Application of FXR inhibitors in the preparation of drugs for the prevention and / or treatment of lung cancer lymph node metastasis

Abstract

This invention discloses the use of FXR inhibitors in the preparation of drugs for the prevention and / or treatment of lung cancer lymph node metastasis, wherein the FXR inhibitor is Z-Guggulsterone or DY268. The drug comprises a pharmaceutically acceptable carrier and an FXR inhibitor. This invention corrects the pathological remodeling of high endothelial venules (HEVs) within draining lymph nodes by blocking the “FXR-HEV-MDSC” signaling axis with an FXR inhibitor, thereby reducing myeloid-derived suppressor cell (MDSC) recruitment and inhibiting lung cancer lymph node metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of FXR inhibitors in the preparation of drugs for the prevention and / or treatment of lung cancer lymph node metastasis. Background Technology

[0002] Lung cancer is currently the leading cause of cancer-related death worldwide, with the highest mortality rate among all cancers. In clinical practice, non-small cell lung cancer (NSCLC) accounts for approximately 80%-85% of all cases. Despite significant advancements in surgical resection, radiotherapy, chemotherapy, and emerging targeted and immunotherapies (such as PD-1 / PD-L1 inhibitors) in recent years, the five-year survival rate for lung cancer patients remains unsatisfactory.

[0003] Tumor metastasis is a leading cause of death in lung cancer patients. Lymph node metastasis (LNM) is not only the most common early metastatic route for lung cancer, but also the most critical indicator for clinical staging, determining the scope of surgery, and assessing prognosis. Once lymph node metastasis occurs, the patient's risk of recurrence increases significantly, and survival is drastically shortened. Therefore, in-depth research into the molecular mechanisms of lung cancer lymph node metastasis and the search for novel drug targets that can block metastasis at an early stage are core issues that urgently need to be addressed in the fields of tumor biology and translational medicine.

[0004] In the process of tumor metastasis, the formation of the pre-metastatic niche is crucial. Myeloid-derived suppressor cells (MDSCs), as a heterogeneous cell population with highly immunosuppressive functions, are a core component in constructing this suppressive microenvironment. MDSCs provide a "breeding ground" for tumor cells to colonize and proliferate in draining lymph nodes by inhibiting the anti-tumor activity of CD8+ T cells.

[0005] High endothelial venules (HEVs) are specialized post-endothelial venous portals through which immune cells enter lymphoid tissues from the bloodstream. Under normal physiological conditions, HEVs are responsible for recruiting immature lymphocytes; however, under tumor pathological conditions, HEVs within draining lymph nodes undergo significant phenotypic and functional remodeling. Studies have shown that tumors can modulate the state of HEVs, transforming them into channels for recruiting immunosuppressive cells (such as MDSCs), thereby promoting lymph node metastasis.

[0006] The farnesoid X receptor (FXR) is a classic ligand-activated nuclear receptor, long thought to regulate bile acid metabolism and lipid homeostasis primarily in the liver and intestine. Existing research (such as relevant literature in Nature Reviews Gastroenterology & Hepatology or the Journal of Hepatology) focuses primarily on its role in metabolic diseases and gastrointestinal tumors. However, in the field of lung cancer, particularly regarding how lung cancer cells regulate HEV remodeling in lymph nodes distally via FXR signaling, thereby inducing massive recruitment of MDSCs, existing technical approaches and theoretical studies remain lacking.

[0007] While existing literature reports that FXR can promote tumor cell migration, invasion, and angiogenesis through IL-6 / STAT3 signaling, thereby participating in distant metastasis, these techniques primarily focus on the malignant phenotype of tumor cells and hematogenous metastasis mechanisms, failing to reveal the role of FXR in high endothelial microvenous remodeling, MDSC targeted recruitment, and pre-metastatic niche formation in tumor-draining lymph nodes. This invention, for the first time, positions the role of FXR in the distal regulatory chain of "tumor cells—HEV—MDSC—lymph node metastasis," addressing the problem of blocking the initiation of lymphatic metastasis. Its site of action, molecular mechanism, pathological process, and technical effects are fundamentally different from existing technologies, and the two do not overlap. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides the application of FXR inhibitors in the preparation of drugs for the prevention and / or treatment of lymph node metastasis in lung cancer. This invention precisely regulates the physical morphology and functional remodeling of high endothelial microveins (HEVs) in tumor draining lymph nodes (TDLNs) by targeting and inhibiting the FXR signaling pathway, thereby blocking the targeted recruitment of myeloid-derived suppressor cells (MDSCs).

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: The primary objective of this invention is to provide the use of FXR inhibitors in the preparation of drugs for the prevention and / or treatment of lymph node metastasis in lung cancer.

[0010] Preferably, the FXR inhibitor is Z-Guggulsterone or DY268.

[0011] A second objective of this invention is to provide a drug for the prevention and / or treatment of lymph node metastasis in lung cancer, comprising a pharmaceutically acceptable carrier and an FXR inhibitor.

[0012] Preferably, the FXR inhibitor is Z-Guggulsterone or DY268.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention achieves precise blocking of the initiation stage of lymph node metastasis in lung cancer, significantly improving prevention and treatment efficacy. Existing technologies mostly target and kill existing metastatic lesions or inhibit conventional angiogenesis (VEGF pathway). This invention, through improvement, focuses the intervention on the nuclear receptor FXR. Experiments have confirmed that FXR is the "upstream switch" inducing HEV phenotypic remodeling in draining lymph nodes. By inhibiting FXR, the portal for recruiting immunosuppressive cells (HEVs) can be "closed" at its source, preventing the large-scale accumulation of MDSCs in lymph nodes. This precise blocking of the pre-metastatic niche initiation stage is more effective than traditional methods in preventing the colonization of micrometastases, demonstrating significantly superior prevention and treatment performance compared to traditional broad-spectrum methods.

[0014] 2. This invention overcomes the shortcomings of existing immunotherapies in treating "cold tumors" and improves tumor immune response performance. Lung cancer often presents as a "cold tumor," partly because lymph nodes are filled with a large number of MDSCs, creating a severely immunosuppressive microenvironment. This invention, by inhibiting FXR signaling, can correct the abnormal recruitment function of HEVs. When HEVs no longer recruit large numbers of MDSCs, the immunosuppressive state in the lymph nodes is relieved, and the activity of anti-tumor T cells is restored. This strategy, which improves portal function rather than simply activating effector cells, provides a novel, high-performance solution for addressing immune resistance in lung cancer.

[0015] 3. This invention provides a multidimensional, combined early warning indicator, significantly improving diagnostic accuracy and prognostic reliability. Single tumor markers often lack sufficient specificity. This invention improves the diagnostic strategy by proposing the combined detection of FXR expression in tumor tissue with HEV markers (such as PNAd) in draining lymph nodes. FXR, as a driving factor, shows elevated expression, indicating that the HEV portal is about to open or has already opened. This multidimensional indicator correlation is more accurate in predicting the risk of lymph node micrometastasis than simply detecting tumor size or conventional markers. It not only improves detection sensitivity but also provides clinicians with a more reliable basis for prognostic judgment.

[0016] 4. This invention offers good feasibility for clinical translation and cost advantages. Compared to developing entirely new biological macromolecular drugs, small molecule antagonists targeting FXR already have a strong research foundation in medicinal chemistry (e.g., some drugs have entered clinical trials for metabolic diseases). This invention explores new applications of FXR in lung cancer lymph node metastasis, drawing on existing drug development experience. This not only significantly reduces the cost and risk of new drug development, but small molecule drugs also typically have better tissue penetration and ease of use (e.g., oral feasibility), making them more suitable for large-scale clinical deployment and production quality control.

[0017] 5. This invention reduces the toxic side effects of systemic immune interventions, resulting in improved safety. Systemic immunomodulatory drugs often lead to immune-related adverse events (irAEs). This invention targets the HEV remodeling process in lymph nodes under specific pathological conditions (lung cancer-induced). Because FXR has strong spatial specificity in regulating HEVs, intervention targeting this signaling axis can act more concentratedly on the draining lymph node region, reducing indiscriminate attack on normal immune cells in the circulatory system. The advantage of this technology is a significant reduction in systemic toxic side effects and improved patient safety. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the "FXR-HEV-MDSC" axis in lung cancer lymph node metastasis and the preventive effect of FXR inhibitors in this invention.

[0019] Figure 2 This is a flow cytometry gating strategy for MDSCs (top) and their subsets (bottom) in tumor draining lymph nodes (TDLN). Figure 2 A and 2D are schematic diagrams of cell population delineation using FSC-A and SSC-A scatter plots, excluding cell debris and impurities; Figure 2 B and 2E are schematic diagrams of screening single cells using FSC-H and FSC-A to remove cell aggregates; Figure 2 C is the identification of MDSCs by CD11b and Gr-1 double staining (CD11b) + Gr-1 + Schematic diagram of cell population; Figure 2 F and 2G are in CD11b + Based on cellular data, a schematic diagram further distinguishes MDSC subsets according to Ly6G and Ly6C expression, including polymorphic MDSCs (PMN-MDSCs, Ly6G...). + Ly6C low ) and single-core MDSCs (M-MDSCs, Ly6G) - Ly6C high ).

[0020] Figure 3A shows the WB detection of FXR protein expression level in this invention. The results show that compared with the control group (Control, shNC), the FXR knockdown (shFXR) group showed a significant decrease in FXR protein expression, with GAPDH as an internal reference; the right side shows the quantitative analysis of protein grayscale values.

[0021] Figure 3 B shows the appearance of solid tumors in each group of tumor-bearing mice in this invention, indicating that the tumor volume in the shFXR group is smaller than that in the control group.

[0022] Figure 3 The left figure (C) shows the tumor growth curve in this invention, recording changes in tumor volume at different time points. Figure 3 Figure C on the right shows the statistical analysis of the ratio of tumor weight to body weight (Tumor weight / Body weight) at the experimental endpoint in this invention.

[0023] Figure 3 D is the immunohistochemical (IHC) staining image of tumor tissue in this invention, comparing the expression levels of cytokeratin-related indicators in the shNC group and the shFXR group (×200).

[0024] Figure 3 E represents the distribution of fluorescence signals in the lymph node regions of mice in each group, detected by in vivo imaging in this invention, reflecting the metastasis of tumor cells to the lymph nodes.

[0025] Figure 3 The left figure (F) shows the relative quantitative analysis of fluorescence intensity in the lymph node region in this invention. Figure 3 Figure F on the right shows the quantitative analysis of the positive area of ​​cytokeratin in lymph node tissue in this invention, used to assess tumor metastasis burden.

[0026] Figure 3 In the ns interval, the difference is not statistically significant. *P<0.05, **P<0.01, ***P<0.001.

[0027] Figure 4 A is a representative flow cytometry scatter plot from this invention. In single-cell populations, MDSCs were identified using CD11b and Gr-1 markers (CD11b...). + Gr-1 + Cell populations were compared between the shNC group and the shFXR group.

[0028] Figure 4 B represents the MDSC (CD11b) in each group of TDLN in this invention. + Gr-1 + Quantitative analysis of the proportion.

[0029] Figure 4C is a flow cytometry-gated diagram representing MDSC subsets in this invention. Mononuclear MDSCs (M-MDSCs, CD11b) are distinguished based on Ly6G and Ly6C expression levels. + Ly6G - Ly6Chigh) and polymorphic MDSCs (PMN-MDSC, CD11b) + Ly6G + Ly6C^low).

[0030] Figure 4 D represents the quantitative analysis of the ratio of M-MDSC and PMN-MDSC in this invention.

[0031] Figure 4 E is a representative flow cytometry scatter plot used in this invention to detect CD4 in TDLN. + T cells and CD8 + T cell ratio.

[0032] Figure 4 F represents CD4 in this invention. + T cells and CD8 + Quantitative analysis of the proportion of T cells.

[0033] Figure 4 All samples were subjected to the same gating strategy, and the results shown in the figure are representative. *P<0.05, **P<0.01, ***P<0.001.

[0034] Figure 5 This invention utilizes immunofluorescence staining to detect the expression of MECA-79, a marker of high endothelial venules (HEVs), in different groups of TDLNs. DAPI (blue) was used to label cell nuclei, and MECA-79 (red) was used to label HEV structures. Compared with the control group (shNC), the FXR knockdown group (shFXR) showed significantly enhanced MECA-79 positive signal, exhibiting a more continuous and structurally complete vascular-like distribution, indicating the recovery of HEV morphology. Merge plots show the spatial relationship between MECA-79 signal and cell nucleus distribution. Scale bar = 20 μm.

[0035] Figure 6 This invention uses immunofluorescence staining to detect the expression of MECA-79, a marker of high endothelial venules (HEVs), in TDLNs of different groups. DAPI (blue) was used to label cell nuclei, and MECA-79 (red) was used to label HEV structures. Compared with the low FXR expression group, the HEVs in the high FXR expression group showed abnormal morphology and enlarged lumens. Scale bar = 20 μm.

[0036] Figure 7 A shows representative multiplex immunofluorescence images of lymph node tissues in different states in this invention. Scale bar: 100 μm.

[0037] Figure 7 B represents the linear correlation analysis of FXR and CD33 expression levels in this invention. The scatter plot shows the distribution of FXR expression (X-axis) and CD33 expression percentage (Y-axis) in the clinical cohort sample. The solid red line represents the linear regression line, and the shaded red area represents the 95% confidence interval. CD33 is used in human specimens to assess the level of myeloid cell infiltration associated with MDSCs; NLN: normal lymph nodes; NMLN: non-metastatic lymph nodes in lung cancer patients; MLN: metastatic lymph nodes in lung cancer patients.

[0038] Figure 8 A is the forest plot for multivariate regression analysis in this invention, showing the influence of each variable on the outcome event, its effect size (such as OR or HR), and the 95% confidence interval. Different colors represent different variables, the points represent the effect size, and the horizontal lines represent the confidence interval range.

[0039] Figure 8 B represents the nomogram in this invention, used to construct a predictive model for the outcome event. A total score is obtained by summing the scores corresponding to each variable (such as CD33, FXR, etc.), which is then used to predict the probability of an individual experiencing the outcome event (Risk of outcome).

[0040] Figure 8 C represents the receiver operating characteristic (ROC) curve in this invention, used to evaluate the model's discriminative ability, while the area under the curve (AUC) is used to quantify the model's predictive efficacy.

[0041] Figure 8 D is the calibration curve in this invention, used to evaluate the consistency between the model's predicted probability and the actual observation results. Ideally, the curve is close to a 45° diagonal.

[0042] Figure 8 E represents the decision curve analysis (DCA) in this invention, used to evaluate the clinical net benefit of the model under different threshold probabilities and compare the clinical application value of the model with the "treat all" and "treat none" strategies. The results show that the predictive model has good discriminative ability, calibration accuracy, and clinical application value. Detailed Implementation

[0043] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0044] The experimental conditions are standardized as follows in the examples below: (1) Cell culture conditions: Lewis lung cancer (LLC) cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics at 37 ℃, 5% CO2, and saturated humidity. Cells were inoculated when they were in the logarithmic growth phase.

[0045] (2) Animal experimental conditions: C57BL / 6 mice were housed in an SPF-grade animal room with an ambient temperature of 22 ± 2 ℃, a relative humidity of 50% ± 10%, a 12 h light / dark cycle, and free access to food and water. All animal experiments were approved by the laboratory animal ethics committee of our unit, with ethics approval number: RYE2025071901.

[0046] (3) Tissue and flow cytometry experimental conditions: lymph node tissue sampling, fixation, section thickness, antibody incubation time, number of washings and flow cytometry voltage parameters were kept consistent across groups.

[0047] (4) Statistical analysis: Data are expressed as mean ± standard deviation (x̄ ± σ̄). Mean ± s. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA and Tukey's post-hoc test were used for comparisons among multiple groups. Chi-square test or Fisher's exact test was used for comparisons of categorical variables. P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPadPrism 9.0 or SPSS 26.0 software.

[0048] The following examples demonstrate how molecular-level regulation of the "FXR-HEV-MDSC" core axis can prevent lymph node metastasis. Figure 1 As shown, its scientific principle is as follows: Nuclear receptor transcriptional antagonism mechanism: FXR is a key transcription factor regulating the cellular secretory proteome. Inhibitors interfere with the recruitment of FXR to coactivators by binding to its ligand-binding domain (LBD), thereby specifically downregulating pro-HEV remodeling factors (such as certain chemokines or angiogenesis regulators) released by lung cancer cells.

[0049] The principle of HEV morphological repair and physical barrier reconstruction: Normal HEVs have tall columnar endothelium and extremely narrow lumens, exhibiting high selectivity. Tumor-activated FXR signaling leads to endothelial cell flattening and abnormal lumen dilation. This invention inhibits FXR, promoting endothelial cell cytoskeleton reorganization, restoring its tall columnar physiological structure, and physically tightening the "immunosuppressive cell recruitment gateway."

[0050] Cell adhesion kinetics intervention principle: MDSCs enter lymph nodes through the HEV wall depending on the interaction between L-selectin and PNAd. This invention reduces the frequency of "rolling" and "adhesion" of circulating MDSCs on the inner surface of the HEV by decreasing FXR-induced overexpression of endothelial adhesion molecules.

[0051] The principle of immune homeostasis rebalancing: As physical channels are closed, the influx of MDSCs decreases. The concentration of inhibitory cytokines such as IL-10 and TGF-beta in the lymph node parenchyma decreases, allowing CD8+ T cells that were previously exhausted due to MDSC suppression to be reactivated and proliferate, restoring the lymph node's ability as an immune sentinel to clear metastatic cancer cells.

[0052] Example 1: Drugs for the prevention and / or treatment of lung cancer lymph node metastasis.

[0053] (1) FXR inhibitors: Small molecule chemicals: Highly selective FXR antagonists Z-Guggulsterone or DY268. Physicochemical properties: Purity ≥ 99.0% (detected by HPLC), endotoxin < 0.1 EU / mg.

[0054] Nucleic acid intervention sequence: shRNA interference fragment designed targeting the conserved region of human NR1H4 gene (FXR) mRNA (target sequence selected from bases 240-260 or 350-370 of the coding region), constructed in pHBLV-U6-MCS-CMV-IE-ZsGreen-Puro lentiviral vector.

[0055] (2) Drug solvent and sustained-release carrier: The compound solvent is 5% DMSO (molecular biology grade) + 40% PEG300 (polyethylene glycol) + 5% Tween-80 + 50% PBS (phosphate buffer, pH 7.4).

[0056] Local delivery carrier: Chitosan-polylactic acid (CS-PLA) nanospheres with a particle size distribution between 150-250 nm, used to encapsulate nucleic acid sequences to achieve sustained release in the drainage region.

[0057] (3) HEV and MDSC functional monitoring probes: Endothelial marker: Rat anti-mouse PNAd (MECA-79 clone) monoclonal antibody, used to identify HEV.

[0058] Effector cell markers: VioBlue-labeled anti-CD11b and PerCP-Cy5.5-labeled anti-Gr-1 antibody.

[0059] Example 2: Methods and process conditions for using the drug.

[0060] (1) Patient subtyping and target activation assessment process: Procedure: Immunohistochemistry (IHC) was used to quantitatively detect the expression of FXR in the primary tumor.

[0061] Parameters: Tissue fixative was 4% paraformaldehyde (PFA), antigen retrieval was performed using an autoclave (121℃, 3 minutes), and the retrieval solution was EDTA buffer (pH 9.0).

[0062] Judgment criteria: The IRS scoring method (staining intensity multiplied by positive rate) is used. When the product is greater than or equal to 6 points and accompanied by an average diameter of more than 35 μm (showing a lumen-like morphology) of PND-positive vessels in the draining lymph nodes, it is confirmed as an advantageous intervention target of the present invention.

[0063] (2) Drug preparation and titration process: Procedure: Dissolve Z-Guggulsterone in DMSO preheated to 37°C under light-protected conditions, and add PEG300 and Tween-80 in sequence.

[0064] Operating conditions: The final drug concentration is titrated to 10 mg / mL (for injection). In vivo administration pressure is maintained at a constant rate to ensure a stable concentration gradient of the drug in the local lymph node drainage area (target effective concentration ≥ 20 μM).

[0065] (3) Perioperative sequential intervention process: Neoadjuvant blocking phase: 72-120 hours before surgery, administer FXR inhibitors via intraperitoneal injection (ip) or intravenous infusion (iv) at a frequency of QD (once daily) and a dose of 25-50 mg / kg.

[0066] In situ local targeting stage: During the primary lesion resection, after clearing the hilar and mediastinal lymph node areas, the tumor bed and drainage channels are sprayed with lentiviral fluid carrying shRNA (total titer greater than or equal to 2 x 10⁸ TU), and then sealed in situ with biological protein glue to prevent residual tumor signals from continuously inducing HEV remodeling after surgery.

[0067] (4) Lymph node HEV remodeling and MDSC flow monitoring process: Histological quantification: 3D images of mediastinal lymph nodes with PNAd / CD31 double staining were obtained using laser confocal scanning microscopy (CLSM).

[0068] Quantitative indicators: The tortuosity, branch point density, and endothelial height of HEVs were measured. The normalization criterion was that the endothelial cell height recovered from a flattened state to a tall columnar morphology greater than 12 μm.

[0069] Flow cytometry: Collect single-cell suspensions from lymph nodes, centrifuge at 1500 rpm for 5 minutes, and incubate with antibodies at 4°C for 30 minutes.

[0070] Example 3: Procedure for in vitro non-therapeutic inhibition of lung cancer lymph node metastasis.

[0071] (1) Flow cytometry was used to detect the distribution of MDSCs and their subgroups in lymph node tissues of each group.

[0072] Mouse lymph node tissue was collected, mechanically ground, and filtered through a 70 μm filter to prepare a single-cell suspension. After treatment with erythrocyte lysis buffer, the cells were washed with PBS and counted. An appropriate amount of cells was incubated with an Fc receptor blocker, followed by the addition of fluorescently labeled antibodies: CD11b, Gr-1, Ly6G, and Ly6C, and incubated in the dark for 30 min. After washing, the cells were resuspended in PBS and analyzed by flow cytometry.

[0073] Data analysis was performed using FlowJo software. First, fragmentation was removed using FSC / SSC, followed by exclusion of dual-TS cells using FSC-A / FSC-H, and then CD11b... + Gr-1 + The total MDSC was defined in the population, and further divided into PMN-MDSC (Ly6G-MDSC) based on Ly6G and Ly6C expression. + Ly6Clow) and M-MDSC (Ly6G) - Ly6Chigh). T cell subsets were analyzed using CD4 and CD8 antibodies. For example... Figure 2As shown, MDSCs and their subgroups were detected in all groups, with clear gating and good reproducibility. Each subgroup accounted for a certain proportion of the total MDSCs, and the distribution proportions varied among different treatment groups (details are provided in the subsequent quantitative analysis).

[0074] (2) In vitro and in vivo experiments on the inhibition of tumor growth and lymph node metastasis by FXR knockdown.

[0075] Western blot detection of FXR expression: Total protein was extracted from tissues or cells, and protein concentration was determined using the BCA method. Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder, and FXR and GAPDH primary antibodies were added, followed by incubation overnight at 4°C. The next day, HRP-labeled secondary antibody was added, and ECL staining and imaging were performed. Grayscale analysis was performed using ImageJ software.

[0076] like Figure 3 As shown, compared with the control group, the expression of FXR protein was significantly reduced in the FXR knockdown group ( Figure 3 A). In vivo tumorigenesis experiments showed that the tumor growth rate was slowed in the shFXR group mice, and the terminal tumor volume and weight were lower than those in the control group ( Figure 3 B–C). Immunohistochemical results showed that the area of ​​cytokeratin-positive tumor tissue was reduced in the shFXR group. Figure 3 D). In vivo imaging results further showed that the fluorescence signal intensity was reduced in the shFXR group mice ( Figure 3 E), quantitative analysis showed that its fluorescence intensity was significantly lower than that of the control group (E), Figure 3 F). In summary, FXR knockdown can suppress tumor growth and metastasis-related phenotypes.

[0077] (3) Further flow cytometry was used to analyze the infiltration of immune cells in the lymph nodes of each group.

[0078] like Figure 4 As shown, FXR knockdown reduces MDSC infiltration in TDLN and modulates T cell subset distribution: compared with the shNC group, the shFXR group showed a decrease in CD11b... + Gr-1 + The proportion of MDSCs decreased significantly. Figure 4 A–B). In the MDSC subgroup, M-MDSC (Ly6G) - Ly6Chigh) and PMN-MDSC (Ly6G) + The proportion of Ly6Clow has decreased significantly. Figure 4 C–D). Simultaneously, analysis of T cell subsets revealed that CD4+ was present in the shFXR group. + T cells and CD8 + The proportion of T cells was significantly increased ( Figure 4 E–F).

[0079] (4) Immunofluorescence staining (HEV and colocalization analysis).

[0080] Lymph node tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4 μm). After dewaxing and hydration, the sections underwent antigen retrieval (citrate buffer, pH 6.0). After blocking at room temperature, primary antibodies MECA-79 (HEV marker), FXR, and CD33 were added, and the sections were incubated overnight at 4°C. The following day, the corresponding fluorescently labeled secondary antibodies were added, and the sections were incubated at room temperature in the dark for 1 h. Cell nuclei were counterstained with DAPI. Images were acquired using a fluorescence microscope. Semi-quantitative analysis of fluorescence signal intensity or positive area was performed using ImageJ software.

[0081] like Figure 5 As shown, FXR knockdown promotes the morphological restoration of high endothelial venules (HEVs) in tumor draining lymph nodes (TDLNs): compared with the shNC group, the shFXR group showed significant changes in HEV morphology, characterized by more regular and continuous vascular structures and reduced abnormal dilation. Combined images showed a more uniform distribution of MECA-79 positive areas, with an overall morphology approaching normal. These results suggest that FXR knockdown can improve abnormal HEV structures, restoring them to a relatively normal state.

[0082] (5) Further, the effect of FXR expression level on HEV structure was detected by immunofluorescence staining.

[0083] like Figure 6 As shown, high FXR expression is associated with enlarged lumens and abnormal morphology of high endothelial venules (HEVs) in tumor draining lymph nodes (TDLNs): in the low FXR expression group, MECA-79-labeled HEV structures exhibited continuous and regular tubular morphology; while in the high FXR expression group, HEV structures were significantly abnormal, showing irregular vascular morphology, localized clumps, or dilation. These results indicate that FXR expression levels are closely related to HEV structural status; high FXR expression can exacerbate HEV structural abnormalities, while low FXR expression helps maintain or restore the relatively normal morphology of HEVs.

[0084] (6) Further analysis of FXR and MDSC infiltration in different lymph node tissues was conducted by immunofluorescence staining.

[0085] like Figure 7 As shown, the expression characteristics and correlation analysis of FXR and CD33 in clinical lymph node samples: Compared with the non-lymph node metastasis group (NLN), the number of FXR and CD33 positive cells (used to label MDSC cells) was significantly increased in the lymph node metastasis group (MLN), while the number of adjacent metastatic lymph nodes (NMLN) was at an intermediate level. Figure 7 A). Correlation analysis results showed that FXR expression levels were related to CD33. + The degree of cell infiltration was significantly positively correlated (r = 0.65, P < 0.001). Figure 7 B).

[0086] (7) Constructing a lymph node metastasis prediction model based on CD33 and FXR and evaluating its performance.

[0087] Based on clinical or experimental data, multivariate logistic regression analysis was used to screen independent influencing factors, and a predictive model was constructed. A nomogram was used for visualization. Figure 8 (A–B) The results showed that CD33 and FXR were both important predictors of the model and made a high contribution to the outcome.

[0088] Nomograms were plotted using the "rms" package in R software (version 4.2.0). Model performance was evaluated using receiver operating characteristic (ROC) curves, and the area under the curve (AUC) was calculated. Analysis of the model's discriminative ability showed that the AUC was 0.836 (95% CI: 0.764–0.907), indicating that the model has good discriminative ability. Figure 8 C).

[0089] Calibration curves were used to assess predictive consistency, and internal validation was performed using the bootstrap method. Decision curve analysis (DCA) was used to evaluate the clinical net benefit of the model at different threshold probabilities. Calibration curve results showed that the model's predicted probabilities were largely consistent with the actual probabilities, indicating a good fit. Figure 8 D). Decision curve analysis (DCA) shows that, over a wider range of threshold probabilities, the model has a higher net benefit than the "full intervention" or "no intervention" strategies. Figure 8 E).

[0090] The following application examples verify the exact effects of FXR inhibition on regulating HEV morphology, blocking MDSC recruitment, and inhibiting lung cancer lymph node metastasis through specific experimental schemes and data.

[0091] Application Example 1: Intervention experiment of FXR inhibitor Z-Guggulsterone on lymph node metastasis in lung cancer.

[0092] 1. Experimental objective: To verify the reversal effect of different doses of FXR inhibitors on HEV luminal remodeling in draining lymph nodes (TDLN) and their blocking effect on MDSC recruitment.

[0093] 2. Experimental subjects: 6-8 week old C57BL / 6 mice, with Lewis lung cancer (LLC) cells (1x10⁻¹) subcutaneously injected into the left paw pad. 6 indivual).

[0094] Dosing group (selecting point values ​​within the protection range): Low-dose group: 10 mg / kg Z-Guggulsterone; Medium-dose group: 30 mg / kg Z-Guggulsterone; High-dose group: 50 mg / kg Z-Guggulsterone; Control group: Equal volume of solvent (5% DMSO + 40% PEG300 + 5% Tween-80 + 50% PBS).

[0095] Administration procedure: Starting from the 7th day after tumor inoculation, administer the drug via intraperitoneal injection once daily for 21 consecutive days.

[0096] 3. Performance Characterization Data and Results: HEV morphological markers (PNAd staining): Normal group: age-matched C57BL / 6 mice that were not inoculated with tumors or given any drugs, with an average lumen diameter of 15.2 μm.

[0097] Control group: HEVs in lymph nodes showed significant dilation, with an average lumen diameter of 45.3 μm, and endothelial cells showed flattening.

[0098] In the 10 mg / kg group, the degree of HEV expansion was alleviated, and the average lumen diameter was 32.5 μm.

[0099] In the 30 mg / kg group, HEV morphology was significantly improved, with the lumen diameter reduced to 20.2 μm and the endothelial cells regaining their tall columnar characteristics.

[0100] 50 mg / kg group: HEV basically restored physiological tightness, with an average lumen diameter of 16.8 μm.

[0101] MDSC recruitment rate (CD11b+Gr1+ as measured by flow cytometry): The flow cytometry gating strategy is as follows: First, debris is excluded based on FSC-A / SSC-A; second, single cell populations are selected using FSC-H / FSC-A; CD11b+ myeloid cells are delineated from the single cell populations; Gr-1+ cells are further analyzed in CD11b+ cells as the total MDSC population, and all groups use completely consistent compensation parameters, negative controls, and gating thresholds.

[0102] The proportion of MDSC in the control group TDLN was 18.6%; the proportion of MDSC in the 30 mg / kg intervention group decreased to 5.1% (a decrease of 72.6%, P<0.01).

[0103] Metastasis inhibition effect: The number of draining lymph node metastases was significantly reduced in the 30 mg / kg group and the 50 mg / kg group at the end of the observation period (day 28) compared with the control group.

[0104] Application Example 2: Experiment on the blocking effect of lentivirus-mediated shRNA intervention on the FXR-HEV axis.

[0105] 1. Experimental materials: Intervention tool: shRNA lentiviral vector packaged with a sequence targeting the human FXR gene (NR1H4).

[0106] Process conditions: Three titer gradient values ​​were set for intervention: 1x10^7 TU / mL, 5x10^7 TU / mL, and 1x10^8 TU / mL.

[0107] 2. Experimental Procedure: A mouse model of lung cancer was established by local infiltration injection at multiple points in the primary tumor and draining lymph node areas, with injection volumes of 50 uL, 100 uL, and 150 uL, respectively. Mediastinal lymph nodes were harvested for histological evaluation 14 days later.

[0108] 3. Experimental Results and Principle Verification: FXR expression level: 1x10 8 The expression level of FXR protein in tumor tissue of the TU / mL group decreased by 82.3% compared with that of the NC group.

[0109] HEV functional remodeling: Immunofluorescence showed that the mean fluorescence intensity (MFI) distribution in PNAd-positive areas of draining lymph nodes tended to be more uniform as FXR expression decreased. At high titers (1x10⁻¹), 8 Under intervention (TU / mL), the expression of adhesion factors on the surface of HEV endothelial cells was downregulated, and they no longer formed "expansion portals" that supported the large-scale entry of MDSCs.

[0110] Immune microenvironment reversal: Experimental observation of CD8 in TDLN + T and CD4 + The ratio of T cells to MDSCs (T / MDSC ratio) increased significantly, indicating a rearrangement of the microenvironment from "immunosuppression" to "immunoactivation".

[0111] Application Example 3: Detection experiment for predicting lymph node metastasis risk based on FXR expression level.

[0112] 1. Detection method: Correlation analysis was performed on the expression of FXR (IHC score) and the degree of HEV dilation in draining lymph nodes in surgical resection specimens from lung cancer patients.

[0113] 2. Data Points and Conclusions: Low FXR score group (IRS = 3): HEV morphology in draining lymph nodes is mostly physiological (diameter <20 μm), with less MDSC infiltration.

[0114] In the FXR score group (IRS = 6): HEVs began to expand initially, and the risk of local lymph node metastasis was moderately increased.

[0115] High FXR score group (IRS = 9): HEVs were extremely dilated (diameter > 40 μm) with large-scale infiltration of MDSCs (area > 10%). The postoperative lymph node recurrence rate in this group was significantly higher than that in the low score group.

[0116] Conclusion: This invention, by detecting the activation status of the FXR-HEV axis, can serve as an effective assessment tool for lung cancer lymph node micrometastasis and drug resistance prognosis.

[0117] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of FXR inhibitors in the preparation of drugs for the prevention and / or treatment of lymph node metastasis in lung cancer.

2. The application according to claim 1, characterized in that, The FXR inhibitor is Z-Guggulsterone or DY268.

3. A drug for the prevention and / or treatment of lung cancer lymph node metastasis, characterized in that, This includes pharmaceutically acceptable carriers and FXR inhibitors.

4. The drug according to claim 2, characterized in that, The FXR inhibitor is Z-Guggulsterone or DY268.

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