Application of CFD as target spot in gastric cancer peritoneal metastasis

Abstract

The invention relates to application of CFD as a target spot in gastric cancer peritoneal metastasis, and belongs to the technical field of biological medicine. Through single cell transcriptome sequencing, it is found that CFDmesothelial cells in a microenvironment before peritoneal metastasis of a gastric cancer patient are remarkably amplified, and it is proved that CFD promotes gastric cancer cell invasion and metastasis by inducing mesothelium-mesenchymal transition (MMT). Targeted CFD can inhibit formation of MMT and peritoneal metastasis, the invention discloses a molecular mechanism that CFD regulates microenvironment mediated metastasis before peritoneal metastasis, and provides a new target and strategy for early warning and targeted therapy of gastric cancer peritoneal metastasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of CFD as a target in peritoneal metastasis of gastric cancer. Background Art

[0002] Gastric cancer is the fifth most common malignant tumor in the world and the third leading cause of cancer-related death in the world, posing a serious threat to human health. In my country, the incidence and mortality of gastric cancer rank third and second among malignant tumors, respectively. Most gastric cancer patients are already in the middle and late stages when diagnosed, and tumor metastasis becomes the main problem in treatment. With the widespread popularity of standard radical resection for gastric cancer, local lymph node metastasis is no longer the main cause of gastric cancer recurrence, and peritoneal metastasis has become an important risk factor affecting the prognosis of gastric cancer. However, the current understanding of the pathogenesis of peritoneal metastasis of gastric cancer is still unclear, especially the dynamic evolution of the microenvironment before peritoneal metastasis and its driving role in tumor metastasis have not been fully elucidated, which makes it difficult to detect and intervene in peritoneal metastasis at an early stage, limiting the treatment effect.

[0003] Previous studies have shown that even in the early stages of gastric cancer, before peritoneal metastasis occurs, the peritoneal microenvironment has already changed. However, because these studies used exome and transcriptome sequencing, they were unable to precisely analyze tumor heterogeneity and cell-cell interactions.

[0004] Complement Factor D (CFD) is a key serine protease in the alternative pathway of the complement system, playing a central role in the initial activation of the alternative pathway. As a core component of innate immunity, aberrant activation of the complement system's alternative pathway has been shown to be closely associated with tumor development, progression, and immune escape. Previous research on CFD has primarily focused on its effects on tumor cells, but its role in remodeling the peritoneal niche prior to metastasis remains unclear. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention provides the application of CFD as a target in peritoneal metastasis of gastric cancer.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The present invention discloses the use of a CFD inhibitor in preparing a medicine for preventing or treating peritoneal metastasis of gastric cancer.

[0008] Furthermore, the CFD inhibitor includes inhibiting CFD expression or activity.

[0009] Preferably, the method for inhibiting CFD expression comprises siRNA or shRNA targeting CFD, and the sequence of the siRNA is shown in SEQ ID NO. 1-2.

[0010] Preferably, the inhibiting CFD activity comprises a small molecule inhibitor targeting CFD.

[0011] The small molecule inhibitor of CFD is Pelecopan.

[0012] The present invention also discloses a drug for inhibiting peritoneal metastasis of gastric cancer, comprising a CFD inhibitor and a pharmaceutically acceptable carrier.

[0013] Furthermore, the CFD inhibitor includes inhibiting CFD expression or activity.

[0014] Preferably, the method for inhibiting CFD expression comprises siRNA or shRNA targeting CFD, and the sequence of the siRNA is shown in SEQ ID NO. 1-2.

[0015] Preferably, the inhibiting CFD activity comprises a small molecule inhibitor targeting CFD.

[0016] The present invention also discloses the use of a CFD inhibitor in the preparation of a drug for inhibiting mesothelial-mesenchymal transition (MMT).

[0017] Compared with the prior art, the present invention has the following beneficial effects.

[0018] This study focuses on the clinical challenge of peritoneal metastasis of gastric cancer and systematically studies the role of complement factor D (CFD) in the regulation of the pre-metastatic microenvironment and its molecular mechanism for the first time. Currently, pre-metastatic niche research focuses on the regulation of exosomes or immune cells, while the role of the complement system is still underrecognized. This study first discovered CFD through the analysis of single-cell sequencing data of the greater omentum of gastric cancer patients (dynamic cohort of early-mid-late stage). + The specific expansion of mesothelial cell subsets has not been reported before. Based on this, the inventors will further explore the molecular mechanism by which CFD mediates gastric cancer peritoneal metastasis by regulating mesothelial-mesenchymal transition (MMT) and remodeling the immune microenvironment, filling the gap in CFD research on peritoneal metastasis.

[0019] This invention not only deeply reveals the mechanism of action of the complement system in peritoneal metastasis of gastric cancer, but also provides new ideas for targeted therapy. It is expected to achieve precise intervention of peritoneal metastasis, shifting from "passive treatment of metastasis" to "active prevention of metastasis", and will provide theoretical basis and technical support for early warning and treatment of peritoneal metastasis of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Single-cell transcriptome atlas of the omental microenvironment. (A) Experimental design. (B) UMAP plot showing the distribution of major cell types in patients. (C) UMAP plot showing the expression of markers for major cell types in each population. (D) Proportion of each cell type in early-stage, mid-stage, and late-stage patient samples. (E) Comparison of the number and strength of cell-cell interactions in early-stage, mid-stage, and late-stage patient samples.

[0021] Figure 2 Mesothelial cells in omentum tissue differentiate toward fibroblasts. (A) UMAP images show epithelial cell subpopulation. (B) Proportion of each cell population in tissues from patients at early, mid, and late stages of disease. (C) CNV analysis of epithelial cells. (D) Comparison of EMT scores between epithelial cells in peritoneal tissue and those in primary tumors. (E) Pseudo-time analysis of mesothelial cells and fibroblasts.

[0022] Figure 3 CFD-induced mesothelial cell differentiation into fibroblasts. (A and B) Human mesothelial cells Sv5 were cocultured with gastric cancer cells for 24 and 48 hours. (C) qRT-PCR analysis of differential gene expression in mesothelial cells after 24 and 48 hours of coculture. (D) Western blot analysis of CFD and MMT indicator proteins. (E) siCFD was added to the coculture system. (F) Western blot analysis of CFD and MMT indicator proteins. (G) After knockdown of CFD in mesothelial cells, the invasion and migration abilities of gastric cancer cells were assessed using a Transwell assay.

[0023] Figure 4 The CFD inhibitor pelecopan inhibits MMT in mesothelial cells. (A) 10 nM of the CFD inhibitor pelecopan was added to the co-culture system of human mesothelial cell line Sv5 cells and tumor cells. (B) The effects of CFD recombinant protein on MMT were compared with and without the CFD inhibitor.

[0024] Figure 5 This nude mouse model demonstrates that CFD promotes peritoneal metastasis of gastric cancer cells. (A) Schematic diagram of nude mouse tumors with peritoneal metastasis of gastric cancer and tumor size. (B) HE staining analysis of tumor area. (C) Immunohistochemical analysis of CFD expression in tumor tissue. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0026] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0027] 1. Single-cell transcriptome atlas of the omental microenvironment.

[0028] 1. Experimental materials.

[0029] Greater omentum tissue from patients with early, middle and late (with metastatic nodules) stages of gastric cancer.

[0030] 2. Experimental method.

[0031] (1) Tissue dissociation and single-cell suspension preparation.

[0032] The obtained tissue was transferred to a culture dish pre-added with 1×PBS (without RNase, Ca, and Mg ions) (the culture dish was placed on wet ice) and the tissue was cut into 0.5 mm 2The washed tissue pieces were then repeatedly washed with 1× PBS to remove surface deposits such as blood stains and grease. The washed tissue pieces were then added with dissociation solvent (0.35% collagenase IV5, 2 mg / mL papain, 120 units / mL DNase I) and incubated at 37°C in a shaking waterbath (100 rpm) for 20 minutes. Dissociation was then terminated by adding 1× PBS (containing 10% fetal bovine serum (FBS)). The cell suspension was pipetted up and down 5–10 times (gently to prevent shear stress that may cause cell death). The cell suspension was filtered through a 70–30 μm cell sieve and centrifuged at 300 g for 5 minutes at 4°C. The cell pellet was collected and resuspended in 100 μL of 1× PBS (0.04% BSA). To remove erythrocytes, 1 mL of erythrocyte lysis buffer (MACS 130-094-183, 10×) was added and lysed for 2–10 minutes at room temperature or on wet ice. After lysis, centrifuge at 300g for 5 minutes at room temperature. Collect the cell pellet and add 100μL of dead cell removal reagent: Dead Cell Removal MicroBeads (MACS130-090-101) and remove dead cells using the Miltenyi® Dead Cell Removal Kit (MACS130-090-101). After the reaction, remove the reagent by centrifugation. Resuspend the pellet in 1× PBS (0.04% BSA) and centrifuge at 300g for 3 minutes at 4°C (repeat twice). After tissue dissociation, erythrocyte lysis, and dead cell removal, prepare a cell suspension in 50μL of 1× PBS (0.04% BSA). Cell viability was assessed using trypan blue staining, with a viability requirement of >85%. Count cells using a hemocytometer or automated cell counter (Countess II Automated Cell Counter). The cell concentration should be 700-1200 cells / μL.

[0033] (2) Library construction and sequencing.

[0034] The resulting cell suspension was loaded onto a 10x Chromium instrument. Cell capture (5,000 target cells captured), cDNA amplification, and library construction were performed according to the official library construction kit (10XGenomics Chromium Single-Cell 3' kit, V3). After library construction, sequencing was performed using the NovaSeq 6000 sequencing platform (paired-end multiplexing runs, 150 bp) with a required sequencing depth of 20,000 reads per cell. All technologies were provided by LC-Bio Technology Co., Ltd., Hangzhou, China.

[0035] 3. Experimental results.

[0036] In order to systematically explore and analyze the cell map and dynamic changes of various cells in the peritoneal microenvironment during peritoneal metastasis of gastric cancer, clinical samples from 17 gastric cancer patients were collected. Among them, there were 7 early-stage, 7 mid-stage and 3 late-stage patients. Except for the 3 late-stage patients who had metastatic lesions colonized in the greater omentum tissue, the other 14 cases were all omentum tissue. The experiments were conducted according to unified and standard single-cell suspension preparation, single-cell library construction and library sequencing experimental protocols to reduce the bias and batch effect caused by experimental operations. The 10x Genomics ChromiumTM Next GEM Single Cell 3ʹ-end single-cell RNA sequencing platform was used to perform single-cell RNA sequencing on each tissue sample. After strict quality control and screening, we screened and finally obtained 111,068 high-quality single-cell data ( Figure 1 A). Based on the expression of the PTPRC (CD45) gene, cells can be clearly divided into immune cells and non-immune cells. Based on known marker genes and differentially expressed genes in cell subpopulations, we identified 6 major cell subpopulations. The UMAP diagram shows that there are certain differences in the distribution of cells among the groups ( Figure 2B and 2C). For immune cells, the identification results include T cells (T Cell: CD3D and CD3E), myeloid cells (MyeloidCell: CD14), B / plasma cells (B Cell: MS4A1 (Plasma Cell: XBP1 and MZB1) For the annotation of non-immune cells, we identified epithelial (mesothelial) cells (EPCAM and CLDN4), fibroblasts (Fibroblast: COL3A1 and NNMT) and endothelial cells (Endothelial Cell: VWF). It was found that with the progression of the disease (early-middle-late metastasis), the proportion of cells changed significantly, especially in the middle stage, the proportion of CAFs increased significantly, while the proportion of mesothelial cells decreased significantly. In addition, the intercellular communication network was significantly enhanced in patients in the middle stage without metastasis (P<0.01), and further strengthened in the late stage ( Figure 1 These results confirm the existence of pre-metastatic niche remodeling.

[0037] 2. Mesothelial cells in peritoneal tissue differentiate into fibroblasts.

[0038] 1. Experimental method.

[0039] Bioinformatics Analysis: Sequencing data were parsed using Illumina bcl2fastq software (version 2.20) and converted to FASTQ format. We used 10x Genomics' official analysis software, CellRanger (https: / / support.10xgenomics.com / single-cell-geneexpression / software / pipelines / latest / what-is-cell-ranger, version 6.1.1), to analyze the raw sequencing data for each sample and align them to the Ensembl genome GRCh38 / GRCm38 reference genome. The analysis results showed that a total of 30,000 cells were captured from five healthy and five diseased donor samples. CellRanger analysis files were imported into Seurat software (version 3.1.1) for data dimensionality reduction, cluster analysis, and expression matrix analysis. A total of 25,000 high-quality cells were obtained through quality control filtering. The quality control thresholds were: all genes were detected in at least three cells, the number of genes expressed in a single cell was between 500 and 5000, the number of UMIs was greater than or equal to 500, and the mitochondrial gene expression ratio was less than 25%. Seurat software was used to perform dimensionality reduction analysis on the filtered 18,339 cells, and t-SNE visualization was performed.

[0040] The analysis process included the following steps: 1. After removing low-quality cells, expression levels were normalized using the LogNormalize method in the "Normalization" function of Seurat software. 2. Dimensionality reduction was performed using principal component analysis to reduce the number of variables, and then PCA (Principal Component Analysis) was performed using the normalized expression values. The top 10 principal components from the PCA analysis were selected for subsequent clustering and subpopulation analysis. 3. Cell clusters were identified using a clustering algorithm optimized using the shared nearest neighbor (SNN) module. Seurat's FindAllMarkers toolkit used the bimodal likelihood ratio statistical test to identify differentially expressed marker genes across cell populations. Marker gene analysis criteria required that the gene be expressed in at least 10% of cells in either the target or control subpopulations and that the gene expression fold change logFC be ≥ 0.26.

[0041] 2. Experimental results.

[0042] Mesothelial cells were repopulated, such as Figure 2 As shown in A, 9 cell populations were obtained. It was found that C7_CFD mesothelial cells were significantly increased in the omentum tissue of patients in the middle stage compared with the early stage tissue, and continued to increase in the late stage tissue ( Figure 2 B). CNV analysis of T cells, B cells, and epithelial cells was performed using the "inferCNV" algorithm. The results showed that compared to normal cells, all C5_LYZ epithelial cells showed significant abnormal amplification or deletion of large-scale chromosome copy number, suggesting that C5_LYZ cells are tumor cells. This group of cells also showed a specific increase in late-stage metastatic lesions ( Figure 2 C). Single-cell data from gastric cancer primary lesions were included to score cell EMT, and it was found that the EMT of mesothelial cells in peritoneal tissue was significantly higher than that in primary lesions. Pseudo-time analysis further confirmed the differentiation of mesothelial cells into fibroblasts ( Figure 2 D and 2E). It is worth noting that the C7_CFD mesothelial cells that significantly increased in the mid-stage are the intermediate part of the MMT process, suggesting that this group of cells may be an important intermediate in the process of mesothelial to fibroblast transformation.

[0043] 3. CFD induces mesothelial cells to differentiate into tumor-associated fibroblasts to promote gastric cancer cell invasion and migration.

[0044] 1. Experimental materials.

[0045] (1) Cells: Human gastric cancer cell lines HGC-27 and MGC-803; human epithelial cells Sv5 (Shanghai Cell Culture Bank, Chinese Academy of Sciences).

[0046] (2) Cell culture: Dulbecco's modified Eagle's medium (DMEM, Hyclone, USA); fetal bovine serum (FBS) (Ecosine, China); RPMI-1640 medium (Hyclone, USA); trypsin (Biological Industries, USA); dimethyl sulfoxide (DMSO) (Dingguo Changsheng, Beijing); puromycin (Selleck Chemicals, USA).

[0047] (3) RNA extraction and RT-qPCR reagents: Trizol (Dingguo Changsheng, Beijing); chloroform (Beijing Chemical Plant, Beijing); isopropanol (Fuyu, Tianjin); DEPC water (Dingguo Changsheng, Beijing); reverse transcription kit (Takara, Japan); SYBR (Takara, Japan); primers (BGI, Beijing).

[0048] (4) Other reagents: cell lysis buffer (Beyotime, Shanghai); secondary antibodies: HRP-goat anti-rabbit, HRP-goat anti-mouse (Beijing Zhongshan Company).

[0049] 2. Experimental method.

[0050] 2.1 Cell co-culture.

[0051] Gastric cancer cells MGC-803 and HGC-27 were cultured for 48 h, and the tumor cell supernatant was collected in a 15 mL centrifuge tube and centrifuged at 500 × g for 5 min. The supernatant was added to human mesothelial cells Sv5 and cultured for 24 or 48 h. Changes in mesothelial cell morphology were observed under an inverted microscope.

[0052] 2.2 Extraction of total cell RNA.

[0053] (1) Take the culture dish full of cells out of the incubator, discard the cell culture medium, wash it with PBS three times, add 500 μL Trizol to a disposable culture dish, lyse the cells, and repeatedly pipette until the adherent cells are completely detached.

[0054] (2) Transfer the cell lysate into a 1.5 mL siliconized EP tube, add 100 μL of chloroform, shake gently, vortex for 5 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes.

[0055] (3) After centrifugation, the liquid in the EP tube is divided into three layers. 200 μL of the upper aqueous phase is transferred to a new 1.5 mL siliconized EP tube. Then, an equal volume of isopropanol is added. The tube is allowed to stand at room temperature for 30 min. Centrifuge at 4°C at 12,000 rpm for 10 min. A white precipitate is observed at the bottom of the siliconized EP tube. Discard the supernatant.

[0056] (4) Add 500 μL of 75% ethanol (prepared with DEPC water) to the siliconized EP tube, gently flick the bottom precipitate, centrifuge at 12,000 rpm at 4°C for 5 minutes, and discard the supernatant. Repeat step (4) once.

[0057] (5) Air-dry the tube to evaporate the remaining ethanol. Dissolve the precipitate in 30 μL of DEPC water. Measure the concentration and observe the OD260 / OD280 values. Store at -80°C.

[0058] 2.3RNA reverse transcription and real-time quantitative immunofluorescence analysis.

[0059] (1) Calculate the required volumes of RNA and RNase-free ddH2O based on a 1 μg system.

[0060] .

[0061] Incubate at room temperature for 5 minutes or at 42°C for 2 minutes.

[0062] (2) Configure the reverse transcription reaction system.

[0063] .

[0064] (3)PCR procedure.

[0065] .

[0066] The cDNA product was stored at -20°C and used as a PCR template.

[0067] Forward and reverse primers for RT-qPCR.

[0068] .

[0069] (4) Fluorescence quantitative reaction system (20 μL).

[0070] .

[0071] (5) Fluorescence quantification procedure: 95℃ 1min, 55℃ 30s, 72℃ 1min; 40 cycles.

[0072] 2.4 Cell protein extraction and Western-blotting.

[0073] (1) Collect cells: Discard the culture medium of the cells to be collected and add 2 mL of PBS to wash them three times. Discard the PBS completely for the last time and place the treated cells on ice.

[0074] (2) Cell lysis: Add the prepared RIPA lysis buffer (with protease inhibitors added in proportion) evenly to the culture dish according to the number of cells (generally 6-8 times the volume of the cell pellet) and lyse on ice for 20 minutes. Scrape the lysed cell mixture with a cell scraper and transfer it to a 1.5 mL EP tube. Ultrasonicate the mixture 5 times and then let it stand on ice for 5 minutes.

[0075] (3) Centrifugation: Centrifuge at 4°C, 12,000 rpm, for 20 min. Transfer the supernatant to a clean 1.5 mL EP tube and label it.

[0076] (4) Concentration measurement and sample preparation: Determine the total cellular protein concentration using the G250 Coomassie Brilliant Blue method. Prepare samples at a mass of 30 μg per sample. Add the appropriate amount of 6× Loading Buffer and make up to a certain volume with ddH2O. Boil in boiling water for 5 min, remove, and set aside.

[0077] (5) Glue preparation: Clean the glass plate and comb and let them dry. Prepare a 12% concentration of lower separation gel, mix well and add it to the glass plate. Add a small amount of 95% alcohol to flatten the liquid surface and let it stand for 30 minutes. Pour out the alcohol and let it dry. Prepare concentrated gel according to the proportion, add the concentrated gel and insert the comb into the hole. Let it stand for 10-15 minutes until it solidifies.

[0078] SDS-PAGE separation gel (12% / 15mL): .

[0079] SDS-PAGE stacking gel (6 mL): .

[0080] (6) Electrophoresis: Prepare 1 L of running buffer, assemble the electrophoresis tank, add the sample, and run the stacking gel at 90 V and the separating gel at 110 V. Stop the electrophoresis when the protein sample moves to the bottom of the gel plate.

[0081] (7) Transfer: Place the PVDF membrane in a clean box, add 100 mL of methanol, let it soak for 1 min, prepare 1 L of transfer buffer, and place it in the transfer tank in the order of anode - sponge - filter paper - PVDF membrane - gel - filter paper - sponge - cathode, 4°C, 80 V, 2 h.

[0082] (8) Blocking: After the transfer is completed, the membrane is cut according to the desired protein position and blocked with 5% skim milk powder at room temperature for 1 hour.

[0083] (9) Primary antibody: Use specific primary antibody prepared in TBST and incubate overnight at 4°C (antibody concentration: E-cadherin, N-cadherin, CFD and Vimentin 1:1000; GAPDH 1:10000).

[0084] (10) Membrane washing: Take out the membrane the next day and wash it three times with TBST, each time for 10 minutes.

[0085] (11) Secondary antibody: Incubate with the corresponding secondary antibody labeled with horseradish peroxidase (1:10000) at room temperature for 1 h, goat anti-rabbit (Dingguo, Beijing, 1:10000); goat anti-mouse (Dingguo, Beijing, 1:10000).

[0086] (12) Membrane washing: Wash the membrane with TBST three times, each time for 10 min.

[0087] (13) Luminescence: Prepare ECL luminescence solution (substrate: buffer solution = 1:1, prepared immediately before use), and use an ECL luminometer to acquire and save images.

[0088] 2.5 Transwell assay

[0089] The Transwell chamber was rehydrated with Matrigel and DMEM (serum-free) at 37°C for 2 h, with a mixing ratio of 1:9, and 30 μL of the mixture was added to each chamber. 3 30 μL of cells were plated in Transwell chambers and cultured for 4 h. Then 500 μL of culture medium containing 10% FBS was added to the 24-well plate and 2×10 4 50 µL of tumor cell (AGS) suspension was added to the chamber. After 18 hours, the cells were fixed with 95% ethanol for 15 minutes and stained with trypan blue for 20 minutes. Excess cells were removed with a cotton swab. Migrated cells were counted under a microscope and photographed, which was considered to be the cell's migratory capacity.

[0090] 2.6 Virus transfection.

[0091] CFD-RNAi lentivirus was constructed using GenePharma (Suzhou, China). Sv5 cells (2 × 10 5 ) were replaced by 20 μL of lentiviral suspension (3 × 10 8 TU / mL) and polybrene was used according to the manufacturer's instructions. Infected cells were selected with 5 μg / mL puromycin 72 h later.

[0092] .

[0093] 3. Experimental results.

[0094] To explore the role of CFD in the transformation of mesothelial cells into fibroblasts, we first co-cultured human mesothelial cell line Sv5 cells with human gastric cancer cells MGC-803 and HGC-27. We found that with the extension of co-culture time, the morphology of mesothelial cells changed significantly, from cobblestone to spindle-shaped, suggesting that co-culture leads to the transformation of mesothelial cells into fibroblasts ( Figure 3 A and 3B). Next, the co-cultured mesothelial cells were extracted and the differential gene expression of mesothelial cells in the single-cell transcriptome sequencing of Result 2 was verified one by one. It was found that CFD expression increased 20-fold after 24 hours of co-culture with tumor cells and continued to increase at 48 hours ( Figure 3 C). Western blot experiments also confirmed the increase of CFD and N-cadherin protein and the decrease of E-cadherin protein expression. This indicates that after co-culture with tumor cells, Sv5 mesothelial cells undergo MMT ( Figure 3D). In a co-culture system with tumor cells, siCFD was added and it was found that knocking down the expression of CFD in mesothelial cells could maintain the cobblestone shape of mesothelial cells and significantly inhibit the occurrence of MMT in mesothelial cells ( Figure 3 E and 3F). Next, the mesothelial cells with CFD knockdown were plated in Transwell chambers, and gastric cancer cells were added to observe their invasion and migration abilities. It was found that knockdown of CFD in mesothelial cells could inhibit the invasion and migration of gastric cancer cells ( Figure 3 G). The above results strongly confirm that CFD induces mesothelial cells to differentiate into tumor-associated fibroblasts, thereby promoting the invasion and migration of gastric cancer cells.

[0095] 4. The CFD inhibitor Pelecopan (CAS No.: 2378380-49-3) inhibits the occurrence of MMT in mesothelial cells.

[0096] 1. Experimental materials.

[0097] (1) Pelecopan, a CFD inhibitor; recombinant protein CFD (MCE, USA).

[0098] (2) Human gastric cancer cell line MGC-803; human epithelial cell line Sv5 (Shanghai Cell Culture Collection, Chinese Academy of Sciences).

[0099] 2. Experimental method.

[0100] Gastric cancer cells MGC-803 and HGC-27 were cultured for 48 h, and the tumor cell supernatant was collected in a 15 mL centrifuge tube and centrifuged at 500 × g for 5 min. CFD recombinant protein (1 μg / mL), CFD inhibitor Pelecopan (10 nM), and tumor supernatant were added to human mesothelial cells Sv5 cells, respectively. After culture for 24 or 48 h, changes in mesothelial cell morphology were observed under an inverted microscope.

[0101] 3. Experimental results.

[0102] In December 2024, AstraZeneca's CFD inhibitor was approved by the FDA for marketing in combination with C5 antibody to treat extravascular hemolysis. Next, the CFD inhibitor was used to observe whether it could also inhibit the transformation of mesothelial cells into tumor-associated fibroblasts. Figure 4 As shown in A, the CFD inhibitor pelecopan can effectively inhibit the mesothelial-mesenchymal transition of mesothelial cells induced by tumor supernatant. Next, the recombinant protein of CFD was used to replace the tumor cell co-culture system. It was found that even in the absence of tumor cells, the addition of recombinant CFD protein can induce the transformation of mesothelial cells into tumor-associated fibroblasts ( Figure 4 B) Therefore, it can be seen that pelecopan can effectively inhibit the occurrence of MMT in mesothelial cells.

[0103] 5. The peritoneal metastasis nude mouse model confirmed that CFD promotes peritoneal metastasis of gastric cancer cells.

[0104] 1. Experimental materials.

[0105] (1) BALB / c female mice (Beijing Weitonglihua Co., Ltd.).

[0106] (2) Histochemical and HE staining reagents: xylene (Fuyu, Tianjin), anhydrous ethanol (Hengxing, Tianjin), 3% hydrogen peroxide, EDTA, citric acid, goat serum, hematoxylin, eosin, histochemical secondary antibody, DAB chromogen (Maixin, Fuzhou), hydrochloric acid (Hengxing, Tianjin), resin glue (Solarbio, Beijing), cover glass (Dingguo Changsheng, Beijing), slide (Dingguo Changsheng, Beijing).

[0107] 2. Experimental method.

[0108] 2.1 Mouse gastric cancer peritoneal metastasis model.

[0109] Fifteen 5-week-old BALB / c female mice were randomly divided into three groups, with 5 mice in each group. 5 5 × 10 control or siCFD mesothelial cells were added 5 MGC-803 gastric cancer cells were thoroughly mixed and injected intraperitoneally into mice. Four weeks after injection, the mice were sacrificed and peritoneal tumor tissue was obtained and embedded in paraffin. Tumor size and volume were compared and statistically analyzed.

[0110] 2.2 HE staining.

[0111] (1) Dewaxing: bake the slides at 65°C for 4 h; then place them in three xylene tanks in sequence, each for 15 min.

[0112] (2) Hydration: Pass through the ethanol tank in descending order of concentration (100%, 95%, 90%, 80% and 70%), with the high concentration lasting 8-10 minutes and the low concentration lasting 5 minutes.

[0113] (3) Rehydration: single distilled water, 3min×1.

[0114] (4) Hematoxylin staining: 50 μL per slide, 3 min, rinse with distilled water to terminate the reaction.

[0115] (5) Hydrochloric acid alcohol for 30 seconds, then rinse with tap water for 10 minutes.

[0116] (6) Stain with eosin for 2 minutes and rinse with distilled water to stop the reaction.

[0117] (7) Dehydration: Alcohol (from low to high concentrations).

[0118] (8) Transparency: three cylinders of xylene, 3 minutes each.

[0119] (9) Mounting: Mount the slides with neutral resin and let them dry. Take photos.

[0120] 2.3 Immunohistochemistry and scoring

[0121] (1) Dewaxing: bake the slides at 65°C for 4 h; then place them in three xylene tanks in sequence, each for 15 min.

[0122] (2) Hydration: Pass through the ethanol tank in descending order of concentration (100%, 95%, 90%, 80% and 70%), with the high concentration lasting 8-10 minutes and the low concentration lasting 5 minutes.

[0123] (3) Washing: single distilled water, 3 min × 1; PBS, 3 min × 3.

[0124] (4) Elimination of endogenous peroxidase and biotin 20 mL 3% H2O2 + 180 mL methanol, room temperature for 20 min; PBS, 3 min × 3.

[0125] (5) Antigen repair (high pressure): After the water boils, place the cylinder containing the slide and repair solution into the water, cover the pot, and start timing for 90 seconds when the maximum air pressure is reached. After turning off the power, take out the repair solution cylinder, immerse it in cold water, and cool it to room temperature.

[0126] (6) Histological stroke circle (1 mm from the edge); PBS, 3 min × 3.

[0127] (7) Blocking: Add 50 μL of BSA (bovine serum albumin) to each slide and incubate at room temperature for 30 min.

[0128] (8) Primary antibody application: Shake off the BSA and directly add 40 μL of primary antibody diluted in PBS (the antibody concentration depends on the experiment) and incubate in a humidified chamber at 4°C overnight.

[0129] (9) Rewarming: On the second day, remove the wet box and rewarm at room temperature for 30 minutes.

[0130] (10) Washing: PBS, 3 min × 3.

[0131] (11) Secondary antibody application: Enhancement solution for 20 min; PBS, 3 min × 3; Secondary antibody for 30 min; PBS, 3 min × 3.

[0132] (12) DAB color development: Dissolve one drop each of A, B, and C in 850 μL of double-distilled water, 50 μL per slide. Record the color development time and keep it consistent for all slides. Stop the reaction in tap water.

[0133] (13) Hematoxylin staining: 50 μL per slide, 90 s, rinse with tap water to terminate the reaction.

[0134] (14) Use hydrochloric acid alcohol for 10 seconds, then rinse with tap water for 10 minutes to return to blue.

[0135] (15) Dehydration: Alcohol (from low to high concentrations).

[0136] (16) Xylene, three tanks, 3 minutes each.

[0137] (17) Mounting: Mount the slides with neutral resin and let them dry. Take photos.

[0138] (18) Scoring: IHC staining was assessed based on the percentage of positive cells (0: <5%, 1: 5%–25%, 2: 25%–50%, 3: 50%–75%, and 4: >75%) multiplied by a score based on staining intensity (0: colorless, 1: light yellow, 2: brown, and 3: dark brown).

[0139] 3. Experimental results.

[0140] A mixture of human mesothelial cell line Sv5 cells and gastric cancer cells MGC-803 was injected into the abdominal cavity of nude mice to establish a gastric cancer peritoneal metastasis nude mouse model. After 4 weeks, the mice were killed and the abdominal metastases were weighed. Compared with the control group, the tumor weight of the mice was significantly reduced after silencing CFD in mesothelial cells ( Figure 5 A). Further objective analysis of tumor area using immunohistochemistry yielded the same conclusion. Immunohistochemical staining of tumor tissue for CFD revealed that siCFD effectively inhibited CFD expression in the tissue. Therefore, this animal model also confirms that CFD promotes the formation of peritoneal metastasis in gastric cancer.

[0141] In summary, the present invention focuses on the dynamic changes in the remodeling of the pre-metastatic microenvironmental niche, conducts research on the molecular mechanism of CFD regulating gastric cancer peritoneal metastasis, and uses single-cell transcriptomics, molecular biology, and small molecule drug screening to systematically verify from three levels: cellular, animal, and clinical, that "CFD mediates and regulates gastric cancer peritoneal metastasis through the dual effects of regulating the immune microenvironment and participating in MMT", providing a new theoretical basis and candidate drugs for the precise treatment of gastric cancer peritoneal metastasis.

[0142] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of CFD inhibitors in the preparation of drugs for preventing or treating peritoneal metastasis of gastric cancer.

2. The use according to claim 1, characterized in that The CFD inhibitor includes inhibiting CFD expression or activity.

3. The use according to claim 2, characterized in that The method for inhibiting CFD expression includes siRNA and shRNA targeting CFD, and the sequence of the siRNA is shown in SEQ ID NO. 1-2.

4. The use according to claim 2, characterized in that The inhibition of CFD activity includes small molecule inhibitors targeting CFD.

5. The use according to claim 4, characterized in that The small molecule inhibitor of CFD is Pelecopan.

6. A drug for inhibiting peritoneal metastasis of gastric cancer, comprising a CFD inhibitor and a pharmaceutically acceptable carrier.

7. The drug according to claim 6, characterized in that The CFD inhibitor includes inhibiting CFD expression or activity.

8. The drug according to claim 7, wherein The method for inhibiting CFD expression includes siRNA and shRNA targeting CFD, and the sequence of the siRNA is shown in SEQ ID NO. 1-2.

9. The drug according to claim 7, wherein The inhibition of CFD activity includes small molecule inhibitors targeting CFD.

10. Application of CFD inhibitors in the preparation of drugs for inhibiting mesothelial-mesenchymal transition (MMT).

Images