacrifice, we performed only IHC, which could account for discrepancies between immunostaining and flow cytometry results. Additionally, tumor evaluation was not feasible for tumor-free mice in the treatment groups. In such cases, evaluation of immune cells in other lymphoid organs could serve as a substitute for their evaluation in the tumors, regardless of the effect of the tumor microenvironment, and might be useful for assessing tumor-free mice. Second, the mechanism by which the combination therapy of anti-PD-1 plus arginine and citrulline induces more CD8\u003csup\u003e+\u003c/sup\u003e lymphocytes remains unclear. In vitro experiments and bioinformatic analyses may be useful in elucidating the interactions between tumors and immune cells and should be performed in future. Finally, our study did not include an anti-PD-1 plus citrulline group. In future studies, this group should be compared with the anti-PD-1 plus arginine group or the anti-PD-1 plus arginine and citrulline combination group for more clarity on the effects of arginine and citrulline.\u003c/p\u003e \u003cp\u003eIn conclusion, we demonstrate that arginine and citrulline supplementation with anti-PD-1 therapy suppresses lung cancer growth in mice and improves progression-free survival. This combination therapy enhances CD8\u003csup\u003e+\u003c/sup\u003e lymphocyte infiltration and the efficacy of anti-PD-1 drugs. Future studies should be conducted to investigate the mechanisms through which arginine and citrulline combination therapy induces CD8\u0026thinsp;+\u0026thinsp;lymphocytes.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003e\u003cFunding\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNo funding was received.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNM and HT designed the study. NM performed the experiments and wrote the manuscript. NM and MY conducted the animal experiments. HO performed histological analysis. ST and TO analyzed the data. HT, MY, and KY supervised the study.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request. All data generated or analyzed in this study are included in this published article.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll experiments were performed in accordance with the guidelines established by the Tokushima University Committee on Animal Care and Use. All experimental protocols were reviewed and approved by the Animal Research Committee of the University of Tokushima (Approval No.: T202422).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConsent to publish\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgarwal U, Didelija IC, Yuan Y, Wang X, Marini JC (2017) Supplemental citrulline is more efficient than arginine in increasing systemic arginine availability in mice. J Nutr 147:596\u0026ndash;602. doi:10.3945/jn.116.240382\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE and Rosenberg SA (2009) Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood 114:1537\u0026ndash;1544. doi:10.1182/blood-2008-12-195792\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexander M, Kim SY and Cheng H (2020) Update 2020. Management of non-small cell lung cancer. Lung 198:897\u0026ndash;907.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarile MF, Leventhal BG (1968) Possible mechanism for mycoplasma inhibition of lymphocyte transformation induced by phytohaemagglutinin. Nature 219:750\u0026ndash;752. doi:10.1038/219751a0\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonavida B (2020) Sensitizing activities of nitric oxide donors for cancer resistance to anticancer therapeutic drugs. Biochem Pharmacol 176:113913. doi:10.1016/j.bcp.2020.113913\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuriel TJ, Coukos G, Zou L, et al (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10:942\u0026ndash;949. doi:10.1038/nm1093\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Hattab AWE, Hsu JW, Emrick LT, Wong LJC, Craigen WJ, Jahoor F, Scaglia F (2012) Restoration of impaired nitric oxide production in MELAS syndrome with citrulline and arginine supplementation. Mol Genet Metab 105:607\u0026ndash;614. doi:10.1016/j.ymgme.2012.01.016\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFigueroa A, Alvarez-Alvarado S, Jaime SJ, Kalfon R (2016) l-Citrulline supplementation attenuates blood pressure, wave reflection and arterial stiffness responses to metaboreflex and cold stress in overweight men. Br J Nutr 116:279\u0026ndash;285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeng Y, Shao Y, He W, Hu W, Xu Y, Chen J, Wu C, Jiang J (2015) Prognostic role of tumor-infiltrating lymphocytes in lung cancer: A meta-analysis. 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Oncotarget 7:13765\u0026ndash;13781.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"anti-PD-1, immunotherapy, CD8 T cells, non-small cell lung cancer, arginine, citrulline supplementation","lastPublishedDoi":"10.21203/rs.3.rs-5294074/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5294074/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study aimed to examine the role of citrulline in the lung cancer microenvironment and its potential synergistic effects with other therapies.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMurine lung cancer cells (CMT167) were subcutaneously implanted into mice to establish tumor models, followed by treatment with the anti-PD-1 antibody either alone or in combination with arginine or arginine and citrulline. Tumor growth, survival rate, cytokine levels, immune cell populations, and metabolic marker expression were assessed using histological, immunostaining, flow cytometry, and serum analyses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMice in the treatment groups exhibited significantly lower tumor volumes than those in the control group (control, 1161.59\u0026thinsp;\u0026plusmn;\u0026thinsp;294.73; anti-PD-1, 427.38\u0026thinsp;\u0026plusmn;\u0026thinsp;355.34; anti-PD-1 plus arginine, 452.10\u0026thinsp;\u0026plusmn;\u0026thinsp;332.04; anti-PD-1 plus arginine and citrulline, 198.45\u0026thinsp;\u0026plusmn;\u0026thinsp;236.22 mm\u003csup\u003e3\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Furthermore, the anti-PD-1 plus arginine and citrulline group exhibited significantly improved progression-free survival compared with that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00039). The anti-PD-1 plus arginine and citrulline group also showed a significantly higher number of tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e lymphocytes per high-power field (hpf) than the control group (control, 24.22\u0026thinsp;\u0026plusmn;\u0026thinsp;9.13; anti-PD-1, 29.20\u0026thinsp;\u0026plusmn;\u0026thinsp;9.41; anti-PD-1 plus arginine, 34.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.81; anti-PD-1 plus arginine and citrulline, 46.56\u0026thinsp;\u0026plusmn;\u0026thinsp;10.01 cells/hpf).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eArginine and citrulline supplementation facilitated CD8\u003csup\u003e+\u003c/sup\u003e lymphocyte infiltration into the tumor microenvironment, thereby augmenting the efficacy of lung cancer immunotherapy.\u003c/p\u003e","manuscriptTitle":"Enhancement of anti-programmed cell death protein-1 immunotherapy in non-small cell lung cancer using arginine and citrulline supplementation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-30 11:46:01","doi":"10.21203/rs.3.rs-5294074/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a011e9fd-e322-4060-b265-2e257283b5f9","owner":[],"postedDate":"October 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-06T14:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-30 11:46:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5294074","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5294074","identity":"rs-5294074","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}