IDO1 Inhibitor Enhances the Effectiveness of PD-1 Blockade in Microsatellite Stable Colorectal Cancer by Promoting Macrophage Pro-Inflammatory Phenotype Polarization

IDO1 Inhibitor Enhances the Effectiveness of PD-1 Blockade in Microsatellite Stable Colorectal Cancer by Promoting Macrophage Pro-Inflammatory Phenotype Polarization | 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 IDO1 Inhibitor Enhances the Effectiveness of PD-1 Blockade in Microsatellite Stable Colorectal Cancer by Promoting Macrophage Pro-Inflammatory Phenotype Polarization Lv Guangzhao, Wang Xin, Wu Miaoqing, Ma Wenjuan, Liu Ranyi, Pan Zhizhong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5080703/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 10 You are reading this latest preprint version Abstract Microsatellite stable (MSS) colorectal cancer (CRC) is a subtype of CRC that generally exhibits resistance to immunotherapy, particularly immune checkpoint inhibitors such as PD-1 blockade. This study investigates the effects and underlying mechanisms of combining PD-1 blockade with IDO1 inhibition in MSS CRC. Bioinformatics analyses of TCGA-COAD and TCGA-READ cohorts revealed significantly elevated IDO1 expression in CRC tumors, correlating with tumor mutation burden across TCGA datasets. In vivo experiments demonstrated that the combination of IDO1 inhibition and PD-1 blockade significantly reduced tumor growth and increased immune cell infiltration, particularly pro-inflammatory macrophages and CD8 + T cells. IDO1 knockdown in CRC cell lines impaired tolerance to interferon-γ and increased apoptosis in vitro , while IDO1 knockdown in MSS CRC enhanced the effectiveness of PD-1 blockade therapy in vivo . IDO1-knockdown CRC cells promoted pro-inflammatory macrophage polarization and enhanced phagocytic activity via the JAK2-STAT3-IL6 signaling pathway. These findings highlight the role of IDO1 in modulating the tumor immune microenvironment in MSS CRC and suggest that combining PD-1 blockade with IDO1 inhibition could enhance therapeutic efficacy by promoting macrophage pro-inflammatory polarization and infiltration through the JAK2-STAT3-IL6 pathway. immunotherapy microsatellite stable colorectal cancer IDO1 PD-1 blockade macrophage polarization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Colorectal cancer (CRC) remains a major global health challenge, being one of the most prevalent cancers worldwide and imposing a significant burden on healthcare systems. Statistics reported by Chinese National Cancer Center demonstrated that the incidence of colorectal cancer had risen for the past decades to be the second leading cause of cancer mortality in Chinese population. [ 1 ] Despite advances in treatment strategies, the efficacy of current therapies varies, particularly in the context of immune checkpoint inhibitor (ICI) therapy. Among all kinds of ICIs, targeting programmed cell death protein 1 (PD-1) have revolutionized the treatment of CRC, demonstrating remarkable efficacy in a subset of patients with microsatellite instability-high (MSI-H) tumors. [ 2 ] However, this benefit is not universally observed, particularly in patients with microsatellite stable (MSS) tumors, who constitute the majority and exhibit limited response to immunotherapy. [ 3 ] The search for strategies to enhance the response of MSS CRCs to ICI therapy has been vigorous, with oncologists exploring combinations of ICI with other treatments, such as radiotherapy, or the combination of HDAC inhibitors and VEGF antibody to promote a pro-inflammatory tumor microenvironment conducive to immune activation. [ 4 , 5 ] But the application of immunotherapy in MSS CRCs is still full of obstacles. Indoleamine 2,3-dioxygenase 1 (IDO1), functioning as a suppressive immune checkpoint, which is pivotal in regulating the tumor microenvironment. [ 6 ] By catalyzing the conversion of tryptophan to kynurenine, IDO1 effectively depletes tryptophan, which was considered vital for maintaining T cell function. This depletion, alongside activation of the aryl hydrocarbon receptor (AhR) pathway, fosters an immunosuppressive milieu conducive to tumor growth. [ 7 ] In most theories, IDO1 expression is upregulated by pro-inflammatory cytokines, notably interferon-γ (IFN-γ) and was considered a protective “brake” that prevent uncontrollable inflammatory response. [ 8 , 9 ] Clinical studies targeting IDO1 inhibition have shown variable efficacy, highlighting its potential in combination therapies aimed at enhancing anti-tumor immune responses. [ 10 ]. Some potential has been observed when IDO1 inhibitors are combined with other immune checkpoint inhibitors such as Pembrolizumab (the most widely used PD-1 monoclonal antibody). [ 11 ] But most of these studies focus mainly on the MSI-H CRCs. This study is positioned at the intersection of these exploratory avenues, focusing on the role of IDO1 inhibition in conjunction with PD-1 blockade to enhance immunotherapy efficacy in MSS CRC. By hypothesizing that IDO1 inhibition could modulate the tumor microenvironment, specifically by impacting myeloid immune cell activity, such as macrophage polarization and antigen processing behaviors. This research endeavors to elucidate the interplay between IDO1 inhibition, macrophage behavior and modulation of immune cell infiltration, offering insights that could inform the development of more effective, personalized treatment strategies for CRC patients unresponsive to current immunotherapies. Materials and Methods Bioinformatic analysis ESTIMATE and TIMER2.0 were used to predict the immune infiltration status in TCGA-COAD and TCGA-READ datasets. [ 12 , 13 ] R package ESTIMATE (version 1.0.13) and IOBR(version 0.99.9) were used for immune infiltration status. [ 14 ] The simple nucleotide variation datasets were downloaded from TCGA, and the tumor mutation burden (TMB) of each sample were calculated using R package maftools. [ 15 ] RNA-seq expression matrix was imported into the GSEA software (Broad, v4.3.2), and the HALLMARK 2023.2 gene sets was used for gene set enrichment analysis. [ 16 ] Cell culture CT26, MC38, HCT116, HT29, THP-1 and RAW264.7 were purchased from ATCC. CT26 and THP-1 were cultured with RPMI-1640 with 10% fetal bovine serum (FBS), MC38 and RAW264.7 was cultured with DMEM with 10% FBS, HCT116, HT29 was culture with Mcoy-5A medium with 10% FBS. All cells were detected mycoplasma free and were maintained anti-bacterial with 1× penicillin and streptomycin solution (Solarbio Inc, Beijing). Small interfering RNA transfection Small interfering RNAs (siRNAs) were dissolved in RNase free water to reach a final concentration of 100nmol/µL. Lipofectamine 3000 (Invitrogen) and siRNAs were mixed in Opti-MEM(Gibco) and incubated in room temperature for 20 minutes to compose transfection liposome added on the cells. Culture medium was removed 8 hours after transfection and fresh culture medium with FBS was added subsequently. Total RNA or total protein was extracted 48 hours after transfection for downstream analyses. The RNAi sequences used in this study were listed in Supplementary Table 1. IDO1 knockdown lentivirus transfection LVRU6GP plasmids with RNAi sequences were constructed by GeneCopoeia Inc. Lentivirus was packaged in HEK293T cells via co-transfection of target plasmids, psPAX2 and pMD2G using Lipofectamine 3000. The lentivirus-containing supernatant was purified by centrifugation and filtration. Stable cells were selected with puromycin at predetermined concentrations. Quantitative RT-PCR Total RNA was extracted using TRIzol (Invitrogen) according to the standard procedure. The RNA was then reverse-transcribed into cDNA using reverse transcription kits (Yeason, Shanghai) following the manufacturer's instructions. RT-qPCR was performed using the SYBR Green mix (Yeason, Shanghai) and specific primers according to the recommended protocol in GTEx (BioRad). The primer sequences are listed in Supplementary Table 2. Western Blot Cells were lysed in RIPA buffer with 1% PMSF on ice for 30 minutes. Protein concentration was determined using a BCA kit. Proteins were separated by SDS-PAGE and transferred to membranes. Following blocking with skimmed milk, membranes were incubated with primary antibodies, then HRP-conjugated secondary antibodies for chemiluminescence detection. The expression levels were compared using chemiluminescence blotting. For nuclear and cytoplasmic protein separation, the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027) was used according to the manufacturer’s instructions. Antibodies used are listed in Supplementary Table 3. Animal experiment The animal experiment was approved by the Ethics Committee of Sun Yat-Sen University Cancer Center (Approval ID: 025503202112028). C57BL/6 or Balb/c mice were purchased from Guangdong Yaokang Biotech Ltd or the Experimental Animal Center of Sun Yat-Sen University Cancer Center. A total of 1×10⁵ tumor cells were inoculated subcutaneously into the left axillary area. Anti-mouse PD-1 (10 mg/kg, InVivoMAb, clone ID: RMP1-14, Cat # BE0146) was administered intraperitoneally every 3 days, and epacadostat (10 mg/kg, INCB 024360, MedChemExpress) was given daily. [ 17 , 18 ] Mice were sacrificed 15 to 17 days after the final treatment. Flow cytometry Tumor tissues were cut into 2 mm pieces and dissociated into single-cell suspension using the Tumor Dissociation Kit (Miltenyi, Cat# 130-096-730) and Single Cell Suspension Dissociator (RWD, China) following the manufacturer’s instructions. [ 19 ] Erythrocytes were lysed with BD Pharm Lyse™ solution (Cat # 555899). After Fc receptor blockade with TruStain FcX™ (Biolegend, Cat# 101319), surface markers were stained with antibodies listed in Table 3. Cells were then permeabilized and fixed using True-Nuclear™ Transcription Factor Buffer Set (Biolegend, Cat# 424401) for nuclear marker staining. Single fluorescence-stained Compensation Beads (Biolegend, Cat# 424602) were used for compensation adjustment. The stained cell suspension was analyzed using a CytoFLEX LX Flow Cytometer (Beckman Coulter). Cell markers were determined as described in previous studies. [ 20 ]Fluorescence labeled antibody for FCS analysis were listed in Supplementary Table 4. In vitro cell function assays In vitro cell proliferation was assessed using the CCK-8 viability kit. Briefly, 1×10⁴ cells were cultured in a 96-well plate and incubated under standard conditions. Medium was replaced with 10% CCK-8 in serum-free medium and incubated at 37°C for 45 minutes. Absorbance at 450 nm was measured using a microplate reader. For the colony formation assay, 500 cells per well were plated in 6-well plates and cultured with 10% FBS medium for 10–14 days to form colonies. For migration and invasion assays, 2×10⁵ cells in 250 µL serum-free medium were placed on the upper side of an 8 µm transwell membrane coated with Matrigel. Transwell inserts were placed in 24-well plates containing 10% FBS medium in the lower chamber. After 24 hours of incubation, cells on the upper surface were removed. Cells from both assays were stained with crystal violet, photographed, and quantified using ImageJ (1.54g). Immune Fluorescence Staining Cells were incubated on 15 mm diameter round coverslips and fixed with 4% paraformaldehyde. Plasma membranes were permeabilized using Triton X-100. Blocking was performed with 5% bovine serum albumin (BSA). Primary antibodies, diluted as listed in Table 3, were applied overnight at 4°C. Fluorescence-labeled secondary antibodies were then incubated for 1 hour at room temperature in a light-protected environment. Finally, coverslips were mounted with an anti-fading agent, and fluorescence images were acquired using a laser confocal microscope (Olympus). FITC-labelled dextran uptake assay Phagocytic activity of macrophages was examined using FITC-labeled dextran (MW 4,000, Beyotime, Cat# ST2930). THP-1 cells were differentiated into M0 macrophages by treating with 100 ng/mL PMA (Beyotime, Cat# S1819) for 24 hours. FITC-labeled dextran was dissolved in Hank’s balanced salt solution (HBSS) at a stock concentration of 100 mg/mL. Macrophages were incubated with serum-free RPMI 1640 medium containing 1 mg/mL FITC-labeled dextran at 37°C with 5% CO2 for 2 hours. Cells were then washed three times with PBS and resuspended in PBS. Fluorescence images were captured, and flow cytometry was used to analyze the mean fluorescence intensity in the FITC channel, quantifying the dextran uptake by macrophages. [ 21 ] Statistical analysis GraphPad Prism9 was applied for the presentation of figures. ANOVA was used for multiple sample comparison and two-sided Dunnett-t test was applied for one-to-one comparison. The p value was recorded in the figures with exact numbers. Results IDO1 upregulation is associated with increased immune infiltration in colorectal cancer. We analyzed the role of IDO1 in colorectal cancer using the TCGA database, including its correlation with immune checkpoints, expression in cancer vs. normal tissues, mutation levels, and impact on tumor mutation burden and immune cell infiltration. IDO1 showed positive correlations with PDCD1 (PD-1), CD274 (PD-L1), CTLA4, and other immune checkpoints like TIGIT, TGFB1, CXCL9, CXCL10, and IFNG. (Fig. 1 A, Supplementary Fig. 1A). IDO1 expression was higher in cancer tissues compared to normal epithelium in a wide range of adenocarcinomas (Fig. 1 B). IDO1 mutation rates were low, at 1.8% in COAD and 1.1% in READ (Fig. 1 C). Pan-cancer analysis revealed a strong correlation between IDO1 expression and tumor mutation burden (Fig. 1 D). Using ESTIMATE and TIMER algorithms, we found positive correlations between IDO1 expression and immune infiltration scores in CRC (Fig. 1 E). Increased IDO1 expression in CRC was associated with heightened interferon responses and activation of the IL-6, JAK-STAT signaling pathway, and inflammatory response (Fig. 1 F). CRC tissue samples showed concurrent elevation of IDO1 and immune cell markers (CD4, CD8, CD68, CD206) (Fig. 1 G). Upregulation of IDO1 in colorectal cancer cell lines and paired cancer-normal tissue samples was confirmed (Figs. 1 H, 1 I). Higher IDO1 expression was also linked to a greater overall mutation frequency (Supplementary Fig. 1B). IDO1 inhibition sensitized PD-1 blockade therapy in MSS colorectal cancer. We evaluated whether IDO1 inhibition enhances the effectiveness of PD-1 blockade in colorectal cancer using an animal model (Fig. 2 A). Two mouse-derived cell lines were used: CT26 (MSS, Balb/c mice) initially resistant to PD-1 blockade, and MC38 (MSI-H, C57 mice) (Fig. 2 A). [ 22 , 23 ] Tumor cells were inoculated superficially in mice, followed by treatments with PD-1 blockade, IDO1 inhibition (epacadostat), or their combination. Results showed that epacadostat was effective in CT26, especially when combined with PD-1 blockade (Fig. 2 B, C). IDO1 expression and macrophage markers (F4/80, CD206, IL-6) were examined, revealing IDO1 upregulation with PD-1 treatment. CD206 increased in the PD-1 group, while IL-6 was elevated with epacadostat, with or without PD-1 blockade (Fig. 2 D). Leukocyte infiltration analysis indicated increased CD8 + T cells and a higher M1/M2 ratio with epacadostat (Fig. 2 E, F). Epacadostat also showed combined anti-tumor effects with PD-1 blockade in MC38 cell lines (Supplementary Fig. 2). Knockdown of IDO1 impairs resistance to interferon-γ in CRC cell lines via AHR-CXCL9/CXCL10 axis. We used RNAi to knockdown IDO1 in MSS cell lines CT26 and HT29 to examine its impact on cell growth and invasion (Fig. 3 A). No significant changes were observed in cell duplication, migration, or invasiveness after IDO1 suppression (Fig. 3 B-F). As IDO1 is IFN-inducible, we treated HT29 cells with 50 ng/mL human recombinant interferon gamma (rhIFN-γ, R&D, Cat# 285-IF-100) for 6 hours to assess whether IDO1 inhibition affects tumor resistance to IFN stimulation. IFN-γ significantly increased apoptosis in IDO1 knockdown HT29 cells (Fig. 3 G). We investigated the impact of IDO1 knockdown on AHR activation by performing nuclear-cytoplasmic protein separation on IFN-γ treated cancer cells, finding a significant reduction in AHR nuclear translocation (ARNT) in IDO1 knockdown cells. The reduced ARNT could be restored by an AHR ligand 6-Formylindolo (3,2-b)carbazole (FICZ, CAS #172922-91-7). (Fig. 3 H) FICZ could also restore the tolerance of IFN-γ in CRC cells by reducing IFN-γ induced apoptosis. (Fig. 3 I) Additionally, IDO1 knockdown resulted in decreased overall AHR expression and increased levels of cleaved caspase 3 and PD-L1 (Fig. 3 J). Knockdown of IDO1 in MSS CRC improves sensitivity to PD-1 blockade therapy in vivo . We used IDO1 knockdown CT26 cells to assess the effectiveness of PD-1 blockade in vivo (Fig. 4 A). To achieve long-term IDO1 inhibition at the transcription level, we constructed shIDO1 plasmids and generated stable knockdown CT26 cells via lentivirus infection (Fig. 4 B). Due to GFP expression in CT26 cells post-infection (Fig. 4 C), we utilized new cell marker antibodies. As expected, IDO1 knockdown suppressed tumor growth under PD-1 blockade therapy (Fig. 4 D & E). We also assessed macrophage marker expression, finding elevated pro-inflammatory IL-6 and reduced anti-inflammatory CD206 in IDO1 knockdown CT26 cells (Fig. 4 F). Flow cytometry revealed increased leukocyte infiltration, particularly T cells, with a notable decrease in regulatory T cells (Fig. 4 G, H). Additionally, IDO1 knockdown in MC38 cells enhanced response to PD-1 blockade therapy (Supplementary Fig. 3). IDO1 KD in cancer cell promote macrophage pro-inflammatory phenotype polarization via JAK2-STAT3-IL6 pathway. We used a coculture method to examine interactions between cancer cells and macrophages (RAW264.7). When cocultured with CT26 cells, macrophages exhibited high CD163 and low IL-6 levels in the presence of shNC tumor cells. Knocking down IDO1 in tumor cells increased IL-6 and decreased CD163 in macrophages (Fig. 5 A). In THP-1 cells treated with tumor cell supernatant and recombinant human IFN-γ, the shNC group showed CD163 positivity and iNOS negativity, while the shIDO1 group showed the opposite (Fig. 5 B). FITC-labelled dextran uptake in THP-1 cells was higher when stimulated with rhIFN-γ loaded shIDO1 HT29 cell supernatant (Fig. 5 C, D). Flow cytometry confirmed higher IL-6 expression in THP-1 cells stimulated by shIDO1 cell supernatant (Fig. 5 E). JAK2 and STAT3 mRNA expression increased in THP-1 cells stimulated by IDO1-knockdown HT29 cells (Fig. 5 F). Protein analysis showed increased JAK2 and STAT3 phosphorylation in IDO1 knockdown HT29 cells (Fig. 5 G). Discussion Immunotherapy, despite its effectiveness, often fails in MSS colorectal cancer due to the suppressive tumor immune microenvironment (TIME). This environment is characterized by the infiltration of suppressive immune cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), along with reduced levels of stimulatory cytokines like CXCL9 and CXCL10. These factors contribute to an immune-suppressive region in MSS CRC, inhibiting antigen recognition by antigen-presenting cells (APCs) and the generation of cancer-targeting cytotoxic T cells, thereby diminishing the efficacy of immune-based treatments. [ 24 ] IDO1 is known as a suppressive immune checkpoint for catalyzing the initial and rate-limiting step in the kynurenine pathway. IDO1 depletes tryptophan and produces immunosuppressive metabolites, including kynurenine. This activity can create a local immunosuppressive microenvironment conducive to tumor growth and survival. The upregulation of IDO1 in these cells has been associated with the suppression of T cell responses and the promotion of regulatory T cell (Treg) development, further contributing to immune evasion by tumors. [ 25 , 26 ] Inhibiting IDO1 activity is hypothesized to restore local and systemic immune responses against tumor cells by reversing tryptophan depletion, reducing kynurenine levels, and promoting a more immunogenic tumor microenvironment. Clinical trials have investigated IDO1 inhibitors, both as monotherapies and in combination with other immunotherapeutic agents, such as PD-1/PD-L1 inhibitors, to enhance anti-tumor immunity. While results from these trials have been mixed, none of these researches focused on its potentials in MSS CRC. The joint effect of IDO1 inhibitors and PD-1 blockade required more evidence. In our study, we discovered that IDO1 is up-regulated in tumor versus normal tissues. Despite IDO1 is not a frequently mutated gene, its expression was closely correlated with the overall mutation burden and pro-inflammatory status of the TIME. Collectively, these results suggested that IDO1 also played a pivotal role in mediating the tumor microenvironment in CRCs, playing an internal agent against inflammation. We then conducted an in vivo assay to test whether IDO1 inhibition benefits PD-1 blockade immunotherapy. The results were highly encouraging, demonstrating the good synergistic effect of IDO1 inhibitor and PD-1 blockade in MSS CRCs. Notably, epacadostat significantly regulated the distribution of immune cells within the tumor microenvironment. A marked increase in the infiltration of cytotoxic CD8 + T cells and pro-inflammatory macrophages was witnessed, which are known to play a key role in activated antitumor immunity. Our studies also uncovered a significant role for IDO1 in mediating cancer cell resistance to interferon-gamma (IFN-γ). The abnormal upregulation of IDO1 suggested a complex interplay where cancer cells utilize IDO1 to counteract the immune-activating effects of IFN-γ. This mechanism not only illustrates IDO1's role in creating an immunosuppressive environment but also underscores its potential as a therapeutic target to enhance cancer immunotherapy efficacy. While IDO1 expression itself may not directly influence the proliferative or metastatic capabilities of cancer cells in vitro , its role in mediating IFN-γ resistance positions its importance to immune resistance. The inhibition of IDO1 presents a promising strategy not only by impacting the metabolic functions of cancer cells but also by modulating the behavior of immune cells within the tumor microenvironment. This study revealed that while IDO1 knockdown did not significantly alter the growth rate of CT26 tumors in vivo , it notably enhanced the effectiveness of PD-1 blockade therapy. This suggests that the primary role of IDO1 in these tumors may not be in promoting tumor cell proliferation directly but in modulating the immune microenvironment to favor tumor survival. Further investigation into the immune profiles of these tumors showed a marked increase in the infiltration of cytotoxic CD8 + T cells in the IDO1 knockdown models treated with PD-1 inhibitors suggesting that IDO1 activity in the tumor cells might contribute to an immune-exclusion phenotype. Finally, we sought to uncover the mechanism of IDO1 in cancer cell and the influence of tumor IDO1 expression to macrophage. We found that IDO1 plays pivotal role in maintaining the immunosuppressive characteristics of tumor associated macrophages (TAMs). By silencing the expression of IDO1 in cancer cell, the contacted macrophages tend to present pro-inflammatory phenotype, the expression of IL-6 increased with the coculture of IDO1 knockdown cancer cells. And macrophages also presented increased phagocytic activity by indirect stimulation of IDO1-knockdown cancer cells, which indicated that the antigen presentation was activated. Collectively, these results illustrated that, IDO1 not only give resistance to inflammatory stimuli to cancer cell but also allowed secretion of specific agents that regulate macrophage polarization. IL6 is one of the mediated cytokines that was regulated by local IDO1 level. Previous results showed that IL6 was mediated via JAK-STAT pathway. Collectively, these results highlight the dual role of IDO1 in cancer progression—both as a shield against immune attack directly on cancer cells and as a regulator of the immune landscape via macrophage polarization. Our findings underscore the potential therapeutic benefits of targeting IDO1 in cancer treatment, not only to inhibit tumors’ intrinsic pathways but also to reprogram the immunological milieu of the tumor to enhance the efficacy of existing and emerging therapies. These insights pave the way for novel therapeutic strategies that aim at disrupting the IDO1-mediated immunosuppressive network within tumors, potentially leading to more effective immunotherapy outcomes. However, this study also has certain limitations, such as not thoroughly exploring whether changes in the levels of downstream metabolites of IDO1 affect the local conditions of the tumor immune microenvironment, and whether sensitizing the effect of IDO1 blockade in MSS CRC is achieved by reshaping the changes in metabolite levels caused by IDO1. Additionally, the regulatory effect of tumor cells on stromal cells and the mediators inducing this regulation were not clarified in this study. In future research, the sensitizing mechanisms of IDO1 inhibitors on ICIs require further elucidation. Conclusions Our study introduces a novel approach to enhance the efficacy of PD-1 blockade therapy in MSS colorectal cancer. By combining an IDO1 inhibitor with PD-1 blockade, we demonstrated significant therapeutic benefits in a pre-clinical MSS cancer model. Specifically, inhibition of IDO1 in CRC cells led to a significant alteration in the distribution of tumor-infiltrating lymphocytes (TILs) and remodeled the immune microenvironment into a pro-inflammatory state. This transformation effectively "lit up" the tumors, rendering them more responsive to PD-1 blockade therapy. This strategy not only highlights the potential of IDO1 inhibitors in modifying the tumor microenvironment but also underscores their role in improving the outcomes of existing immunotherapies. (Fig. 6 ) Declarations Declaration of interest statement The authors declare no conflicts of interest. Author Contribution Lv G, Wang X and Wu M contributed equally to this work by performing the in vitro and in vivo experiments.Ma W was responsible for constructing the IDO1 gene modification plasmid and primers.Liu R assisted with the study design and provided statistical analysis support.Pan Z supervised the colorectal surgery experimental platform.Zhang R and Chen G are the principal supervisors of the study, overseeing the research design, data analysis, and manuscript preparation.All authors contributed to the drafting and revision of the manuscript and approved the final version for publication. Acknowledgement The work was supported by grants from Guangdong Basic and Applied Basic Research Foundation (Grant No. 2023A1515010243), Chinese Society of Clinical Oncology Foundation (Grant Nos. Y-HR2018-319, Y-L2017-002, and Y-JS2019-009), Sun Yat-sen University Basic Research Fund (Grant No. 19ykpy180), and the open research funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital (202011-103, 202301-314). Data availability statement The generated original data during the current study are not publicly available but will be deposited on the Research Data Depot (RDD) system of our institute. These data will be available from the corresponding author on reasonable request. For access, please contact Dr. Chen Gong at [email protected] . References Bray F et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin Diaz LA Jr. et al (2022) Pembrolizumab versus chemotherapy for microsatellite instability-high or mismatch repair-deficient metastatic colorectal cancer (KEYNOTE-177): final analysis of a randomised, open-label, phase 3 study. 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Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 03 Jan, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 17 Oct, 2024 Reviews received at journal 15 Oct, 2024 Reviews received at journal 10 Oct, 2024 Reviewers agreed at journal 02 Oct, 2024 Reviewers agreed at journal 02 Oct, 2024 Reviewers agreed at journal 01 Oct, 2024 Reviewers invited by journal 30 Sep, 2024 Editor assigned by journal 14 Sep, 2024 Submission checks completed at journal 14 Sep, 2024 First submitted to journal 12 Sep, 2024 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. 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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-5080703","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":367394612,"identity":"aebb68cd-fcd7-41d2-8090-c5bc71baaeb1","order_by":0,"name":"Lv Guangzhao","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Lv","middleName":"","lastName":"Guangzhao","suffix":""},{"id":367394613,"identity":"812f10c5-845a-4045-b727-c34d8db48b16","order_by":1,"name":"Wang Xin","email":"","orcid":"","institution":"The Affiliated Qingyuan Hospital (Qingyuan People's Hospital), Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Xin","suffix":""},{"id":367394618,"identity":"417ecb2f-75e5-431e-8eff-13ba6e4bfa7a","order_by":2,"name":"Wu Miaoqing","email":"","orcid":"","institution":"The Seventh Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Wu","middleName":"","lastName":"Miaoqing","suffix":""},{"id":367394619,"identity":"27be72f9-4c72-411a-aac9-68a6f290d569","order_by":3,"name":"Ma Wenjuan","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Ma","middleName":"","lastName":"Wenjuan","suffix":""},{"id":367394620,"identity":"7aed84ee-71c9-490c-b15f-30ec7eaed90d","order_by":4,"name":"Liu Ranyi","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Ranyi","suffix":""},{"id":367394621,"identity":"9c36d87d-e7ba-413b-aab9-60d41fb53b6e","order_by":5,"name":"Pan Zhizhong","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Pan","middleName":"","lastName":"Zhizhong","suffix":""},{"id":367394622,"identity":"975df77c-b1e0-48d8-81ac-d4a3e97cfdfa","order_by":6,"name":"Zhang Rongxin","email":"","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Rongxin","suffix":""},{"id":367394623,"identity":"b57565f5-7fad-46fd-b0d2-bbd9d7afc337","order_by":7,"name":"Chen Gong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACCSjNT7oWyQaStRgcIFYH/+zmYw+/VNyx23z+jPFnnho7Bn7p4xcYfu7AY8mdY+nGMmeeJW+7kWMmzXMsmUGyL6eAsfcMbi0GEkCVkm2Hk81u8Jgx8zYcYDA4w5PAzNiGT0v+N7AW436gw4jUksMm+bHtsJ0BQ46BNEQL+wG8WiRupJlJM5w5nABklEnOOZbMI9nDw3CwF48W/hnJzyR/VBy25+8/vPnDmxo7OX4e9ocPfuLRAgLMPAwMiQ0MHAYgDpDNQziOGH8wMNgzMLA/gPLhjFEwCkbBKBgFYAAA1UdOHZQdxicAAAAASUVORK5CYII=","orcid":"","institution":"Sun Yat-sen University Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Chen","middleName":"","lastName":"Gong","suffix":""}],"badges":[],"createdAt":"2024-09-13 03:25:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5080703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5080703/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-024-03925-w","type":"published","date":"2025-01-03T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69898342,"identity":"60b981b4-0cb8-4ab8-860d-d35a350874ea","added_by":"auto","created_at":"2024-11-26 11:37:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14279252,"visible":true,"origin":"","legend":"\u003cp\u003eIDO1 upregulation is associated with increased immune infiltration in colorectal cancer.\u003c/p\u003e\n\u003cp\u003eA: Scatterplots illustrating the correlation between the immune checkpoint genes PDCD1 (encoding PD-1), CD274 (encoding PD-L1), and CTLA4, and the IDO1 gene within the TCGA-COAD transcriptomic datasets.\u003c/p\u003e\n\u003cp\u003eB: Expression of IDO1 in tumor versus adjacent normal tissues in TCGA adenocarcinoma datasets, highlighted COAD and READ.\u003c/p\u003e\n\u003cp\u003eC: The mutation sites, types, and frequencies of the IDO1 gene in TCGA-COAD and TCGA-READ.\u003c/p\u003e\n\u003cp\u003eD: Correlation of IDO1 expression and tumor mutation burden (TMB) in TCGA pan-cancer database.\u003c/p\u003e\n\u003cp\u003eE: Correlation between immune infiltration and IDO1 in TCGA-COAD and TCGA-READ. Overall immune infiltration status was predicted by ESTIMATE, the infiltration of CD8+ T cell, CD4+ T cell and Macrophages were predicted by IPS.\u003c/p\u003e\n\u003cp\u003eF: Gene set enrichment analysis (GSEA) of TCGA-COAD and TCGA-READ, presenting up-regulation of inflammatory associated pathways in IDO1\u003csup\u003ehigh \u003c/sup\u003ecancer datasets.\u003c/p\u003e\n\u003cp\u003eG: Immunohistochemistry staining of tumor infiltrating leukocytes: CD4, CD8a, CD68 and CD206 in tumor versus normal epithelium.\u003c/p\u003e\n\u003cp\u003eH: Expression of IDO1 in clinically collected colon cancer and adjacent normal tissue (n=4).\u003c/p\u003e\n\u003cp\u003eI: Expression of IDO1 expression in multiple human derived colorectal cancer cell lines versus colon epithelial cell (CCD841con).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5080703/v1/46408ed691bf4766a7d2086e.png"},{"id":69898346,"identity":"ed42613c-093f-44bb-8aed-429513c55941","added_by":"auto","created_at":"2024-11-26 11:37:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17114483,"visible":true,"origin":"","legend":"\u003cp\u003eIDO1 inhibition sensitized ICI therapy in MSS colorectal cancer.\u003c/p\u003e\n\u003cp\u003eA: Schema of in vivo analysis of the effect to combined therapy of PD-1 blockage and IDO1 inhibition in superficial inoculated MSS or MSI-H colon cancer.\u003c/p\u003e\n\u003cp\u003eB: Snapshot of tumor gross morphologies at day 16 post-inoculation.\u003c/p\u003e\n\u003cp\u003eC: Tumor size statistics of CT26 and the increase of tumor volume with time post-inoculation. (n=5, data was presented as mean ± SEM)\u003c/p\u003e\n\u003cp\u003eD: Immunohistochemistry staining of IDO1, the mouse monocyte/macrophage biomarker F4/80, the anti-inflammatory marker CD206, and the pro-inflammatory marker IL-6 in different treatment groups.\u003c/p\u003e\n\u003cp\u003eE: Scatterplots of flow cytometry analysis of CT26 tumor receiving different treatments.\u003c/p\u003e\n\u003cp\u003eF: Distribution of different tumor infiltrated leukocytes (total leukocytes, total T cells, CTLs, helper Ts, Tregs, Macrophages and M1/M2 in CT26 tumor-bearing mice receiving different treatments. (n=5, data was presented as mean ± SEM)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5080703/v1/d4e029279088529bc158eb31.png"},{"id":69899408,"identity":"18838a03-5c68-4bd4-b47f-7818df9b46e3","added_by":"auto","created_at":"2024-11-26 11:45:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5993500,"visible":true,"origin":"","legend":"\u003cp\u003eIDO1 knockdown suppresses tumor resistance to IFN-γ\u003c/p\u003e\n\u003cp\u003eA: Western blot showing the interfering efficiency in knockdown of IDO1 in CT26 with siRNA and HT29 using lentiviral infection.\u003c/p\u003e\n\u003cp\u003eB \u0026amp; C: Wound healing assay of CT26 and HT29 after knockdown of IDO1 expression in vitro. (n=3, data was presented as mean ± SEM)\u003c/p\u003e\n\u003cp\u003eD \u0026amp; E: Cell invasiveness assay with 8μm transwell in IDO1 knockdown cell lines, with statistical analysis. (n=3, data was presented as mean ± SEM)\u003c/p\u003e\n\u003cp\u003eF: In vitro proliferation assay by CCK-8 in CT29 and HT29 with modified expression of IDO1. (n=6, data was presented as mean ± SEM)\u003c/p\u003e\n\u003cp\u003eG: Apoptotic and dead cells detected by Annexin V/PI stain in HT29 after IFN-γ stimulation (50ng/mL) for 12 hours.\u003c/p\u003e\n\u003cp\u003eH: Nuclear and cytoplasmic protein presenting the decrease of AHR nuclear translocation in IDO1-knockdown HT29 after IFN-γ treatment, which could be restored by AhR ligand FICZ.\u003c/p\u003e\n\u003cp\u003eI: Apoptotic and dead cells detected by Annexin V/PI stain in HT29 cells after co-treatment of IFN-γ (50ng/mL) and FICZ(100nM).\u003c/p\u003e\n\u003cp\u003eJ: Expression of AHR, caspase 3, cleaved caspase 3, PD-L1 in IFN-γ treated HT29 cells.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5080703/v1/4a579674d0528a2beb16129f.png"},{"id":69899873,"identity":"ab695387-5cf0-4741-9ced-a3b267027fcc","added_by":"auto","created_at":"2024-11-26 11:53:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12481015,"visible":true,"origin":"","legend":"\u003cp\u003eIDO1 knockdown in tumor cell promote immunotherapy sensitivity in MSS CRC.\u003c/p\u003e\n\u003cp\u003eA: Schema of \u003cem\u003ein vivo\u003c/em\u003e analysis of the effect on knocking down IDO1 in tumor cell combined with PD-1 blockage in superficial inoculated MSS colon cancer;\u003c/p\u003e\n\u003cp\u003eB: Expression of IDO1 in lentivirus infected CT26 cell lines presented by Western Blot or qRT-CPR;\u003c/p\u003e\n\u003cp\u003eC: 488 excitation channel presenting the GFP expression of shIDO1 paired cells.\u003c/p\u003e\n\u003cp\u003eD: Snapshot of tumor gross morphologies at day 17 post-inoculation;\u003c/p\u003e\n\u003cp\u003eE: Tumor size statistics of CT26 and the increase of tumor volume with time post-inoculation;\u003c/p\u003e\n\u003cp\u003eF: Immunohistochemistry staining of IDO1, the mouse monocyte/macrophage biomarker F4/80, the anti-inflammatory marker CD206, and the pro-inflammatory marker IL-6 in different treatment groups.\u003c/p\u003e\n\u003cp\u003eG: Scatterplots of flow cytometry analysis of Ido1-sh2 and Ido1-shNC mice receiving different treatments.\u003c/p\u003e\n\u003cp\u003eH: Distribution of different tumor infiltrated leukocytes (total leukocytes, total T cells, CTLs, Th Ts, Tregs, Macrophages and M1/M2 in CT26 tumor-bearing mice receiving different treatments. (n=5, data was presented as mean ± SEM)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5080703/v1/a1bd4373280c4c0a985f9f9a.png"},{"id":69898347,"identity":"6b703c92-b77c-4c48-a4cb-4f38719d33d2","added_by":"auto","created_at":"2024-11-26 11:37:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4702068,"visible":true,"origin":"","legend":"\u003cp\u003eIDO1 knockdown in tumor cell potentiate pro-inflammatory macrophage polarization with elevated phagocytic activity.\u003c/p\u003e\n\u003cp\u003eA: Co-culture of RAW264.7 cells with CT26-shIDO1 cells shows increased IL-6 expression and decreased CD163 expression in THP-1 cells.\u003c/p\u003e\n\u003cp\u003eB: Western Blot of THP-1 protein that present the expression of M1 marker iNOS and M2 marker CD163, and the corresponding expression of AHR after stimulated by IFN-γ treated HT29 cell lines.\u003c/p\u003e\n\u003cp\u003eB: IL-6 abundance of THP-1 after stimulated by IFN-γ treated culture medium of HT29-shNC and HT29-shIDO1.\u003c/p\u003e\n\u003cp\u003eC: FITC channel of THP-1 stimulated by IFN-γ treated culture medium of HT29-shNC and HT29-shIDO1.\u003c/p\u003e\n\u003cp\u003eD: Fluorescence intensity distribution and mean fluorescence intensity of FITC-dextran in THP-1, stimulated by IFN-γ treated culture medium of HT29-shNC and HT29-shIDO1. (n=3, data was presented as mean ± SEM)\u003c/p\u003e\n\u003cp\u003eE: Fluorescence intensity distribution and mean fluorescence intensity(MFI) of Alexa Fluor 647 labelled IL-6, stimulated by IFN-γ treated culture medium of HT29-shNC and HT29-shIDO1. (n=3, data was presented as mean ± SEM)\u003c/p\u003e\n\u003cp\u003eF: mRNA abundance of JAK2 and STAT3 in THP-1 after stimulated by IFN-γ treated culture medium of HT29-shNC and HT29-shIDO1. (n=,4 data was presented as mean ± SEM)\u003c/p\u003e\n\u003cp\u003eG: Expression of JAK2, STAT3, IL6 in THP-1 stimulated by IFN-γ treated culture medium of HT29-shNC and HT29-shIDO1.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5080703/v1/0497804b1a99842acb6c9e30.png"},{"id":69901200,"identity":"0614de23-b179-4a5d-8ec5-b9de5dbba095","added_by":"auto","created_at":"2024-11-26 12:01:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4988049,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of IDO1 inhibitors in enhancing PD-1 blockade therapy in MSS CRCs. (Generated by Figdraw)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5080703/v1/1ee05d4b63164e1ec776a2e7.png"},{"id":73093317,"identity":"3956fc4b-316b-4b17-8504-014065d25e5e","added_by":"auto","created_at":"2025-01-06 16:13:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":60926434,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5080703/v1/9a9788b2-18b7-4bd8-9591-2dfd70ab001d.pdf"},{"id":69898340,"identity":"75054102-8106-4973-a49e-33b8beedbbe4","added_by":"auto","created_at":"2024-11-26 11:37:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2537637,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5080703/v1/0a54c22d129b730c09ad810f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"IDO1 Inhibitor Enhances the Effectiveness of PD-1 Blockade in Microsatellite Stable Colorectal Cancer by Promoting Macrophage Pro-Inflammatory Phenotype Polarization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) remains a major global health challenge, being one of the most prevalent cancers worldwide and imposing a significant burden on healthcare systems. Statistics reported by Chinese National Cancer Center demonstrated that the incidence of colorectal cancer had risen for the past decades to be the second leading cause of cancer mortality in Chinese population. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Despite advances in treatment strategies, the efficacy of current therapies varies, particularly in the context of immune checkpoint inhibitor (ICI) therapy. Among all kinds of ICIs, targeting programmed cell death protein 1 (PD-1) have revolutionized the treatment of CRC, demonstrating remarkable efficacy in a subset of patients with microsatellite instability-high (MSI-H) tumors. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] However, this benefit is not universally observed, particularly in patients with microsatellite stable (MSS) tumors, who constitute the majority and exhibit limited response to immunotherapy. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] The search for strategies to enhance the response of MSS CRCs to ICI therapy has been vigorous, with oncologists exploring combinations of ICI with other treatments, such as radiotherapy, or the combination of HDAC inhibitors and VEGF antibody to promote a pro-inflammatory tumor microenvironment conducive to immune activation. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] But the application of immunotherapy in MSS CRCs is still full of obstacles.\u003c/p\u003e \u003cp\u003eIndoleamine 2,3-dioxygenase 1 (IDO1), functioning as a suppressive immune checkpoint, which is pivotal in regulating the tumor microenvironment. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] By catalyzing the conversion of tryptophan to kynurenine, IDO1 effectively depletes tryptophan, which was considered vital for maintaining T cell function. This depletion, alongside activation of the aryl hydrocarbon receptor (AhR) pathway, fosters an immunosuppressive milieu conducive to tumor growth. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] In most theories, IDO1 expression is upregulated by pro-inflammatory cytokines, notably interferon-γ (IFN-γ) and was considered a protective \u0026ldquo;brake\u0026rdquo; that prevent uncontrollable inflammatory response. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] Clinical studies targeting IDO1 inhibition have shown variable efficacy, highlighting its potential in combination therapies aimed at enhancing anti-tumor immune responses. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Some potential has been observed when IDO1 inhibitors are combined with other immune checkpoint inhibitors such as Pembrolizumab (the most widely used PD-1 monoclonal antibody). [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] But most of these studies focus mainly on the MSI-H CRCs.\u003c/p\u003e \u003cp\u003eThis study is positioned at the intersection of these exploratory avenues, focusing on the role of IDO1 inhibition in conjunction with PD-1 blockade to enhance immunotherapy efficacy in MSS CRC. By hypothesizing that IDO1 inhibition could modulate the tumor microenvironment, specifically by impacting myeloid immune cell activity, such as macrophage polarization and antigen processing behaviors. This research endeavors to elucidate the interplay between IDO1 inhibition, macrophage behavior and modulation of immune cell infiltration, offering insights that could inform the development of more effective, personalized treatment strategies for CRC patients unresponsive to current immunotherapies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eBioinformatic analysis\u003c/p\u003e \u003cp\u003eESTIMATE and TIMER2.0 were used to predict the immune infiltration status in TCGA-COAD and TCGA-READ datasets. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] R package ESTIMATE (version 1.0.13) and IOBR(version 0.99.9) were used for immune infiltration status. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] The simple nucleotide variation datasets were downloaded from TCGA, and the tumor mutation burden (TMB) of each sample were calculated using R package maftools. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] RNA-seq expression matrix was imported into the GSEA software (Broad, v4.3.2), and the HALLMARK 2023.2 gene sets was used for gene set enrichment analysis. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCell culture\u003c/p\u003e \u003cp\u003eCT26, MC38, HCT116, HT29, THP-1 and RAW264.7 were purchased from ATCC. CT26 and THP-1 were cultured with RPMI-1640 with 10% fetal bovine serum (FBS), MC38 and RAW264.7 was cultured with DMEM with 10% FBS, HCT116, HT29 was culture with Mcoy-5A medium with 10% FBS. All cells were detected mycoplasma free and were maintained anti-bacterial with 1\u0026times; penicillin and streptomycin solution (Solarbio Inc, Beijing).\u003c/p\u003e \u003cp\u003eSmall interfering RNA transfection\u003c/p\u003e \u003cp\u003eSmall interfering RNAs (siRNAs) were dissolved in RNase free water to reach a final concentration of 100nmol/\u0026micro;L. Lipofectamine 3000 (Invitrogen) and siRNAs were mixed in Opti-MEM(Gibco) and incubated in room temperature for 20 minutes to compose transfection liposome added on the cells. Culture medium was removed 8 hours after transfection and fresh culture medium with FBS was added subsequently. Total RNA or total protein was extracted 48 hours after transfection for downstream analyses. The RNAi sequences used in this study were listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eIDO1 knockdown lentivirus transfection\u003c/p\u003e \u003cp\u003eLVRU6GP plasmids with RNAi sequences were constructed by GeneCopoeia Inc. Lentivirus was packaged in HEK293T cells via co-transfection of target plasmids, psPAX2 and pMD2G using Lipofectamine 3000. The lentivirus-containing supernatant was purified by centrifugation and filtration. Stable cells were selected with puromycin at predetermined concentrations.\u003c/p\u003e \u003cp\u003eQuantitative RT-PCR\u003c/p\u003e \u003cp\u003eTotal RNA was extracted using TRIzol (Invitrogen) according to the standard procedure. The RNA was then reverse-transcribed into cDNA using reverse transcription kits (Yeason, Shanghai) following the manufacturer's instructions. RT-qPCR was performed using the SYBR Green mix (Yeason, Shanghai) and specific primers according to the recommended protocol in GTEx (BioRad). The primer sequences are listed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eWestern Blot\u003c/p\u003e \u003cp\u003eCells were lysed in RIPA buffer with 1% PMSF on ice for 30 minutes. Protein concentration was determined using a BCA kit. Proteins were separated by SDS-PAGE and transferred to membranes. Following blocking with skimmed milk, membranes were incubated with primary antibodies, then HRP-conjugated secondary antibodies for chemiluminescence detection. The expression levels were compared using chemiluminescence blotting. For nuclear and cytoplasmic protein separation, the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027) was used according to the manufacturer\u0026rsquo;s instructions. Antibodies used are listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003cp\u003eAnimal experiment\u003c/p\u003e \u003cp\u003e The animal experiment was approved by the Ethics Committee of Sun Yat-Sen University Cancer Center (Approval ID: 025503202112028). C57BL/6 or Balb/c mice were purchased from Guangdong Yaokang Biotech Ltd or the Experimental Animal Center of Sun Yat-Sen University Cancer Center. A total of 1\u0026times;10⁵ tumor cells were inoculated subcutaneously into the left axillary area. Anti-mouse PD-1 (10 mg/kg, InVivoMAb, clone ID: RMP1-14, Cat # BE0146) was administered intraperitoneally every 3 days, and epacadostat (10 mg/kg, INCB 024360, MedChemExpress) was given daily. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] Mice were sacrificed 15 to 17 days after the final treatment.\u003c/p\u003e \u003cp\u003eFlow cytometry\u003c/p\u003e \u003cp\u003eTumor tissues were cut into 2 mm pieces and dissociated into single-cell suspension using the Tumor Dissociation Kit (Miltenyi, Cat# 130-096-730) and Single Cell Suspension Dissociator (RWD, China) following the manufacturer\u0026rsquo;s instructions. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Erythrocytes were lysed with BD Pharm Lyse\u0026trade; solution (Cat # 555899). After Fc receptor blockade with TruStain FcX\u0026trade; (Biolegend, Cat# 101319), surface markers were stained with antibodies listed in Table\u0026nbsp;3. Cells were then permeabilized and fixed using True-Nuclear\u0026trade; Transcription Factor Buffer Set (Biolegend, Cat# 424401) for nuclear marker staining. Single fluorescence-stained Compensation Beads (Biolegend, Cat# 424602) were used for compensation adjustment. The stained cell suspension was analyzed using a CytoFLEX LX Flow Cytometer (Beckman Coulter). Cell markers were determined as described in previous studies. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]Fluorescence labeled antibody for FCS analysis were listed in Supplementary Table\u0026nbsp;4.\u003c/p\u003e \u003cp\u003eIn vitro cell function assays\u003c/p\u003e \u003cp\u003eIn vitro cell proliferation was assessed using the CCK-8 viability kit. Briefly, 1\u0026times;10⁴ cells were cultured in a 96-well plate and incubated under standard conditions. Medium was replaced with 10% CCK-8 in serum-free medium and incubated at 37\u0026deg;C for 45 minutes. Absorbance at 450 nm was measured using a microplate reader. For the colony formation assay, 500 cells per well were plated in 6-well plates and cultured with 10% FBS medium for 10\u0026ndash;14 days to form colonies. For migration and invasion assays, 2\u0026times;10⁵ cells in 250 \u0026micro;L serum-free medium were placed on the upper side of an 8 \u0026micro;m transwell membrane coated with Matrigel. Transwell inserts were placed in 24-well plates containing 10% FBS medium in the lower chamber. After 24 hours of incubation, cells on the upper surface were removed. Cells from both assays were stained with crystal violet, photographed, and quantified using ImageJ (1.54g).\u003c/p\u003e \u003cp\u003eImmune Fluorescence Staining\u003c/p\u003e \u003cp\u003eCells were incubated on 15 mm diameter round coverslips and fixed with 4% paraformaldehyde. Plasma membranes were permeabilized using Triton X-100. Blocking was performed with 5% bovine serum albumin (BSA). Primary antibodies, diluted as listed in Table\u0026nbsp;3, were applied overnight at 4\u0026deg;C. Fluorescence-labeled secondary antibodies were then incubated for 1 hour at room temperature in a light-protected environment. Finally, coverslips were mounted with an anti-fading agent, and fluorescence images were acquired using a laser confocal microscope (Olympus).\u003c/p\u003e \u003cp\u003eFITC-labelled dextran uptake assay\u003c/p\u003e \u003cp\u003ePhagocytic activity of macrophages was examined using FITC-labeled dextran (MW 4,000, Beyotime, Cat# ST2930). THP-1 cells were differentiated into M0 macrophages by treating with 100 ng/mL PMA (Beyotime, Cat# S1819) for 24 hours. FITC-labeled dextran was dissolved in Hank\u0026rsquo;s balanced salt solution (HBSS) at a stock concentration of 100 mg/mL. Macrophages were incubated with serum-free RPMI 1640 medium containing 1 mg/mL FITC-labeled dextran at 37\u0026deg;C with 5% CO2 for 2 hours. Cells were then washed three times with PBS and resuspended in PBS. Fluorescence images were captured, and flow cytometry was used to analyze the mean fluorescence intensity in the FITC channel, quantifying the dextran uptake by macrophages. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism9 was applied for the presentation of figures. ANOVA was used for multiple sample comparison and two-sided Dunnett-t test was applied for one-to-one comparison. The p value was recorded in the figures with exact numbers.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIDO1 upregulation is associated with increased immune infiltration in colorectal cancer.\u003c/p\u003e \u003cp\u003eWe analyzed the role of IDO1 in colorectal cancer using the TCGA database, including its correlation with immune checkpoints, expression in cancer vs. normal tissues, mutation levels, and impact on tumor mutation burden and immune cell infiltration. IDO1 showed positive correlations with PDCD1 (PD-1), CD274 (PD-L1), CTLA4, and other immune checkpoints like TIGIT, TGFB1, CXCL9, CXCL10, and IFNG. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;1A). IDO1 expression was higher in cancer tissues compared to normal epithelium in a wide range of adenocarcinomas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). IDO1 mutation rates were low, at 1.8% in COAD and 1.1% in READ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Pan-cancer analysis revealed a strong correlation between IDO1 expression and tumor mutation burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Using ESTIMATE and TIMER algorithms, we found positive correlations between IDO1 expression and immune infiltration scores in CRC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Increased IDO1 expression in CRC was associated with heightened interferon responses and activation of the IL-6, JAK-STAT signaling pathway, and inflammatory response (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). CRC tissue samples showed concurrent elevation of IDO1 and immune cell markers (CD4, CD8, CD68, CD206) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Upregulation of IDO1 in colorectal cancer cell lines and paired cancer-normal tissue samples was confirmed (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Higher IDO1 expression was also linked to a greater overall mutation frequency (Supplementary Fig.\u0026nbsp;1B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIDO1 inhibition sensitized PD-1 blockade therapy in MSS colorectal cancer.\u003c/p\u003e \u003cp\u003eWe evaluated whether IDO1 inhibition enhances the effectiveness of PD-1 blockade in colorectal cancer using an animal model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Two mouse-derived cell lines were used: CT26 (MSS, Balb/c mice) initially resistant to PD-1 blockade, and MC38 (MSI-H, C57 mice) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Tumor cells were inoculated superficially in mice, followed by treatments with PD-1 blockade, IDO1 inhibition (epacadostat), or their combination. Results showed that epacadostat was effective in CT26, especially when combined with PD-1 blockade (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). IDO1 expression and macrophage markers (F4/80, CD206, IL-6) were examined, revealing IDO1 upregulation with PD-1 treatment. CD206 increased in the PD-1 group, while IL-6 was elevated with epacadostat, with or without PD-1 blockade (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Leukocyte infiltration analysis indicated increased CD8\u0026thinsp;+\u0026thinsp;T cells and a higher M1/M2 ratio with epacadostat (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). Epacadostat also showed combined anti-tumor effects with PD-1 blockade in MC38 cell lines (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKnockdown of IDO1 impairs resistance to interferon-γ in CRC cell lines via AHR-CXCL9/CXCL10 axis.\u003c/p\u003e \u003cp\u003eWe used RNAi to knockdown IDO1 in MSS cell lines CT26 and HT29 to examine its impact on cell growth and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). No significant changes were observed in cell duplication, migration, or invasiveness after IDO1 suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-F). As IDO1 is IFN-inducible, we treated HT29 cells with 50 ng/mL human recombinant interferon gamma (rhIFN-γ, R\u0026amp;D, Cat# 285-IF-100) for 6 hours to assess whether IDO1 inhibition affects tumor resistance to IFN stimulation. IFN-γ significantly increased apoptosis in IDO1 knockdown HT29 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). We investigated the impact of IDO1 knockdown on AHR activation by performing nuclear-cytoplasmic protein separation on IFN-γ treated cancer cells, finding a significant reduction in AHR nuclear translocation (ARNT) in IDO1 knockdown cells. The reduced ARNT could be restored by an AHR ligand 6-Formylindolo (3,2-b)carbazole (FICZ, CAS #172922-91-7). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) FICZ could also restore the tolerance of IFN-γ in CRC cells by reducing IFN-γ induced apoptosis. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI) Additionally, IDO1 knockdown resulted in decreased overall AHR expression and increased levels of cleaved caspase 3 and PD-L1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKnockdown of IDO1 in MSS CRC improves sensitivity to PD-1 blockade therapy \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe used IDO1 knockdown CT26 cells to assess the effectiveness of PD-1 blockade \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To achieve long-term IDO1 inhibition at the transcription level, we constructed shIDO1 plasmids and generated stable knockdown CT26 cells via lentivirus infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Due to GFP expression in CT26 cells post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), we utilized new cell marker antibodies. As expected, IDO1 knockdown suppressed tumor growth under PD-1 blockade therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD \u0026amp; E). We also assessed macrophage marker expression, finding elevated pro-inflammatory IL-6 and reduced anti-inflammatory CD206 in IDO1 knockdown CT26 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Flow cytometry revealed increased leukocyte infiltration, particularly T cells, with a notable decrease in regulatory T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H). Additionally, IDO1 knockdown in MC38 cells enhanced response to PD-1 blockade therapy (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIDO1 KD in cancer cell promote macrophage pro-inflammatory phenotype polarization via JAK2-STAT3-IL6 pathway.\u003c/p\u003e \u003cp\u003eWe used a coculture method to examine interactions between cancer cells and macrophages (RAW264.7). When cocultured with CT26 cells, macrophages exhibited high CD163 and low IL-6 levels in the presence of shNC tumor cells. Knocking down IDO1 in tumor cells increased IL-6 and decreased CD163 in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In THP-1 cells treated with tumor cell supernatant and recombinant human IFN-γ, the shNC group showed CD163 positivity and iNOS negativity, while the shIDO1 group showed the opposite (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). FITC-labelled dextran uptake in THP-1 cells was higher when stimulated with rhIFN-γ loaded shIDO1 HT29 cell supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Flow cytometry confirmed higher IL-6 expression in THP-1 cells stimulated by shIDO1 cell supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). JAK2 and STAT3 mRNA expression increased in THP-1 cells stimulated by IDO1-knockdown HT29 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Protein analysis showed increased JAK2 and STAT3 phosphorylation in IDO1 knockdown HT29 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImmunotherapy, despite its effectiveness, often fails in MSS colorectal cancer due to the suppressive tumor immune microenvironment (TIME). This environment is characterized by the infiltration of suppressive immune cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), along with reduced levels of stimulatory cytokines like CXCL9 and CXCL10. These factors contribute to an immune-suppressive region in MSS CRC, inhibiting antigen recognition by antigen-presenting cells (APCs) and the generation of cancer-targeting cytotoxic T cells, thereby diminishing the efficacy of immune-based treatments. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIDO1 is known as a suppressive immune checkpoint for catalyzing the initial and rate-limiting step in the kynurenine pathway. IDO1 depletes tryptophan and produces immunosuppressive metabolites, including kynurenine. This activity can create a local immunosuppressive microenvironment conducive to tumor growth and survival. The upregulation of IDO1 in these cells has been associated with the suppression of T cell responses and the promotion of regulatory T cell (Treg) development, further contributing to immune evasion by tumors. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eInhibiting IDO1 activity is hypothesized to restore local and systemic immune responses against tumor cells by reversing tryptophan depletion, reducing kynurenine levels, and promoting a more immunogenic tumor microenvironment. Clinical trials have investigated IDO1 inhibitors, both as monotherapies and in combination with other immunotherapeutic agents, such as PD-1/PD-L1 inhibitors, to enhance anti-tumor immunity. While results from these trials have been mixed, none of these researches focused on its potentials in MSS CRC. The joint effect of IDO1 inhibitors and PD-1 blockade required more evidence.\u003c/p\u003e \u003cp\u003eIn our study, we discovered that IDO1 is up-regulated in tumor versus normal tissues. Despite IDO1 is not a frequently mutated gene, its expression was closely correlated with the overall mutation burden and pro-inflammatory status of the TIME. Collectively, these results suggested that IDO1 also played a pivotal role in mediating the tumor microenvironment in CRCs, playing an internal agent against inflammation.\u003c/p\u003e \u003cp\u003eWe then conducted an \u003cem\u003ein vivo\u003c/em\u003e assay to test whether IDO1 inhibition benefits PD-1 blockade immunotherapy. The results were highly encouraging, demonstrating the good synergistic effect of IDO1 inhibitor and PD-1 blockade in MSS CRCs. Notably, epacadostat significantly regulated the distribution of immune cells within the tumor microenvironment. A marked increase in the infiltration of cytotoxic CD8\u0026thinsp;+\u0026thinsp;T cells and pro-inflammatory macrophages was witnessed, which are known to play a key role in activated antitumor immunity.\u003c/p\u003e \u003cp\u003eOur studies also uncovered a significant role for IDO1 in mediating cancer cell resistance to interferon-gamma (IFN-γ). The abnormal upregulation of IDO1 suggested a complex interplay where cancer cells utilize IDO1 to counteract the immune-activating effects of IFN-γ. This mechanism not only illustrates IDO1's role in creating an immunosuppressive environment but also underscores its potential as a therapeutic target to enhance cancer immunotherapy efficacy. While IDO1 expression itself may not directly influence the proliferative or metastatic capabilities of cancer cells \u003cem\u003ein vitro\u003c/em\u003e, its role in mediating IFN-γ resistance positions its importance to immune resistance.\u003c/p\u003e \u003cp\u003eThe inhibition of IDO1 presents a promising strategy not only by impacting the metabolic functions of cancer cells but also by modulating the behavior of immune cells within the tumor microenvironment. This study revealed that while IDO1 knockdown did not significantly alter the growth rate of CT26 tumors \u003cem\u003ein vivo\u003c/em\u003e, it notably enhanced the effectiveness of PD-1 blockade therapy. This suggests that the primary role of IDO1 in these tumors may not be in promoting tumor cell proliferation directly but in modulating the immune microenvironment to favor tumor survival. Further investigation into the immune profiles of these tumors showed a marked increase in the infiltration of cytotoxic CD8\u0026thinsp;+\u0026thinsp;T cells in the IDO1 knockdown models treated with PD-1 inhibitors suggesting that IDO1 activity in the tumor cells might contribute to an immune-exclusion phenotype.\u003c/p\u003e \u003cp\u003eFinally, we sought to uncover the mechanism of IDO1 in cancer cell and the influence of tumor IDO1 expression to macrophage. We found that IDO1 plays pivotal role in maintaining the immunosuppressive characteristics of tumor associated macrophages (TAMs). By silencing the expression of IDO1 in cancer cell, the contacted macrophages tend to present pro-inflammatory phenotype, the expression of IL-6 increased with the coculture of IDO1 knockdown cancer cells. And macrophages also presented increased phagocytic activity by indirect stimulation of IDO1-knockdown cancer cells, which indicated that the antigen presentation was activated. Collectively, these results illustrated that, IDO1 not only give resistance to inflammatory stimuli to cancer cell but also allowed secretion of specific agents that regulate macrophage polarization. IL6 is one of the mediated cytokines that was regulated by local IDO1 level. Previous results showed that IL6 was mediated via JAK-STAT pathway.\u003c/p\u003e \u003cp\u003eCollectively, these results highlight the dual role of IDO1 in cancer progression\u0026mdash;both as a shield against immune attack directly on cancer cells and as a regulator of the immune landscape via macrophage polarization. Our findings underscore the potential therapeutic benefits of targeting IDO1 in cancer treatment, not only to inhibit tumors\u0026rsquo; intrinsic pathways but also to reprogram the immunological milieu of the tumor to enhance the efficacy of existing and emerging therapies. These insights pave the way for novel therapeutic strategies that aim at disrupting the IDO1-mediated immunosuppressive network within tumors, potentially leading to more effective immunotherapy outcomes.\u003c/p\u003e \u003cp\u003eHowever, this study also has certain limitations, such as not thoroughly exploring whether changes in the levels of downstream metabolites of IDO1 affect the local conditions of the tumor immune microenvironment, and whether sensitizing the effect of IDO1 blockade in MSS CRC is achieved by reshaping the changes in metabolite levels caused by IDO1. Additionally, the regulatory effect of tumor cells on stromal cells and the mediators inducing this regulation were not clarified in this study. In future research, the sensitizing mechanisms of IDO1 inhibitors on ICIs require further elucidation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study introduces a novel approach to enhance the efficacy of PD-1 blockade therapy in MSS colorectal cancer. By combining an IDO1 inhibitor with PD-1 blockade, we demonstrated significant therapeutic benefits in a pre-clinical MSS cancer model. Specifically, inhibition of IDO1 in CRC cells led to a significant alteration in the distribution of tumor-infiltrating lymphocytes (TILs) and remodeled the immune microenvironment into a pro-inflammatory state. This transformation effectively \"lit up\" the tumors, rendering them more responsive to PD-1 blockade therapy. This strategy not only highlights the potential of IDO1 inhibitors in modifying the tumor microenvironment but also underscores their role in improving the outcomes of existing immunotherapies. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of interest statement\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLv G, Wang X and Wu M contributed equally to this work by performing the in vitro and in vivo experiments.Ma W was responsible for constructing the IDO1 gene modification plasmid and primers.Liu R assisted with the study design and provided statistical analysis support.Pan Z supervised the colorectal surgery experimental platform.Zhang R and Chen G are the principal supervisors of the study, overseeing the research design, data analysis, and manuscript preparation.All authors contributed to the drafting and revision of the manuscript and approved the final version for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe work was supported by grants from Guangdong Basic and Applied Basic Research Foundation (Grant No. 2023A1515010243), Chinese Society of Clinical Oncology Foundation (Grant Nos. Y-HR2018-319, Y-L2017-002, and Y-JS2019-009), Sun Yat-sen University Basic Research Fund (Grant No. 19ykpy180), and the open research funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital (202011-103, 202301-314).\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe generated original data during the current study are not publicly available but will be deposited on the Research Data Depot (RDD) system of our institute. These data will be available from the corresponding author on reasonable request. For access, please contact Dr. Chen Gong at [email protected].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F et al (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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J Exp Clin Cancer Res 40(1):288\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRandrian V, Evrard C, Tougeron D (2021) Microsatellite Instability in Colorectal Cancers: Carcinogenesis, Neo-Antigens, Immuno-Resistance and Emerging Therapies. Cancers (Basel), 13(12)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHill M et al (2007) IDO expands human CD4\u0026thinsp;+\u0026thinsp;CD25high regulatory T cells by promoting maturation of LPS-treated dendritic cells. Eur J Immunol 37(11):3054\u0026ndash;3062\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei C et al (2016) 1-Methyl-tryptophan attenuates regulatory T cells differentiation due to the inhibition of estrogen-IDO1-MRC2 axis in endometriosis. Cell Death Dis 7(12):e2489\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"immunotherapy, microsatellite stable colorectal cancer, IDO1, PD-1 blockade, macrophage polarization","lastPublishedDoi":"10.21203/rs.3.rs-5080703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5080703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrosatellite stable (MSS) colorectal cancer (CRC) is a subtype of CRC that generally exhibits resistance to immunotherapy, particularly immune checkpoint inhibitors such as PD-1 blockade. This study investigates the effects and underlying mechanisms of combining PD-1 blockade with IDO1 inhibition in MSS CRC. Bioinformatics analyses of TCGA-COAD and TCGA-READ cohorts revealed significantly elevated IDO1 expression in CRC tumors, correlating with tumor mutation burden across TCGA datasets. \u003cem\u003eIn vivo\u003c/em\u003e experiments demonstrated that the combination of IDO1 inhibition and PD-1 blockade significantly reduced tumor growth and increased immune cell infiltration, particularly pro-inflammatory macrophages and CD8\u0026thinsp;+\u0026thinsp;T cells. IDO1 knockdown in CRC cell lines impaired tolerance to interferon-γ and increased apoptosis \u003cem\u003ein vitro\u003c/em\u003e, while IDO1 knockdown in MSS CRC enhanced the effectiveness of PD-1 blockade therapy \u003cem\u003ein vivo\u003c/em\u003e. IDO1-knockdown CRC cells promoted pro-inflammatory macrophage polarization and enhanced phagocytic activity via the JAK2-STAT3-IL6 signaling pathway. These findings highlight the role of IDO1 in modulating the tumor immune microenvironment in MSS CRC and suggest that combining PD-1 blockade with IDO1 inhibition could enhance therapeutic efficacy by promoting macrophage pro-inflammatory polarization and infiltration through the JAK2-STAT3-IL6 pathway.\u003c/p\u003e","manuscriptTitle":"IDO1 Inhibitor Enhances the Effectiveness of PD-1 Blockade in Microsatellite Stable Colorectal Cancer by Promoting Macrophage Pro-Inflammatory Phenotype Polarization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 11:37:30","doi":"10.21203/rs.3.rs-5080703/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-17T20:17:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-15T10:07:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-10T07:40:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154955581511107468235408770221388461572","date":"2024-10-03T01:43:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256069832984283407357032993043700952828","date":"2024-10-02T20:41:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62497743786829140004307106668916149643","date":"2024-10-02T02:29:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-30T20:38:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-14T05:11:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-14T05:11:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2024-09-13T03:24:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"65dd687d-729e-411e-9b04-ceb41d10b2b1","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-06T16:01:18+00:00","versionOfRecord":{"articleIdentity":"rs-5080703","link":"https://doi.org/10.1007/s00262-024-03925-w","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2025-01-03 15:57:21","publishedOnDateReadable":"January 3rd, 2025"},"versionCreatedAt":"2024-11-26 11:37:30","video":"","vorDoi":"10.1007/s00262-024-03925-w","vorDoiUrl":"https://doi.org/10.1007/s00262-024-03925-w","workflowStages":[]},"version":"v1","identity":"rs-5080703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5080703","identity":"rs-5080703","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}