Use of DUSP4 in preparation of a marker for predicting efficacy of immunotherapy for hepatocellular carcinoma and a sensitizing drug

Abstract

The application discloses application of DUSP4 in preparation of a marker for predicting the curative effect of immunotherapy of hepatocellular carcinoma and a sensitizing drug, and belongs to the technical field of biological medicine.The application finds that high expression of dual specificity phosphatase 4 (DUSP4) is significantly related to high response rate and long survival period of an immunological checkpoint blocking therapy for a hepatocellular carcinoma patient.DUSP4 promotes CD8+ T cell and NK cell infiltration and remodels an immune microenvironment by inhibiting a TGF-beta signal path, down-regulating an immunosuppressive factor and up-regulating an antigen presenting molecule and a chemotactic factor.The application provides a kit and a method for detecting the expression level of DUSP4 to predict an immunotherapy response, and a combined drug composition comprising a DUSP4 activator or a TGF-beta inhibitor and an immunological checkpoint inhibitor.Experiments prove that up-regulation of the expression of DUSP4 can significantly enhance T cell killing function, and produces a synergistic anti-tumor effect with an anti-PD-1 antibody.The application provides a new strategy for precise stratified treatment and overcoming immunological drug resistance of hepatocellular carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and tumor immunotherapy technology. Specifically, this invention relates to the application of the bispecific phosphatase 4 (DUSP4) gene and its expression products in the diagnosis and treatment of hepatocellular carcinoma (HCC), particularly as a biomarker for predicting the efficacy of immune checkpoint blockade (ICB) therapy, and therapeutic strategies for reshaping the tumor immune microenvironment, inhibiting the TGF-β signaling pathway, and enhancing the anti-tumor activity of T cells by regulating DUSP4 expression levels or activity. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a highly prevalent and deadly malignant tumor worldwide. In recent years, immune checkpoint blockade (ICB) therapy, represented by anti-PD-1 / PD-L1 antibodies, has brought new hope to patients with advanced HCC. However, clinical data show that only about 15%-20% of HCC patients achieve an objective response to ICB monotherapy, with most patients exhibiting primary or secondary resistance. This difference in efficacy is mainly attributed to the highly heterogeneous tumor immune microenvironment (TIME) of HCC, particularly the "cold tumor" characteristics (i.e., insufficient effector T cell infiltration) and the overactivity of immunosuppressive factors.

[0003] The transforming growth factor-β (TGF-β) signaling pathway plays a dual role in HCC progression: suppressing tumors in the early stages and promoting epithelial-mesenchymal transition (EMT), angiogenesis, and immune escape in the later stages. High levels of TGF-β inhibit the proliferation and killing function of CD8+ T cells and recruit regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), thereby constructing an immunosuppressive microenvironment. Although TGF-β inhibitors are under development, their clinical application is limited due to their systemic toxicity and the lack of precise patient stratification methods.

[0004] Bispecific phosphatase 4 (DUSP4) is a member of the mitogen-activated protein kinase (MAPK) phosphatase family. It is known to function as a tumor suppressor or oncogene in certain cancers, with specific functions exhibiting tissue specificity. Previous research by the applicant found that DUSP4 can regulate sorafenib-induced ferroptosis, but its specific mechanism of action in the regulation of the HCC immune microenvironment and immunotherapy response remains unclear. Currently, there is a lack of specific molecular markers in clinical practice that can accurately predict the response of HCC patients to ICB therapy, and there is also a lack of effective combination therapy strategies based on specific molecular mechanisms to enhance T cell infiltration.

[0005] Therefore, it is urgent to discover new key molecular targets, elucidate their mechanisms of regulating the HCC immune microenvironment, and develop corresponding diagnostic biomarkers and treatment regimens to improve the accuracy and effectiveness of HCC immunotherapy. Summary of the Invention

[0006] The primary objective of this invention is to provide DUSP4 as a biomarker for predicting the response of hepatocellular carcinoma patients to immune checkpoint blockade therapy (ICB).

[0007] Another objective of this invention is to reveal the molecular mechanism by which DUSP4 remodels the tumor immune microenvironment and enhances the anti-tumor activity of T cells by inhibiting the TGF-β signaling pathway.

[0008] Another object of the present invention is to provide a patient screening method, a detection kit, and a pharmaceutical composition that enhances the efficacy of immunotherapy based on DUSP4 expression levels.

[0009] An application of a DUSP4 detection reagent designed for this purpose in the preparation of a product for predicting the response of hepatocellular carcinoma patients to immune checkpoint blockade therapy, wherein the product makes predictions by detecting the expression level of DUSP4 in the patient's biological sample based on the following logic; When the expression level of DUSP4 is higher than a preset threshold, the patient is determined to be sensitive to the immune checkpoint blockade therapy or have a high probability of response. When the expression level of DUSP4 is lower than or equal to the preset threshold, the patient is determined to be insensitive to the immune checkpoint blockade therapy or have a low probability of response.

[0010] The immune checkpoint blockade therapy includes administering one or more inhibitors selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.

[0011] The biological samples were selected from tumor tissue sections, fresh frozen tissue, peripheral blood mononuclear cells, circulating tumor cells, or exosomes; The detection indicators for DUSP4 include the transcriptional level of DUSP4 mRNA or the expression level of DUSP4 protein. The detection reagents are selected from primer pairs that specifically amplify the DUSP4 gene, probes that specifically hybridize the DUSP4 gene, or antibodies that specifically bind to the DUSP4 protein.

[0012] A kit for predicting the efficacy of immunotherapy for hepatocellular carcinoma, comprising: (a) Specific reagents for detecting DUSP4 expression levels; (b) Internal reference gene detection reagent; (c) The judgment criteria description, which describes the correspondence between DUSP4 expression level and immunotherapy response, indicating that high DUSP4 expression predicts a good treatment prognosis.

[0013] The use of a DUSP4 activator in the preparation of a pharmaceutical composition for enhancing T cell-mediated antitumor immunity. The DUSP4 activator promotes the infiltration, proliferation and killing function of effector T cells by increasing the expression level or activity of DUSP4 in tumor cells and inhibiting the TGF-β signaling pathway.

[0014] The DUSP4 activator is selected from: (I) A gene overexpression vector containing the DUSP4 coding sequence, preferably an adenovirus vector, a lentivirus vector, or an AAV vector; (II) DUSP4 protein or its functional fragments; (III) Small molecule compounds that can upregulate DUSP4 transcription or translation; (IV) Inhibitors that can stabilize DUSP4 protein.

[0015] A pharmaceutical composition for treating hepatocellular carcinoma, comprising a therapeutically effective amount of: (A) Immune checkpoint inhibitors, and (B) DUSP4 expression upregulators; Components (A) and (B) are formulated as individual formulations, mixed formulations, or sequential administration formulations.

[0016] The pharmaceutical composition also contains a TGF-β signaling pathway inhibitor, used to screen patients with low DUSP4 expression before treatment, or to simulate the immune microenvironment state of high DUSP4 expression by directly blocking the TGF-β pathway when DUSP4 cannot be directly upregulated.

[0017] A method for screening hepatocellular carcinoma patients suitable for adoptive cell therapy (ACT) includes the following steps: (1) Obtain liver cancer samples from the individuals to be tested; (2) Determine the expression level of DUSP4 in the sample; (3) Compare the expression level with a reference threshold; (4) If the expression level is higher than the reference threshold, it is recommended that the individual receive chimeric antigen receptor T-cell (CAR-T) therapy or tumor-infiltrating lymphocyte (TIL) therapy; (5) If the expression level is lower than the reference threshold, it is recommended to first perform DUSP4 upregulation treatment or combined TGF-β inhibitor treatment before implementing adoptive cell therapy. A method for assessing the state of the tumor immune microenvironment in hepatocellular carcinoma uses the expression level of DUSP4 as a surrogate indicator to infer the status of the following parameters in the tumor microenvironment: (I) Infiltration density of CD8+ T cells and NK cells; (II) Expression activity of genes related to antigen processing and presentation; (III) The degree of activation of the TGF-β signaling pathway; (IV) Enrichment of myeloid-derived suppressor cells (MDSCs) and M2 macrophages; Among them, high DUSP4 expression indicates a state of high infiltration of effector immune cells, active antigen presentation, suppression of the TGF-β pathway, and a reduction in immunosuppressive cells.

[0018] In summary, the present invention adopts the following technical solution: Firstly, it provides the application of DUSP4 detection reagents in the preparation of products for predicting the response of hepatocellular carcinoma patients to immune checkpoint blockade therapy.

[0019] The immune checkpoint blockade therapy includes, but is not limited to, treatment using anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, or combinations thereof.

[0020] The prediction logic is as follows: detect the expression level of DUSP4 in the patient's tumor sample. If the DUSP4 expression level is higher than the preset cut-off value, the patient is determined to be sensitive to the immune checkpoint blockade therapy or have a high probability of response. Conversely, if the DUSP4 expression level is lower than or equal to the cut-off value, the patient may be resistant to the drug or have a low response rate.

[0021] Secondly, a method for enhancing T cell-mediated anti-tumor immunity is provided, including administering an effective amount of DUSP4 activator or DUSP4 expression vector to the subject to increase the expression level of DUSP4 in tumor cells, thereby inhibiting the TGF-β signaling pathway and promoting effector T cell infiltration and activation.

[0022] Thirdly, a pharmaceutical composition for treating hepatocellular carcinoma is provided, comprising: (a) Immune checkpoint inhibitors, and (b) DUSP4 expression upregulators (such as DUSP4 gene overexpression vectors, DUSP4 protein or peptides, or small molecule activators). Alternatively, for treatment-resistant patients with low DUSP4 expression, the pharmaceutical composition comprises: (a) Immune checkpoint inhibitors, and (b) TGF-β signaling pathway inhibitors.

[0023] Fourthly, a method for screening hepatocellular carcinoma patients suitable for immunotherapy is provided, including the following steps: (1) Obtain liver cancer tissue samples or peripheral blood samples from the individual to be tested; (2) Use specific primers, probes or antibodies to detect the mRNA or protein expression level of DUSP4 in the sample; (3) Compare the test results with the standard reference values ​​and output treatment suggestions based on the comparison results.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. High predictive accuracy: This invention is the first to demonstrate that high DUSP4 expression is significantly associated with high objective response rate (ORR) and long survival (OS / PFS) in HCC patients treated with ICB. ROC curve analysis shows that its AUC value is superior to traditional clinical indicators and can be used as an independent prognostic predictor.

[0025] 2. The complete molecular axis of "DUSP4 high expression - inhibition of TGF-β signaling pathway - downregulation of immunosuppressive factors / upregulation of antigen-presenting molecules - promotion of CD8+ T cell / NK cell infiltration and activation - enhancement of anti-tumor immunity" was elucidated, providing a solid theoretical basis for combination therapy.

[0026] 3. Significantly enhanced therapeutic efficacy: In vivo and in vitro experiments have demonstrated that upregulating DUSP4 expression can significantly enhance the killing activity of CAR-T cells and ordinary T cells, and produce a synergistic anti-tumor effect with anti-PD-1 antibody, providing a new strategy for solving ICB resistance.

[0027] 4. Great potential for clinical translation: The provided detection methods and kits are easy to operate and suitable for clinical pathological sections or liquid biopsies, which helps to achieve precise stratified treatment of liver cancer. Attached Figure Description

[0028] Figure 1 The results of a correlation analysis between DUSP4 expression levels and clinical response and prognosis of hepatocellular carcinoma (HCC) patients treated with immune checkpoint blockade (ICB) were presented.

[0029] in, Figure 1 AB: Box plot showing the difference in DUSP4 expression between the "Responder" and "Non-responder" groups treated with ICB in the GSE202069 dataset.

[0030] Figure 1 C: Receiver operating characteristic (ROC) curves for predicting ICB treatment response using DUSP4 expression levels.

[0031] Figure 1 D: Distribution of DUSP4 expression in patients with different efficacy evaluations (CR / PR / SD / PD) in the GSE140901 dataset.

[0032] Figure 1 E: Scatter plot of the correlation between DUSP4 expression level and immune score in the TCGA cohort.

[0033] Figure 1 FI: Bar chart comparing the DUSP4 high / low expression groups across four dimensions of the immunophenotypic score (IPS): MHC molecules, immune checkpoints, effector cells, and suppressor cells.

[0034] Figure 1 JK: Kaplan-Meier curves of overall survival (OS) in pan-cancer analysis of DUSP4 high / low expression groups after receiving anti-PD-1 / CTLA-4 treatment.

[0035] Figure 2 The results of in vitro experiments show that DUSP4 enhances the anti-tumor function of T cells and CAR-T cells.

[0036] in, Figure 2 A: Schematic diagram of the co-culture experiment of liver cancer cells with T cells / CAR-T cells.

[0037] Figure 2 BC: Bar chart showing the effect of DUSP4 overexpression (OE) or knockout (KO) on the efficiency of GPC3-CAR-T cells in killing liver cancer cells (different effector-target ratios).

[0038] Figure 2 DE: Flow cytometry and statistical graphs showing the effect of DUSP4 regulation on T cell proliferation in co-culture systems (CFSE dilution method).

[0039] Figure 2 FG: Flow cytometry was used to detect the double positivity rate of CD25 and CD69, activation markers on the surface of T cells after co-culture.

[0040] Figure 2 HK: ELISA and flow cytometry were used to detect the expression levels of effector molecules (IFN-γ, GZMB) and degranulation markers (CD107a) in co-culture supernatants or intracellular cells.

[0041] Figure 3 The transcriptomic analysis results of key downstream genes and signaling pathways regulated by DUSP4 are presented.

[0042] in, Figure 3 A: Volcano plot of differentially expressed genes (DEGs) between the DUSP4 overexpression group and the control group.

[0043] Figure 3 B: Heatmap clustering analysis of key differentially expressed immune-related genes (such as the TGF-β family, antigen-presenting genes, and chemokines).

[0044] Figure 3C: Bubble chart of KEGG pathway enrichment analysis, highlighting significantly enriched pathways such as "TGF-β signaling pathway" and "antigen processing and presentation".

[0045] Figure 4 The results of the Rescue experiment demonstrate the mechanism by which DUSP4 regulates T cell function via the TGF-β pathway.

[0046] Figure 4 AD: Comparison of T cell killing rate, proliferation rate, activation markers and cytokine secretion levels after the addition of TGF-β neutralizing antibody in the DUSP4 knockout (sgDUSP4) background.

[0047] Figure 4 E: A comparison of the results of reversing the above-mentioned immune enhancement effect after exogenous addition of recombinant TGF-β protein in the context of DUSP4 overexpression.

[0048] Figure 5 Flow cytometry analysis results show that DUSP4 remodels the tumor immune microenvironment in an in vivo mouse xenograft model.

[0049] in, Figure 5 A: Schematic diagram of the grouping and treatment process for mouse subcutaneous xenograft tumor experiments.

[0050] Figure 5 BF: Flow cytometry analysis of the infiltration ratio and absolute number of effector immune cells (CD8+PD-1+ T cells, CD3+ T cells, NK cells, CD11c+ DC cells) in tumor tissues of each group.

[0051] Figure 5 GJ: Statistical graph showing the infiltration ratio and absolute number of immunosuppressive cells (MDSCs, TAMs) in tumor tissues of each group as detected by flow cytometry.

[0052] Figure 6 This study demonstrated the in vivo efficacy of combination therapy that enhances the sensitivity of hepatocellular carcinoma to immune checkpoint blockade (anti-PD-1) therapy by high DUSP4 expression.

[0053] in, Figure 6 AB: A heatmap showing the correlation between DUSP4 expression and immune cell infiltration abundance in a public database, and immunohistochemical (IHC) staining images of clinical samples (DUSP4 and CD8 co-stained).

[0054] Figure 6 C: Subcutaneous tumor volume growth curves in mice under different treatment groups (Vector, Dusp4-OE, Vector+α-PD-1, Dusp4-OE+α-PD-1).

[0055] Figure 6 D: Box plot comparing tumor weight of mice in each group at the end of the experiment.

[0056] Figure 6 E: Graphical summary of animal research.

[0057] Figure 6 FH: Comparison of survival rates among different groups of mice. Detailed Implementation

[0058] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0059] The present invention will be further described below with reference to the accompanying drawings and embodiments. Example

[0060] See Figure 1 Bioinformatics analysis and clinical validation of DUSP4 as a predictive biomarker for ICB efficacy.

[0061] Experimental methods and data processing: Transcriptome data were collected from HCC patients receiving anti-PD-1 monotherapy (GSE202069 cohort, n=17; GSE140901 cohort, n=25). Patients were divided into response (CR+PR) and non-response (SD+PD) groups according to RECIST 1.1 criteria. The expression difference of DUSP4 between the two groups was analyzed using the limma package in R. ROC curves were constructed to assess predictive efficacy. Immune scores were calculated using the ESTIMATE algorithm on TCGA-LIHC data, and IPS scores were obtained from the TCIA database. Pan-cancer survival analysis was performed.

[0062] Experimental results: like Figure 1 As shown in AB, in the GSE202069 cohort, the expression level of DUSP4 mRNA in tumor tissues of patients in the response group was significantly higher than that in the non-response group (P<0.01). Figure 1 C shows that the area under the ROC curve (AUC) for predicting ICB response using DUSP4 expression level as the cutoff value is 0.7816, indicating that it has good discriminative power.

[0063] In the independent verification queue GSE140901 ( Figure 1DUSP4 expression was highest in patients with complete remission (CR) and lowest in patients with disease progression (PD), showing a clear gradient difference.

[0064] Further analysis of TCGA data ( Figure 1 E), and found that DUSP4 expression level was significantly positively correlated with immune score (R=0.45, P<0.001). Figure 1 FI showed that the DUSP4 high expression group scored significantly higher than the low expression group in all four dimensions of the IPS score (MHC molecules, immune checkpoints, effector cells, and suppressor cells), suggesting that it has stronger immunogenicity.

[0065] Figure 1 JK's pan-cancer survival analysis showed that, in a variety of solid tumors, patients with high DUSP4 expression had significantly prolonged overall survival (OS) after receiving anti-PD-1 / CTLA-4 therapy (Log-rank P<0.05).

[0066] In this embodiment, Figure 1 illustrates the relationship between DUSP4 expression and resistance to immune checkpoint inhibitor (ICB) therapy.

[0067] In Figure 1, (A) the volcano plot shows that DUSP4 expression levels were elevated in the response group of HCC patients receiving anti-PD-1 monotherapy in the GSE202069 dataset. (B) the box plot shows the difference in DUSP4 expression levels between the non-response and response groups in the GSE202069 dataset. (C) the ROC curve shows the predictive power of DUSP4 levels for HCC treatment response. (D) the bar chart shows DUSP4 expression levels in patients with complete remission (CR) and partial remission (PR) in the GSE140901 dataset. (E) the association between DUSP4 expression levels (high / low) quantified by the ESTIMATE algorithm and tumor immune scores in the TCGA-LIHC cohort. (F-I) the association between DUSP4 expression and immune profile (IPS) in HCC patients (TCIA database). (J-K) Overall survival of pan-cancer patients with high and low DUSP4 expression levels after receiving anti-PD-1 or anti-CTLA-4 immunotherapy.

[0068] in conclusion: High DUSP4 expression is an independent biomarker for predicting good response to ICB treatment and survival benefit in HCC patients. Example

[0069] See Figure 2Experimental study on the regulation of T cell killing function by DUSP4 in vitro.

[0070] Experimental methods: We constructed the HepG2 human hepatocellular carcinoma cell line with stable DUSP4 overexpression (OE-DUSP4) and gene knockout (sgDUSP4). We prepared GPC3-specific CAR-T cells and activated T cells derived from healthy individuals. We established Transwell or direct co-culture systems (effect-target ratio E:T = 5:1, 10:1, 20:1).

[0071] The tumor cell killing rate was detected by LDH release assay; T cell proliferation was detected by CFSE dye labeling; the expression of CD25, CD69, and CD107a on the surface of T cells was detected by flow cytometry; and the concentrations of IFN-γ and Granzyme B (GZMB) in the supernatant were detected by ELISA.

[0072] Experimental results: like Figure 2 As shown in BC, compared with the control group (NC), the OE-DUSP4 group of liver cancer cells showed significantly enhanced sensitivity to GPC3-CAR-T cells, with the killing rate increasing from 35% to 65% at E:T=10:1 (P<0.001); while the sgDUSP4 group showed obvious drug resistance, with the killing rate decreasing to 15%.

[0073] Figure 2 CFSE flow cytometry results from DE showed that the T cell proliferation index and cell division number increased significantly when co-cultured with OE-DUSP4 cells.

[0074] Figure 2 FG showed that the proportion of CD25+CD69+ double-positive activation in T cells co-cultured in the OE-DUSP4 group was significantly increased (approximately 45% vs. 25% in the control group).

[0075] Figure 2 HK showed that the OE-DUSP4 group induced T cells to secrete higher levels of IFN-γ and GZMB, and upregulated CD107a degranulation expression, indicating enhanced T cell effector function. Conversely, DUSP4 knockdown significantly inhibited these indicators.

[0076] In this embodiment, Figure 2 shows that DUSP4 can enhance T cell effector responses in vitro.

[0077] In Figure 2, (A) co-culture of liver cancer cells with activated T cells to establish a co-culture system. (BC) Cells overexpressing DUSP4 (OE) or knockout DUSP4 (sgDUSP4) were co-cultured with T cells at different effector-to-target cell ratios compared to control cells (NC or sgNC). (n = 3; presented as mean ± standard error; two-way ANOVA). (DE) Cells overexpressing or knockout DUSP4 were co-cultured with T cells carrying Cell Trace Violet (CTV) dye compared to control cells (NC or sgNC). Representative histograms of CTV solution dilutions and calculated proliferation indices are shown (n = 3; presented as mean ± standard error; two-tailed unpaired t-test). (FG) T cells co-cultured with cells overexpressing or knockout DUSP4 were assessed by CD25 and CD69 expression, and representative flow cytometry plots (left panel) and quantitative results (right panel) are shown (n = 3; presented as mean ± standard error; two-tailed unpaired t-test). (HI). T cells co-cultured with cells overexpressing or knockout DUSP4 were assessed by IFNγ and GZMB expression, and representative flow cytometry plots (left panel) and quantitative results (right panel) are shown (n = 3; presented as mean ± standard error; two-tailed unpaired t-test). (G) T cells were co-cultured with OE cells or sgDUSP4 cells and assessed by CD107A expression, and representative flow cytometry plots (left panel) and quantitative results (right panel) are shown (n = 3; presented as mean ± standard error; two-tailed unpaired t-test). in conclusion: High expression of DUSP4 in tumor cells can significantly enhance the in vitro killing, proliferation and activation functions of T cells and CAR-T cells. Example

[0078] See Figure 3 The mechanism by which DUSP4 exerts its effects by inhibiting the TGF-β signaling pathway is elucidated.

[0079] Experimental methods: Total RNA was extracted from OE-DUSP4 and NC HepG2 cells for high-throughput sequencing (RNA-seq). Differentially expressed genes (|log2FC|>1, FDR<0.05) were screened using the DESeq2 package. GO function and KEGG pathway enrichment analyses were performed. Western blotting was used to detect changes in the expression of key proteins in the TGF-β pathway (p-SMAD2, p-SMAD3, Total-SMAD2 / 3).

[0080] Experimental results: like Figure 3 As shown in Volcano Plot A, DUSP4 overexpression leads to significant changes in a large number of immune-related genes. Figure 3 The B-type heatmap showed that immunosuppressive factors (such as TGFB1 and SERPINE1) were significantly downregulated, while antigen processing and presenting genes (HLA-A, HLA-B, B2M, TAP1) and T cell chemokines (CXCL9 and CXCL10) were significantly upregulated.

[0081] Figure 3 KEGG enrichment analysis of C showed that the "TGF-β signaling pathway" was one of the most significantly enriched pathways (FDR<0.001).

[0082] Western Blot results (supporting) Figure 3 (Conclusion) confirmed that the levels of p-SMAD2 and p-SMAD3 proteins were significantly reduced in OE-DUSP4 cells, indicating that DUSP4 inhibited the phosphorylation activation of the TGF-β pathway.

[0083] In this embodiment, Figure 3 identifies the immunosuppressive cytokine TGF-β as a key gene controlled by DUSP4.

[0084] Figure 3 shows: (A) a volcano plot of differentially expressed genes (DEGs) in HepG2 cells based on RNA-seq analysis: control group (NC) and DUSP4 knock-up group (DUSP4 OE). Red dots represent upregulated genes; blue dots represent downregulated genes; gray dots represent genes with no significant difference. (B) a heatmap showing key genes controlled by DUSP4. (C) the top 10 KEGG signaling pathways identified by enrichment analysis using Cytoscape. Pathways and their constituent genes within the same group are coded with the same color; shared genes in different pathways are connected by lines, and the size of the circle is negatively correlated with the p-value.

[0085] in conclusion: DUSP4 reshapes the tumor's immune phenotype by negatively regulating the TGF-β / SMAD signaling pathway, thereby relieving its inhibition of immune genes. Example

[0086] See Figure 4 Validation of DUSP4-dependent TGF-β pathway regulation of T cell function.

[0087] Experimental Method (Rescue Experiment): Four co-cultivation systems were set up: (1) Control group: NC liver cancer cells + T cells; (2) KD group: sgDUSP4 liver cancer cells + T cells; (3) KD+Ab group: sgDUSP4 liver cancer cells + T cells + TGF-β neutralizing antibody (10 μg / mL); (4) OE+rTGF group: OE-DUSP4 liver cancer cells + T cells + recombinant human TGF-β1 protein (5 ng / mL).

[0088] The detection indicators are the same as in Example 2 (kill rate, proliferation, activation, cytokines).

[0089] Experimental results: like Figure 4 As shown in AD, the reduced T cell killing, proliferation inhibition, and decreased activation caused by sgDUSP4 were significantly reversed after the addition of TGF-β neutralizing antibody (KD+Ab group), and all indicators recovered to levels close to those of the Control group.

[0090] like Figure 4 As shown in E, the addition of exogenous recombinant TGF-β protein (OE+rTGF group) to the OE-DUSP4 system successfully counteracted the immune enhancement effect brought about by DUSP4 overexpression, and T cell function was suppressed again, with the indicators falling back to low levels.

[0091] In this embodiment, Figure 4 This study demonstrates that DUSP4 impairs T cell function in a TGF-β-dependent manner.

[0092] That Figure 4In the following sections, (A) sgDUSP4 and sgNC cells were incubated with T cells at different effector-to-target cell ratios. (n = 3; presented as mean ± standard error; two-way ANOVA). (B) sgDUSP4 and sgNC cells were incubated with T cells carrying Cell TraceViolet (CTV) dye. Representative histograms of the CTV solution and calculated proliferation indices are shown (n = 3; presented as mean ± standard error; two-tailed unpaired t-test). (C) T cells cultured with sgDUSP4 or sgNC cells were evaluated by CD25 and CD69 expression. Representative flow cytometry plots (left panel) and quantitative results (right panel) are shown (n = 3; presented as mean ± standard error; two-tailed unpaired t-test). (D) T cells cultured with sgDUSP4 or sgNC cells were evaluated by IFNγ and GZMB expression, and representative flow cytometry plots (left panel) and quantitative results (right panel) are shown (n = 3; presented as mean ± standard error; two-tailed unpaired t-test). (E) T cells were co-cultured with sgDUSP4 or sgNC cells and evaluated by CD107A expression, and representative flow cytometry plots (left panel) and quantitative results (right panel) are shown (n = 3; presented as mean ± standard error; two-tailed unpaired t-test). Cells were treated with 50 ng / mL of anti-TGF-β antibody or were untreated.

[0093] in conclusion: The TGF-β signaling pathway is a key downstream effector molecule that regulates T cell function by DUSP4. DUSP4 exerts its immunosensitizing effect by inhibiting this pathway. Example

[0094] See Figure 5 Flow cytometry analysis of how DUSP4 remodels the immune microenvironment in vivo.

[0095] Experimental methods: A C57BL / 6 mouse subcutaneous xenograft model was constructed (inoculated with Hepa1-6 cells, divided into three groups: Vector, Dusp4-OE, and Dusp4-KO, n=8 / group). Mice were sacrificed when the tumor reached 150 mm³, and tumor tissue was dissected to prepare single-cell suspensions. Multicolor flow cytometry was used to detect tumor-infiltrating lymphocyte subsets.

[0096] Experimental results: like Figure 5 Experimental design shown in Figure A. Flow cytometry analysis results ( Figure 5 BF showed that, compared with the Vector group, the Dusp4-OE group had significantly increased infiltration rate and absolute number of CD8+PD-1+ effector T cells, total number of CD3+ T cells, NK cells (NK1.1+) and dendritic cells (CD11c+MHC-II+) in tumors (P<0.01).

[0097] on the contrary, Figure 5 GJ showed that the infiltration of myeloid-derived suppressor cells (MDSCs, CD11b+Gr-1+) and M2 tumor-associated macrophages (TAMs, F4 / 80+CD206+) was significantly reduced in the Dusp4-OE group. The Dusp4-KO group, on the other hand, exhibited the opposite immunosuppressive phenotype.

[0098] In this embodiment, Figure 5 This study demonstrates the role of DUSP4 in remodeling the immune microenvironment in vivo.

[0099] That Figure 5 (A) Graphical summary of animal studies. (B) Representative histograms used to identify CD8⁺PD-1⁺T cells in the tumor microenvironment. (C) Quantitative analysis of the frequency of tumor-infiltrating CD8⁺PD-1⁺T cells between the OE group and the NC group, and between the sgDUSP4 group and the sgNC group (representative histograms used to identify CD3⁺T cells (D), CD11c⁺CD11b- (DC) cells (F), CD11b-NK1.1⁺ (NK) cells (H), F4 / 80 low Gr-1 high (MDSC) cells (J), and F4 / 80 high Gr-1 low (TAM) cells (J)). Quantitative analysis of the frequencies of tumor-infiltrating CD3⁺ T cells (E), CD11c⁺CD11b-(DC) cells (G), CD11b-NK1.1⁺ (NK) cells (I), F4 / 80 low Gr-1 high (MDSC) cells (K), and F4 / 80 high Gr-1 low (TAM) cells (L) between the OE group and the NC group, and between the sgDUSP4 group and the sgNC group. (I) Absolute number of immune cells per gram of tumor tissue (n = 6; presented as mean ± standard error; two-sided unpaired t-test).

[0100] in conclusion: DUSP4 can effectively remodel the tumor immune microenvironment in vivo, promote the infiltration of effector immune cells and reduce the aggregation of inhibitory cells. Example

[0101] See Figure 6 DUSP4 enhances the sensitivity of liver cancer to immune checkpoint blockade therapy.

[0102] Experimental methods: A mouse model of Hepa1-6 subcutaneous xenograft tumor was established and randomly divided into four groups (n=10 / group): (1) Vector control group; (2) Dusp4-OE group; (3) Vector+α-PD-1 group (intraperitoneal injection of anti-PD-1 antibody, 10 mg / kg, once every 3 days); (4) Dusp4-OE+α-PD-1 group.

[0103] Tumor volume changes were monitored, and mouse survival time was recorded. Simultaneously, clinical HCC samples were collected for immunohistochemical co-staining validation of DUSP4 and CD8.

[0104] Experimental results: Figure 6 Analysis of public data from AB and IHC staining of clinical samples showed that the DUSP4 high-expression region highly overlapped with the CD8+ T cell high-infiltration region.

[0105] Results of in vivo treatment experiments ( Figure 6 C) shows that Dusp4-OE or α-PD-1 alone can inhibit tumor growth to some extent, but the effect is limited. However, the Dusp4-OE + α-PD-1 combination group showed a significant synergistic anti-tumor effect, with the smallest tumor volume and complete tumor regression in some mice.

[0106] Figure 6 D showed that the tumor weight at the endpoint of the combined group was significantly lower than that of other groups (P<0.001).

[0107] Figure 6 E survival curves showed that the median survival time of mice in the combined group was significantly prolonged, and the long-term survival rate (>60 days) was the highest.

[0108] In this embodiment, Figure 6 illustrates how DUSP4 mobilizes T cell infiltration to enhance the response to immune checkpoint blockade.

[0109] That Figure 6(A) Quantitative maps of immune cell infiltration in LIHCs with high and low DUSP4 expression, analyzed using MCPcounter. (B) Correlation between DUSP4 expression and activated CD8⁺ T cell levels in LIHCs. Data were analyzed using the TISIDB database (an integrated portal for tumor-immune system interactions). (C) Representative immunohistochemical images of DUSP4 and CD3 expression in HCC tissues. (D) Correlation analysis of DUSP4 immunohistochemical staining intensity and CD3⁺ cell count per unit area. (E) Graphical summary of animal studies. (F) Tumor volume in subcutaneous tumor models (n = 6; expressed as mean ± standard error; two-way ANOVA).

[0110] in conclusion: High expression of DUSP4 can transform "cold tumors" into "hot tumors," significantly enhancing the sensitivity of liver cancer to PD-1 blockade therapy. The combination strategy has great potential for clinical application.

[0111] The above describes the preferred embodiments of the present invention, illustrating and describing the basic principles, main features, and advantages of the invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as defined by the appended claims and their equivalents.

Claims

1. The use of a DUSP4 detection reagent in the preparation of a product for predicting the response of hepatocellular carcinoma patients to immune checkpoint blockade therapy, characterized in that: The product makes predictions based on the following logic by detecting the expression level of DUSP4 in patient biological samples; When the expression level of DUSP4 is higher than a preset threshold, the patient is determined to be sensitive to the immune checkpoint blockade therapy or have a high probability of response. When the expression level of DUSP4 is lower than or equal to the preset threshold, the patient is determined to be insensitive to the immune checkpoint blockade therapy or have a low probability of response.

2. The use of the DUSP4 detection reagent according to claim 1 in the preparation of a product for predicting the response of hepatocellular carcinoma patients to immune checkpoint blockade therapy, characterized in that: The immune checkpoint blockade therapy includes administering one or more inhibitors selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.

3. The use of the DUSP4 detection reagent according to claim 1 in the preparation of a product for predicting the response of hepatocellular carcinoma patients to immune checkpoint blockade therapy, characterized in that: The biological samples were selected from tumor tissue sections, fresh frozen tissue, peripheral blood mononuclear cells, circulating tumor cells, or exosomes; The detection indicators for DUSP4 include the transcriptional level of DUSP4 mRNA or the expression level of DUSP4 protein. The detection reagents are selected from primer pairs that specifically amplify the DUSP4 gene, probes that specifically hybridize the DUSP4 gene, or antibodies that specifically bind to the DUSP4 protein.

4. A kit for predicting the efficacy of immunotherapy for hepatocellular carcinoma, characterized in that: Include: (a) Specific reagents for detecting DUSP4 expression levels; (b) Internal reference gene detection reagent; (c) The judgment criteria description, which describes the correspondence between DUSP4 expression level and immunotherapy response, indicating that high DUSP4 expression predicts a good treatment prognosis.

5. The use of a DUSP4 activator in the preparation of a pharmaceutical composition for enhancing T cell-mediated antitumor immunity, characterized in that: The DUSP4 activator promotes the infiltration, proliferation, and killing function of effector T cells by increasing the expression level or activity of DUSP4 in tumor cells and inhibiting the TGF-β signaling pathway.

6. The use of the DUSP4 activator according to claim 5 in the preparation of a pharmaceutical composition for enhancing T cell-mediated antitumor immunity, characterized in that: The DUSP4 activator is selected from: (I) A gene overexpression vector containing the DUSP4 coding sequence, preferably an adenovirus vector, a lentivirus vector, or an AAV vector; (II) DUSP4 protein or its functional fragments; (III) Small molecule compounds that can upregulate DUSP4 transcription or translation; (IV) Inhibitors that can stabilize DUSP4 protein.

7. A pharmaceutical composition for treating hepatocellular carcinoma, characterized in that: Including therapeutically effective doses: (A) Immune checkpoint inhibitors, and (B) DUSP4 expression upregulators; Components (A) and (B) are formulated as individual formulations, mixed formulations, or sequential administration formulations.

8. The pharmaceutical composition for treating hepatocellular carcinoma according to claim 7, characterized in that: The pharmaceutical composition also contains a TGF-β signaling pathway inhibitor, used to screen patients with low DUSP4 expression before treatment, or to simulate the immune microenvironment state of high DUSP4 expression by directly blocking the TGF-β pathway when DUSP4 cannot be directly upregulated.

9. A method for screening hepatocellular carcinoma patients suitable for adoptive cell therapy (ACT), characterized in that: Includes the following steps: (1) Obtain liver cancer samples from the individuals to be tested; (2) Determine the expression level of DUSP4 in the sample; (3) Compare the expression level with a reference threshold; (4) If the expression level is higher than the reference threshold, it is recommended that the individual receive chimeric antigen receptor T-cell (CAR-T) therapy or tumor-infiltrating lymphocyte (TIL) therapy; (5) If the expression level is lower than the reference threshold, it is recommended to first perform DUSP4 upregulation treatment or combined TGF-β inhibitor treatment before implementing adoptive cell therapy.

10. A method for assessing the state of the tumor immune microenvironment in hepatocellular carcinoma, characterized in that: By detecting DUSP4 expression levels as a proxy indicator, the status of the following parameters in the tumor microenvironment can be inferred: (I) Infiltration density of CD8+ T cells and NK cells; (II) Expression activity of genes related to antigen processing and presentation; (III) The degree of activation of the TGF-β signaling pathway; (IV) Enrichment of myeloid-derived suppressor cells (MDSCs) and M2 macrophages; Among them, high DUSP4 expression indicates a state of high infiltration of effector immune cells, active antigen presentation, suppression of the TGF-β pathway, and a reduction in immunosuppressive cells.

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