MS275 induces tumor immunosuppression by up-regulating PD-L1 and enhances the efficacy of anti-PD-1 immunotherapy in colorectal cancer

MS275 induces tumor immunosuppression by up-regulating PD-L1 and enhances the efficacy of anti-PD-1 immunotherapy in colorectal cancer | 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 MS275 induces tumor immunosuppression by up-regulating PD-L1 and enhances the efficacy of anti-PD-1 immunotherapy in colorectal cancer Sihan Chen, Zhigang Mao, Deng Tang, Mi Su, Meng lai, Xiya Yan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4133957/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 11 You are reading this latest preprint version Abstract The histone deacetylase inhibitor MS275 (Entinostat) demonstrates anti-tumor effects against various types of solid tumors in vitro . But its effectiveness in clinical trials is limited. The underlying reasons remain to be determined. The purpose of this study was to explore how to enhance the anti-tumor effects of MS275 in colorectal cancer(CRC). Our data showed that MS275 inhibited CRC cell proliferation and induced apoptosis, irrespective of gene mutation status. However, MS275 did not effectively suppress tumor growth in the AOM-DSS CRC model as observed in vitro . MS275 decreased CD3 + T cell tumor infiltration and created an anti-tumor immunosuppressive microenvironment in the AOM-DSS CRC model. MS275 also decreased the percentage of CD8 + T cells while increasing the percentage of CD4 + T cells in tumor-lymphocyte mixed culture. Reshaping tumor immune response may contribute to the less pronounced anti-tumor effect of MS275 observed in vivo compared to in vitro . Further study show that the increased PD-L1 expression in CRC both in vivo and in vitro following MS275 treatment. Moreover, the anti-tumor effects of MS275 were enhanced by combining it with an anti-PD-1 antibody. This combination treatment also increased CD3 + T cell tumor infiltration and M1 macrophage polarization in the AOM-DSS CRC model, thereby leading to an anti-tumor immune response. Therefore, the combination of MS275 and anti-PD-1 immunotherapy represents a potential strategy for low PD-L1 expression tumors and should be considered a promising treatment approach for colon cancer. HDACi MS275 PD-L1 Colorectal Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer as well as the third leading cause of cancer-related deaths. Environmental risk factors including lifestyle, together with the inherited genetic background, may lead to the development of the tumor 1 . Although genetic mutations (K-Ras, ß-catenin, p53, et.al) undoubtedly play a significant role in CRC initiation and development, CRC progression is also driven by epigenetic modifications, including DNA methylation, histone protein modifications, and non-coding RNA-mediated gene silencing. Histone deacetylase (HDAC) plays a critical role in the development of cancer by reversibly modulating the acetylation status of histone and nonhistone proteins 2 . Recent studies suggest that HDACs, particularly HDAC-1, -2, and − 3, are overexpressed in CRC and their increased expression is correlated with a poorer prognosis 3 . This highlights the potential of HDACs as therapeutic targets in CRC. Histone deacetylase inhibitors (HDACIs) may block tumor cell proliferation by restoring the balance of histone acetylation, thus resulting in proper gene expression. They can induce growth arrest, apoptosis, and/or differentiation of transformed cells both in vitro and in vivo 2 . Furthermore, HDACIs have been shown to modulate the tumor immune microenvironment 4 , affecting immune responses such as antigen presentation, T cell activation, and differentiation of regulatory T cells (Tregs). This immunomodulatory effect suggests that HDACIs could also impact the efficacy of immunotherapy in cancer. However, initial clinical trials employing HDACIs as monotherapy against CRC revealed either limited responses or no response at all 5 . These trials were also hindered by clinical adverse events due to HDACIs toxicity, which was mainly attributed to global histone acetylation, a phenomenon not limited to cancer cells 1 . Therefore the search for HDAC inhibitors with high specificity is the main direction for the treatment of targeted HDACs. N-(2-aminophenyl)-4-[N- (pyridine-3yl-methoxy-carbonyl) aminomethyl] benzamide (MS-275) is a class I histone deacetylase inhibitor (HDAC) that selectively targets HDAC1 and HDAC2. MS275 has shown promising results, especially against advanced breast cancer 6 and colon cancer in vitro 7 . However, clinic phase trials indicate that neither monotherapy nor combination therapy with MS275 was well tolerated and showed no evident activity in metastatic CRC 5 . Further studies of MS275 are warranted to enhance its efficacy through combination with other novel agents, like PD-1/PD-L1 inhibitors. In our study, we observed that MS275 inhibits CRC proliferation both in vitro and in vivo . However, it also suppresses the anti-tumor immune microenvironment by up-regulating the expression of PD-L1. Additionally, We found that there is a synergistic effect with MS275 and PD-1 immunotherapy in the treatment of CRC. Combining MS275 with immunotherapy and antiangiogenic therapies may hold greater promise for improving treatment for CRC. Materials and methods Cell lines and reagents The human colon cancer cell lines HCT116 Mut , HCT116 WT , DLD1 Mut and DLD1 WT were obtained from were kindly provided by Dr. Kevin Haigis (Beth Israel Deaconess Medical Center, Boston, USA). The mouse colon cancer cell lines CT26 was purchased from Kunming Cell Bank (KCB, China). All cells were cultured in high- glucose DMEM with 5% FBS. and maintained under humidified condition (37°C, 5% CO2), and continual culture did not exceed 2 months. Each human cell line was authenticated using the Human STR human cell line authentication service (ATCC). MS275 was purchased from Selleck Chemicals (Houston, TX, USA). An anti-PD-1 antibody was purchased from Biolegend (San Diego, CA, USA). Antibodies The following antibodies (Abs) were used for Western blotting: anti-STAT3(#9139), anti-p-STAT3(#9145), anti-cleaved-PARP(#5625), anti-Arginase-1(#93668), anti-iNOS(#13120), anti-PD-L1(#13684), anti-rabbit antibody(#7074), all of these were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-HDAC1(sc-81598) and anti-HDAC3(sc-17795) were purchased from Santa Cruz. Anti-HDAC2(abs136328) and goat anti-mouse antibody were purchased from Absin. Anti-β-actin(#A2228) was purchased from Sigma-Aldrich. The following antibodies (Abs) were used for Immunochemistry (IHC): anti-F4/80(#70076), anti-CD3(#78588), anti-CD19(#90176), anti-CD11C(#97585), Brdu(#5292), all were purchased from Cell Signaling Technology (Danvers, MA, USA). Cell Proliferation Assays MTS assay was performed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA). Briefly, cells were seeded into 96-well plates at 75% confluence and incubated for 24 hours. The cells were treated with MS275, and an equal amount of DMSO was added to the control. After 24 hours, cell viability was measured using the MTS assay. Apoptosis analysis Apoptosis in colorectal cancer cells treated with MS275 was analyzed using an Annexin V-FITC Apoptosis Detection Kit II (BD). The results were measured using the BD Acuri C6. The percentage of apoptosis was then calculated based on the cells in the region of Q2 and Q4. RNA and RT-qPCR RNA was extracted using TRIzol (Thermo Fisher Scientific), and cDNA was synthesized using a PrimeScriptTM RT Reagent Kit (TaKaRa). The qPCR assays were performed using SYBR® Premix Ex TaqTM II (TaKaRa) and a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used simultaneously as the internal control. The data was analyzed using the 2–ΔΔCt method. Western blotting Cell lysis buffer (100 mM NaCl, 10 mM EDTA (pH 8.0), 50 mM Tris–Cl (pH 8.0) and 0.5% (v/v) Triton X-100) with EDTA-free complete protease and phosphatase inhibitors (Roche) was used for protein extraction. The lysates were separated on a 10% SDS-PAGE gel and transferred onto PVDF membranes. The targets were detected using the ImageQuant LAS 4000 (GE Healthcare Life Sciences). Actin was used as the loading control. AOM-DSS CRC mouse model Male BALB/c mice were placed in an SPF-grade standard environment for one week of adaptation feeding. To establish the CAC mouse model, an intraperitoneal injection of (10 mg/kg) of azoxymethane (AOM) was administered on the first day. After 7 days, mice were provided with drinking water containing 1.5% dextran sulfate sodium (DSS) for a week. followed by regular drinking water for another two weeks, constituting one cycle of water administration. A total of three cycles of AOM-DSS treatment were performed. Four weeks after the completion of the AOM-DSS cycles, mice were orally administered with MS275 (20 mg/kg) per day, lasting for 21 days.. The colons were collected, and the tumors were counted and measured. Immunochemistry (IHC) Paraffin-embedded tissues were used for IHC staining. After deparaffinized in xylene for 10 min, the slides were immersed in 3% H 2 O 2 for 20 min to block the endogenous peroxidase and blocked in goat serum blocking solution for 30 min. After being incubated at 4°C overnight with primary antibodies, the slides were incubated with secondary HRP-conjugated antibodies (Thermo Fisher Scientific) for 30 min at RT. The primary antibodies were shown above. IHC staining was examined with microscopy. Elisa Supernatants of cultured cells were collected and centrifuged. ELISA kits for mouse TNF-a (NOVUS,VAL609) and mouse IFN-g (NOVUS,VAL607) were used according to the manufacturer’s instructions. The absorbance of the samples was measured using Synergy™ HT microplate reader (BioTek). The concentration of cytokines in the supernatants was calculated based on the standard curve generated using the provided cytokines standard. Mixed lymphocyte tumor cell culture (MLTC) Mouse spleen mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. After treatment of CT26 cells with MS275 (2.5 µM) for 24 hours, the cells were co-cultured in 12-well plates with lymphocytes (2X106): CT26 cells = 10:1, and cultured in a mixed culture for 48 hours. The proportion of CD3+, CD4 + and CD8 + T cells were assayed by flow cytometry for assaying T-cell differentiation. Flow cytometry analysis The following mouse Abs were used for flow cytometry analyses: Anti-PD-L1 PE (BD, 557924), anti-CD3e PerCP-CyTM5.5 (BD, 551163), anti-CD4 FITC (BD, 553046), and anti-CD8 alpha PE (BD, 553046). Gates were determined using isotype control Ab staining. Data were acquired with BD C6 flow cytometry. Statistical analysis The data were analyzed by two-tailed Student’s t test or ANOVA using GraphPad Prism software (La Jolla, CA). Comparisons between groups are presented as the mean ± SEM. Values of p < 0.05 were considered statistically significant. Results 1. MS275 inhibits proliferation of colorectal cancer cells and induces apoptosis MS275 is a class I selective HDACi that targets HDAC1 and HDAC2 in cells. MS275 (1 µM, 2.5 µM and 5 µM) was applied to CRC cells for 48 hours, Western Blot assay showed that MS275 dose-dependently decreased the intracellular HDAC3 protein expression level, while not affecting the HDAC1 and HDAC2 protein expression levels (Fig. 1A). Two pairs of cell lines, HCT116 Mut and HCT116 WT , as well as DLD-1 Mut and DLD-1 WT , shared the same genetic background but differed in KRas mutation status. HCT116 Mut showed more sensitivity to MS275 compared to HCT116 WT (Fig. 1B), while the reverse was observed in the DLD-1 cell line pair (Fig. 1C). MS275 inhibited cell proliferation regardless of KRAS mutation status. Additionally, we treated a panel of colorectal cell lines with MS275 at indicated concentrations (0.1–10 µM) for 48 hours. The MTS assay revealed a dose-dependent reduction in cell viability (Fig. 1D). Among these cell lines, HCT116 Mut , DLD1 Mut , and SW480 carry active KRAS mutations (G13D), whereas HT29 harbors BRaf V600 mutations. Interestingly, the sensitivity to the drug did not correlate with gene mutations across different cell lines. To determine whether MS275 reduced cell proliferation by inducing apoptosis, we conducted apoptosis assessment using the Annexin V-PI staining assay, with results shown in Fig. 1E. Following treatment with MS275, there was a 31.8% increase in apoptosis in the HCT116 Mut cell lines, and MS275 induced apoptosis in a dose-dependent manner. Additionally, cleavage of PARP, a hallmark of apoptosis, was observed after treatment with the indicated concentrations of MS275 in both HCT116 Mut and HCT116 WT cell lines (Fig. 1F). The induction of apoptosis by MS275 was further confirmed in a panel of CRC cell lines (Fig. 1G). Figure 1. MS275 inhibited proliferation, and induced apoptosis in colorectal cancer cells. A. Western blot analysis of HDAC1, HDAC2, and HDAC3 levels in CRC cells following treatment with increased concentrations of MS-275. β-actin levels were determined as controls; B. The impact of MS275 on cell proliferation was assessed in DLD1 Mut and DLD1 WT cells; C. The impact of MS275 on cell proliferation was assessed in HCT116 Mut and HCT116 WT cells; D. The cell proliferation of a panel of CRC cell lines was evaluated following treatment with MS275 for 48 hours; E. Apoptosis was examined using an Annexin V/PI assay; F. The levels of cleaved PARP were determined by Western blot; G. Effect of apoptosis induced by MS275 was analyzed in a panel of the CRC cell lines by flow cytometry, following 48 hours of incubation with MS275. *** p < 0.001. Data plotted are mean ± SEM (n = 3). 2. MS275 showed anti-tumor activity against colon cancer in vivo To elucidate the tumor-suppressing effect of MS275 in vivo , we established a classical mouse colorectal cancer model (see Materials and Methods section). Four weeks after the completion of the AOM- DSS cycle, mice received MS275 at a dose of 20 mg/kg via gavage. After 3 weeks of treatment, the mice were euthanized, and their colons were examined, shown in Fig. 2 A. Following treatment of colorectal cancer mice with MS275, the total size of all tumors in the intestinal segment did not show a significant reduction (Fig. 2 B). However, the number of colon tumors decreased by 23.6% compared to that of the saline group (control group) (Fig. 2 C). This suggests that MS275 could moderately inhibit the proliferation of colorectal tumors in mice, but not as effectively as observed in vitro . Immunohistochemistry was utilized to assess the expression of BrdU in intestinal tumors of mice from each group. The results revealed that the intensity of local BrdU staining in tumors of mice in the control group was higher than that in the MS275-treated group (Fig. 2 D), indicating that MS275 could inhibit tumor cell proliferation. Furthermore, TUNEL staining results indicated apoptosis of tumor tissues in the MS275-treated group (Fig. 2 E). 3. MS275 treatment suppresses tumor immune microenvironment AOM-DSS CAC tumor models were used to investigate the microenvironmental changes following MS275 administration. Immunohistochemical analysis revealed that F4/80-positive cells predominantly infiltrated the tumor microenvironment in the mouse colorectal cancer model (Fig. 3 A), with fewer CD3 + T cells (Fig. 3 B). A small clusters of CD19 + cells primarily present within the interstitial space of tissues, predominantly expressing intra-lymphatic node expression. Upon MS275 treatment, CD11C + and other immune cell populations remained largely unchanged (data not shown). Additionally, pro-inflammatory cytokines TNF-α and IFN-γ were both upregulated in mesenteric lymphocytes isolated from AOM-DSS mice treated with MS275, indicating their potential role in tumor development (Fig. 3 C and 3 D). To further elucidate the effect of MS275 on T lymphocyte differentiation, we established an co-culture system using CT26 cells and spleen T lymphocytes from BALB/c mice. Following MS275 administration to CT26 cells, co-cultured with T lymphocytes for 24 hours, the expression levels of surface marker molecules on T lymphocytes were assessed using flow cytometry. The results indicated that MS275 influenced T cell differentiation into various subpopulations. In the co-culture system of CT26 tumor cells and T cells, the percentage of CD3 + positive cells decreased (Fig. 3 E). Specifically, the proportion of CD3 + CD4 + cells significantly increased(Fig. 3 F and 3 H), while CD3 + CD8 + cells decreased compared to the T cells alone group(Fig. 3 G and 3 I). 4. MS275 upregulated PD-L1 expression in colorectal cancer both in vitro and in vivo To understand how MS275 reshapes the tumor microenvironment and its subsequent impact on the anti-tumor effect, we analyzed the effects of MS275 on PD-L1 expression in CRC cells. Following treatment with MS275, qRT-PCR (Fig. 4 A-C) and Western blotting assays (Fig. 4 D-F) revealed an upregulation of PD-L1 at both the mRNA and protein levels. These findings were further confirmed by evaluating PD-L1 expression using flow cytometry (Fig. 4 G). The ability of MS275 to upregulate PD-L1 in vivo was also investigated in tumor tissue from the AOM-DSS CAC model. After treatment with MS275, PD-L1 expression in the tumors was assessed by Western blotting. Consistent with the in vitro data, MS275 treatment significantly increased PD-L1 expression in tumors (Fig. 4 H). 5. Anti-tumor effects of MS275 in combination with an anti‐PD‐1 antibody Given that MS275 inhibits tumor immune response by upregulating PD-L1 expression in tumor cells, we further explored the possibility of enhancing the anticancer effect of MS275 by blocking the PD-1/PD-L1 signaling pathway using a PD-1 monoclonal antibody. In AOM-DSS CRC model mice, we evaluated the anti-tumor effects following 3 weeks of treatment with both MS275 (20 mg/kg/day) and PD-1 monoclonal antibody (5 mg/kg/3 days). The results showed that the co-treatment significantly enhanced the inhibitory effect on tumor growth compared to treatment with MS275 alone (Fig. 5 A). Moreover, the number of tumors in either the combination treatment group or the treatment alone group was significantly decreased compared to the control group (Fig. 5 B). While treatment with either MS275 or PD-1 monoclonal antibody alone tended to result in smaller tumor volumes than the control group, the combination of MS275 and PD-1 monoclonal antibody treatment led to the most significant tumor suppression, with lower tumor volumes than those observed with either treatment alone (Fig. 5 C). The proliferation or apoptosis was assessed after administration with MS275. Intra-tumoral proliferation rates were decreased after MS275 treatment, suggesting that MS275 inhibits epithelial proliferation (5D), likely attributable to increased intra-tumoral apoptosis (Fig. 5 E). A significant increase in CD3 + T-cell tumor infiltration was observed in the tumor micro-environment (Fig. 5 F), whereas there was no significant change in CD19 + cell tumor infiltration (data not shown). Despite an overall decrease tumor infiltration of F4/80 cells, Western blot results showed an increase expression of iNOS and a decrease expression of Arg-1 in co-treatment groups (Fig. 5 G), indicating a potential shift towards a higher proportion of M1-type macrophages. 6. Stat3 activation is required for MS275 upregulation of PD-L1 in colorectal cancer cells It has been demonstrated that MS275 activates STAT3 in gut epithelial cells. To elucidate if MS275 regulates PD-L1 transcriptionally, we assessed phosphorylated STAT3 protein levels in colorectal cell lines and a CRC mouse model. MS275 significantly up-regulated the expression levels of phosphorylated STAT3 protein in HCT116 Mut and HCT116 WT cell lines, along with increased PD-L1 expression (Fig. 6 A and 6 B). The same results were obtained in the AOM-DSS CRC mouse model. The phosphorylated Stat3 was increased in MS275 and anti PD-1 co-treatment group compared with the control group (Fig. 6 C). The role of STAT3 was investigated using the STAT3 inhibitor Stattic. Pre-treatment of CRC cells with Stattic(5µM) for 60 min, phosphorylated STAT3 in both HCT116 Mut and HCT116 WT cell lines were inhibited. The levels of PD-L1 gene and protein expression remained unchanged following subsequent treatment with MS275 for 24 or 48 hours(Fig. 6 D). These results suggest that activated STAT3 is likely a central regulator of PD-L1 gene induction by MS275. Discussion MS275, a benzamide that specifically inhibits class I HDAC, has shown preclinic anti-tumor effects in a variety of cancers, including colorectal cancer, breast cancer, hematological malignancies 8 , and others. Consistently, our study also confirmed that MS275 can inhibit proliferation and viability and promote apoptosis in colorectal cancer cells. Notably, the inhibitory effect of MS275 on colorectal cancer cells appeared independent of the specific gene mutations carried by the cells, suggesting that MS275's impact on tumor cells is influenced by deacetylation-mediated epigenetic modifications. However, the inhibition of tumor growth by MS275 in the AOM-DSS CAC mouse model was not as effective as observed in vitro . The efficacy of MS275 as a single agent therapy remains limited 9 . In a phase II study of MS275 monotherapy in relapsed/refractory Hodgkin lymphoma, the overall response rate was modest (12%) 10 . Studies have explored strategies to combine MS275 with other drugs to enhance therapeutic outcomes. For instance, traditional chemotherapy drugs like 5-fluorouracil (5-FU) 11 , and oxaliplatin 12 , 13 were combined with MS275 to augment chemotherapy efficacy. Additionally, combining MS275 with inhibitors targeting the EGFR pathway (such as cetuximab or panitumumab) in colorectal cancer 14 or with HER2-targeted therapies in breast cancer 15 may enhance anti-tumor effects. Furthermore, MS275 may be combined with other epigenetic modifiers. However, the clinical phase outcomes of these combinations have not met expectations. The combination of regorafenib, HCQ, and MS275 was poorly tolerated without evident activity in metastatic CRC 5 . In addition to directly suppressing tumor cells, HDAC inhibitors have been reported to modulate the immune system 4 , 16 , 17 . We also found a reduction in CD3 + cell tumor infiltration following MS275 treatment. Moreover, levels of the pro-inflammatory cytokines TNF-α and IFN-γ were both increased in mesenteric lymphocytes isolated from AOM-DSS mice treated with MS275. Results from in vitro lymphocyte-tumor cell co-culture experiments indicated a significant decrease in the proportion of CD3 + CD8 + T cells, coupled with an increase in the proportion of CD3 + CD4 + T cells in MS275-treated tumor cells. The enhancement of immune suppression by MS275 and its contribution to tumor immune escape explains the lesser effectiveness of MS275 in vivo compared to in vitro , which in turn, leads to tumor immune tolerance or escape, leading to its limitations in clinical treatment. Immune checkpoints play a crucial role in regulating tumor immune environments. Among them, the upregulation of PD-1/PD-L1 has gained significant attention due to its role in immune suppression, making it a prominent target for tumor immunotherapy. Accumulating evidence indicates that histone deacetylase (HDAC) activation could induce PD-L1 expression in various types of cancer 18 . We also found that PD-L1 is upregulated on tumor cells following MS275 treatment. Furthermore, combining MS275 with anti-PD-1 in AOM-DSS mouse tumor models increased CD3 + T cell infiltration and induced an M1-like phenotype characterized by diminished immunosuppressive function. This results in a significant reduction in mouse tumor burden and a notable prolongation of survival compared to control mice. These findings suggest that reprogramming immunosuppressive responses via MS275 has the potential to overcome the limitations of current checkpoint blockade-based immunotherapy. Although inhibition of class I HDACs has been associated with increased PD-L1 expression 4 , 19 , the precise mechanism of PD-L1 regulation remains unclear. Our research revealed elevated levels of STAT3 phosphorylation in mouse colorectal cancer tissues or cells cultured in vitro following MS275 treatment. Inhibiting Stat3 activation can down-regulate the expression of PD-L1 20 . These findings collectively suggest that STAT3-mediated transcriptional activation by MS275 leads to PD-L1 upregulation. In CRC, the balance between anti-tumoral and pro-tumoral immune functions is largely dependent on the level of PD-L1 expression. CRC with both high PD-1 and PD-L1 levels may have an active immune checkpoint activity and may therefore represent the subset that benefits from anti-PD-1 therapy 21 . However, the overall response rates to PD-1 inhibitors in CRC remain modest, with only a subset of patients experiencing durable responses 22 . Although the upregulation of PD-L1 following MS275 treatment negatively regulates T cell responses and facilitates immune escape, emerging evidence suggests that MS275-induced upregulation of PD-L1 can enhance T cell infiltration, thereby improving the efficacy of anti-PD-1 immunotherapy. This highlights the potential for combining MS275 with anti-PD-1 therapy as an effective strategy to enhance immunotherapy efficacy in CRC. Declarations Acknowledgments We thank Dr.Ling GU for providing for useful discussion. We thank Dr. Tao Wang for revising the manuscript. We thank the National Natural Science Foundation of China for its support. Funding This work was supported by grants from the National Natural Science Foundation of China (Grant No. 82173104 and 81871233) Competing Interests “The authors have no relevant financial or non-financial interests to disclose.” Author Contributions Yufang Wang designed the research; Sihan Chen, Zhigang Mao and Deng Tang carried out most of the experiments; Mi Su, Meng Lai, Xiya Yan, and Ruiting Yan provided extra technical assistance; Siqi Lan, Meng Lai, Deng Tang, Mi Su, Xiya Yan nd Ruiting Yan performed material preparation; the first draft of the manuscript was written by Yufang Wang and Ji Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 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Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 14 Jun, 2024 Reviews received at journal 14 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviews received at journal 13 May, 2024 Reviewers agreed at journal 01 May, 2024 Reviewers agreed at journal 30 Apr, 2024 Reviewers invited by journal 25 Mar, 2024 Submission checks completed at journal 20 Mar, 2024 Editor assigned by journal 20 Mar, 2024 First submitted to journal 19 Mar, 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-4133957","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281996769,"identity":"11cecf12-b078-4125-9354-0e64c99a2ac6","order_by":0,"name":"Sihan Chen","email":"","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Sihan","middleName":"","lastName":"Chen","suffix":""},{"id":281996770,"identity":"ff51e8f3-7137-4fb8-b820-7ec0105acefe","order_by":1,"name":"Zhigang Mao","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Zhigang","middleName":"","lastName":"Mao","suffix":""},{"id":281996771,"identity":"2534e0d9-6982-43bb-b5b0-52df72398517","order_by":2,"name":"Deng Tang","email":"","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Deng","middleName":"","lastName":"Tang","suffix":""},{"id":281996772,"identity":"496c93fc-b9fc-4318-b462-6f472c2c289b","order_by":3,"name":"Mi Su","email":"","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Mi","middleName":"","lastName":"Su","suffix":""},{"id":281996773,"identity":"0995b405-b94c-4962-972e-94e3648b5e7c","order_by":4,"name":"Meng lai","email":"","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"lai","suffix":""},{"id":281996774,"identity":"7f5f5642-a8bd-4b59-a51e-a2c9a10de14e","order_by":5,"name":"Xiya Yan","email":"","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xiya","middleName":"","lastName":"Yan","suffix":""},{"id":281996775,"identity":"44bc5fdf-dbe3-45b8-a187-fc90ae68ffc4","order_by":6,"name":"Ruiting Yan","email":"","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Ruiting","middleName":"","lastName":"Yan","suffix":""},{"id":281996777,"identity":"03c4e7c1-3655-44f5-ab1b-462df6ebec7a","order_by":7,"name":"Siqi Lan","email":"","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Lan","suffix":""},{"id":281996781,"identity":"8cb4e663-d583-43fd-8214-adc287e21428","order_by":8,"name":"Ji Zhang","email":"","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"","lastName":"Zhang","suffix":""},{"id":281996784,"identity":"eb9944f9-f6a8-4a74-909a-22f422c9326f","order_by":9,"name":"Yufang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYPACOSBmPgBhHyBOizEQsyWQrIXHgDgtBsfPHn7N22Ygb86/5vOHn20Mcnw3Ehg/F+DTciYvzRqoxXDnjLfbJHvbGIwlbyQwS8/Ao8XsQI6ZMW/bH8YNN85uY2ZsY0jccCOBjZkHn5bzb0BaDOw33Djz+DNQSz1hLTdyjB8DtSRuON/DIA3UkmBASIv9jTdmjHPOGSRvuMFmJtlzTsJw5pmHzdL4tEj25xh/eFNmYLvh/OHHH36U2cjzHU8++BmfFiBgkwIrkEgAk0DM2IBfAzChfPwBovgPEFI4CkbBKBgFIxUAAKZVUIMEV4BaAAAAAElFTkSuQmCC","orcid":"","institution":"West China School of Basic Medical Science \u0026 Forensic Medicine, Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Yufang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-03-20 03:59:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4133957/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4133957/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-025-04004-4","type":"published","date":"2025-03-17T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53221000,"identity":"cf8bed4c-2e5d-4923-a0c0-3ff906074c96","added_by":"auto","created_at":"2024-03-22 04:56:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":392474,"visible":true,"origin":"","legend":"\u003cp\u003eMS275 inhibited proliferation, and induced apoptosis in colorectal cancer cells. \u0026nbsp;A. Western blot analysis of HDAC1, HDAC2, and HDAC3 levels in CRC cells following treatment with increased concentrations of MS-275. β-actin levels were determined as controls; B. The impact of MS275 on cell proliferation was assessed in DLD1\u003csup\u003eMut \u003c/sup\u003eand DLD1\u003csup\u003eWT\u003c/sup\u003e cells; C. The impact of MS275 on cell proliferation was assessed in HCT116\u003csup\u003eMut \u003c/sup\u003eand HCT116\u003csup\u003eWT\u003c/sup\u003e cells; D. The cell proliferation of a panel of CRC cell lines was evaluated following treatment with MS275 for 48 hours; E. Apoptosis was examined using an Annexin V/PI assay; F. The levels of cleaved PARP were determined by Western blot; G. Effect of apoptosis induced by MS275 was analyzed in a panel of the CRC cell lines by flow cytometry, following 48 hours of incubation with MS275. \u0026nbsp;*** p \u0026lt; 0.001. Data plotted are mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4133957/v1/c3323d09054d750ace26b5f3.png"},{"id":53221239,"identity":"683dbf1b-5e9d-49b5-b56c-2c986926ebf2","added_by":"auto","created_at":"2024-03-22 05:04:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1345256,"visible":true,"origin":"","legend":"\u003cp\u003eMS275 inhibits AOM/DSS-induced CRC tumorigenesis. \u0026nbsp;A. Representative pictures of colon tumors in the different groups at week 14 after tumor initiation with/without MS275 treatment; Tumor volumes (B) and numbers(C) of AOM/DSS-treated mice (AOM/DSS) and AOM/DSS/MS275-treated mice (MS275); D. Epithelial proliferation was measured via Brdu staining of the colon sections of mice (upper, ×100 magnification; lower, ×400 magnification). E. Apoptosis was measured via TUNEL staining of the colon sections of mice (upper, ×100 magnification; lower, ×400 magnification). * p \u0026lt; 0.05. Data plotted are mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4133957/v1/2eab62e9ba12adc164271c3b.png"},{"id":53221002,"identity":"0fa86fdc-e9bb-4487-810b-225f51844332","added_by":"auto","created_at":"2024-03-22 04:56:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1724992,"visible":true,"origin":"","legend":"\u003cp\u003eMS275 treatment suppresses tumor immune microenvironment. \u0026nbsp;A and B. Immunohistochemical features of tumor infiltrating immune/inflammatory cells. The representative photos are shown: F4/80(A) and CD3+(B); C-D. IFN-γ (C)and TNF-α (D) concentrations in culture mesenteric lymphocytes by ELISA.; E-H. Flow cytometry analysis of levels of CD3+ CD4+ and CD3+ CD8+ T cells in co-cultured CT26 cells and spleen T lymphocytes; The distribution of CD3+ T cells was analyzed(E). Gated CD3 positive events were analyzed for CD4(F) and CD8(G) distribution. The graph of flow cytometry represents data from three experiments with similar results; H and I were the proportion of CD3+CD4+ and CD3+ CD8+ cells. * p \u0026lt; 0.05; ** p \u0026lt; 0.01 and *** p \u0026lt; 0.001. Data plotted are mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4133957/v1/0bd05c9867d4919460fa2ad0.png"},{"id":53221005,"identity":"78959a57-4eb6-452e-a8b3-586199c96ba8","added_by":"auto","created_at":"2024-03-22 04:56:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":280544,"visible":true,"origin":"","legend":"\u003cp\u003eMS275 upregulated PD‐L1 expression in colorectal cancer. \u0026nbsp;A-C. q-RT-PCR of PD-L1 in CRC cells treated with MS275 for 24 hours, GAPDH as controls; D-F. Western blot analysis of PD-L1 expression in CRC cells treated with MS275 for 24 hours, β-actin levels were determined as controls; G. FACS analysis of cell-surface expression of PD-L1 in colorectal cancer cells following MS275 treatment for 24h; H. Western blot analysis of PD-L1 expression in tumor from AOM-DSS CRC model with/without MS275 treatment for 3 weeks. * p \u0026lt; 0.05; ** p \u0026lt; 0.01 and *** p \u0026lt; 0.001. Data plotted are mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4133957/v1/978b298a9e52f235f606947f.png"},{"id":53221003,"identity":"3b5b0b8f-1f0f-41b6-89b6-c3fb838a556a","added_by":"auto","created_at":"2024-03-22 04:56:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1552793,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced anti-tumor efficacy through co-treatment with MS275 and PD-1 monoantibody. A. Representative pictures of colon tumors in AOM-DSS mouse model with MS275/PD-1 alone or together treatment. B and C. Tumor numbers (B) and volumes (C) of colon tumors in AOM-DSS mouse model with MS275/PD-1 alone or together treatment; D. Brdu staining for proliferation of the colon sections of mice (upper, ×100 magnification; lower, ×400 magnification); E. Apoptosis was measured via TUNEL staining of the colon sections of mice (upper, ×100 magnification; lower, ×400 magnification); F. Representative CD3+ staining for immunohistochemistry of tumor infiltrating lymphocytes, upper, ×100 magnification; lower, ×400 magnification; G. iNos and Arg-1 expressions in each group of mice were estimated by WB analysis. β-actin levels were determined as controls. * p \u0026lt; 0.05; ** p \u0026lt; 0.01 and *** p \u0026lt; 0.001. Data plotted are mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4133957/v1/86d5b290ac31afd69fa4714b.png"},{"id":53221004,"identity":"fb2933df-0805-4079-b867-7cd22c99ea9e","added_by":"auto","created_at":"2024-03-22 04:56:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":361828,"visible":true,"origin":"","legend":"\u003cp\u003eMS275 increased PD-L1 expression by activating STAT3 \u0026nbsp;A. Western blot analysis of STAT3 phosphorylation in CRC cells treated with MS275 for 24 hours, β-actin levels as controls; B. Semi-quantitative analysis of phosphorylated STAT3 in CRC cells treated with MS275 for 24 hours; C. Representative STAT3 phosphorylation and PD-L1 expression of colon tumors in AOM-DSS mouse model with MS275/PD-1 alone or together treatment; D. Western blot analysis was performed to assess Stat3 phosphorylation in CRC cells pre-treated with Stattic (5 μM) for 1 hour, followed by treatment with MS275 for 24 hours. * p \u0026lt; 0.05; ** p \u0026lt; 0.01 and *** p \u0026lt; 0.001. Data plotted are mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4133957/v1/25e90a20b66bf931a57b970d.png"},{"id":79120549,"identity":"779003d9-6cb8-4454-a932-c9b22289476e","added_by":"auto","created_at":"2025-03-24 16:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6835270,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4133957/v1/2c8f348a-7cad-4ebb-8532-22594ca99992.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"MS275 induces tumor immunosuppression by up-regulating PD-L1 and enhances the efficacy of anti-PD-1 immunotherapy in colorectal cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the third most commonly diagnosed cancer as well as the third leading cause of cancer-related deaths. Environmental risk factors including lifestyle, together with the inherited genetic background, may lead to the development of the tumor\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough genetic mutations (K-Ras, \u0026szlig;-catenin, p53, et.al) undoubtedly play a significant role in CRC initiation and development, CRC progression is also driven by epigenetic modifications, including DNA methylation, histone protein modifications, and non-coding RNA-mediated gene silencing. Histone deacetylase (HDAC) plays a critical role in the development of cancer by reversibly modulating the acetylation status of histone and nonhistone proteins\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Recent studies suggest that HDACs, particularly HDAC-1, -2, and \u0026minus;\u0026thinsp;3, are overexpressed in CRC and their increased expression is correlated with a poorer prognosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This highlights the potential of HDACs as therapeutic targets in CRC.\u003c/p\u003e \u003cp\u003eHistone deacetylase inhibitors (HDACIs) may block tumor cell proliferation by restoring the balance of histone acetylation, thus resulting in proper gene expression. They can induce growth arrest, apoptosis, and/or differentiation of transformed cells both in vitro and in vivo\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Furthermore, HDACIs have been shown to modulate the tumor immune microenvironment\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, affecting immune responses such as antigen presentation, T cell activation, and differentiation of regulatory T cells (Tregs). This immunomodulatory effect suggests that HDACIs could also impact the efficacy of immunotherapy in cancer.\u003c/p\u003e \u003cp\u003eHowever, initial clinical trials employing HDACIs as monotherapy against CRC revealed either limited responses or no response at all\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These trials were also hindered by clinical adverse events due to HDACIs toxicity, which was mainly attributed to global histone acetylation, a phenomenon not limited to cancer cells\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Therefore the search for HDAC inhibitors with high specificity is the main direction for the treatment of targeted HDACs.\u003c/p\u003e \u003cp\u003eN-(2-aminophenyl)-4-[N- (pyridine-3yl-methoxy-carbonyl) aminomethyl] benzamide (MS-275) is a class I histone deacetylase inhibitor (HDAC) that selectively targets HDAC1 and HDAC2. MS275 has shown promising results, especially against advanced breast cancer\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and colon cancer in vitro\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, clinic phase trials indicate that neither monotherapy nor combination therapy with MS275 was well tolerated and showed no evident activity in metastatic CRC\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Further studies of MS275 are warranted to enhance its efficacy through combination with other novel agents, like PD-1/PD-L1 inhibitors.\u003c/p\u003e \u003cp\u003eIn our study, we observed that MS275 inhibits CRC proliferation both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. However, it also suppresses the anti-tumor immune microenvironment by up-regulating the expression of PD-L1. Additionally, We found that there is a synergistic effect with MS275 and PD-1 immunotherapy in the treatment of CRC. Combining MS275 with immunotherapy and antiangiogenic therapies may hold greater promise for improving treatment for CRC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and reagents\u003c/h2\u003e \u003cp\u003eThe human colon cancer cell lines HCT116\u003csup\u003eMut\u003c/sup\u003e, HCT116\u003csup\u003eWT\u003c/sup\u003e, DLD1\u003csup\u003eMut\u003c/sup\u003e and DLD1\u003csup\u003eWT\u003c/sup\u003e were obtained from were kindly provided by Dr. Kevin Haigis (Beth Israel Deaconess Medical Center, Boston, USA). The mouse colon cancer cell lines CT26 was purchased from Kunming Cell Bank (KCB, China). All cells were cultured in high- glucose DMEM with 5% FBS. and maintained under humidified condition (37\u0026deg;C, 5% CO2), and continual culture did not exceed 2 months. Each human cell line was authenticated using the Human STR human cell line authentication service (ATCC). MS275 was purchased from Selleck Chemicals (Houston, TX, USA). An anti-PD-1 antibody was purchased from Biolegend (San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eThe following antibodies (Abs) were used for Western blotting: anti-STAT3(#9139), anti-p-STAT3(#9145), anti-cleaved-PARP(#5625), anti-Arginase-1(#93668), anti-iNOS(#13120), anti-PD-L1(#13684), anti-rabbit antibody(#7074), all of these were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-HDAC1(sc-81598) and anti-HDAC3(sc-17795) were purchased from Santa Cruz. Anti-HDAC2(abs136328) and goat anti-mouse antibody were purchased from Absin. Anti-β-actin(#A2228) was purchased from Sigma-Aldrich. The following antibodies (Abs) were used for Immunochemistry (IHC): anti-F4/80(#70076), anti-CD3(#78588), anti-CD19(#90176), anti-CD11C(#97585), Brdu(#5292), all were purchased from Cell Signaling Technology (Danvers, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell Proliferation Assays\u003c/h2\u003e \u003cp\u003eMTS assay was performed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA). Briefly, cells were seeded into 96-well plates at 75% confluence and incubated for 24 hours. The cells were treated with MS275, and an equal amount of DMSO was added to the control. After 24 hours, cell viability was measured using the MTS assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis analysis\u003c/h2\u003e \u003cp\u003eApoptosis in colorectal cancer cells treated with MS275 was analyzed using an Annexin V-FITC Apoptosis Detection Kit II (BD). The results were measured using the BD Acuri C6. The percentage of apoptosis was then calculated based on the cells in the region of Q2 and Q4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRNA and RT-qPCR\u003c/h2\u003e \u003cp\u003eRNA was extracted using TRIzol (Thermo Fisher Scientific), and cDNA was synthesized using a PrimeScriptTM RT Reagent Kit (TaKaRa). The qPCR assays were performed using SYBR\u0026reg; Premix Ex TaqTM II (TaKaRa) and a QuantStudioTM 5 Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used simultaneously as the internal control. The data was analyzed using the 2\u0026ndash;ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCell lysis buffer (100 mM NaCl, 10 mM EDTA (pH 8.0), 50 mM Tris\u0026ndash;Cl (pH 8.0) and 0.5% (v/v) Triton X-100) with EDTA-free complete protease and phosphatase inhibitors (Roche) was used for protein extraction. The lysates were separated on a 10% SDS-PAGE gel and transferred onto PVDF membranes. The targets were detected using the ImageQuant LAS 4000 (GE Healthcare Life Sciences). Actin was used as the loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAOM-DSS CRC mouse model\u003c/h2\u003e \u003cp\u003eMale BALB/c mice were placed in an SPF-grade standard environment for one week of adaptation feeding. To establish the CAC mouse model, an intraperitoneal injection of (10 mg/kg) of azoxymethane (AOM) was administered on the first day. After 7 days, mice were provided with drinking water containing 1.5% dextran sulfate sodium (DSS) for a week. followed by regular drinking water for another two weeks, constituting one cycle of water administration. A total of three cycles of AOM-DSS treatment were performed. Four weeks after the completion of the AOM-DSS cycles, mice were orally administered with MS275 (20 mg/kg) per day, lasting for 21 days.. The colons were collected, and the tumors were counted and measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunochemistry (IHC)\u003c/h2\u003e \u003cp\u003eParaffin-embedded tissues were used for IHC staining. After deparaffinized in xylene for 10 min, the slides were immersed in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 20 min to block the endogenous peroxidase and blocked in goat serum blocking solution for 30 min. After being incubated at 4\u0026deg;C overnight with primary antibodies, the slides were incubated with secondary HRP-conjugated antibodies (Thermo Fisher Scientific) for 30 min at RT. The primary antibodies were shown above. IHC staining was examined with microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eElisa\u003c/h2\u003e \u003cp\u003eSupernatants of cultured cells were collected and centrifuged. ELISA kits for mouse TNF-a (NOVUS,VAL609) and mouse IFN-g (NOVUS,VAL607) were used according to the manufacturer\u0026rsquo;s instructions. The absorbance of the samples was measured using Synergy\u0026trade; HT microplate reader (BioTek). The concentration of cytokines in the supernatants was calculated based on the standard curve generated using the provided cytokines standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMixed lymphocyte tumor cell culture (MLTC)\u003c/h2\u003e \u003cp\u003eMouse spleen mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. After treatment of CT26 cells with MS275 (2.5 \u0026micro;M) for 24 hours, the cells were co-cultured in 12-well plates with lymphocytes (2X106): CT26 cells\u0026thinsp;=\u0026thinsp;10:1, and cultured in a mixed culture for 48 hours. The proportion of CD3+, CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells were assayed by flow cytometry for assaying T-cell differentiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eThe following mouse Abs were used for flow cytometry analyses: Anti-PD-L1 PE (BD, 557924), anti-CD3e PerCP-CyTM5.5 (BD, 551163), anti-CD4 FITC (BD, 553046), and anti-CD8 alpha PE (BD, 553046). Gates were determined using isotype control Ab staining. Data were acquired with BD C6 flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed by two-tailed Student\u0026rsquo;s t test or ANOVA using GraphPad Prism software (La Jolla, CA). Comparisons between groups are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Values of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e1. MS275 inhibits proliferation of colorectal cancer cells and induces apoptosis\u003c/p\u003e \u003cp\u003eMS275 is a class I selective HDACi that targets HDAC1 and HDAC2 in cells. MS275 (1 \u0026micro;M, 2.5 \u0026micro;M and 5 \u0026micro;M) was applied to CRC cells for 48 hours, Western Blot assay showed that MS275 dose-dependently decreased the intracellular HDAC3 protein expression level, while not affecting the HDAC1 and HDAC2 protein expression levels (Fig.\u0026nbsp;1A).\u003c/p\u003e \u003cp\u003eTwo pairs of cell lines, HCT116\u003csup\u003eMut\u003c/sup\u003e and HCT116\u003csup\u003eWT\u003c/sup\u003e, as well as DLD-1\u003csup\u003eMut\u003c/sup\u003e and DLD-1\u003csup\u003eWT\u003c/sup\u003e, shared the same genetic background but differed in KRas mutation status. HCT116\u003csup\u003eMut\u003c/sup\u003e showed more sensitivity to MS275 compared to HCT116\u003csup\u003eWT\u003c/sup\u003e(Fig.\u0026nbsp;1B), while the reverse was observed in the DLD-1 cell line pair (Fig.\u0026nbsp;1C). MS275 inhibited cell proliferation regardless of KRAS mutation status. Additionally, we treated a panel of colorectal cell lines with MS275 at indicated concentrations (0.1\u0026ndash;10 \u0026micro;M) for 48 hours. The MTS assay revealed a dose-dependent reduction in cell viability (Fig.\u0026nbsp;1D). Among these cell lines, HCT116 \u003csup\u003eMut\u003c/sup\u003e, DLD1\u003csup\u003eMut\u003c/sup\u003e, and SW480 carry active KRAS mutations (G13D), whereas HT29 harbors BRaf V600 mutations. Interestingly, the sensitivity to the drug did not correlate with gene mutations across different cell lines.\u003c/p\u003e \u003cp\u003eTo determine whether MS275 reduced cell proliferation by inducing apoptosis, we conducted apoptosis assessment using the Annexin V-PI staining assay, with results shown in Fig.\u0026nbsp;1E. Following treatment with MS275, there was a 31.8% increase in apoptosis in the HCT116\u003csup\u003eMut\u003c/sup\u003e cell lines, and MS275 induced apoptosis in a dose-dependent manner. Additionally, cleavage of PARP, a hallmark of apoptosis, was observed after treatment with the indicated concentrations of MS275 in both HCT116\u003csup\u003eMut\u003c/sup\u003e and HCT116\u003csup\u003eWT\u003c/sup\u003e cell lines (Fig.\u0026nbsp;1F). The induction of apoptosis by MS275 was further confirmed in a panel of CRC cell lines (Fig.\u0026nbsp;1G).\u003c/p\u003e \u003cp\u003eFigure 1. MS275 inhibited proliferation, and induced apoptosis in colorectal cancer cells. A. Western blot analysis of HDAC1, HDAC2, and HDAC3 levels in CRC cells following treatment with increased concentrations of MS-275. β-actin levels were determined as controls; B. The impact of MS275 on cell proliferation was assessed in DLD1\u003csup\u003eMut\u003c/sup\u003e and DLD1\u003csup\u003eWT\u003c/sup\u003e cells; C. The impact of MS275 on cell proliferation was assessed in HCT116\u003csup\u003eMut\u003c/sup\u003e and HCT116\u003csup\u003eWT\u003c/sup\u003e cells; D. The cell proliferation of a panel of CRC cell lines was evaluated following treatment with MS275 for 48 hours; E. Apoptosis was examined using an Annexin V/PI assay; F. The levels of cleaved PARP were determined by Western blot; G. Effect of apoptosis induced by MS275 was analyzed in a panel of the CRC cell lines by flow cytometry, following 48 hours of incubation with MS275. *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Data plotted are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003e2. MS275 showed anti-tumor activity against colon cancer \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the tumor-suppressing effect of MS275 \u003cem\u003ein vivo\u003c/em\u003e, we established a classical mouse colorectal cancer model (see \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eMaterials and Methods\u003c/span\u003e section). Four weeks after the completion of the AOM- DSS cycle, mice received MS275 at a dose of 20 mg/kg via gavage. After 3 weeks of treatment, the mice were euthanized, and their colons were examined, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003eFollowing treatment of colorectal cancer mice with MS275, the total size of all tumors in the intestinal segment did not show a significant reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, the number of colon tumors decreased by 23.6% compared to that of the saline group (control group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This suggests that MS275 could moderately inhibit the proliferation of colorectal tumors in mice, but not as effectively as observed \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eImmunohistochemistry was utilized to assess the expression of BrdU in intestinal tumors of mice from each group. The results revealed that the intensity of local BrdU staining in tumors of mice in the control group was higher than that in the MS275-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating that MS275 could inhibit tumor cell proliferation. Furthermore, TUNEL staining results indicated apoptosis of tumor tissues in the MS275-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3. MS275 treatment suppresses tumor immune microenvironment\u003c/p\u003e \u003cp\u003eAOM-DSS CAC tumor models were used to investigate the microenvironmental changes following MS275 administration. Immunohistochemical analysis revealed that F4/80-positive cells predominantly infiltrated the tumor microenvironment in the mouse colorectal cancer model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with fewer CD3\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). A small clusters of CD19\u0026thinsp;+\u0026thinsp;cells primarily present within the interstitial space of tissues, predominantly expressing intra-lymphatic node expression. Upon MS275 treatment, CD11C\u0026thinsp;+\u0026thinsp;and other immune cell populations remained largely unchanged (data not shown).\u003c/p\u003e \u003cp\u003eAdditionally, pro-inflammatory cytokines TNF-α and IFN-γ were both upregulated in mesenteric lymphocytes isolated from AOM-DSS mice treated with MS275, indicating their potential role in tumor development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo further elucidate the effect of MS275 on T lymphocyte differentiation, we established an co-culture system using CT26 cells and spleen T lymphocytes from BALB/c mice. Following MS275 administration to CT26 cells, co-cultured with T lymphocytes for 24 hours, the expression levels of surface marker molecules on T lymphocytes were assessed using flow cytometry.\u003c/p\u003e \u003cp\u003eThe results indicated that MS275 influenced T cell differentiation into various subpopulations. In the co-culture system of CT26 tumor cells and T cells, the percentage of CD3\u0026thinsp;+\u0026thinsp;positive cells decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Specifically, the proportion of CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;cells significantly increased(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), while CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;cells decreased compared to the T cells alone group(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e4. MS275 upregulated PD-L1 expression in colorectal cancer both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e \u003cp\u003eTo understand how MS275 reshapes the tumor microenvironment and its subsequent impact on the anti-tumor effect, we analyzed the effects of MS275 on PD-L1 expression in CRC cells. Following treatment with MS275, qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C) and Western blotting assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F) revealed an upregulation of PD-L1 at both the mRNA and protein levels. These findings were further confirmed by evaluating PD-L1 expression using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eThe ability of MS275 to upregulate PD-L1 \u003cem\u003ein vivo\u003c/em\u003e was also investigated in tumor tissue from the AOM-DSS CAC model. After treatment with MS275, PD-L1 expression in the tumors was assessed by Western blotting. Consistent with the in vitro data, MS275 treatment significantly increased PD-L1 expression in tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e5. Anti-tumor effects of MS275 in combination with an anti‐PD‐1 antibody\u003c/p\u003e \u003cp\u003eGiven that MS275 inhibits tumor immune response by upregulating PD-L1 expression in tumor cells, we further explored the possibility of enhancing the anticancer effect of MS275 by blocking the PD-1/PD-L1 signaling pathway using a PD-1 monoclonal antibody.\u003c/p\u003e \u003cp\u003eIn AOM-DSS CRC model mice, we evaluated the anti-tumor effects following 3 weeks of treatment with both MS275 (20 mg/kg/day) and PD-1 monoclonal antibody (5 mg/kg/3 days). The results showed that the co-treatment significantly enhanced the inhibitory effect on tumor growth compared to treatment with MS275 alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Moreover, the number of tumors in either the combination treatment group or the treatment alone group was significantly decreased compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). While treatment with either MS275 or PD-1 monoclonal antibody alone tended to result in smaller tumor volumes than the control group, the combination of MS275 and PD-1 monoclonal antibody treatment led to the most significant tumor suppression, with lower tumor volumes than those observed with either treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe proliferation or apoptosis was assessed after administration with MS275. Intra-tumoral proliferation rates were decreased after MS275 treatment, suggesting that MS275 inhibits epithelial proliferation (5D), likely attributable to increased intra-tumoral apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eA significant increase in CD3\u0026thinsp;+\u0026thinsp;T-cell tumor infiltration was observed in the tumor micro-environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), whereas there was no significant change in CD19\u0026thinsp;+\u0026thinsp;cell tumor infiltration (data not shown). Despite an overall decrease tumor infiltration of F4/80 cells, Western blot results showed an increase expression of iNOS and a decrease expression of Arg-1 in co-treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), indicating a potential shift towards a higher proportion of M1-type macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e6. Stat3 activation is required for MS275 upregulation of PD-L1 in colorectal cancer cells\u003c/p\u003e \u003cp\u003eIt has been demonstrated that MS275 activates STAT3 in gut epithelial cells. To elucidate if MS275 regulates PD-L1 transcriptionally, we assessed phosphorylated STAT3 protein levels in colorectal cell lines and a CRC mouse model.\u003c/p\u003e \u003cp\u003eMS275 significantly up-regulated the expression levels of phosphorylated STAT3 protein in HCT116 \u003csup\u003eMut\u003c/sup\u003e and HCT116\u003csup\u003eWT\u003c/sup\u003e cell lines, along with increased PD-L1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The same results were obtained in the AOM-DSS CRC mouse model. The phosphorylated Stat3 was increased in MS275 and anti PD-1 co-treatment group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe role of STAT3 was investigated using the STAT3 inhibitor Stattic. Pre-treatment of CRC cells with Stattic(5\u0026micro;M) for 60 min, phosphorylated STAT3 in both HCT116\u003csup\u003eMut\u003c/sup\u003e and HCT116\u003csup\u003eWT\u003c/sup\u003e cell lines were inhibited. The levels of PD-L1 gene and protein expression remained unchanged following subsequent treatment with MS275 for 24 or 48 hours(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results suggest that activated STAT3 is likely a central regulator of PD-L1 gene induction by MS275.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMS275, a benzamide that specifically inhibits class I HDAC, has shown preclinic anti-tumor effects in a variety of cancers, including colorectal cancer, breast cancer, hematological malignancies\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and others. Consistently, our study also confirmed that MS275 can inhibit proliferation and viability and promote apoptosis in colorectal cancer cells. Notably, the inhibitory effect of MS275 on colorectal cancer cells appeared independent of the specific gene mutations carried by the cells, suggesting that MS275's impact on tumor cells is influenced by deacetylation-mediated epigenetic modifications.\u003c/p\u003e \u003cp\u003eHowever, the inhibition of tumor growth by MS275 in the AOM-DSS CAC mouse model was not as effective as observed \u003cem\u003ein vitro\u003c/em\u003e. The efficacy of MS275 as a single agent therapy remains limited\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In a phase II study of MS275 monotherapy in relapsed/refractory Hodgkin lymphoma, the overall response rate was modest (12%) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Studies have explored strategies to combine MS275 with other drugs to enhance therapeutic outcomes. For instance, traditional chemotherapy drugs like 5-fluorouracil (5-FU)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and oxaliplatin\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e were combined with MS275 to augment chemotherapy efficacy. Additionally, combining MS275 with inhibitors targeting the EGFR pathway (such as cetuximab or panitumumab) in colorectal cancer\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e or with HER2-targeted therapies in breast cancer\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e may enhance anti-tumor effects. Furthermore, MS275 may be combined with other epigenetic modifiers. However, the clinical phase outcomes of these combinations have not met expectations. The combination of regorafenib, HCQ, and MS275 was poorly tolerated without evident activity in metastatic CRC\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to directly suppressing tumor cells, HDAC inhibitors have been reported to modulate the immune system\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We also found a reduction in CD3\u0026thinsp;+\u0026thinsp;cell tumor infiltration following MS275 treatment. Moreover, levels of the pro-inflammatory cytokines TNF-α and IFN-γ were both increased in mesenteric lymphocytes isolated from AOM-DSS mice treated with MS275. Results from \u003cem\u003ein vitro\u003c/em\u003e lymphocyte-tumor cell co-culture experiments indicated a significant decrease in the proportion of CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells, coupled with an increase in the proportion of CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T cells in MS275-treated tumor cells. The enhancement of immune suppression by MS275 and its contribution to tumor immune escape explains the lesser effectiveness of MS275 \u003cem\u003ein vivo\u003c/em\u003e compared to \u003cem\u003ein vitro\u003c/em\u003e, which in turn, leads to tumor immune tolerance or escape, leading to its limitations in clinical treatment.\u003c/p\u003e \u003cp\u003eImmune checkpoints play a crucial role in regulating tumor immune environments. Among them, the upregulation of PD-1/PD-L1 has gained significant attention due to its role in immune suppression, making it a prominent target for tumor immunotherapy. Accumulating evidence indicates that histone deacetylase (HDAC) activation could induce PD-L1 expression in various types of cancer\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. We also found that PD-L1 is upregulated on tumor cells following MS275 treatment. Furthermore, combining MS275 with anti-PD-1 in AOM-DSS mouse tumor models increased CD3\u0026thinsp;+\u0026thinsp;T cell infiltration and induced an M1-like phenotype characterized by diminished immunosuppressive function. This results in a significant reduction in mouse tumor burden and a notable prolongation of survival compared to control mice. These findings suggest that reprogramming immunosuppressive responses via MS275 has the potential to overcome the limitations of current checkpoint blockade-based immunotherapy.\u003c/p\u003e \u003cp\u003eAlthough inhibition of class I HDACs has been associated with increased PD-L1 expression\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, the precise mechanism of PD-L1 regulation remains unclear. Our research revealed elevated levels of STAT3 phosphorylation in mouse colorectal cancer tissues or cells cultured in vitro following MS275 treatment. Inhibiting Stat3 activation can down-regulate the expression of PD-L1\u003csup\u003e20\u003c/sup\u003e. These findings collectively suggest that STAT3-mediated transcriptional activation by MS275 leads to PD-L1 upregulation.\u003c/p\u003e \u003cp\u003eIn CRC, the balance between anti-tumoral and pro-tumoral immune functions is largely dependent on the level of PD-L1 expression. CRC with both high PD-1 and PD-L1 levels may have an active immune checkpoint activity and may therefore represent the subset that benefits from anti-PD-1 therapy\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, the overall response rates to PD-1 inhibitors in CRC remain modest, with only a subset of patients experiencing durable responses\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Although the upregulation of PD-L1 following MS275 treatment negatively regulates T cell responses and facilitates immune escape, emerging evidence suggests that MS275-induced upregulation of PD-L1 can enhance T cell infiltration, thereby improving the efficacy of anti-PD-1 immunotherapy. This highlights the potential for combining MS275 with anti-PD-1 therapy as an effective strategy to enhance immunotherapy efficacy in CRC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr.Ling GU for providing for useful discussion. We thank Dr. Tao Wang for revising the manuscript. We thank the National Natural Science Foundation of China for its support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (Grant No. 82173104 and 81871233)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;The authors have no relevant financial or non-financial interests to disclose.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYufang Wang designed the research; Sihan Chen, Zhigang Mao and Deng Tang carried out most of the experiments; Mi Su, Meng Lai, Xiya Yan, and Ruiting Yan provided extra technical assistance; Siqi Lan, Meng Lai,\u0026nbsp;Deng Tang, Mi Su, Xiya Yan nd Ruiting Yan performed material preparation; the first draft of the manuscript was written by Yufang Wang and Ji Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in line with International Guidelines and Protocols. Approval was granted by the Institutional Animal Care and Use Committee at the Institutional Animal Care and Use Committee (IACUC), Sichuan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGarmpis N et al (2022) Histone Deacetylases and their Inhibitors in Colorectal Cancer Therapy: Current Evidence and Future Considerations. 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J Cell Physiol 235:5461\u0026ndash;5475. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1002/jcp.29494\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1002/jcp.29494\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\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":"HDACi, MS275, PD-L1, Colorectal Cancer","lastPublishedDoi":"10.21203/rs.3.rs-4133957/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4133957/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe histone deacetylase inhibitor MS275 (Entinostat) demonstrates anti-tumor effects against various types of solid tumors \u003cem\u003ein vitro\u003c/em\u003e. But its effectiveness in clinical trials is limited. The underlying reasons remain to be determined. The purpose of this study was to explore how to enhance the anti-tumor effects of MS275 in colorectal cancer(CRC). Our data showed that MS275 inhibited CRC cell proliferation and induced apoptosis, irrespective of gene mutation status. However, MS275 did not effectively suppress tumor growth in the AOM-DSS CRC model as observed \u003cem\u003ein vitro\u003c/em\u003e. MS275 decreased CD3\u0026thinsp;+\u0026thinsp;T cell tumor infiltration and created an anti-tumor immunosuppressive microenvironment in the AOM-DSS CRC model. MS275 also decreased the percentage of CD8\u0026thinsp;+\u0026thinsp;T cells while increasing the percentage of CD4\u0026thinsp;+\u0026thinsp;T cells in tumor-lymphocyte mixed culture. Reshaping tumor immune response may contribute to the less pronounced anti-tumor effect of MS275 observed \u003cem\u003ein vivo\u003c/em\u003e compared to \u003cem\u003ein vitro\u003c/em\u003e. Further study show that the increased PD-L1 expression in CRC both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e following MS275 treatment. Moreover, the anti-tumor effects of MS275 were enhanced by combining it with an anti-PD-1 antibody. This combination treatment also increased CD3\u0026thinsp;+\u0026thinsp;T cell tumor infiltration and M1 macrophage polarization in the AOM-DSS CRC model, thereby leading to an anti-tumor immune response. Therefore, the combination of MS275 and anti-PD-1 immunotherapy represents a potential strategy for low PD-L1 expression tumors and should be considered a promising treatment approach for colon cancer.\u003c/p\u003e","manuscriptTitle":"MS275 induces tumor immunosuppression by up-regulating PD-L1 and enhances the efficacy of anti-PD-1 immunotherapy in colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 04:56:45","doi":"10.21203/rs.3.rs-4133957/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-14T21:14:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-14T17:09:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2306634022645211626877745030789932138","date":"2024-06-04T01:11:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27974046767282531245615798232460012837","date":"2024-06-03T15:11:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-13T23:01:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222909337682947619762347218876866702667","date":"2024-05-02T01:17:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281525114954349696353368187734743924519","date":"2024-04-30T20:06:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-25T12:07:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-20T04:58:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-20T04:58:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2024-03-20T03:56:58+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":"10a752eb-1432-4123-8481-366cc3388367","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T16:03:39+00:00","versionOfRecord":{"articleIdentity":"rs-4133957","link":"https://doi.org/10.1007/s00262-025-04004-4","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2025-03-17 15:58:08","publishedOnDateReadable":"March 17th, 2025"},"versionCreatedAt":"2024-03-22 04:56:45","video":"","vorDoi":"10.1007/s00262-025-04004-4","vorDoiUrl":"https://doi.org/10.1007/s00262-025-04004-4","workflowStages":[]},"version":"v1","identity":"rs-4133957","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4133957","identity":"rs-4133957","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}