Application of sTREM2-NCL-AKT / eNOS axis in ovarian cancer vascular permeability and malignant ascites formation

Abstract

The invention belongs to the technical field of cancer treatment, and discloses an application of an sTREM2-NCL-AKT / eNOS axis in the vascular permeability of ovarian cancer and the formation of malignant ascites. The invention proves that the level of the sTREM2 is increased in ascites of an ovarian cancer patient, discloses the effect of the sTREM2 in promoting vascular permeability and driving ascites formation, and puts forward that the sTREM2 has double values of a prognosis biomarker and a potential treatment target. The discovery lays a foundation for developing accurate treatment for ovarian cancer and malignant ascites. In mechanism, the sTREM2 activates an AKT / eNOS signal channel through interaction with a cell surface nucleolin (NCL) protein, so that the integrity of an endothelial barrier is damaged. Targeting or blocking sTREM2 effectively mitigates vascular leakage and ascites production. According to the discovery, the sTREM2 is determined as a core driving factor of ascites pathogenesis, and the potential of the sTREM2 as an ovarian cancer treatment target is highlighted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of ovarian cancer ascites treatment, more specifically, relates to the application of sTREM2-NCL-AKT / eNOS axis in ovarian cancer vascular permeability and malignant ascites formation. BACKGROUND

[0002] Ovarian cancer (OC) is one of the most lethal gynecological malignancies, with treatment challenges and poor prognosis at the advanced stage being particularly prominent. Malignant ascites, a typical feature of advanced OC, not only severely impairs patients' quality of life and increases the complexity of surgical intervention, but also promotes disease progression by facilitating tumor metastasis and chemotherapy resistance. Current treatment methods mainly rely on palliative methods such as abdominal puncture and diuretics, which fail to target the underlying pathophysiological mechanisms and are associated with the risk of infection and metabolic disorders. These limitations highlight the urgent need to elucidate the molecular mechanisms driving ascites formation to develop targeted therapies.

[0003] Managing malignant ascites remains a major challenge in OC. Increased vascular permeability is a key pathophysiological driver, and both cellular (tumor cells, macrophages) and non-cellular (especially VEGF) components in ascites can directly or indirectly regulate vascular permeability. Although primary disease treatment and VEGF inhibitors (such as bevacizumab) can reduce ascites, recurrence and treatment-related adverse reactions are often observed. Therefore, it is imperative to identify and block new targets for vascular leakage. Previous work has demonstrated the role of macrophage blockade, particularly VLA4 hi and PLIN2 hi subpopulations, in the pathogenesis of ascites. However, interventions targeting these cellular pathways are either significantly toxic or have limited efficacy, which may be due to the complexity and heterogeneity of the tumor microenvironment.

[0004] Soluble TREM2 (sTREM2) is a soluble protein formed by hydrolytic cleavage, which can be detected in blood, cerebrospinal fluid, and other body fluids, and mediates various biological functions. For example, in optic neuritis, sTREM2 induces optic nerve dysfunction by interacting with heat shock protein 70; in Alzheimer's disease, it improves cognitive function by acting on transglutaminase 2; and in breast cancer, it promotes disease progression by acting on transglutaminase 2. Despite these insights, the role of sTREM2 in the pathogenesis of OC, particularly the specific mechanisms in ascites formation, remains unclear. SUMMARY

[0005] To overcome the shortcomings of the prior art, the present application aims to deepen the understanding of the pathogenesis of ovarian cancer ascites and develop new treatment methods for ovarian cancer and malignant ascites.

[0006] The object of the present application is achieved by the following technical solutions:

[0007] The present study identified sTREM2 as a key mediator of vascular hyperpermeability and malignant ascites formation in OC. We demonstrated that ascites sTREM2 levels were directly correlated with the severity of OC.

[0008] Therefore, the present application first provides the use of sTREM2 in developing and screening functional products for reducing malignant ascites of ovarian cancer. In this way, sTREM2 can be used as a target to develop and screen substances that block sTREM2 for reducing the formation of malignant ascites of ovarian cancer.

[0009] Preferably, the functional product comprises any one of the following:

[0010] (i) small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid targeting sTREM2 or NCL or AKT or eNOS, or sTREM2 or NCL or AKT or eNOS transcript, and capable of inhibiting the expression or gene transcription of sTREM2 or NCL or AKT or eNOS gene expression product;

[0011] (ii) capable of expressing or forming the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid construct described in (i);

[0012] (iii) a construct containing sTREM2 or NCL or AKT or eNOS, or sTREM2 or NCL or AKT or eNOS complementary sequence and capable of forming an interfering molecule that inhibits the expression or gene transcription of sTREM2 gene expression product after being transferred into the body;

[0013] (iv) immune-related cells, differentiated cells or constructs after inhibiting or knocking out sTREM2 or NCL or AKT or eNOS gene sequence.

[0014] More preferably, the functional product is a scFv targeting the extracellular domain of TREM2, or a small interfering RNA targeting NCL, or an AKT inhibitor MK2206, or an eNOS inhibitor L-NAME.

[0015] To evaluate whether targeting sTREM2 can reduce OC ascites formation and disease progression, in the ID8 mouse model, scFv antibody was administered three weeks after tumor inoculation, which significantly reduced ascites volume and tumor burden compared with the control group. Notably, sTREM2 levels in ascites of scFv-treated mice were reduced, and sTREM2 levels were still positively correlated with ascites volume. These results indicate that sTREM2 blockade effectively alleviates vascular leakage and inhibits malignant ascites formation.

[0016] In addition, the sTREM2 content in the ovarian cancer ascites has a significant correlation with the ascites volume, and therefore the application also provides an application of sTREM2 in the development and screening of functional products for the prognosis analysis of malignant ascites of ovarian cancer.

[0017] Based on the above findings, the application studies the relationship between sTREM2 and vascular endothelial cell permeability. The application directly processes endothelial cells by recombinant sTREM2, and the results show that sTREM2 can induce vascular leakage. In Trem2 MacKO The administration of sTREM2 to mice significantly increases the permeability of the peritoneal blood vessels. These results show that sTREM2 directly damages the endothelial barrier function. It is later found that NCL is a functional receptor of sTREM2, which is essential for the permeability effect of sTREM2. Further cell experiments verify that sTREM2 enhances the phosphorylation of AKT and eNOS in endothelial cells. In addition, this activation effect is eliminated by NCL knockdown. At the same time, cell experiments also confirm that scFv treatment reduces sTREM2-induced endothelial leakage.

[0018] In the signal pathway verification experiment, we process endothelial cells with the eNOS inhibitor L-NAME, which can eliminate the high permeability of endothelial cells mediated by sTREM2. Similarly, the inhibition of AKT phosphorylation with MK2206 produces an effect comparable to L-NAME. In addition, in vivo permeability experiments show that intraperitoneal injection of L-NAME effectively inhibits sTREM2-induced vascular leakage. In summary, these results suggest that sTREM2 promotes vascular permeability by activating the NCL-dependent AKT / eNOS signaling pathway.

[0019] Therefore, the application also provides an application of sTREM2 in the development and screening of functional products for reducing the permeability of endothelial cells.

[0020] Preferably, the functional product comprises any one of the following:

[0021] (i) small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid that can inhibit the expression of sTREM2 or NCL or AKT or eNOS gene expression product or gene transcription with sTREM2 or NCL or AKT or eNOS or sTREM2 or NCL or AKT or eNOS transcript as a target sequence;

[0022] (ii) can express or form the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid construct in (i);

[0023] (iii) a construct containing sTREM2 or NCL or AKT or eNOS, or a sequence complementary to sTREM2 or NCL or AKT or eNOS and capable of forming an interfering molecule that inhibits the expression of the sTREM2 gene expression product or gene transcription after being introduced into the body;

[0024] (iv) immune-related cells, differentiated cells or constructs after inhibiting or knocking out sTREM2 or NCL or AKT or eNOS gene sequences.

[0025] More preferably, the functional product is an scFv targeting the extracellular domain of TREM2, or a small interfering RNA targeting NCL, or an AKT inhibitor MK2206, or an eNOS inhibitor L-NAME.

[0026] The present application identifies a soluble mediator sTREM2 as a key driver of malignant ascites. Therapeutic targeting of sTREM2 can offer unique pharmacological advantages compared to targeting cells or membrane-bound proteins, including enhanced bioavailability in the fluid compartment, reduced potential for cytotoxicity, favorable pharmacokinetic properties, and the ability to monitor circulating sTREM2 levels as a biomarker of drug efficacy and measure of therapeutic response.

[0027] Importantly, administration of sTREM2-neutralizing scFv antibodies alleviated ascites production in preclinical models, demonstrating their therapeutic efficacy. The compact structure of scFv antibodies enhances tissue penetration and reduces immunogenicity. Their transient systemic exposure enables precise dose control - a safety advantage over persistent biologies - and facilitates streamlined prokaryotic production and engineering. Crucially, their therapeutic value is amplified by functional specificity. Targeting sTREM2 precisely disrupts tumor-associated macrophage-endothelial crosstalk, potentially circumventing systemic effects of VEGF inhibitors. This novel mechanism offers an alternative strategy beyond anti-VEGF therapy. Preliminary in vivo studies detected no toxicity, supporting good preliminary safety.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] This study confirmed the elevated levels of sTREM2 in ascites and revealed its functional role in promoting vascular permeability and driving ascites formation. Furthermore, we propose that sTREM2 holds dual value as a prognostic biomarker and potential therapeutic target. These findings deepen our understanding of the pathogenesis of malignant ascites and lay the foundation for developing precision therapeutic strategies against ovarian cancer and malignant ascites.

[0030] Mechanistically, sTREM2 activated AKT / eNOS signaling pathway by interacting with the cell surface nucleolin (NCL) protein, thereby impairing endothelial barrier integrity. Importantly, pharmacological targeting or blocking sTREM2 effectively attenuated vascular leakage and ascites production. These findings established sTREM2 as a central driver of ascites pathogenesis, highlighting its potential as a therapeutic target for ovarian cancer. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 sTREM2 levels were shown to be elevated in malignant ascites of ovarian cancer; (A) sTREM2 content detection in malignant (n=8 cases) and benign (n=11 cases) ascites; (B) sTREM2 concentration stratified by ascites volume (ELISA detection) (< 500 mL, n=7 cases; ≥ 500 mL, n=4 cases); Ctrl, control group; OC, ovarian cancer group; data are presented as mean ± SEM; *P<0.05; ****P<0.001; two-tailed Student’s t test was used for A and B;

[0032] Figure 2 sTREM2 was shown to promote vascular permeability; (A) TRITC-dextran leakage detection of HUVECs cell barrier treated with recombinant sTREM2 or PBS (n=3); (B) HUVECs cell barrier function detection exposed to sTREM2 or PBS (n=3); (C, D) Western blot analysis (C) and quantification (D) of p-VE-cadherin (Tyr658) and VE-cadherin in HUVECs treated with sTREM2 or PBS, β-actin was used as a loading control (n=3); (E, F) Immunofluorescence analysis (E) and quantification (F) of p-VE-cadherin (Tyr658) (green) in HUVECs treated with sTREM2 or PBS; nuclei were stained with DAPI (blue); MFI, mean fluorescence intensity; scale bar, 20 μm (n=3); (G) Schematic diagram of sTREM2 administration regimen in mice; (H) Peritoneal vascular permeability of mice injected with sTREM2 and control group (n=7); data are presented as mean ± SEM; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-tailed Student’s t test was used for A, D, F and H, two-way ANOVA analysis was used for B;

[0033] Figure 3sTREM2 drives malignant ascites formation in the ovary; (A-D) Representative images and quantification of ascites volume (A, C) and tumor burden (B, D) in mice injected with recombinant sTREM2 versus PBS with ID8 cells (n = 6); (E) ELISA measurement of sTREM2 in ascites supernatant of mice; (F) Correlation of ascites volume with sTREM2 levels; (G, H) Immunofluorescence analysis (G) and quantification (H) of p-VE-cadherin (Tyr658) (green) in mesenteric blood vessels and tumor vasculature (CD31, red); nuclei were counterstained with DAPI (blue), MFI is mean fluorescence intensity; scale bar: 20 pm (n = 6); data are presented as mean ± SEM; ***P < 0.001; ****P < 0.0001; two-tailed Student’s t test for C-E, H panels, Pearson correlation analysis for F panel;

[0034] Figure 4NCL as a functional cell surface receptor for sTREM2 in endothelial cells; (A) Workflow of pull-down assay combined with mass spectrometry analysis, proteins identified are ranked by abundance; (B) Western blot showing NCL distribution in different cellular fractions (nuclei: Nuc; plasma membrane: MF; cytoplasm: Cyto); (C) Bio-layer interferometry analysis of the binding kinetics of biotinylated sTREM2 (sTREM2-biotin) to recombinant NCL; (D, E) Co- immunoprecipitation using anti-TREM2-biotin antibody (D) or anti-NCL antibody (E) confirming the interaction of sTREM2-biotin with endogenous NCL in HUVEC membrane fractions; (F) TRITC-dextran leakage assay in NCL-knocked down HUVECs upon sTREM2 treatment (n = 3); (G, H) Western blot analysis (G) and quantification (H) of p-VE-cadherin (Tyr658) in the same experimental groups as in (F), β-actin was used as a loading control (n = 4); (I) Molecular docking model of the sTREM2-NCL interaction; (J) Domain composition of FLAG-tagged NCL constructs: full-length (NCL-FL 1-710), ΔGAR (NCLΔGAR 1-652), ΔC-terminus (NCLΔC 1-269), ΔN-terminus (NCLΔN 270-710), and RNA-binding domain (NCL-RBD 270-652); (K) Co- immunoprecipitation analysis of sTREM2-biotin binding to NCL truncation mutants; (L) TRITC-dextran leakage assay in NCL-knocked down HUVECs upon sTREM2 treatment after rescue with NCL truncation mutants (n = 4); (M) Western blot analysis of p-VE-cadherin (Tyr658) in the same experimental groups as in (L), β-actin was used as a loading control; (N, O) Immunofluorescence analysis (N) and quantification (O) of p-VE-cadherin (Tyr658) (green) in the same experimental groups as in (L); nuclei were stained with DAPI (blue); MFI is mean fluorescence intensity; scale bar: 20 μm (n = 3); data are presented as mean ± SEM; *P < 0.05; **P < 0.01; F, H, L, N graphs use two-tailed Student’s t-test;

[0035] Figure 5sTREM2 regulates vascular permeability through AKT / eNOS signaling pathway; (A) KEGG pathway analysis of differentially expressed genes in sTREM2-treated and untreated HUVECs (RNA-seq); (B, C) Western blot analysis (B) and quantification (C) of phosphorylated AKT (p-AKT) and phosphorylated eNOS (p-eNOS) in sTREM2-treated HUVECs, β-actin was used as a loading control (n=4); (D-G) Western blot analysis (D) and quantification (E-G) of p-AKT, p-eNOS and NCL in NCL-knocked down or control HUVECs treated with PBS or sTREM2, β-actin was used as a loading control (n=4); (H) Leakage detection of TRITC-dextran in HUVECs treated with sTREM2 and eNOS inhibitor L-NAME; (I, J) Western blot analysis (I) and quantification (J) of p-VE-cadherin (Tyr658) in the same experimental groups as (H), β-actin was used as a loading control (n=3); (K, L) Immunofluorescence analysis (K) and quantification (L) of p-VE-cadherin (Tyr658) (green) in HUVECs treated with sTREM2 and eNOS inhibitor L-NAME, nuclei were counterstained with DAPI (blue), MFI is mean fluorescence intensity, scale bar: 20 μm (n=3); (M) Schematic diagram of in vivo L-NAME treatment scheme; (N) Peritoneal vascular permeability of mice treated with sTREM2 and L-NAME by intraperitoneal injection (n=6); Data are presented as mean ± SEM; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; Two-tailed Student’s t test was used for C, L panels, one-way ANOVA analysis was used for E-H, J, N panels;

[0036] Figure 6Pharmacological inhibition of the AKT / eNOS signaling pathway blocks sTREM2-mediated effects; (A, B) Western blot analysis (A) and quantification (B) of p-AKT and p-eNOS in sTREM2-treated C166 endothelial cells, β-actin was used as a loading control (n = 4); (C, D) Western blot analysis (C) and quantification (D) of L-NAME on eNOS phosphorylation inhibition, β-actin was used as a loading control (n = 3); (E-G) Western blot analysis (E) and quantification (F, G) of MK2206 on AKT and eNOS phosphorylation inhibition, β-actin was used as a loading control (n = 4); (H) Leakage assay of TRITC-dextran in HUVECs treated with sTREM2 ± AKT inhibitor MK2206 (n = 4); Data are expressed as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Two-tailed Student’s t-test was used for B graph, one-way ANOVA analysis was used for D, F-H graphs;

[0037] Figure 7Targeting sTREM2 inhibits ovarian cancer ascites formation; (A) ELISA quantification of sTREM2 depletion efficiency in ovarian cancer ascites; (B) TRITC-dextran permeability assay of HUVECs treated with sTREM2-depleted and control ascites (n = 3 patients); (C, D) Western blot analysis (C) and quantification (D) of p-VE-cadherin (Tyr658) in HUVECs exposed to sTREM2-depleted or non-depleted ascites, β-actin was used as a loading control (n = 3 patients); (E) TRITC-dextran flux of C166 endothelial cells treated with sTREM2 ± anti-sTREM2 scFv (n = 4); (F) Schematic of anti-sTREM2 scFv treatment regimen in vivo; (G) Peritoneal vascular permeability in mice injected i.p. with sTREM2 ± anti-sTREM2 scFv (n = 7); (H-K) Representative images (H, I) and quantification (J, K) of ascites volume and tumor burden in mice injected i.p. with ID8 and PBS or ID8 and anti-sTREM2 scFv (n = 6); (L) ELISA measurement of sTREM2 levels in mouse ascites supernatant; (M) Correlation analysis of ascites volume and sTREM2 content; (N, O) Immunofluorescence analysis (N) and quantification (O) of p-VE-cadherin (Tyr658) (green) in mesenteric blood vessels and tumor vasculature (CD31, red); Nuclei were counterstained with DAPI (blue); MFI is mean fluorescence intensity; Scale bar: 20 μm (n = 6), data are presented as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Two-tailed Student’s t test was used for A, B, D, J-L, O graphs, Pearson correlation analysis was used for M graph; One-way ANOVA analysis was used for E, G graphs. DETAILED DESCRIPTION

[0038] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0039] METHOD

[0040] Human specimen collection: The specimens used in this study include: peritoneal lavage fluid from 8 cases of benign controls and malignant ascites from 11 cases of OC patients (approval numbers: 2022-K319-1 and 2025-011).

[0041] Cell lines and cell culture: Human umbilical vein endothelial cells (HUVECs) were purchased from SciencCell (USA); ID8 mouse ovarian epithelial cancer cells were purchased from Zhijiaoxinzhou (China). All cell lines were cultured in a humidified 5% CO2 environment at 37 °C. HUVECs were cultured in Endothelial Cell Medium (SciencCell, USA) supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin / streptomycin (100 U / mL penicillin and 100 pg / mL streptomycin). ID8 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0042] Permeability assay: Transwell-based permeability assay was used to assess endothelial barrier integrity. A confluent endothelial barrier was established by seeding HUVECs (1 × 10 5 cells / well) into the upper chamber of a 24-well Transwell plate (0.4 pm pore size; Corning, USA), and experimental treatments were added after the barrier was established. After washing twice with phosphate-buffered saline (PBS, 100 pL each time), 500 pL PBS was added to the lower chamber, and 200 pL TRITC-dextran (2 mg / mL, 70 kDa; Sigma-Aldrich, USA) was added to the upper chamber. After incubation at 37 °C for 2 h, the lower chamber fluid was collected, and the fluorescence intensity was measured using a PerkinElmer EnVision plate reader (USA).

[0043] Real-time monitoring of cell-cell interaction: The xCELLigence RTCA DP system (ACEA Biosciences, USA) was used to monitor cell-cell interaction in real time. Endothelial cells were seeded on E-Plates at a density of 2 × 10 4 cells / well. After 24 h, experimental treatments were added to each well, co-cultured with HUVECs, and the changes in cell index were monitored. The normalized cell index was calculated by dividing the cell index value at each time point by the value at the initial time point.

[0044] Enzyme-linked immunosorbent assay: The concentration of sTREM2 was quantified under standardized conditions using commercial enzyme-linked immunosorbent assay (ELISA) kits. For human samples, human-specific ELISA kits from Abeam (USA) and Join Biotech (China) were used to measure sTREM2 levels in OC patient ascites supernatant. Mouse-specific ELISA kits from Jianglai Biotech (China) were used to quantify mouse sTREM2 levels in ascites of ovarian cancer mouse models. All procedures were strictly followed the kit instructions. Briefly, a standard curve was established using the provided calibrators, followed by sample preparation and optical density measurement. The sTREM2 concentration was calculated according to the standard curve.

[0045] Western blot analysis: Total cellular proteins were extracted using protein lysis buffer (Beyotime, China). Membrane, cytoplasmic and nuclear fractions were isolated using protein extraction and cell fraction separation kit (Invent, USA). Protein concentration was quantified using BCA method protein quantification kit (Beyotime, China). Equal amounts of protein lysates were separated by SDS-PAGE (7.5% or 10% gel) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Germany). After blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with the corresponding horseradish peroxidase-labeled secondary antibodies (Proteintech, China) at room temperature. Protein bands were visualized using enhanced chemiluminescence substrate and detected by ChemiDoc XRS imaging system (Bio-Rad, USA). The corresponding internal reference proteins were used as loading controls for each fraction. Band intensities were quantified using ImageJ software (version 1.51j8; National Institutes of Health, USA).

[0046] Immunofluorescence staining: HUVECs were cultured on confocal dishes and treated with sTREM2 or other indicated conditions. Cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100 (Solarbio, China), and blocked with PBS containing 1% BSA (Solarbio, China) for 1 hour at room temperature. After incubation with anti-p-VE-cadherin (Tyr658; Thermo Fisher, USA, cat# 44-1144G) primary antibody overnight at 4°C, samples were washed and incubated with DyLight 488-labeled goat anti-rabbit secondary antibody (Abbkine, cat# A23220, China) or DyLight 649-labeled goat anti-rabbit secondary antibody (Abbkine, cat# A23620, China) for 1 hour at room temperature. Mouse tissue samples were embedded with OCT compound (Sakura Finetek, USA) immediately after collection. Frozen sections were prepared and after removal of OCT compound, the sections were blocked and permeabilized with PBS containing 5% BSA and 0.3% Triton X-100 for 1 hour. Sections were incubated with the following primary antibodies overnight at 4°C: anti-CD31 (R&D Systems, USA, cat# AF3628) and anti-p-VE-cadherin (Tyr658; Thermo Fisher, USA, cat# 44-1144G). All sections were subsequently incubated with the corresponding secondary antibodies: donkey anti-rabbit IgG DyLight 488 (Boster, China, cat# BA1146) and rabbit anti-goat IgG DyLight 649 (Abbkine, China, cat# A23630), respectively. Nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI; Solarbio, China) for 10 minutes at room temperature. Images were acquired using a confocal microscope (Zeiss, Germany). Immunofluorescence signal intensity was quantified using ImageJ software.

[0047] Streptavidin pull-down and mass spectrometry analysis: After washing with PBS for three times (5 minutes each), cells were lysed to prepare whole cell lysates or subcellular fractions. Biotinylated recombinant human TREM2 protein (Sino Biological, China; cat# 11084-H49H-B) was incubated with proteins at 4°C for 2 hours. Pierce TMStreptavidin agarose beads (ThermoFisher Scientific, USA; cat# 20347) were used to immunoprecipitate biotinylated sTREM2 and its interacting proteins from whole cell lysates or membrane fractions. The bound protein complexes were eluted, analyzed by SDS-PAGE, and then subjected to liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis (APTBIO, China).

[0048] Co-immunoprecipitation analysis: Co-immunoprecipitation experiments were performed to investigate protein-protein interactions. Cells were lysed on ice for 30 min using Western and immunoprecipitation lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitor cocktail (Beyotime, China). The lysates were then centrifuged at 12,000 x g for 20 min at 4 °C. The supernatants were incubated with the indicated primary antibodies overnight at 4 °C: anti-nucleolin (NCL; CST, USA; cat# 14574S), anti-biotin (CST, USA; cat# 7075S), anti-Flag (Proteintech, China, cat# 20543-1-AP; ABclonal, China, cat# AE005), or normal rabbit / mouse IgG control antibodies (CST, USA; cat# 3900S, 5415S). Protein A / G agarose beads (Santa Cruz Biotechnology, USA) were added to the lysate-antibody mixture and incubated with gentle rotation at 4 °C for 2 h. The beads were washed three times with pre-chilled PBS, and the bound proteins were eluted by boiling in 2x SDS loading buffer at 95 °C for 5 min. The eluted proteins were then separated by SDS-PAGE and analyzed by Western blotting to identify potential interaction partners.

[0049] Bio-layer interferometry: Bio-layer interferometry (BLI) experiments were performed using an Octet R8 system (Sartorius, Germany). All experiments were performed in PBS supplemented with 0.02% (v / v) Tween-20 (pH 7.4) at 25 °C. Biotinylated sTREM2 was freshly prepared and immobilized on streptavidin biosensors. A range of concentrations of NCL were analyzed to characterize the binding and dissociation kinetics, generating real-time binding curves.

[0050] Molecular docking: To elucidate the potential interaction between sTREM2 (PDB: 1FJ7) and NCL (PDB: 9CB5), we performed protein-protein interaction simulations using the Prime module of the Schrödinger suite. The computational model revealed a favorable binding interface between sTREM2 and NCL, suggesting a stable interaction. Specifically, Ser35 and Thr94 of sTREM2 were predicted to form hydrogen bonds with Arg342 of NCL, while Asp87 of sTREM2 established a hydrogen bond with Asn308 of NCL. These hydrogen bond interactions can contribute to stabilizing the sTREM2-NCL complex, providing structural insights into their potential functional association.

[0051] In vitro sTREM2 depletion: To deplete the protein of interest, the conditioned media was incubated with specific antibodies, including anti-TREM2 (Thermo Fisher Scientific, USA; cat# PA5119690), anti-HA (Proteintech, China; cat# 51064-2-AP), or anti-His (ABclonal, China; cat# AE003) at a concentration of 10 pg / mL for 2 hours at 37°C. Subsequently, 100 pL of Protein A / G agarose beads was added and incubated gently at 4°C for 6 hours. The immune complexes were pelleted by centrifugation at 400 x g for 5 minutes, and the supernatant was collected. To assess the effect of single-chain antibody fragments (scFv) on sTREM2 detection, His-tagged scFv was incubated with the test supernatant at room temperature for 2 hours. Anti-His antibody and 100 pL of Protein A / G agarose beads were then added sequentially. After the final immunoprecipitation step, the clarified supernatant was taken for ELISA analysis.

[0052] Recombinant proteins and scFv antibodies acquisition: All recombinant proteins and scFv antibodies were commercially purchased, including the following: human sTREM2-His (Sino Biological, China; cat# 11084-H08H), mouse sTREM2-His (Sino Biological; cat# 50149-M08H), and biotinylated human sTREM2-His (Sino Biological; cat# 11084-H49H-B); human NCL recombinant protein (OriGene, USA; cat# TP319082); anti-sTREM2 scFv-His (KAIRUI Biotech, China; patent WO2020123664A1); and mouse sTREM2-Flag (Tsingke, China; UniProt KB Q99NH8, residues 19-171).

[0053] Animals: All animals were housed under specific pathogen-free (SPF) conditions at the Guangdong Provincial Engineering Research Center for Molecular Imaging. Female C57BL / 6 wild-type (WT) and macrophage-specific TREM2 knockout (Trem2MacKO) mice (approval numbers: 00353 and 00585) on C57BL / 6 background were used in this study. Trem2 MacKO Mice were generously gifted by Professor Huang Xi (Sun Yat-Sen University).

[0054] In vivo model permeability experiment: To evaluate the effect of sTREM2, female Trem2 MacKO Mice (6-8 weeks old) were injected intraperitoneally (100 μL) with PBS (control group) or recombinant sTREM2 (200 μg / mouse in PBS) every other day. On day 9, mice were intravenously injected with 100 μL TRITC-dextran (10 mg / mL, 70 kDa, Sigma-Aldrich, USA) via the tail vein. After 30 min of circulation, intraperitoneal lavage was performed using 2.5 mL ice-cold PBS. The lavage fluid was centrifuged, and the fluorescence intensity of the supernatant was measured. For L-NAME treatment, Trem2 MacKO Mice were divided into four groups and received: PBS, L-NAME (625 μg / mouse; Sellect, USA), sTREM2 (200 μg / mouse), or a combination of sTREM2 (200 μg / mouse) and L-NAME (625 μg / mouse; Sellect, USA). sTREM2 was administered every other day, and L-NAME was administered daily. Vascular permeability was evaluated on day 9 using the method described above. For scFv treatment, Trem2 MacKOMice were divided into three groups and dosed every other day: PBS, sTREM2 (200 pg / mouse), or a combination of sTREM2 and scFv (200 pg / mouse). Vascular permeability was assessed on day 9.

[0055] Establishment of the OC mouse model: On day 1, female Trem2 MacKO Mice (6-8 weeks old) were intraperitoneally inoculated with 1 x 10 6 ID8 mouse ovarian cancer cells. Subsequently, recombinant sTREM2 (200 pg / mouse in 100 pL PBS) or PBS was administered by intraperitoneal injection on days 1, 8, 15, and 22, once a week. To evaluate the therapeutic potential of the sTREM2-targeting scFv, WT mice with established ID8 tumors (established by inoculation with 1 x 10 6 ID8 mouse ovarian cancer cells on day 1) were intraperitoneally injected with scFv (200 pg / dose in 100 pL PBS) or PBS every week starting from day 21. At the end of the experiment, animals were euthanized. Mesenteric tissue, ascites, and tumor tissue were collected. Ascites cells and ascites supernatant were collected by centrifugation. We quantified the ascites volume, evaluated the tumor burden, and performed ELISA analysis on ascites supernatant.

[0056] Statistics: All quantitative data are presented as mean ± standard error of the mean (SEM) of at least three independent biological replicates. All statistical analyses were performed using GraphPad Prism (version 8.2.1). Comparisons between two groups were made using a two-tailed Student’s t-test. Comparisons between more than two groups were made using one-way ANOVA. To assess the influence of two independent variables, a two-way ANOVA was employed. Correlation analysis used the Pearson correlation coefficient (for normally distributed data) and the Spearman rank correlation coefficient (for non-normally distributed data). A p-value less than 0.05 (p < 0.05) was considered statistically significant. Immunofluorescence and Western blot images were quantified using ImageJ software (version 1.51j8; National Institutes of Health, USA).

[0057] Example 1 sTREM2 drives ascites formation in mouse ovarian cancer

[0058] I. sTREM2 is significantly elevated in ovarian cancer ascites

[0059] sTREM2 has been reported to be involved in the pathogenesis of various diseases, and we quantified sTREM2 levels in ascites supernatant by ELISA. sTREM2 was significantly elevated in malignant ascites and correlated with increased ascites volume (Figure 1 A, B). This clinical finding was further supported by published proteomic data showing that sTREM2 was significantly enriched in malignant ascites ( Figure 1 C). Taken together, these data suggest that sTREM2 is upregulated in malignant ascites.

[0060] II. sTREM2 promotes vascular permeability

[0061] To investigate whether sTREM2 directly regulates vascular permeability, we treated endothelial cells directly with recombinant sTREM2, which resulted in induction of vascular leakage ( Figure 2 A, B), and promotion of VE-cadherin phosphorylation ( Figure 2 C-F). Importantly, in Trem2 MacKO In vivo administration of sTREM2 to mice significantly increased vascular permeability ( Figure 2 G, H). Together, these results suggest that sTREM2 directly impairs endothelial barrier function.

[0062] III. sTREM2 drives ascites formation in mouse ovarian cancer

[0063] To directly assess the role of sTREM2 in ovarian cancer ascites generation, ID8 tumor cells were injected into Trem2 MacKO mice, which were subsequently administered sTREM2 or PBS intraperitoneally. The results showed that sTREM2 induced more ascites and intraperitoneal tumors than the PBS control group ( Figure 3 A-D). In addition, the ascites sTREM2 content was positively correlated with the ascites volume ( Figure 3 E, F). Immunofluorescence analysis of mesenteric blood vessels and tumor blood vessels in these mice showed enhanced VE-cadherin phosphorylation ( Figure 3 G, H), reproducing the in vitro findings. Together, these results suggest that sTREM2 promotes vascular hyperpermeability and drives OC ascites generation.

[0064] Example 2 NCL is a functional receptor for sTREM2 in endothelial cells

[0065] Although the function of sTREM2 in macrophages, neurons, and tumor cells has been extensively characterized, its biological effects on endothelial cells are not well understood. To identify the endothelial cell receptor for sTREM2, we performed a pull-down assay combined with mass spectrometry in HUVECs. Interestingly, nucleolin (NCL) was identified as the most abundant sTREM2 cell surface binding protein ( Figure 4 A). Although NCL is primarily localized to the nucleolus, it also binds to oncogenic and angiogenic ligands as a cell surface receptor.

[0066] To verify NCL as the functional receptor for sTREM2, we first performed a subcellular fractionation experiment, confirming the presence of NCL on the plasma membrane of HUVECs ( Figure 4 B). Next, a biolayer interferometry assay revealed the affinity between sTREM2 and NCL ( Figure 4 C). Immunoprecipitation analysis and Pull-down assay of membrane proteins further confirmed the interaction between sTREM2 and NCL ( Figure 4 D, E). In addition, knockdown of NCL significantly attenuated sTREM2-mediated endothelial hyperpermeability ( Figure 4 F-H).

[0067] NCL is a multi-domain protein, containing an N-terminal domain, four central RNA-binding domains (RBDs), and a C-terminal glycine-arginine-rich domain. To identify the specific NCL domains that mediate sTREM2 binding, we first performed molecular docking, predicting the RBDs as potential binding sites for sTREM2 ( Figure 4 I). To verify this prediction, we transiently transfected cells with FLAG-tagged full-length NCL or various truncated constructs ( Figure 4 J), and performed immunoprecipitation. Notably, a C-terminal deletion mutant that lacked only the RBDs failed to bind to sTREM2 ( Figure 4 K). Crucially, in NCL-knockdown cells, transfection of full-length NCL enabled observation of the sTREM2-induced pro-permeability phenotype, but transfection of the C-terminal deletion mutant did not ( Figure 4 L-O). In summary, these results indicate that the RBDs of cell-surface NCL are critical for sTREM2 to exert its permeability effect.

[0068] Example 3 sTREM2 regulates vascular permeability by mediating AKT / eNOS signaling

[0069] To elucidate the mechanism by which sTREM2 mediates vascular permeability, we performed RNA sequencing on sTREM2-treated HUVECs. KEGG pathway enrichment analysis of differentially expressed genes showed significant enrichment of the phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) signaling pathway ( Figure 5 A). Although the interaction of NCL with the PI3K / AKT pathway in malignant cells has been reported, their role in endothelial cells is poorly understood. In addition, endothelial nitric oxide synthase (eNOS), as a key downstream effector of the PI3K / AKT signal, is known to regulate vascular permeability by promoting VE-cadherin phosphorylation. Based on these findings, we hypothesized that sTREM2 functions through the AKT / eNOS signaling axis.

[0070] Consistent with the hypothesis, we observed that sTREM2 enhanced phosphorylation of AKT and eNOS in endothelial cells ( Figure 5 B, C; Figure 6 A, B). Moreover, this activating effect could be abolished by NCL knockdown ( Figure 5 D-G). To functionally validate this mechanism, we treated endothelial cells with the eNOS inhibitor L-NAME, which abolished sTREM2-mediated endothelial hyperpermeability ( Figure 5 H-L; Figure 6 C, D). Similarly, inhibiting AKT phosphorylation with MK2206 had an equivalent effect to L-NAME ( Figure 6 E-H). Furthermore, in vivo permeability experiments showed that intraperitoneal injection of L-NAME effectively inhibited sTREM2-induced vascular leakage ( Figure 5 M, N). Together, these results suggest that sTREM2 promotes vascular permeability by activating an NCL-dependent AKT / eNOS signaling pathway.

[0071] Example 4 Targeting sTREM2 can inhibit ovarian cancer ascites formation

[0072] To assess whether targeting sTREM2 can attenuate OC ascites formation and disease progression, we first depleted sTREM2 in patient-derived ascites supernatants using a neutralizing antibody ( Figure 7 A). Incubation of endothelial cells with sTREM2-depleted ascites supernatants significantly reduced TRITC-dextran leakage and VE-cadherin phosphorylation compared to control IgG-treated supernatants ( Figure 7 B-D). Next, we evaluated whether sTREM2 neutralization inhibits in vivo vascular leakage using a previously reported single-chain antibody (scFv) targeting the mouse TREM2 ectodomain. Permeability experiments confirmed that scFv treatment attenuated sTREM2-induced endothelial leakage ( Figure 7 E). Crucially, in vivo studies showed that mice receiving scFv treatment exhibited significantly reduced vascular permeability compared to controls ( Figure 7 F, G).

[0073] Finally, in the ID8 mouse model, scFv antibody administration was initiated three weeks after tumor inoculation, significantly reducing ascites volume and tumor burden compared to controls ( Figure 7 H-K). Notably, scFv-treated mice exhibited reduced ascites sTREM2 levels ( Figure 7 L), and sTREM2 levels remained positively correlated with ascites volume ( Figure 7 M). Moreover, immunofluorescence analysis of metastatic tissue showed attenuated VE-cadherin phosphorylation in scFv-treated miceFigure 7 N, O). In summary, these results show that sTREM2 blockade effectively mitigates vascular leakage and inhibits malignant ascites formation.

[0074] sTREM2 is generated by receptor shedding or alternative splicing and can be detected in plasma and cerebrospinal fluid. sTREM2 has thus been validated as a diagnostic and prognostic biomarker for various pathological conditions, including non-alcoholic fatty liver disease, Alzheimer’s disease, and primary angiitis of the central nervous system. In the present study, we detected sTREM2 in ascites and established a significant correlation between sTREM2 levels and ascites volume. This finding opens new avenues for OC and ascites research. Mechanistically, how sTREM2 is involved in multiple pathologies remains unclear. Current studies have mainly focused on its immunomodulatory role, while neglecting its impact on non-immune components of the microenvironment, especially the vasculature, which is an important knowledge gap. To address this blind spot, we discovered a new pathway in which sTREM2 directly targets endothelial cells by binding NCL, thereby disrupting barrier integrity. This finding establishes the sTREM2-endothelial axis as a novel regulatory mechanism in the tumor microenvironment and identifies sTREM2 as a new functional ligand for NCL. NCL is a multifunctional protein widely expressed in eukaryotic cells. Although mainly localized in the nucleolus, NCL can dynamically shuttle between the nucleoplasm, cytoplasm, and plasma membrane, and its subcellular translocation determines functional specificity. Our study identifies NCL as a functional receptor for sTREM2 in endothelial cells, redefining it as a key regulator of vascular permeability.

[0075] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Use of sTREM2 in developing and screening functional products for reducing malignant ascites of ovarian cancer.

2. Use according to claim 1, characterized in that, The functional products include any of the following: (i) small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid targeting sTREM2 or NCL or AKT or eNOS, or sTREM2 or NCL or AKT or eNOS transcript, and capable of inhibiting the expression or gene transcription of sTREM2 or NCL or AKT or eNOS gene expression product; (ii) small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid construct capable of expressing or forming the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid in (i); (iii) construct containing sTREM2 or NCL or AKT or eNOS, or sTREM2 or NCL or AKT or eNOS complementary sequence, and capable of forming an interference molecule inhibiting the expression or gene transcription of sTREM2 gene expression product after being transferred into the body; (iv) immune-related cells, differentiated cells or constructs after inhibiting or knocking out sTREM2 or NCL or AKT or eNOS gene sequence.

3. Use according to claim 2, characterized in that, The functional products are scFv targeting the extracellular domain of TREM2, or small interfering RNA targeting NCL, or AKT inhibitor MK2206, or eNOS inhibitor L-NAME.

4. Use of sTREM2 in developing and screening functional products for prognosis analysis of malignant ascites of ovarian cancer.

5. Use of sTREM2 in developing and screening functional products for reducing endothelial cell permeability.

6. Use according to claim 5, characterized in that, The functional products include any of the following: (i) small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid targeting sTREM2 or NCL or AKT or eNOS, or sTREM2 or NCL or AKT or eNOS transcript, and capable of inhibiting the expression or gene transcription of sTREM2 or NCL or AKT or eNOS gene expression product; (ii) small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid construct capable of expressing or forming the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid in (i); (iii) construct containing sTREM2 or NCL or AKT or eNOS, or sTREM2 or NCL or AKT or eNOS complementary sequence, and capable of forming an interference molecule inhibiting the expression or gene transcription of sTREM2 gene expression product after being transferred into the body; (iv) immune-related cells, differentiated cells or constructs after inhibiting or knocking out sTREM2 or NCL or AKT or eNOS gene sequence.

7. Use according to claim 6, characterized in that, The functional products are scFv targeting the extracellular domain of TREM2, or small interfering RNA targeting NCL, or AKT inhibitor MK2206, or eNOS inhibitor L-NAME.

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