The mechanistic study of novel immuno-adjuvant NCL-P2 improve the effectiveness of anti- PD-1 in colorectal cancer treatment through suppressing the expression of USP2

The mechanistic study of novel immuno-adjuvant NCL-P2 improve the effectiveness of anti- PD-1 in colorectal cancer treatment through suppressing the expression of USP2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The mechanistic study of novel immuno-adjuvant NCL-P2 improve the effectiveness of anti- PD-1 in colorectal cancer treatment through suppressing the expression of USP2 Wu Shan, Zhang Miao, Wang Huaqing This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7274573/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: Colorectal cancer (CRC) has risen to second place in the incidence rate of malignant tumors in China. However, treatments for advanced CRC are not currently effective, andtreatment of anti-PD-1 monoclonal antibodies alone has no significant effect on improving the overall survival rate of CRC patients. Methods: Our research group has discovered a novel immune adjuvant NCL-P2 that can synergistically enhance the anti-CRC therapeutic effect of anti-PD-1 antibody. Results: Preliminary data shows that NCL-P2 can inhibit the expression of PD-L1 on the surface of CRC cells, activate dendritic cells, promote the secretion of cytokines such as TNFα and IL-1β , present tumor antigens, and then initiate downstream cytotoxic T cells to kill tumor cells. We found that USP2 is one of the key proteins that down regulate the expression of PD-L1 on the surface of CRC cells by NCL-P2. This project takes the CRC mouse model as the research object and explores the molecular mechanism of USP2 's impact on the expression of PD-L1 in CRC cells by activating or knocking out USP2. Conclusion: This research explores how USP2 participates in the immune activation of NCL-P2 in CRC and enhances the anti-CRC efficacy of anti-PD-1 antibody, which provides new targets and theoretical support for the clinical treatment of colorectal cancer. Biological sciences/Cancer Biological sciences/Immunology Health sciences/Oncology immune checkpoint treatment micro-environment immuno-adjuvant anti-PD-1 antibody antigen-presenting cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colorectal cancer (CRC), as the third most common cause of cancer death in the world [1], has been on the rise in China, ranking second in the incidence of malignant tumors since 2022, reaching as high as 37 per 100,000 people [2, 3]. Up to date, the main treatment for CRC still relies on the combination of surgery, adjuvant chemotherapy and radiotherapy; however, traditional chemotherapy drugs commonly have issues such as multiple side effects, short half-life, low solubility, and unstable efficacy [4]. In recent years, a series of antibody immunotherapy drugs and molecular targeted drugs have emerged in the field of anticancer treatment; among them, the rapid development and widespread clinical application of immune checkpoint inhibitors (ICIs), including PD-1/PD-L1 antibodies, have provided new hope for the treatment of many cancer patients [5-7]. PD-1 monoclonal antibody is a type of immunotherapy drug that can bind to the PD-1 receptor on the surface of T cells, competitively antagonizing the binding of the PD-L1 ligands on the surface of tumor cells to this receptor [8-12]. However, the use of ICIs in the treatment of advanced CRC patients remains controversial; current clinical studies suggest that PD-1 monoclonal antibody is more effective for patients with microsatellite instability-high CRC [13], but it does not significantly improve the overall survival of CRC patients [14]. In addition, effective treatment for patients with metastatic or inoperable CRC is still in searching [15]. Therefore, the search for new, safe, and effective treatments is of great clinical significance and social value in improving the survival rate and quality of life of CRC patients. In recent years, the application of immunological adjuvants in anti-tumor therapy has gradually become one of the popular research areas in cancer treatment to enhance the anti-tumor effect of immune checkpoint inhibitors [4, 14, 16-18]. In a phase 1/2 clinical trial for metastatic melanoma, research data indicated that the anti-tumor therapeutic effect of a novel IDO/PD- L1 peptide combined with nivolumab is higher than nivolumab alone [19]; this peptide acts as an adjuvant to induce the body to produce specific T cells targeting IDO and PD-L1, thereby killing PD-L1-expressing tumor cells, and activating immune cells to produce anti-tumor factors, further enhancing the anti-tumor activity of nivolumab [20]. Although immunological adjuvants can activate the body's immune system to produce a series of cytokines, most adjuvants lack strong specificity and have low efficacy, leading to insufficient T cell activation capacity and an inability to effectively improve the anti-tumor therapeutic effect of ICIs [4, 21]. Therefore, exploring adjuvant that can effectively activate the body's immune cells to exert anti-tumor effects can significantly enhance the therapeutic effect of PD-1 monoclonal antibodies in CRC. A recent study on colorectal cancer reported a ubiquitin-specific processing protease 2, USP2, closely associated with the surface expression of PD-L1 on colorectal cancer cells; the study indicated that USP2 is a novel regulatory factor that promotes stable surface expression of PD-L1, aiding tumor cells in evading the killing effect of CD8+ T cells; moreover, the study confirmed that reducing intracellular expression of USP2 can enhance the anti-tumor effect of PD-1 monoclonal antibodies [22]. In this study, we revealed a novel immuno-adjuvant, NCL-P2, discovered by our research group, which effectively activates the body's immune system through inducing dendritic cell activation and secretion of anti-tumor cytokines, promoting antigen presentation, initiating downstream T cell killing of tumor cells, and synergistically enhancing the anti-tumor effects of PD-1 monoclonal antibodies in in-vivo experiments. Additionally, NCL-P2 can inhibit the expression of PD-L1 in CRC cells; we identified USP2 as one of the key proteins through which NCL-P2 affects the expression of PD-L1 in CRC cells. In in-vivo experiments in mice, we found that NCL-P2 down regulates the expression of PD- L1 on the surface of tumor cells by inhibiting USP2 expression, activating the body's immune response, and further confirmed USP2 as a key molecule for NCL-P2 to activate the immune response in CRC. This study elucidated the molecular mechanism by which NCL-P2 enhances the efficacy of PD-1 monoclonal antibodies in CRC, providing a theoretical basis for the application of NCL-P2 in the treatment of CRC, with the aim of providing new methods and important theoretical support for the clinical treatment of CRC. Materials and Methods 1.Materials CT26 colon cancer cell line purchased from abcam; DMEM high glucose culture medium, RPMI-1640 culture medium, fetal bovine serum, trypsin, phosphate buffered saline (PBS) purchased from American Gibco company; inducing agents IL-2, IL-7, IL-15, IL-18, IL-21, OKT3 purchased from American R&D company; TNF and IL-1ß cytokine detection ELISA kits purchased from American Santa Cruz company; antibodies used for flow cytometry purchased from American abcam; CCK-8 kit purchased from Chinese Dojindo Biological company. Mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., free access to food and water during the experiment; animal handling during the experiment conforms to animal ethics standards. Animal experiments were approved by the Experimental Animal Management and Ethics Committee of Tianjin Union Medical Center of Nankai University.All experimental procedures involving live vertebrates were reviewed and approved by the Experimental Animal Management and Ethics Committee of Tianjin Union Medical Center of Nankai University.(Ethical code:2024-SYDWLL-000063. Approval Protocol Number:[2022033]). The experimental procedures were conducted following the National Institutes of Health (NIH) Guidelines for the Protection and Use of Laboratory Animals (NIH Publication No., as per regulations 85-23, updated in 1996. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. 2. Methods: 2.1. Peripheral blood mononuclear cells (PBMCs) separation: PBMCs were separated by Ficoll density gradient centrifugation method, collected and centrifuged to obtain plasma. Monocytes, macrophages, dendritic cells, etc. were differentiated and subsequent cell experiments were conducted using the white membrane layer. Microscopic counting was performed, along with flow cytometry and protein imprinting analysis. 2.2. Establishment and grouping of mouse colorectal cancer models: Log-phase CT26 cells were taken, digested with trypsin to make single-cell suspension, and injected subcutaneously into the skin of the hind limbs of mice at a concentration of 2 × 106 cells/each (125 μL). Mice were randomly divided into the anti PD-1 antibody group (PD-1 group) and the PBS group. In the PD-1 group, mice were given anti PD-1 antibody and control peptide segment by intraperitoneal injection on the 6th and 9th days after tumor bearing, respectively. Tumor measurement and recording were started on the 7th day after tumor bearing, and tumor volume was calculated every day ( ).Animals were euthanized if tumors exceeded 1,500 mm³ or 20 mm in any dimension. Mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (10 mg/kg). For euthanasia, deeply anesthetized mice received an overdose of sodium pentobarbital (150 mg/kg, IP) followed by bilateral thoracotomy to ensure death. All procedures were approved by the Experimental Animal Management and Ethics Committee of Tianjin Union Medical Center of Nankai University IACUC and adhered to AVMA Euthanasia Guidelines (2020). 2.3. Flow cytometry: Flow cytometric analysis was performed using immunofluorescence labeling. Immune cells to be tested were suspended in PBS after washing twice, incubated with target antibodies diluted in 2% FBS/PBS (incubation conditions: 4°C, protected from light, 30 min). Flow cytometry analysis was performed after PBS washing. 2.4. Western blotting experiment: Extract tumor tissue or collect immune cells to be detected, use RIPA lysis buffer to extract total protein, separate proteins by SDS-PAGE gel electrophoresis, transfer to PVDF membrane, block with 5% skim milk in TBST for 1h after transfer; incubate with primary antibody overnight at 4oC, wash the membrane with TBST for 3 times, then incubate with the secondary antibody conjugated with horseradish peroxidase;finally, expose and visualize the target protein using Bio-Rad instrument. 2.5. Immunofluorescence Staining: The target cells to be detected were seeded on cover slips. After 24h, the cells were washed once with PBS, fixed with 4% paraformaldehyde at room temperature for 30min, permeabilized with 0.2% Triton X-100 for 5min washed three times with PBS for 10min each time, and then blocked with 5% BSA at room temperature for 30 min. After blocking, the cells were incubated with the primary antibody at 4 o C overnight. After washing with PBS, the secondary antibody was added in the dark for 30min, followed by three washes with PBS before staining the cell nuclei with DAPI, and then observed under a fluorescence microscope. 2.6. Statistical methods: Statistical analysis was performed using Graph Pad Prism 10.0 software. Data conforming to a normal distribution are presented as mean (average, AVG) ±standard deviation (SD). Group comparisons were made using independent sample t-test, and differences between groups were analyzed using two-way ANOVA. P<0.05 was considered statistically significant. Results 1. The novel immune adjuvant NCL-P2 can activate various antigen-presenting cells (APC). The novel immune adjuvant NCL-P2 effectively stimulates various APCs to produce pro-inflammatory cytokines, particularly TNFα. Across soluble peptide concentrations (Figure 1.1a), NCL-P2 showed superior potency compared to NCL-P1 and NCL-P3. At 10 μg/mL, all peptides induced minimal TNFα production. At 20 μg/mL, NCL-P1 reached ~680 pg/mL, while NCL-P2 remained lower (~430 pg/mL). A significant boost was observed at 40 μg/mL, where NCL-P2 produced 14,040 pg/mL, more than double NCL-P1 (~7,000 pg/mL). Higher concentrations (80 and 100 μg/mL) showed NCL-P2 maintaining TNFα production levels of ~41,040 and ~42,040 pg/mL, surpassing NCL-P3.Similarly, in surface-coated peptide experiments (Figure 1.1b), NCL-P2 induced greater TNFα generation than its counterparts. At 10 μg/mL, TNFα production was minimal (~98 pg/mL for NCL-P2 and ~10 pg/mL for NCL-P1), while NCL-P3 remained inactive. At 40 μg/mL, NCL-P2 elicited 1,110 pg/mL, nearly triple NCL-P1 (~380 pg/mL). At 160 μg/mL, NCL-P2 reached 1,240 pg/mL, compared to NCL-P1 (~410 pg/mL), confirming NCL-P2's superior immune activation. NCL-P3 consistently showed negligible responses in both conditions, reinforcing the potent pro-inflammatory role of NCL-P2 in immune modulation. This immune adjuvant also induces the transformation of immature dendritic cells (imDCs) into mature dendritic cells (mDCs), enhancing APC function and promoting downstream T cell activation (Figure 1.2). Under NCL-P2 stimulation, the surface expression of key markers, including CD83, CD86, and MHCII, significantly increased, signifying the maturation process. 2. NCL-P2 can enter immune cells through passive transportation. NCL-P2, a novel immune adjuvant, exhibits the ability to penetrate cell membranes and enter the cytoplasm and nucleus of immune cells. Experimental results demonstrate that NCL-P2 can effectively enter peripheral blood mononuclear cells (PBMCs). This was confirmed by the use of a recombinant protein composed of a 66 kDa medium-sized antigen, streptavidin, which was tagged with NCL-P2. Importantly, the penetration of NCL-P2 into PBMCs occurred under both cold (4°C) and physiological (37°C) conditions, suggesting that the transport mechanism may not be temperature-dependent. These findings support the notion that NCL-P2 can be delivered into immune cells, potentially enhancing its adjuvant activity and facilitating immune modulation at the cellular level. (Figure 2). 3. NCL-P2 in combination with PD-1 monoclonal antibody can inhibit the growth of tumors in CRC model mice and activate immune cells. In vivo experiments using a CRC mouse model demonstrated that the combination of NCL-P2 and PD-1 monoclonal antibody significantly suppressed tumor growth compared to treatment with either agent alone or the control group. Tumor size measurements (Figure 3.1) indicated that the control group exhibited a continuous and rapid increase, starting from 0.4 cm³ and peaking at approximately 1.8 cm³. NCL-P2 treatment alone reduced tumor growth, reaching a maximum of 1.4 cm³. Anti-PD-1 monoclonal antibody treatment resulted in a further reduction, with tumor size reaching 1.2 cm³. The combined NCL-P2 + anti-PD-1 treatment showed the greatest inhibition, with tumor growth reaching only 1.1 cm³. Although all groups exhibited a gradual upward trend in tumor size over time, the combination therapy clearly demonstrated superior efficacy in limiting tumor progression (Figure 3.1), Further immune cell profiling (Figure 3.2) revealed enhanced immune responses with combination treatment. The migration of monocytes and CD4+ T cells to CT26 cells was markedly increased, reflecting enhanced immune surveillance and response. Additionally, the activation of CD8+ T cells was significantly promoted, indicating that NCL-P2 synergizes with PD-1 blockade to stimulate cytotoxic T cell-mediated antitumor activity. These findings suggest that NCL-P2 amplifies the immune activation initiated by PD-1 inhibition, providing a potent therapeutic strategy for CRC by simultaneously targeting tumor growth and enhancing immune cell engagement. (Figure 3.2). 4. NCL-P2 down regulates the expression of PD-L1 on the surface of CT26 cells. NCL-P2 effectively downregulates PD-L1 expression on the surface of CT26 cells. Pre-experimental results showed that treating CT26 cells with various concentrations of NCL-P2 reduced PD-L1 levels, as illustrated in Figure 4.1. PD-L1 expression was assessed using western blotting, flow cytometry, and confocal staining. The x-axis represents NCL-P2 concentrations (10 μg/ml, 50 μg/ml, 100 μg/ml, and 200 μg/ml), with the control group receiving no treatment (Figure 4.1a-c). PD-L1 detection peaked at 50 μg/ml, followed by 10 μg/ml and 100 μg/ml. In contrast, GADP1[5] expression remained consistently high across all conditions except for a notable decrease at 50 μg/ml. These results highlight the concentration-dependent regulatory effect of NCL-P2 on PD-L1 expression, potentially mediated by USP2 inhibition (Figure 4.1); Flow cytometry analysis (Figure 4.2b) demonstrated a dose-dependent decrease in PD-L1 Mean Fluorescence Intensity (MFI), with NCL-P2 treatment resulting in progressive downregulation of PD-L1. USP2 expression, as measured by MFI, was highest in the control group (approximately 400) but was markedly reduced with increasing concentrations of NCL-P2, peaking at 680 MFI for treated cells, confirming the inhibitory effect of NCL-P2 on USP2 expression. The control group maintained a baseline fold change of 1.0. Treatment with 10 μg/ml NCL-P2 showed minimal impact, with mRNA expression levels remaining similar to the control. However, exposure to 50 μg/ml significantly reduced mRNA expression to 0.8-fold, and further decreases were observed at 100 μg/ml (0.5-fold) and 200 μg/ml (0.45-fold). These data demonstrate that increasing NCL-P2 concentrations effectively inhibit USP2 expression, correlating with reduced PD-L1 levels. This suggests a mechanistic pathway where NCL-P2 downregulates PD-L1 expression through the suppression of USP2, highlighting its potential therapeutic role in immune modulation.(Figure 4.2). Discussion This study investigates the molecular mechanisms by which the novel immune adjuvant NCL-P2 enhances the efficacy of PD-1 monoclonal antibodies in CRC [33–35]. ICIs, such as PD-1/PD-L1 monoclonal antibodies, have transformed cancer immunotherapy by reinvigorating exhausted T cells and restoring antitumor immunity [34]. However, their clinical effectiveness in CRC remains limited due to immune evasion mechanisms and insufficient activation of CTLs [36]. One of the key challenges in improving ICI efficacy is overcoming the persistent expression of PD-L1 on tumor cells, which binds to PD-1 on T cells, inhibiting their cytotoxic function and facilitating tumor progression [37]. NCL-P2, a peptide-based immune adjuvant identified in our research, offers a promising solution by modulating the immune microenvironment and enhancing T cell-mediated immune responses. Based on bioinformatics analysis and previous experimental data, we hypothesize that NCL-P2 inhibits the expression of USP2, a critical regulator of PD-L1 stability on the tumor cell surface [31, 38]. USP2 stabilizes PD-L1 by preventing its degradation, thereby allowing tumor cells to escape immune surveillance. Our results demonstrate that NCL-P2 reduces PD-L1 surface expression in CRC cells by suppressing USP2, effectively reversing immune suppression and enabling robust CTL activation [31]. This mechanism represents a significant advancement in the strategic use of immune adjuvants to boost the efficacy of ICIs. Furthermore, the ability of NCL-P2 to penetrate cells and directly influence intracellular pathways distinguishes it from traditional immune adjuvants, which primarily act on surface receptors. Unlike other adjuvants with single-target mechanisms, NCL-P2 activates multiple signaling pathways through TLRs on antigen-presenting cells [39], enhancing dendritic cell maturation and promoting the expression of costimulatory molecules [40, 41]. These findings highlight the multifunctional nature of NCL-P2, which addresses the limitations of conventional ICIs, including low response rates and variable patient outcomes. By integrating NCL-P2 with PD-1 monoclonal antibodies, this study opens new therapeutic avenues for CRC, especially in patients with advanced or metastatic disease where treatment options are limited. The mechanistic insights provided by our research not only underscore the therapeutic potential of targeting USP2 to regulate PD-L1 but also pave the way for broader applications in immunotherapy. This combination strategy could significantly improve immune checkpoint blockade outcomes, offering a more comprehensive and effective approach to harnessing the immune system for cancer treatment. Immunotherapy and PD-1/PD-L1 Inhibition in Cancer Treatment In recent years, ICIs targeting the PD-1/PD-L1 axis have revolutionized cancer therapy by unleashing the body's immune system to attack tumors. However, the efficacy of PD-1 monoclonal antibodies is often limited by tumor-mediated immune evasion mechanisms. Tumors upregulate PD-L1 on their cell surfaces to bind with PD-1 on T cells, thereby inhibiting T-cell activation and suppressing anti-tumor immunity [5]. This immune evasion mechanism is particularly prominent in CRC, where high PD-L1 expression correlates with poor prognosis [1]. Therefore, targeting the regulatory mechanisms of PD-1/PD-L1 interaction is critical for enhancing the effectiveness of PD-1-based therapies. In CRC, USP2 has been identified as a key regulator of PD-L1 stabilization on the tumor cell surface. USP2 prevents the degradation of PD-L1 by deubiquitinating it, thus promoting immune escape by inhibiting CTL-mediated killing of tumor cells [31, 42]. Inhibition of USP2 in preclinical models has shown promise in reducing PD-L1 levels and reactivating CTLs, resulting in reduced tumor growth and improved immune response [43]. Our study builds upon these findings, demonstrating that NCL-P2 inhibits USP2 expression, thus destabilizing PD-L1 and enhancing CTL activation. These results are consistent with the hypothesis that USP2 inhibition is a viable therapeutic strategy to potentiate PD-1/PD-L1 immunotherapy. The Role of Immune Adjuvants in Enhancing Anti-Tumor Immunity While PD-1/PD-L1 inhibitors have shown clinical success in a variety of cancers, including melanoma, non-small cell lung cancer, and RCC, their efficacy in CRC has been inconsistent. This variability may be due to differences in the tumor microenvironment, immune cell infiltration, and the lack of effective immune adjuvants to stimulate a robust immune response. Traditional immune adjuvants, such as CpG-ODN (TLR9 activator) and Imiquimod (TLR7 activator), have been explored to enhance immune responses but are limited by their narrow target activation profiles and unstable efficacy [39, 43–50]. Moreover, these adjuvants often activate only a subset of immune pathways, resulting in suboptimal immune activation and therapeutic outcomes. To address these limitations, NCL-P2 represents a promising alternative. Our previous work identified NCL-P2, a 36-amino acid peptide, which activates multiple APCs[43, 51], including DCs[52], and induces the production of pro-inflammatory cytokines, maturation of DCs, and the generation of specific immune responses [38]. Notably, NCL-P2 activates Toll-like receptors (TLRs) on immune cells, promoting the maturation of imDCs[43, 53] into mDCs [54] and upregulating costimulatory molecules such as CD80, CD83, CD86, and MHCII [51, 53, 55]. This enhances the capacity of DCs to activate T cells, driving CTL differentiation and anti-tumor immunity. In preclinical CRC mouse models, the combination of NCL-P2 and PD-1 monoclonal antibodies significantly inhibited tumor growth compared to PD-1 treatment alone, providing strong evidence for the potential of NCL-P2 as an adjuvant in CRC immunotherapy. Furthermore, NCL-P2 demonstrates a unique advantage over conventional immune adjuvants by not only activating immune cell surface TLRs but also penetrating cell membranes and entering the cytoplasm and nucleus. This dual function allows NCL-P2 to exert a more profound and sustained immune activation, offering a potential therapeutic strategy that could overcome the limitations of traditional adjuvants [56]. Despite the promising results of combining NCL-P2 with PD-1 monoclonal antibodies, several limitations need to be addressed. First, the precise molecular mechanisms by which NCL-P2 inhibits USP2 and destabilizes PD-L1 remain to be fully elucidated. Future studies should focus on identifying the specific molecular pathways through which NCL-P2 interacts with USP2 and PD-L1, as well as any potential off-target effects. Additionally, while our study demonstrates the efficacy of NCL-P2 in mouse models, further research is needed to assess its safety and efficacy in human clinical trials. One challenge in translating these results to the clinic is the potential variability in immune responses across different patient populations. Factors such as tumor heterogeneity, immune cell composition, and PD-L1 expression levels may influence the therapeutic outcome, highlighting the need for personalized approaches in CRC immunotherapy. Moreover, the use of NCL-P2 as an immune adjuvant could benefit from further optimization. While NCL-P2 shows promise in activating multiple immune pathways, its clinical efficacy may be enhanced by combining it with other immune modulators, such as checkpoint inhibitors targeting CTLA-4 or TIM-3, or cytokines such as IL-2. Combination therapies have the potential to produce synergistic effects by targeting multiple immune checkpoints and enhancing the overall immune response against CRC. Conclusion This study demonstrates the potential of NCL-P2 as an immune adjuvant to enhance the efficacy of PD-1 monoclonal antibodies in CRC. Our results confirm that NCL-P2 can penetrate cells and significantly downregulate PD-L1 expression on CRC cells, a key mechanism for immune evasion. Through RNA sequencing and bioinformatics analysis of CT26 cells, we identified USP2 as a key protein involved in this process, suggesting that NCL-P2 targets USP2 to inhibit PD-L1 expression. Current reshearch highlight the potential of NCL-P2 to improve immune responses in CRC, particularly for advanced or metastatic cases. By reducing PD-L1 levels and promoting CTL activation, NCL-P2 could enhance the therapeutic effects of PD-1 monoclonal antibodies, improving survival rates and quality of life for CRC patients. However, Future studies will further explore the molecular mechanisms of NCL-P2 in CRC mouse models and validate its clinical efficacy. This research offers promising prospects for improving CRC treatment and potentially expanding the use of immune therapies in other cancers. Declarations Funding: Youth Foundation of Tianjin Municipal Bureau of Science and Technology (Grant number： 22JCQNJC00520); the National Natural Science Foundation of China (Grant No. 82070206); Tianjin Key Medical Discipline (Specialty) Construction Project (Grant No.TJYXZDXK-053B) Author Contribution Dr. Wu Shan is mainly responsible for conceptualization, data curation, investigation, formal analysis and writing original draft. Dr. Zhang Miao is responsible for investigation, methodology and project administration. Dr. Wang Huaqing is the corresponding author and is mainly responsible for supervision, funding acquisition and manuscript editing. Data Availability Processed analytical datasets are available in Supplementary Materials, and restricted data are available from the corresponding author on reasonable request. The datasets generated during the current study are temporarily unavailable in public repositories due to ongoing analysis/project-specific restrictions, but are available from the corresponding author on reasonable request. Processed analytical datasets are available in the Supplementary Materials, and restricted data may be accessible through collaboration requests subject to institutional approvals. References Siegel RL, Miller KD, Fedewa SA, et al (2017) Colorectal cancer statistics, 2017. CA Cancer J Clin 67:177–193 Zhou J, Zheng R, Zhang S, et al (2021) Colorectal cancer burden and trends: comparison between China and major burden countries in the world. Chinese J cancer Res 33:1 Mahmoud NN (2022) Colorectal cancer: preoperative evaluation and staging. 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J Transl Med 12:97. https://doi.org/10.1186/1479-5876-12-97 Wu S, Chen J, Teo BHD, et al (2023) The axis of complement C1 and nucleolus in antinuclear autoimmunity. Front Immunol 14:1196544 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigureS1.xlsx SupplementaryFigureS2.xlsx SupplementarydataforELISA.xlsx FACSdataofFigure6andFigure4b.zip ConfocalpictureofPDL1expressionandpeptidepenetration.zip OriginalblotforFigure4.1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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02:02:56","extension":"html","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125364,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/2f3681d55ac6b2388d720f89.html"},{"id":93726130,"identity":"27b614ea-ddad-443f-adfd-01dfc9aa04b0","added_by":"auto","created_at":"2025-10-17 02:02:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":139423,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 1.1 (A) TNFα production by monocytes stimulated with NCL-P1, NCL-P2, and NCL-P3 at soluble peptide concentrations (10, 20, 40, 80, and 100 μg/mL). NCL-P2 shows the highest TNFα output at intermediate and higher concentrations, highlighting its superior immune-stimulating capacity.(B) NFα production by monocytes exposed to surface-coated peptides (10, 40, and 160 μg/mL). NCL-P2 consistently outperforms NCL-P1 and NCL-P3, demonstrating enhanced immune activation and dose-dependent pro-inflammatory effects.\u003c/p\u003e","description":"","filename":"Figure1.1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/9f33d44aca5daad1cba1bbd4.jpg"},{"id":93728324,"identity":"95c024f2-ce85-4d1e-83c7-262f8e950297","added_by":"auto","created_at":"2025-10-17 02:10:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":203873,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 1.2 Under the stimulation of NCL-P2, the expression levels of CD83, CD86 and MHCII on the surface of DC cells were significantly increased, inducing imDC to become mDC.(A). Prior to stimulation, high levels of CD1a and CD14 confirmed the immature state of DCs; (B) Stimulation with NCL-P2 resulted in pronounced upregulation of CD83, CD86, and MHCII, demonstrating the potent ability of NCL-P2 to induce dendritic cell maturation, a critical step for efficient APC-mediated T cell activation.\u003c/p\u003e","description":"","filename":"Figure1.2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/3aa4ddb73f0c7b711dbd53c7.jpg"},{"id":93726133,"identity":"a399ed85-9dcc-44c3-bcf6-96cd14c39c3c","added_by":"auto","created_at":"2025-10-17 02:02:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":262492,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 2. NCL-P2 and streptavidin recombinant protein can penetrate into PBMC in both 4 ℃ and 37℃\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/71bedcffe7de470d6b7d9537.jpg"},{"id":93728326,"identity":"95af4e97-fe58-45c0-840c-1a8dd1634a0f","added_by":"auto","created_at":"2025-10-17 02:10:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132065,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 3.1 Tumor growth suppression in CRC model mice treated with NCL-P2 and PD-1 monoclonal antibody. Tumor size (cm³) was measured over time for the control, NCL-P2 alone, anti-PD-1 alone, and combined NCL-P2 + anti-PD-1 groups. The combination treatment showed the most significant reduction in tumor size compared to the other groups.\u003c/p\u003e","description":"","filename":"Figure3.1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/413803dc9e9cc094fc9ad5e3.jpg"},{"id":93728337,"identity":"1bca08c9-7a5e-4474-be23-4b6b2f74639a","added_by":"auto","created_at":"2025-10-17 02:10:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":142574,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 3.2 Immune cell activation and migration in CRC model mice treated with NCL-P2 and PD-1 monoclonal antibody. The combination therapy promoted monocyte and CD4+ T cell migration to CT26 cells and increased CD8+ T cell activation.\u003c/p\u003e","description":"","filename":"Figure3.2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/ec5fab21a1ca4b6850dd5921.jpg"},{"id":93726200,"identity":"09784053-8c82-4548-a4bd-7fdf81d70926","added_by":"auto","created_at":"2025-10-17 02:02:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":163954,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 4.1 Detection of PD-L1 expression in CT26 cells treated with different concentrations of NCL-P2 (0, 10, 50, 100, and 200 μg/ml). PD-L1 expression was evaluated using western blotting (a), flow cytometry (b), and confocal staining (c). The results showed that PD-L1 expression was significantly reduced in cells treated with NCL-P2, with the extent of downregulation positively correlated with NCL-P2 concentration. Flow cytometry analysis indicated a concentration-dependent reduction, while western blot and confocal imaging confirmed this trend, highlighting the most notable decrease at 50 μg/ml.\u003c/p\u003e","description":"","filename":"Figure4.1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/346d6c184ca512f9533b2c9f.jpg"},{"id":93728331,"identity":"32ff59c7-bd56-4451-952a-dba48b4fdd5e","added_by":"auto","created_at":"2025-10-17 02:10:55","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":194381,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 4.2 Data analysis of CT26 cells treated with NCL-P2. (a) Flow cytometry results plot PD-L1 MFI on the y-axis and NCL-P2 concentrations on the x-axis, highlighting dose-dependent suppression of USP2. (b) Bar graph showing the fold change in USP2 mRNA expression relative to control at different NCL-P2 concentrations (10, 50, 100, and 200 μg/ml).\u003c/p\u003e","description":"","filename":"Figure4.2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/a9a03254515ca1c3ec8189f5.jpg"},{"id":104782467,"identity":"4efb68c5-6c45-4b84-b4db-5c4ecfd53637","added_by":"auto","created_at":"2026-03-17 07:57:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1698338,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/dc1032d5-0be0-46a4-818f-f35e6fa7c170.pdf"},{"id":93726185,"identity":"5cd47032-1d98-4250-9d7d-c519b4a38e73","added_by":"auto","created_at":"2025-10-17 02:02:56","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5191959,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/dd62e59ac4bd1beaf857073a.xlsx"},{"id":93726131,"identity":"9231373e-6fc2-4d47-875e-b59eda9d511b","added_by":"auto","created_at":"2025-10-17 02:02:53","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":160105,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/561ded6bdf011216f2de9882.xlsx"},{"id":93726182,"identity":"8cc50a04-8db1-494a-b550-9aef2c2250fc","added_by":"auto","created_at":"2025-10-17 02:02:56","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11615,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarydataforELISA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/4a4320b53cffc9f28ddb96d0.xlsx"},{"id":93726146,"identity":"92fdda7e-5e6b-4eff-a959-9dfa6800ba6d","added_by":"auto","created_at":"2025-10-17 02:02:54","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7436769,"visible":true,"origin":"","legend":"","description":"","filename":"FACSdataofFigure6andFigure4b.zip","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/bf0c42e1e09600aeff8e894d.zip"},{"id":93728355,"identity":"c5fe6473-ea78-4a26-a701-2ac633e1fdc9","added_by":"auto","created_at":"2025-10-17 02:10:58","extension":"zip","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":24185339,"visible":true,"origin":"","legend":"","description":"","filename":"ConfocalpictureofPDL1expressionandpeptidepenetration.zip","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/2108cf150ee9bb653d08c862.zip"},{"id":93726137,"identity":"0beb104a-6218-40eb-8079-5eb71d3a5fdc","added_by":"auto","created_at":"2025-10-17 02:02:53","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":482410,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalblotforFigure4.1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7274573/v1/2c7028deb0c4fab20299f8f1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The mechanistic study of novel immuno-adjuvant NCL-P2 improve the effectiveness of anti- PD-1 in colorectal cancer treatment through suppressing the expression of USP2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal\u0026nbsp;cancer\u0026nbsp;(CRC),\u0026nbsp;as\u0026nbsp;the\u0026nbsp;third\u0026nbsp;most\u0026nbsp;common\u0026nbsp;cause\u0026nbsp;of\u0026nbsp;cancer\u0026nbsp;death\u0026nbsp;in\u0026nbsp;the\u0026nbsp;world\u0026nbsp;[1],\u0026nbsp; has been on the rise in China, ranking second in the incidence of malignant tumors since 2022, reaching as high as 37 per 100,000 people [2, 3]. Up to date, the main treatment for CRC still\u0026nbsp; relies \u0026nbsp; on the \u0026nbsp;combination \u0026nbsp; of \u0026nbsp;surgery, adjuvant chemotherapy and radiotherapy; however,\u0026nbsp; traditional chemotherapy drugs commonly have issues such as multiple side effects, short\u0026nbsp; \u0026nbsp;half-life, low solubility, and unstable efficacy [4]. In recent years, a series of antibody\u0026nbsp; \u0026nbsp;immunotherapy drugs and molecular targeted drugs have emerged in the field of anticancer\u0026nbsp; treatment; among them, the rapid development and widespread clinical application of immune\u0026nbsp; checkpoint inhibitors (ICIs), including PD-1/PD-L1 antibodies, have provided new hope for the\u0026nbsp; treatment of many cancer patients [5-7]. PD-1 monoclonal antibody is a type of\u0026nbsp; \u0026nbsp;immunotherapy drug that can bind to the PD-1 receptor on the surface of T cells,\u0026nbsp; competitively antagonizing the binding of the PD-L1 ligands on the surface of tumor cells to\u0026nbsp; this receptor [8-12]. However, the use of ICIs in the treatment of advanced CRC patients\u0026nbsp; \u0026nbsp;remains controversial; current clinical studies suggest that PD-1 monoclonal antibody is more\u0026nbsp; \u0026nbsp;effective for patients with microsatellite instability-high CRC [13], but it does not significantly\u0026nbsp; improve the overall survival of CRC patients [14]. In addition, effective treatment for patients\u0026nbsp; with metastatic or inoperable CRC is still in searching [15]. Therefore, the search for new, safe,\u0026nbsp; \u0026nbsp;and effective treatments is of great clinical significance and social value in improving the\u0026nbsp; survival rate and quality of life of CRC patients.\u003c/p\u003e\n\u003cp\u003eIn recent years, the application of immunological adjuvants in anti-tumor therapy has gradually become one of the popular research areas in cancer treatment to enhance the anti-tumor effect of immune checkpoint inhibitors [4, 14, 16-18]. In a phase 1/2 clinical trial for metastatic melanoma, research data indicated that the anti-tumor therapeutic effect of a novel IDO/PD- L1 peptide combined with nivolumab is higher than nivolumab alone [19]; this peptide acts as an adjuvant to induce the body to produce specific T cells targeting IDO and PD-L1, thereby killing \u0026nbsp; PD-L1-expressing \u0026nbsp;tumor \u0026nbsp;cells, \u0026nbsp; and activating immune \u0026nbsp;cells \u0026nbsp;to \u0026nbsp; produce \u0026nbsp;anti-tumor factors, further enhancing the anti-tumor activity of nivolumab [20]. Although immunological adjuvants can activate the body\u0026apos;s immune system to produce a series of cytokines, most adjuvants lack strong specificity and have low efficacy, leading to insufficient T cell activation \u0026nbsp;capacity and an inability to effectively improve the anti-tumor therapeutic effect of ICIs [4, 21]. Therefore, exploring adjuvant that can effectively activate the body\u0026apos;s immune cells to exert anti-tumor \u0026nbsp;effects \u0026nbsp;can \u0026nbsp; significantly \u0026nbsp;enhance \u0026nbsp;the \u0026nbsp; therapeutic \u0026nbsp;effect \u0026nbsp;of \u0026nbsp; \u0026nbsp;PD-1 \u0026nbsp; monoclonal antibodies \u0026nbsp;in \u0026nbsp; CRC. \u0026nbsp;A \u0026nbsp;recent \u0026nbsp; study \u0026nbsp;on \u0026nbsp;colorectal \u0026nbsp; cancer \u0026nbsp;reported \u0026nbsp;a \u0026nbsp; ubiquitin-specific processing protease 2, USP2, closely associated with the surface expression of PD-L1 on colorectal \u0026nbsp; cancer \u0026nbsp;cells; \u0026nbsp;the \u0026nbsp; study \u0026nbsp;indicated \u0026nbsp;that \u0026nbsp; USP2 \u0026nbsp;is \u0026nbsp;a \u0026nbsp; novel \u0026nbsp;regulatory \u0026nbsp;factor \u0026nbsp; that promotes stable surface expression of PD-L1, aiding tumor cells in evading the killing effect of CD8+ T cells; moreover, the study confirmed that reducing intracellular expression of USP2 can enhance the anti-tumor effect of PD-1 monoclonal antibodies [22]. In this study, we revealed a novel immuno-adjuvant, \u0026nbsp;NCL-P2, discovered by our research group, which effectively activates the body\u0026apos;s immune system through inducing dendritic cell activation and secretion of anti-tumor cytokines, \u0026nbsp;promoting \u0026nbsp; antigen \u0026nbsp;presentation, \u0026nbsp;initiating downstream T cell killing of tumor cells, and synergistically enhancing the anti-tumor effects of PD-1 monoclonal antibodies in in-vivo experiments. Additionally, NCL-P2 can inhibit the expression of PD-L1 in CRC cells; we identified USP2 as one of the key proteins through which NCL-P2 affects the expression of PD-L1 in CRC cells. In in-vivo experiments in mice, we found that NCL-P2 down regulates the expression of PD- L1 on the surface of tumor cells by inhibiting USP2 expression, activating the body\u0026apos;s immune response, and further confirmed USP2 as a key molecule for NCL-P2 to activate the immune response \u0026nbsp;in \u0026nbsp;CRC. \u0026nbsp; This \u0026nbsp; study \u0026nbsp;elucidated \u0026nbsp; the \u0026nbsp;molecular \u0026nbsp;mechanism \u0026nbsp; by \u0026nbsp;which \u0026nbsp;NCL-P2 enhances the efficacy of PD-1 monoclonal antibodies in CRC, providing a theoretical basis for the application of NCL-P2 in the treatment of CRC, with the aim of providing new methods and important theoretical support for the clinical treatment of CRC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e1.Materials\u003c/p\u003e\n\u003cp\u003eCT26 colon cancer cell line purchased from abcam; DMEM high glucose culture medium, RPMI-1640 culture medium, fetal bovine serum, trypsin, phosphate buffered saline (PBS) purchased from American Gibco company; inducing agents IL-2, IL-7, IL-15, IL-18, IL-21, OKT3 purchased from American R\u0026amp;D company; TNF and IL-1\u0026szlig; cytokine detection ELISA kits purchased from American Santa Cruz company; antibodies used for flow cytometry purchased from American abcam; CCK-8 kit purchased from Chinese Dojindo Biological company. Mice \u0026nbsp; purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., free access to food and water during the experiment; animal handling during the experiment conforms to animal ethics standards. Animal experiments were approved by the Experimental Animal Management and Ethics Committee of Tianjin Union Medical Center of Nankai University.All experimental procedures involving live vertebrates were reviewed and approved by the Experimental Animal Management and Ethics Committee of Tianjin Union Medical Center of Nankai University.(Ethical code:2024-SYDWLL-000063. Approval Protocol Number:[2022033]). The experimental procedures were conducted following the National Institutes of Health (NIH) Guidelines for the Protection and Use of Laboratory Animals (NIH Publication No., as per regulations 85-23, updated in 1996. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e2. Methods:\u003c/p\u003e\n\u003cp\u003e2.1. Peripheral blood mononuclear cells (PBMCs) separation: PBMCs were separated by Ficoll \u0026nbsp;density gradient centrifugation method, collected and centrifuged to obtain plasma. Monocytes, macrophages, dendritic cells, etc. were differentiated and subsequent cell experiments were conducted using the white membrane layer. Microscopic counting was performed, along with flow cytometry and protein imprinting analysis.\u003c/p\u003e\n\u003cp\u003e2.2. Establishment and grouping of mouse colorectal cancer models: Log-phase CT26 cells were taken, digested with trypsin to make single-cell suspension, and injected subcutaneously into the skin of the hind limbs of mice at a concentration of 2 \u0026times; 106 \u0026nbsp;cells/each (125 \u0026mu;L). Mice were randomly divided into the anti PD-1 antibody group (PD-1 group) and the PBS group. In the PD-1 group, mice were given anti PD-1 antibody and control peptide segment by intraperitoneal injection on the 6th and 9th days after tumor bearing, respectively. Tumor measurement and recording were started on the 7th day after tumor bearing, and tumor \u0026nbsp;volume was calculated every day (\u003cimg width=\"123\" height=\"29\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e).Animals were euthanized if tumors exceeded 1,500 mm\u0026sup3; or 20 mm in any dimension. Mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (10 mg/kg). For euthanasia, deeply anesthetized mice received an overdose of sodium pentobarbital (150 mg/kg, IP) followed by bilateral thoracotomy to ensure death. All procedures were approved by the Experimental Animal Management and Ethics Committee of Tianjin Union Medical Center of Nankai University IACUC and adhered to AVMA Euthanasia Guidelines (2020).\u003c/p\u003e\n\u003cp\u003e2.3. Flow cytometry: Flow cytometric analysis was performed using immunofluorescence labeling. Immune cells to be tested were suspended in PBS after washing twice, incubated with target antibodies diluted in 2% FBS/PBS (incubation conditions: 4\u0026deg;C, protected from light, 30 min). Flow cytometry analysis was performed after PBS washing.\u003c/p\u003e\n\u003cp\u003e2.4. Western blotting experiment: Extract tumor tissue or collect immune cells to be detected, use RIPA lysis buffer to extract total protein, separate proteins by SDS-PAGE gel electrophoresis, transfer to PVDF membrane, block with 5% skim milk in TBST for 1h after transfer; incubate with primary antibody overnight at 4oC, wash the membrane with TBST for 3 times, then incubate with the secondary antibody conjugated with horseradish peroxidase;finally, expose and visualize the target protein using Bio-Rad instrument.\u003c/p\u003e\n\u003cp\u003e2.5. Immunofluorescence Staining: The target cells to be detected were seeded on cover slips. After 24h, the cells were washed once with PBS, fixed with 4% paraformaldehyde at room temperature for 30min, permeabilized with 0.2% Triton X-100 for 5min washed three times with PBS for 10min each time, and then blocked with 5% BSA at room temperature for 30 min. After blocking, the cells were incubated with the primary antibody at 4 \u003csup\u003eo\u003c/sup\u003eC overnight. After washing with PBS, the secondary antibody was added in the dark for 30min, followed by three washes with PBS before staining the cell nuclei with DAPI, and then observed under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e2.6. Statistical methods: Statistical analysis was performed using Graph Pad Prism 10.0 software. Data conforming to a normal distribution are presented as mean (average, AVG) \u0026plusmn;standard deviation (SD). Group comparisons were made using independent sample t-test, and differences between groups were analyzed using two-way ANOVA. P\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. The novel immune adjuvant NCL-P2 can activate various antigen-presenting cells (APC).\u003c/p\u003e\n\u003cp\u003eThe novel immune adjuvant NCL-P2 effectively stimulates various APCs to produce pro-inflammatory cytokines, particularly TNF\u0026alpha;. Across soluble peptide concentrations (Figure 1.1a), NCL-P2 showed superior potency compared to NCL-P1 and NCL-P3. At 10 \u0026mu;g/mL, all peptides induced minimal TNF\u0026alpha; production. At 20 \u0026mu;g/mL, NCL-P1 reached ~680 pg/mL, while NCL-P2 remained lower (~430 pg/mL). A significant boost was observed at 40 \u0026mu;g/mL, where NCL-P2 produced 14,040 pg/mL, more than double NCL-P1 (~7,000 pg/mL). Higher concentrations (80 and 100 \u0026mu;g/mL) showed NCL-P2 maintaining TNF\u0026alpha; production levels of ~41,040 and ~42,040 pg/mL, surpassing NCL-P3.Similarly, in surface-coated peptide experiments (Figure 1.1b), NCL-P2 induced greater TNF\u0026alpha; generation than its counterparts. At 10 \u0026mu;g/mL, TNF\u0026alpha; production was minimal (~98 pg/mL for NCL-P2 and ~10 pg/mL for NCL-P1), while NCL-P3 remained inactive. At 40 \u0026mu;g/mL, NCL-P2 elicited 1,110 pg/mL, nearly triple NCL-P1 (~380 pg/mL). At 160 \u0026mu;g/mL, NCL-P2 reached 1,240 pg/mL, compared to NCL-P1 (~410 pg/mL), confirming NCL-P2\u0026apos;s superior immune activation. NCL-P3 consistently showed negligible responses in both conditions, reinforcing the potent pro-inflammatory role of NCL-P2 in immune modulation. This immune adjuvant also induces the transformation of immature dendritic cells (imDCs) into mature dendritic cells (mDCs), enhancing APC function and promoting downstream T cell activation (Figure 1.2). Under NCL-P2 stimulation, the surface expression of key markers, including CD83, CD86, and MHCII, significantly increased, signifying the maturation process.\u003c/p\u003e\n\u003cp\u003e2. NCL-P2 can enter immune cells through passive transportation.\u003c/p\u003e\n\u003cp\u003eNCL-P2, a novel immune adjuvant, exhibits the ability to penetrate cell membranes and enter the cytoplasm and nucleus of immune cells. Experimental results demonstrate that NCL-P2 can effectively enter peripheral blood mononuclear cells (PBMCs). This was confirmed by the use of a recombinant protein composed of a 66 kDa medium-sized antigen, streptavidin, which was tagged with NCL-P2. Importantly, the penetration of NCL-P2 into PBMCs occurred under both cold (4\u0026deg;C) and physiological (37\u0026deg;C) conditions, suggesting that the transport mechanism may not be temperature-dependent. These findings support the notion that NCL-P2 can be delivered into immune cells, potentially enhancing its adjuvant activity and facilitating immune modulation at the cellular level. (Figure 2).\u003c/p\u003e\n\u003cp\u003e3. NCL-P2 in combination with PD-1 monoclonal antibody can inhibit the growth of tumors in CRC model mice and activate immune cells.\u003c/p\u003e\n\u003cp\u003eIn vivo experiments using a CRC mouse model demonstrated that the combination of NCL-P2 and PD-1 monoclonal antibody significantly suppressed tumor growth compared to treatment with either agent alone or the control group. Tumor size measurements (Figure 3.1) indicated that the control group exhibited a continuous and rapid increase, starting from 0.4 cm\u0026sup3; and peaking at approximately 1.8 cm\u0026sup3;. NCL-P2 treatment alone reduced tumor growth, reaching a maximum of 1.4 cm\u0026sup3;. Anti-PD-1 monoclonal antibody treatment resulted in a further reduction, with tumor size reaching 1.2 cm\u0026sup3;. The combined NCL-P2 + anti-PD-1 treatment showed the greatest inhibition, with tumor growth reaching only 1.1 cm\u0026sup3;. Although all groups exhibited a gradual upward trend in tumor size over time, the combination therapy clearly demonstrated superior efficacy in limiting tumor progression (Figure \u0026nbsp;3.1), \u0026nbsp;Further immune cell profiling (Figure 3.2) revealed enhanced immune responses with combination treatment. The migration of monocytes and CD4+ T cells to CT26 cells was markedly increased, reflecting enhanced immune surveillance and response. Additionally, the activation of CD8+ T cells was significantly promoted, indicating that NCL-P2 synergizes with PD-1 blockade to stimulate cytotoxic T cell-mediated antitumor activity. These findings suggest that NCL-P2 amplifies the immune activation initiated by PD-1 inhibition, providing a potent therapeutic strategy for CRC by simultaneously targeting tumor growth and enhancing immune cell engagement. (Figure 3.2).\u003c/p\u003e\n\u003cp\u003e4. \u0026nbsp;NCL-P2 \u0026nbsp; down \u0026nbsp;regulates \u0026nbsp;the \u0026nbsp; expression \u0026nbsp;of \u0026nbsp;PD-L1 \u0026nbsp; on \u0026nbsp;the \u0026nbsp;surface \u0026nbsp; of \u0026nbsp;CT26 \u0026nbsp;cells. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNCL-P2 effectively downregulates PD-L1 expression on the surface of CT26 cells. Pre-experimental results showed that treating CT26 cells with various concentrations of NCL-P2 reduced PD-L1 levels, as illustrated in Figure 4.1.\u003c/p\u003e\n\u003cp\u003ePD-L1 expression was assessed using western blotting, flow cytometry, and confocal staining. The x-axis represents NCL-P2 concentrations (10\u0026nbsp;\u0026mu;g/ml, 50\u0026nbsp;\u0026mu;g/ml, 100\u0026nbsp;\u0026mu;g/ml, and 200\u0026nbsp;\u0026mu;g/ml), with the control group receiving no treatment (Figure 4.1a-c). PD-L1 detection peaked at 50\u0026nbsp;\u0026mu;g/ml, followed by 10\u0026nbsp;\u0026mu;g/ml and 100\u0026nbsp;\u0026mu;g/ml. In contrast, GADP1[5] expression remained consistently high across all conditions except for a notable decrease at 50\u0026nbsp;\u0026mu;g/ml. These results highlight the concentration-dependent regulatory effect of NCL-P2 on PD-L1 expression, potentially mediated by USP2 inhibition (Figure 4.1);\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFlow cytometry analysis (Figure 4.2b) demonstrated a dose-dependent decrease in PD-L1 Mean Fluorescence Intensity (MFI), with NCL-P2 treatment resulting in progressive downregulation of PD-L1. USP2 expression, as measured by MFI, was highest in the control group (approximately 400) but was markedly reduced with increasing concentrations of NCL-P2, peaking at 680 MFI for treated cells, confirming the inhibitory effect of NCL-P2 on USP2 expression.\u003c/p\u003e\n\u003cp\u003eThe control group maintained a baseline fold change of 1.0. Treatment with 10 \u0026mu;g/ml NCL-P2 showed minimal impact, with mRNA expression levels remaining similar to the control. However, exposure to 50 \u0026mu;g/ml significantly reduced mRNA expression to 0.8-fold, and further decreases were observed at 100 \u0026mu;g/ml (0.5-fold) and 200 \u0026mu;g/ml (0.45-fold). These data demonstrate that increasing NCL-P2 concentrations effectively inhibit USP2 expression, correlating with reduced PD-L1 levels. This suggests a mechanistic pathway where NCL-P2 downregulates PD-L1 expression through the suppression of USP2, highlighting its potential therapeutic role in immune modulation.(Figure 4.2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigates the molecular mechanisms by which the novel immune adjuvant NCL-P2 enhances the efficacy of PD-1 monoclonal antibodies in CRC [33\u0026ndash;35]. ICIs, such as PD-1/PD-L1 monoclonal antibodies, have transformed cancer immunotherapy by reinvigorating exhausted T cells and restoring antitumor immunity [34]. However, their clinical effectiveness in CRC remains limited due to immune evasion mechanisms and insufficient activation of CTLs [36]. One of the key challenges in improving ICI efficacy is overcoming the persistent expression of PD-L1 on tumor cells, which binds to PD-1 on T cells, inhibiting their cytotoxic function and facilitating tumor progression [37].\u003c/p\u003e\n\u003cp\u003eNCL-P2, a peptide-based immune adjuvant identified in our research, offers a promising solution by modulating the immune microenvironment and enhancing T cell-mediated immune responses. Based on bioinformatics analysis and previous experimental data, we hypothesize that NCL-P2 inhibits the expression of USP2, a critical regulator of PD-L1 stability on the tumor cell surface [31, 38]. USP2 stabilizes PD-L1 by preventing its degradation, thereby allowing tumor cells to escape immune surveillance. Our results demonstrate that NCL-P2 reduces PD-L1 surface expression in CRC cells by suppressing USP2, effectively reversing immune suppression and enabling robust CTL activation [31]. This mechanism represents a significant advancement in the strategic use of immune adjuvants to boost the efficacy of ICIs. Furthermore, the ability of NCL-P2 to penetrate cells and directly influence intracellular pathways distinguishes it from traditional immune adjuvants, which primarily act on surface receptors. Unlike other adjuvants with single-target mechanisms, NCL-P2 activates multiple signaling pathways through TLRs on antigen-presenting cells [39], enhancing dendritic cell maturation and promoting the expression of costimulatory molecules [40, 41]. These findings highlight the multifunctional nature of NCL-P2, which addresses the limitations of conventional ICIs, including low response rates and variable patient outcomes.\u003c/p\u003e\n\u003cp\u003eBy integrating NCL-P2 with PD-1 monoclonal antibodies, this study opens new therapeutic avenues for CRC, especially in patients with advanced or metastatic disease where treatment options are limited. The mechanistic insights provided by our research not only underscore the therapeutic potential of targeting USP2 to regulate PD-L1 but also pave the way for broader applications in immunotherapy. This combination strategy could significantly improve immune checkpoint blockade outcomes, offering a more comprehensive and effective approach to harnessing the immune system for cancer treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunotherapy and PD-1/PD-L1 Inhibition in Cancer Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn recent years, ICIs targeting the PD-1/PD-L1 axis have revolutionized cancer therapy by unleashing the body\u0026apos;s immune system to attack tumors. However, the efficacy of PD-1 monoclonal antibodies is often limited by tumor-mediated immune evasion mechanisms. Tumors upregulate PD-L1 on their cell surfaces to bind with PD-1 on T cells, thereby inhibiting T-cell activation and suppressing anti-tumor immunity [5]. This immune evasion mechanism is particularly prominent in CRC, where high PD-L1 expression correlates with poor prognosis [1]. Therefore, targeting the regulatory mechanisms of PD-1/PD-L1 interaction is critical for enhancing the effectiveness of PD-1-based therapies.\u003c/p\u003e\n\u003cp\u003eIn CRC, USP2 has been identified as a key regulator of PD-L1 stabilization on the tumor cell surface. USP2 prevents the degradation of PD-L1 by deubiquitinating it, thus promoting immune escape by inhibiting CTL-mediated killing of tumor cells [31, 42]. Inhibition of USP2 in preclinical models has shown promise in reducing PD-L1 levels and reactivating CTLs, resulting in reduced tumor growth and improved immune response [43]. Our study builds upon these findings, demonstrating that NCL-P2 inhibits USP2 expression, thus destabilizing PD-L1 and enhancing CTL activation. These results are consistent with the hypothesis that USP2 inhibition is a viable therapeutic strategy to potentiate PD-1/PD-L1 immunotherapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Role of Immune Adjuvants in Enhancing Anti-Tumor Immunity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile PD-1/PD-L1 inhibitors have shown clinical success in a variety of cancers, including melanoma, non-small cell lung cancer, and RCC, their efficacy in CRC has been inconsistent. This variability may be due to differences in the tumor microenvironment, immune cell infiltration, and the lack of effective immune adjuvants to stimulate a robust immune response. Traditional immune adjuvants, such as CpG-ODN (TLR9 activator) and Imiquimod (TLR7 activator), have been explored to enhance immune responses but are limited by their narrow target activation profiles and unstable efficacy [39, 43\u0026ndash;50]. Moreover, these adjuvants often activate only a subset of immune pathways, resulting in suboptimal immune activation and therapeutic outcomes.\u003c/p\u003e\n\u003cp\u003eTo address these limitations, NCL-P2 represents a promising alternative. Our previous work identified NCL-P2, a 36-amino acid peptide, which activates multiple APCs[43, 51], including DCs[52], and induces the production of pro-inflammatory cytokines, maturation of DCs, and the generation of specific immune responses [38]. Notably, NCL-P2 activates Toll-like receptors (TLRs) on immune cells, promoting the maturation of imDCs[43, 53] into mDCs [54] and upregulating costimulatory molecules such as CD80, CD83, CD86, and MHCII [51, 53, 55]. This enhances the capacity of DCs to activate T cells, driving CTL differentiation and anti-tumor immunity. In preclinical CRC mouse models, the combination of NCL-P2 and PD-1 monoclonal antibodies significantly inhibited tumor growth compared to PD-1 treatment alone, providing strong evidence for the potential of NCL-P2 as an adjuvant in CRC immunotherapy.\u003c/p\u003e\n\u003cp\u003eFurthermore, NCL-P2 demonstrates a unique advantage over conventional immune adjuvants by not only activating immune cell surface TLRs but also penetrating cell membranes and entering the cytoplasm and nucleus. This dual function allows NCL-P2 to exert a more profound and sustained immune activation, offering a potential therapeutic strategy that could overcome the limitations of traditional adjuvants [56].\u003c/p\u003e\n\u003cp\u003eDespite the promising results of combining NCL-P2 with PD-1 monoclonal antibodies, several limitations need to be addressed. First, the precise molecular mechanisms by which NCL-P2 inhibits USP2 and destabilizes PD-L1 remain to be fully elucidated. Future studies should focus on identifying the specific molecular pathways through which NCL-P2 interacts with USP2 and PD-L1, as well as any potential off-target effects. Additionally, while our study demonstrates the efficacy of NCL-P2 in mouse models, further research is needed to assess its safety and efficacy in human clinical trials. One challenge in translating these results to the clinic is the potential variability in immune responses across different patient populations. Factors such as tumor heterogeneity, immune cell composition, and PD-L1 expression levels may influence the therapeutic outcome, highlighting the need for personalized approaches in CRC immunotherapy.\u003c/p\u003e\n\u003cp\u003eMoreover, the use of NCL-P2 as an immune adjuvant could benefit from further optimization. While NCL-P2 shows promise in activating multiple immune pathways, its clinical efficacy may be enhanced by combining it with other immune modulators, such as checkpoint inhibitors targeting CTLA-4 or TIM-3, or cytokines such as IL-2. Combination therapies have the potential to produce synergistic effects by targeting multiple immune checkpoints and enhancing the overall immune response against CRC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates the potential of NCL-P2 as an immune adjuvant to enhance the efficacy of PD-1 monoclonal antibodies in CRC. Our results confirm that NCL-P2 can penetrate cells and significantly downregulate PD-L1 expression on CRC cells, a key mechanism for immune evasion. Through RNA sequencing and bioinformatics analysis of CT26 cells, we identified USP2 as a key protein involved in this process, suggesting that NCL-P2 targets USP2 to inhibit PD-L1 expression. Current reshearch highlight the potential of NCL-P2 to improve immune responses in CRC, particularly for advanced or metastatic cases. By reducing PD-L1 levels and promoting CTL activation, NCL-P2 could enhance the therapeutic effects of PD-1 monoclonal antibodies, improving survival rates and quality of life for CRC patients. However, Future studies will further explore the molecular mechanisms of NCL-P2 in CRC mouse models and validate its clinical efficacy. This research offers promising prospects for improving CRC treatment and potentially expanding the use of immune therapies in other cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding: Youth Foundation of Tianjin Municipal Bureau of Science and Technology (Grant number： 22JCQNJC00520); the National Natural Science Foundation of China (Grant No. 82070206); Tianjin Key Medical Discipline (Specialty) Construction Project (Grant No.TJYXZDXK-053B)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDr. Wu Shan is mainly responsible for conceptualization, data curation, investigation, formal analysis and writing original draft. Dr. Zhang Miao is responsible for investigation, methodology and project administration. Dr. Wang Huaqing is the corresponding author and is mainly responsible for supervision, funding acquisition and manuscript editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eProcessed analytical datasets are available in Supplementary Materials, and restricted data are available from the corresponding author on reasonable request. The datasets generated during the current study are temporarily unavailable in public repositories due to ongoing analysis/project-specific restrictions, but are available from the corresponding author on reasonable request. Processed analytical datasets are available in the Supplementary Materials, and restricted data may be accessible through collaboration requests subject to institutional approvals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Fedewa SA, et al (2017) Colorectal cancer statistics, 2017. CA Cancer J Clin 67:177\u0026ndash;193\u003c/li\u003e\n\u003cli\u003eZhou J, Zheng R, Zhang S, et al (2021) Colorectal cancer burden and trends: comparison between China and major burden countries in the world. Chinese J cancer Res 33:1\u003c/li\u003e\n\u003cli\u003eMahmoud NN (2022) Colorectal cancer: preoperative evaluation and staging. Surg Oncol Clin 31:127\u0026ndash;141\u003c/li\u003e\n\u003cli\u003eGupta M, Wahi A, Sharma P, et al (2022) Recent advances in cancer vaccines: challenges, achievements, and futuristic prospects. Vaccines 10:2011\u003c/li\u003e\n\u003cli\u003eManzoor U, Ali A, Ali SL, et al (2023) Mutational screening of GDAP1 in dysphonia associated with Charcot-Marie-Tooth disease: clinical insights and phenotypic effects. J Genet Eng Biotechnol 21:. https://doi.org/10.1186/s43141-023-00568-9\u003c/li\u003e\n\u003cli\u003ePareyson D, Saveri P, Pisciotta C (2017) New developments in Charcot\u0026ndash;Marie\u0026ndash;Tooth neuropathy and related diseases. 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J Genet Eng Biotechnol 23:100451\u003c/li\u003e\n\u003cli\u003eAli A, Ali A South African Journal of Botany Integrated Computational modeling and in-silico validation of flavonoids-Alliuocide G and Alliuocide A as therapeutic agents for their multi-target potential : Combination of\u003c/li\u003e\n\u003cli\u003eLorentzen CL, Kjeldsen JW, Ehrnrooth E, et al (2023) Long-term follow-up of anti-PD-1 na\u0026iuml;ve patients with metastatic melanoma treated with IDO/PD-L1 targeting peptide vaccine and nivolumab. J Immunother Cancer 11:\u003c/li\u003e\n\u003cli\u003eKjeldsen JW, Lorentzen CL, Martinenaite E, et al (2021) A phase 1/2 trial of an immune-modulatory vaccine against IDO/PD-L1 in combination with nivolumab in metastatic melanoma. Nat Med 27:2212\u0026ndash;2223\u003c/li\u003e\n\u003cli\u003eHuang T, Liu L, Lv Z, et al (2022) Recent advances in DNA vaccines against lung cancer: A mini review. 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Blood, J Am Soc Hematol 123:678\u0026ndash;686\u003c/li\u003e\n\u003cli\u003eAndreae S, Buisson S, Triebel F (2003) MHC class II signal transduction in human dendritic cells induced by a natural ligand, the LAG-3 protein (CD223). Blood 102:2130\u0026ndash;2137. https://doi.org/10.1182/blood-2003-01-0273\u003c/li\u003e\n\u003cli\u003eCamisaschi C, De Filippo A, Beretta V, et al (2014) Alternative activation of human plasmacytoid DCs in vitro and in melanoma lesions: involvement of LAG-3. J Invest Dermatol 134:1893\u0026ndash;1902. https://doi.org/10.1038/jid.2014.29\u003c/li\u003e\n\u003cli\u003eRomano E, Michielin O, Voelter V, et al (2014) MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial. J Transl Med 12:97. https://doi.org/10.1186/1479-5876-12-97\u003c/li\u003e\n\u003cli\u003eWu S, Chen J, Teo BHD, et al (2023) The axis of complement C1 and nucleolus in antinuclear autoimmunity. Front Immunol 14:1196544\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"immune checkpoint treatment, micro-environment, immuno-adjuvant, anti-PD-1 antibody, antigen-presenting cells","lastPublishedDoi":"10.21203/rs.3.rs-7274573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7274573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eColorectal cancer (CRC) has risen to second place in the incidence rate of malignant tumors in China. However, treatments for advanced CRC are not currently effective, andtreatment of anti-PD-1 monoclonal antibodies alone has no significant effect on improving the overall survival rate of CRC patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eOur research group has discovered a novel immune adjuvant NCL-P2 that can synergistically enhance the anti-CRC therapeutic effect of anti-PD-1 antibody.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003ePreliminary data shows that NCL-P2 can inhibit the expression of PD-L1 on the surface of CRC cells, activate dendritic cells, promote the secretion of cytokines such as TNFα and IL-1β , present tumor antigens, and then initiate downstream cytotoxic T cells to kill tumor cells. We found that USP2 is one of the key proteins that down regulate the expression of PD-L1 on the surface of CRC cells by NCL-P2. This project takes the CRC mouse model as the research object and explores the molecular mechanism of USP2 's impact on the expression of PD-L1 in CRC cells by activating or knocking out USP2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThis research explores how USP2 participates in the immune activation of NCL-P2 in CRC and enhances the anti-CRC efficacy of anti-PD-1 antibody, which provides new targets and theoretical support for the clinical treatment of colorectal cancer.\u003c/p\u003e","manuscriptTitle":"The mechanistic study of novel immuno-adjuvant NCL-P2 improve the effectiveness of anti- PD-1 in colorectal cancer treatment through suppressing the expression of USP2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 02:02:45","doi":"10.21203/rs.3.rs-7274573/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dc8d6ec7-ff59-406a-a576-db416f4d895a","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56161888,"name":"Biological sciences/Cancer"},{"id":56161889,"name":"Biological sciences/Immunology"},{"id":56161890,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-03-16T04:25:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-17 02:02:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7274573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7274573","identity":"rs-7274573","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}