Application of CPS1 inhibitor in preparation of medicine for treating lung cancer metastasis

Abstract

The invention discloses an application of a CPS1 inhibitor in preparation of a medicine for treating lung cancer metastasis, the Gene ID of CPS1 is 1373, and the CPS1 inhibitor is a molecule which takes CPS1 as an action target and has an inhibition effect on CPS1. High expression of CPS1 in a lung cancer metastasis cell line is found for the first time, it is proved that inhibition of CPS1 can inhibit migration and metastasis of lung cancer cells, and new therapeutic targets and strategies are provided for treatment of lung cancer metastasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of CPS1 inhibitors in the preparation of drugs for treating lung cancer metastasis. Background Art

[0002] The urea cycle is an important metabolic pathway in the body, and its function is to transport NH to the liver 3 Through a series of metabolic reactions, it is converted into urea and excreted from the body through urine, playing a role in removing ammonia toxicity. As the starting enzyme and rate-limiting enzyme of the urea cycle, CPS1 plays an important role. Proteins in the urea cycle are dysregulated in a variety of tumors, providing favorable metabolic intermediates for tumor survival, proliferation, and growth.

[0003] Currently, there is no report indicating the association between CPS1 and lung cancer metastasis. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides the application of CPS1 inhibitors in the preparation of drugs for treating lung cancer metastasis.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] Provide the application of CPS1 inhibitors in the preparation of drugs for treating lung cancer metastasis, wherein the Gene ID of CPS1 is 1373, and the CPS1 inhibitor refers to a molecule that has an inhibitory effect on CPS1 with CPS1 as the target.

[0007] Further, the CPS1 inhibitor is an shRNA inhibitor, an siRNA inhibitor, or a small molecule inhibitor.

[0008] Still further, the CPS1 inhibitor is an shRNA inhibitor, and the sequences of the shRNA are shown in SEQ ID NO.1-2:

[0009] 5’-CCGGCGTACTTCAATCAATGTTGTTCTCGAGAACAACATTGATTGAA GTACGTTTTTG-3’ (SEQID NO.1), sense strand;

[0010] 5’-AATTCAAAAACGTACTTCAATCAATGTTGTTCTCGAGAACAACATTG ATTGAAGTACG-3’ (SEQID NO.2), antisense strand.

[0011] Still further, the CPS1 inhibitor is a small molecule inhibitor, and the small molecule inhibitor is H3B-120, with the molecular formula C 19 H24 N4O2S.

[0012] By adopting the above technical solutions, the present invention has the following technical effects compared with the prior art:

[0013] For the first time, the present invention discovers that CPS1 is highly expressed in lung cancer metastatic cell lines, and proves that inhibiting CPS1 can inhibit the migration and metastasis of lung cancer cells, providing a new therapeutic target and strategy for the treatment of lung cancer metastasis. Description of the Drawings

[0014] Figure 1 Showing the results of detecting the migration ability of primary lung cancer cells and metastatic lung cancer cells; among them, (A - B) The Transwell experiment detects the migration ability of primary lung cancer cells and metastatic tumor cells. Equal amounts of primary lung cancer cells H1975, H441, A549 - L0 (L0) and metastatic lung cancer cells H460, A549 - L2 (L2), A549 - L6 (L6) are inoculated on the upper layer of the chamber. After 36 h of cell migration, the chamber is fixed, stained and observed. The scale bar is 50 μm. The experiment is designed with 3 replicates. The experimental results are statistically analyzed, and the unpaired t - test method is used for data significance difference analysis, ***p < 0.001; (C - D) The scratch assay detects the migration ability of primary lung cancer cells and metastatic lung cancer cells. Equal amounts of primary lung cancer cells H1975, H441, A549 - L0 (L0) and metastatic lung cancer cells H460, A549 - L2 (L2), A549 - L6 (L6) are inoculated in a 12 - well plate. Photos are taken of the same scratch at 0 h and 48 h to compare the healing of the scratch at the same position at different times. According to the formula cell migration rate = (S 0h -S xh ) / S 0h ×100% to calculate the migration rate of different cells. The scale bar is 100 μm. The experiment is designed with 3 replicates. The results of the scratch assay are statistically analyzed, and the unpaired t - test method is used for data significance difference analysis, ***p < 0.001.

[0015] Figure 2 Showing the results of the enrichment analysis of differentially expressed proteins in primary lung cancer cells and metastatic lung cancer cells; among them, the primary lung cancer cells H1975, H441, L0 and metastatic lung cancer cells H460, L2, L6 are subjected to proteome detection. The heat map (A) and volcano plot (B) show the proteins with significant changes in the proteome data; the original data is standardized by Z - score, and the changes of differentially expressed genes are shown in two forms of heat map and volcano plot. The fold change of the differentially expressed genes fold change > 2, adjusted P < 0.05; blue represents gene down - regulation, and red represents gene up - regulation.

[0016] Figure 3 It is shown that CPS1 is highly expressed in metastatic lung cancer cells. Among them, (A) Protein immunoblotting was used to detect the expression of CPS1 in different cells: normal lung epithelial cells Beas-2b, primary lung cancer cells H1975, H441, L0, and metastatic lung cancer cells H460, L2, L6 were cultured for 48 h, and cell lysates were collected for protein immunoblotting to detect the expression of CPS1; (B) Immunofluorescence was used to detect the expression of CPS1 in different cells: cells were cultured on coverslips, fixed, and stained. Phalloidin staining was in red, CPS1 antibody staining was in green, and the nucleus was stained with DAPI. The magnification was 60 times, and confocal microscopy was used for photography. The scale bar of the picture was 50 μm.

[0017] Figure 4 It is shown the expression of key enzymes in the urea cycle in primary and metastatic lung cancer tissues in the TCGA and GEO databases. Among them, (A-B) The TCGA database was used to analyze the mRNA expression of key enzymes in the urea cycle in primary lung cancer tissues (M0) and metastatic lung cancer tissues (M1), *p = 0.046; (C) The GEO database was used to analyze the mRNA expression of key enzymes in the urea cycle in primary lung cancer tissues (P) and metastatic lung cancer tissues (M).

[0018] Figure 5 It is shown the experimental results of verifying the knockdown efficiency of CPS1. Among them, (A-B) Stable cell lines with knockdown of CPS1 were constructed in H460, L2, and L6 cells respectively. The expression level of CPS1 was detected by Western blot to determine the knockdown efficiency. The expression level of β-actin was used as the normalization standard. The experiment was designed with three replicates, and unpaired t-tests were performed, *p < 0.05, **p < 0.01, ***p < 0.001.

[0019] Figure 6Knockdown of CPS1 inhibits the proliferation of metastatic lung cancer cells; among them, (A) CCK8 cell proliferation assay was used to detect the proliferation ability of metastatic lung cancer cells after knockdown of CPS1. The same number of shCPS1 stable transfected cells and control group cells were respectively seeded in 96-well plates. CCK8 reagent was added at 0 h, 24 h, 48 h and 72 h. After reacting for 1 h, the OD value at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The relative proliferation ability of different lung cancer cells was statistically analyzed. The experiment was designed with 3 replicates, and two-way ANOVA was used for significant difference analysis, ***p < 0.001; (B-C) Colony formation assay was used to detect the proliferation ability of metastatic lung cancer cells after knockdown of CPS1. The same number of shCPS1 stable transfected cells and control group cells were respectively seeded in 6-well plates. After 7-14 days of cell clone culture, the cells were stained and photographed. The clones were dissolved with 10% acetic acid, and the OD value was read using an ELISA reader. Then the cell survival rate was calculated, and the unpaired t-test method was used for significant difference analysis of the data, ***p < 0.001.

[0020] Figure 7 Knockdown of CPS1 promotes the apoptosis of metastatic lung cancer cells; among them, (A-B) Flow cytometry was used to detect the effect of knockdown of CPS1 on the apoptosis of metastatic lung cancer cells. The same number of shCPS1 stable transfected cells and control group cells were respectively seeded in 6-well plates. The cells were cultured for 48 hours, digested, and stained with Annexin V / PI. The cells were detected and analyzed using a flow analyzer, and the apoptosis of the cells was statistically analyzed. The unpaired t-test method was used for significant difference analysis of the data, ***p < 0.001.

[0021] Figure 8 Knockdown of CPS1 inhibits the migration of metastatic lung cancer cells; among them, (A-B) Cell scratch assay was used to detect the migration ability of metastatic lung cancer cells after knockdown of CPS1. The same number of shCPS1 stable transfected cells and control group cells were respectively seeded in 12-well plates. Photographs of the same scratch were taken at 0 h and 48 h, and the healing of the scratch at the same position at different times was compared. According to the formula cell migration rate = (S 0h -S xh ) / S 0hThe migration rates of different cells were calculated as ×100%, with a scale bar of 100 μm. The experiment was designed with 3 replicates, and the results of the scratch assay were statistically analyzed. The unpaired t-test was used to analyze the significant differences in the data, ***p < 0.001; (C-D) The Transwell assay was used to detect the migration ability of metastatic lung cancer cells after knocking down CPS1. Equal amounts of shCPS1 stably transfected cells and control group cells were seeded on the upper layer of the chamber. After 36 h of cell migration, the chamber was fixed, stained, and observed, with a scale bar of 50 μm. The experiment was designed with 3 replicates, and the experimental results were statistically analyzed. The unpaired t-test was used to analyze the significant differences in the data, ***p < 0.001.

[0022] Figure 9 It was shown that knocking down CPS1 effectively inhibited the metastasis of lung cancer cells in the left ventricular injection mouse metastasis model; among them, (A-B) The left ventricular injection mouse metastasis model was used to detect the metastasis ability of cells after knocking down CPS1: L6-shCPS1-luc and L6-shScr-luc cells were injected into mice by left ventricular injection. The tumor metastasis in mice was detected by in vivo imaging system at 0, 1, 2, 3, 4, and 5 weeks after tumor inoculation. The representative pictures were the fluorescence results detected by the in vivo imaging system at the 5th week; the fluorescence intensity of mice was statistically analyzed, and two-way ANOVA was used for the analysis of significant differences, ***p < 0.001; (C) The survival cycle of mice in the left ventricular injection metastasis model was statistically analyzed, and the log-rank test was used for the analysis of significant differences in the survival cycle, **p < 0.01.

[0023] Figure 10It is shown that knocking down CPS1 effectively inhibits the bone erosion of lung cancer cells in a left ventricular injection mouse metastasis model; among them, (A - B) the mouse spine was taken for Micro-CT scanning and analysis. L6-shCPS1-luc and L6-shScr-luc cells were injected into mice by left ventricular injection. The mice were sacrificed at the 5th week, and the mouse spine was taken for Micro-CT detection. The spine was scanned and analyzed under the condition of 9μm by Micro-CT, and the data were statistically analyzed after obtaining the bone parameters; BMD, bone mineral density; BV / TV, bone volume total / tissue volume; Tb.N, trabecular number (per mm); BS / BV, bone surface / bone volume; Tb.pf, trabecular pattern factor; Tb.Sp, trabecular separation. The unpaired t-test was used for the analysis of significant differences in data, **p < 0.01, ***p < 0.001; (C) the mouse spine was decalcified, paraffin-embedded, sectioned and stained with H&E.

[0024] Figure 11 It is shown that knocking down CPS1 effectively inhibits the metastasis of lung cancer cells in a tail vein injection mouse metastasis model; among them, (A - B) the tail vein injection metastasis model was used to detect the metastasis ability of tumor cells in vivo after knocking down CPS1: CMT167-shCPS1-luc and CMT167-shScr-luc cells were injected into mice by tail vein injection. The tumor metastasis in mice was detected by an in vivo imaging system 3, 6, 9, 12, and 15 days after tumor bearing. The representative pictures are the fluorescence results detected by the in vivo imaging system on the 15th day; the fluorescence intensity of the mice was statistically analyzed, and two-way ANOVA was used for the analysis of significant differences, ***p < 0.001; (C) the tumor metastasis in tissues such as the heart, liver, spleen, kidney, lung, and bone of the mice was detected. 15 days after tumor bearing, the mice were intraperitoneally injected with a luciferase substrate, sacrificed, and the tissues such as the heart, liver, spleen, kidney, lung, and bone were dissected to detect their fluorescence intensity by an in vivo imaging system.

[0025] Figure 12It is shown that knocking down CPS1 effectively inhibits the growth of lung cancer cells in a mouse metastasis model by tibial injection; among them, (A-B) Detection of the growth ability of tumor cells after knocking down CPS1 in the tibial injection model: CMT167-shScr-luc cells and CMT167-shCPS1-luc cells were injected into mice by tibial injection; after tumor-bearing, the growth of tumors in the tibiae of mice was detected by in vivo imaging system every 3 days. The representative pictures are the fluorescence results detected by in vivo imaging system on the 15th day. The fluorescence intensity of mice was statistically analyzed, and two-way ANOVA was used for significant difference analysis, ***p<0.001.

[0026] Figure 13 It is shown that the CPS1 inhibitor H3B-120 can effectively inhibit the proliferation of metastatic lung cancer cells; among them, (A) Detection of the half-maximal inhibitory concentration (IC50) of the CPS1 inhibitor H3B-120 in L2 and L6 cells. L2 and L6 cells were respectively seeded in 96-well plates, and the drug was added at a concentration gradient for treatment. After 48 h, CCK8 reagent was added. After reacting for 1 h, the OD value at 450 nm was measured using a microplate reader, and the IC50 curve of the drug was fitted; (B) CCK8 cell proliferation assay to detect the effect of the CPS1 inhibitor H3B-120 on the proliferation ability of metastatic lung cancer cells. L2 and A549-L6 cells were respectively seeded in 96-well plates, and the drug was added at a concentration gradient for treatment. CCK8 reagent was added at 0 h, 24 h, 48 h, and 72 h respectively. After reacting for 1 h, the OD value at 450 nm was measured using a microplate reader, and the relative proliferation ability of different lung cancer cells was statistically analyzed. The experiment was designed with 3 replicates, and two-way ANOVA was used for significant difference analysis, **p<0.01, ***p<0.001; (C-D) Colony formation assay to detect the effect of the CPS1 inhibitor H3B-120 on the proliferation ability of metastatic lung cancer cells. After cell cloning and culturing for 7-14 days, the cells were stained and photographed. The clones were dissolved with 10% acetic acid, and the OD value at 595 nm was measured using a microplate reader, and then the cell survival rate was calculated. The unpaired t-test method was used for significant difference analysis of the data, ***p<0.001.

[0027] Figure 14 It is shown that the CPS1 inhibitor H3B-120 promotes the apoptosis of metastatic lung cancer cells; among them, (A-B) Flow cytometry was used to detect the effect of the CPS1 inhibitor H3B-120 on the apoptosis of metastatic lung cancer cells. L2 and L6 cells were respectively seeded in 6-well plates, and the drug was added at a concentration gradient for treatment for 24 h. The cells were digested, stained with Annexin V / PI, and analyzed by flow analyzer. The apoptosis of the cells was statistically analyzed, and one-way ANOVA was used for significant difference analysis of the data, *p<0.05, **p<0.01, ***p<0.001.

[0028] Figure 15 It is shown that the CPS1 inhibitor H3B-120 inhibits the migration of metastatic lung cancer cells; among them, (A-B) The Trans-well assay was used to detect the effect of the CPS1 inhibitor H3B-120 on the migration ability of metastatic lung cancer cells. L2 and L6 cells were respectively seeded on the upper layer of the chamber, and the cells were treated with drugs at a concentration gradient for 36 h. The chamber was fixed, stained and observed. The scale bar is 50 μm. The experiment was designed with 3 replicates. The experimental results were statistically analyzed, and the data were analyzed for significant differences using the one-way ANOVA method, *p<0.05, ***p<0.001.

[0029] Figure 16 It is shown that the CPS1 inhibitor H3B-120 effectively inhibits the metastasis of lung cancer cells in the left ventricular injection mouse metastasis model; among them, (A-B) The left ventricular injection metastasis model was used to detect the effect of the CPS1 inhibitor H3B-120 on the metastasis ability of tumor cells: L6-luc cells were injected into mice by left ventricular injection. One week after left ventricular injection, the mice were treated with the drug H3B-120 (40 mg / kg and 80 mg / kg), and the treatment was carried out by intraperitoneal injection every day. The tumor metastasis in mice was detected by an in vivo imaging system at 0, 1, 2, 3, and 4 weeks after tumor inoculation. The representative pictures are the fluorescence results detected by the in vivo imaging system at the 4th week. The fluorescence intensity of the mice was statistically analyzed, and the significant differences were analyzed using two-way ANOVA, ***p<0.001; (C) The survival cycle of the mice in the left ventricular injection metastasis model was statistically analyzed, and the significant differences in the survival cycle were analyzed using the log-rank test, *p<0.05, **p<0.01.

[0030] Figure 17It is shown that the CPS1 inhibitor H3B-120 effectively inhibits the bone erosion of lung cancer cells in a left ventricular injection mouse metastasis model; among them, (A-B) the spine of the mouse was taken for Micro-CT scanning and analysis. The L6 cells were injected into the mouse by left ventricular injection. One week after left ventricular injection, the drug H3B-120 (40 mg / kg and 80 mg / kg) was administered, and the treatment was carried out by intraperitoneal injection every day. The mice were sacrificed at the 4th week, and the spines of the mice were taken for Micro-CT detection. The spine was scanned and analyzed under the condition of 9 μm by Micro-CT, and the data were statistically analyzed after obtaining the bone parameters; BMD, bone mineral density; BV / TV, bone volume total volume-1; Tb.N, trabecular number (per mm). The one-way ANOVA method was used to analyze the significant differences in the data, **p<0.01, ***p<0.001.

[0031] Figure 18 It is shown that the CPS1 inhibitor H3B-120 effectively inhibits the metastasis of lung cancer cells in a tail vein injection mouse metastasis model; among them, (A-B) the tail vein injection metastasis model was used to detect the metastasis ability of tumor cells after treatment with the CPS1 inhibitor H3B-120: The CMT167-luc cells were injected into the mouse by tail vein injection. Three days after tail vein injection, the drug H3B-120 (80 mg / kg) was administered, and the treatment was carried out by intraperitoneal injection every day. The tumor metastasis in the mouse was detected by an in vivo imaging system 3, 6, 9, 12, and 15 days after tumor bearing. The representative picture is the fluorescence result detected by the in vivo imaging system on the 15th day; the fluorescence intensity of the mouse was statistically analyzed, and two-way ANOVA was used for the analysis of significant differences, ***p<0.001; (C) the tumor metastasis in tissues such as the heart, liver, spleen, kidney, lung, and bone of the mouse was detected; 15 days after tumor bearing, the mouse was intraperitoneally injected with a luciferase substrate, and the mouse was sacrificed. The heart, liver, spleen, kidney, lung, bone and other tissues were dissected and the fluorescence intensity was detected by an in vivo imaging system.

[0032] Figure 19It is shown that the CPS1 inhibitor H3B-120 effectively inhibits the growth of lung cancer cells in a tibia injection mouse metastasis model; wherein, (A-B) Detection of the effect of the CPS1 inhibitor H3B-120 on tumor cell growth in a tibia in situ injection model: CMT167-luc cells were injected into mice via the tail vein and treated by intraperitoneal injection every day. The tumor growth at the tibia of mice was detected by an in vivo imaging system on days 0, 3, 6, 9, 12, and 15 after tumor inoculation. The representative picture shows the fluorescence results detected by the in vivo imaging system on day 15. The fluorescence intensity of the mice was statistically analyzed, and two-way ANOVA was used for significant difference analysis, ***p<0.001.

[0033] Figure 20 It is shown that the CPS1 inhibitor H3B-120 effectively inhibits the growth of lung cancer metastatic cells in a lung cancer bone metastasis organoid model; wherein, (A) Construction and verification of lung cancer bone metastasis organoids (PDO): Fresh surgical samples from the bone metastasis sites of lung cancer bone metastasis patients were collected, and the minced fresh surgical samples were digested with tissue digestive fluid to form a cell suspension. After filtration, red blood cells were lysed, and 3D cell culture was performed using organoid matrix gel. Morphological structure observation and IHC staining of lung cancer tumor markers were performed for identification; (B-C) The PDO were inoculated into 96-well plates and treated with drugs at a concentration gradient. The organoids were stained with Calcein-AM fluorescent dye, and the lung cancer bone metastasis cancer organoids were photographed by an optical microscope. The proliferation and growth of tumor organoids were monitored and statistically analyzed for live cells. One-way ANOVA with Tukey’s multiple comparison test was used for significant difference analysis of the data, **p<0.01, ***p<0.001. Detailed implementation manners

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but it is not intended to limit the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0035] Example 1

[0036] Tumor metastasis is a process in which, under the combined action of the tumor microenvironment and internal gene regulation of cancer cells, cancer cells acquire the ability to spread, infiltrate and break through the basement membrane, enter the surrounding tissues, then invade blood vessels or lymphatic vessels, migrate to distant organs or tissues through the circulatory system, and continuously proliferate in the target organs to form metastatic foci. In this study, 6 cell lines were selected, including 3 primary lung cancer cell lines: H441, H1975, A549 (L0), and 3 lung cancer metastatic cell lines: H460, A549-L2, A549-L6. The primary lung cancer cell A549 (L0) and its metastatic cells were both derived from the laboratory of Professor Luo Jian of Tongji University School of Medicine. The human lung cancer cell line A549 was injected into mice through the left ventricle, and different clones of highly metastatic cell lines A549-L2 (hereinafter referred to as L2) and A549-L6 (hereinafter referred to as L6) were isolated and cultured from the spine.

[0037] First, the migration abilities of primary lung cancer cell lines and metastatic cell lines were preliminarily verified: in terms of cell migration, the migration ability of metastatic lung cancer cell lines was significantly enhanced compared with that of primary cell lines ( Figure 1 ), and both Trans-well experiments and cell scratch experiments proved this conclusion.

[0038] Then, proteomics detection and analysis were carried out using the above cell lines. Samples were loaded onto the machine using Q-Exactive, and the raw file was searched using MaxQuant software. 4907 proteins were identified, and differential expression enrichment analysis was performed on these proteins. The top 30 proteins with obvious differences were selected for analysis. From the results of the heat map, it can be seen that CPS1 was most significantly differentially expressed between primary lung cancer cells and lung cancer metastatic cells ( Figure 2 A), and then volcano plot analysis was performed, and the results showed that CPS1 was highly expressed in lung cancer metastatic cell lines ( Figure 2 B).

[0039] In the proteomics results of primary lung cancer cell lines and lung cancer metastatic cell lines, it was found that the key enzyme of the urea cycle, CPS1, was abnormally highly expressed in lung cancer metastatic cell lines. To verify this result, normal lung epithelial cells Beas-2b and the above 6 cell lines were collected, and the expression of CPS1 in these 7 cell lines was detected. Western blot results showed that the expression level of CPS1 in lung cancer metastatic cell lines was generally higher than that in primary lung cancer cell lines, while the expression in normal lung epithelial cells was very low ( Figure 3 A). In a normal organism, CPS1 is only expressed in the liver and small intestine and is not expressed in other organs and tissues, which also verified the accuracy of the non-expression of CPS1 in Beas-2b cells. After the body undergoes metabolic reprogramming, CPS1 is expressed in metastatic cells. This is consistent with the data results obtained by mass spectrometry.

[0040] The results of immunofluorescence staining Figure 3 B) can more intuitively show the expression of CPS1 protein in L0, L2, and L6 cells at the single-cell level. Approximately 30%-50% of the cells with positive CPS1 expression in L0, while the cells with positive CPS1 expression in L2 and L6 are almost close to 100%. Through the staining of phalloidin, it can be clearly seen that the cytoskeletons of L2 and L6 cells are darker in color and mostly distributed at their cell edges, while there are fewer cells with darkly stained cytoskeletons in L0 cells, and the microfilament cytoskeleton is evenly distributed on the cell surface. This also indirectly proves that the cell migration ability of L2 and L6 cells is stronger than that of L0. This is consistent with the previous results Figure 1 ).

[0041] Finally, with the help of the information in the existing database, the expression of CPS1 was verified, attempting to find evidence related to the metastasis of lung cancer from it. The lung cancer transcriptome data from The Cancer Genome Atlas (TCGA), including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Using clinical information, patients were divided into metastatic (M1) and non-metastatic (M0). We found that the mRNA of CPS1 was significantly overexpressed in the primary lung cancer tissues of M1 patients Figure 4 A). The expression of the other two urea cycle enzymes ASS1 and ASL increased slightly, but without statistical significance Figure 4 A). The mRNA expression profiles of these three genes are shown in the box plot of Figure 4 B. From the Gene Expression Omnibus (GEO), we found that the expression of CPS1 mRNA in metastatic lung cancer tissues in the dataset GSE125864 increased compared with that in primary lung cancer tissues Figure 4 C).

[0042] Example 2

[0043] The consistency of high CPS1 expression was verified in cells and clinical tissues. It was hypothesized that high CPS1 expression could promote the metastasis of tumor cells. To verify this hypothesis, a series of cell experiments targeting CPS1 were designed in this invention to verify the function of CPS1 in tumor migration and metastasis.

[0044] 2.1 Knockdown of CPS1 inhibits the proliferation ability of metastatic lung cancer cells

[0045] First, we designed a sequence targeting CPS1 mRNA and constructed an shRNA expression plasmid for the targeting sequence. We infected lung cancer metastatic cell lines H460, L2, and L6 with high CPS1 expression by lentiviral packaging. Through a series of screenings and positive monoclonal amplifications, we obtained H460sh-CPS1, L2sh-CPS1, and L6sh-CPS1 cell lines with stable knockdown of CPS1. A random sequence that does not target any gene was used as the control group (H460sh-Scr, L2sh-Scr, and L6sh-Scr), and the knockdown efficiency was detected by Western blot ( Figure 5 ).

[0046] Next, we compared the differences in cell proliferation ability between lung cancer metastatic cells and their corresponding CPS1-knockdown cells to study the effect of CPS1 knockdown on the proliferation of lung cancer cells. The results of the CCK8 cell proliferation assay showed that the cell proliferation ability was significantly reduced after CPS1 knockdown, and all three knockdown cell lines had the same phenotype, indicating that CPS1 knockdown inhibited the proliferation of lung cancer cells ( Figure 6 A). The colony formation assay showed that the cell proliferation rate was significantly reduced after CPS1 knockdown ( Figure 6 B and Figure 6 C). In summary, CPS1 knockdown effectively inhibited the proliferation ability of lung cancer cells.

[0047] 2.2 CPS1 knockdown promotes apoptosis of metastatic lung cancer cells

[0048] From the above results, we preliminarily concluded that CPS1 knockdown could effectively inhibit the proliferation of metastatic lung cancer cells. There is a certain relationship between the weakened cell proliferation ability and cell apoptosis. Previous studies have shown that some anti-tumor drugs play their roles by inhibiting proliferation and inducing apoptosis. For example, chemotherapy drugs inhibit proliferation and induce apoptosis through DNA damage. Therefore, we detected the apoptosis of cells after CPS1 knockdown by flow cytometry analysis. After plating the same cell amount, the three metastatic lung cancer cell lines and their paired CPS1-knockdown cells were stained and analyzed by flow cytometry 48 hours later. The results showed that CPS1 knockdown promoted the apoptosis of metastatic lung cancer cells ( Figure 7 ).

[0049] 2.3 CPS1 knockdown inhibits the migration of metastatic lung cancer cells

[0050] Previous research results from our group showed that knockdown of CPS1 inhibited cell proliferation. During tumor development, cell proliferation and migration are closely related and jointly promote tumor growth, invasion, and metastasis. The rapid proliferation of tumor cells increases the number of cells, providing an adequate cell source for migration. During cell proliferation, cells may acquire phenotypic changes required for migration, such as epithelial-mesenchymal transition (EMT). Migration is a key step for tumor cells to spread from the primary site to surrounding tissues or distant organs. During migration, tumor cells may secrete matrix metalloproteinases (MMPs) to degrade the extracellular matrix (ECM), creating corresponding space for cell proliferation. Therefore, we conducted relevant cell migration experiments to further explore the effect of CPS1 knockdown on cell migration ability. The results of cell scratch and Trans-well assays showed that knockdown of CPS1 significantly impaired cell migration ability ( Figure 8 ).

[0051] Example 3

[0052] 3.1 Knockdown of CPS1 effectively inhibits lung cancer cell metastasis in a left ventricular injection mouse metastasis model

[0053] Plasmids carrying the luciferase-encoding gene (Luc) were transfected into L6-shscr cells and L6-shCPS1 cells. The cells were injected into the systemic circulation of nude mice via the left ventricle and then distributed to various tissues and organs throughout the body. Fluorescence was generated by the reaction of intracellularly expressed luciferase with intraperitoneally injected luciferase substrate. The location of tumor cells was observed by in vivo imaging system, and the degree of tumor development was analyzed by the intensity of fluorescence. Eight-week-old male nude mice were selected and divided into two groups of 8 mice each. L6shscr-luc and L6shCPS1-luc cells were injected into the left ventricle of each mouse, with 10 5 cells per mouse. After left ventricular injection, the luciferase substrate was intraperitoneally injected into the mice. Three minutes later, if fluorescence spread throughout the body as confirmed by an in vivo imaging instrument (IVIS), it indicated successful injection. Thereafter, the mice were subjected to in vivo imaging and health observation once a week until 5 weeks later, when the fluorescence area of the metastatic foci was significantly enlarged, the fluorescence intensity was significantly increased, and the mice showed symptoms such as emaciation, withered coat color, and hemiplegia. The experiment was terminated at this time, and the main metastatic tissues were retained. The formation of metastatic foci was determined by subsequent hematoxylin-eosin staining (H&E) and micro-CT scanning analysis.

[0054] As can be seen from the results of in vivo imaging 5 weeks after injection into the left ventricle, the fluorescence irradiation range in the mice injected with the L6shscr-luc group was wide, and the fluorescence intensity was stronger than that of the L6shCPS1-luc group. Most of the metastatic foci were concentrated in the brain, spine, chest and other locations( Figure 9 A and Figure 9 B). The spines of the mice were removed and fixed for later use. Subsequently, in the same operation mode, survival analysis of the mice was carried out, and the method was the same as above. The difference was that the mice were not sacrificed. Under the principle of not violating animal ethics and welfare, the death days of each mouse were recorded, and the survival curve of the mice was plotted. It can be seen from the results of survival analysis that the median survival period of the mice in the L6shscr-luc group was 43 days, while that of the mice in the L6-shCPS1-luc group was 77 days( Figure 9 C). These above results suggest that the knockdown of CPS1 inhibits the proliferation and metastasis of tumor cells in vivo and effectively prolongs the survival period of mice.

[0055] 3.2 Knockdown of CPS1 effectively inhibits bone erosion of lung cancer cells in the left ventricular injection mouse metastasis model

[0056] From the imaging data and analysis data of the in vivo imager, we can see that compared with the control group L6-shscr, the metastatic foci formed by knocking down CPS1 cells were small in range and the fluorescence intensity was also significantly weakened. We selected the part of the spine with the strongest fluorescence intensity, removed the excess muscle tissue, fixed it with 4% paraformaldehyde for 72 hours, and then placed the spines of the mice in a small animal CT (Micro-CT) for scanning. The results showed that the metastatic foci of the mice in the L6-shscr group were mainly concentrated in the spine. It can be seen from the 2D cross-sectional view that the trabecular bone at the site of the metastatic foci was significantly reduced, and the spine segment was hollow. In the 3D reconstruction diagram, obvious bone erosion and bone destruction phenomena could be seen, while the spines of the mice in the L6-shCPS1 group were normal( Figure 10 A). Through quantitative analysis of bone mass using CT analysis software, it was found that the bone mineral density (BMD) of the mice in the L6-shscr group was significantly lower than that of the L2-shCPS1 group, the ratio of bone tissue volume to tissue volume (BV / TV) was significantly reduced, the bone surface area density (BS / TV) and the number of trabecular bones (Tb.N) were both significantly reduced, while the trabecular bone separation (Tb.Sp) and the trabecular bone pattern factor (Tb.pf) were significantly increased( Figure 10 B). Subsequently, the spines of the mice were decalcified and stained with H&E, and it was found that in the L6-shscr group, there were significantly more tumor cells, showing obvious enlargement of the cell nucleus, increased number, increased nuclear-cytoplasmic ratio, and relatively reduced cytoplasm( Figure 10 C).

[0057] 3.3 Knockdown of CPS1 effectively inhibits bone erosion of lung cancer cells in a mouse metastasis model by tail vein injection

[0058] In addition to constructing a tumor metastasis model by left ventricular injection, tail vein injection is also a commonly used method for constructing a mouse tumor metastasis model. Cells enter the body through the tail vein and first enter the pulmonary circulation with the blood, and then reach distal organs such as the liver, spleen, and bone through the blood circulation to form metastatic foci. To better simulate the environment in a normal organism, we selected immunocompetent C57BL / 6 mice and constructed mouse cell lines CMTshscr-luc and CMT-shCPS1-luc with CPS1 knockdown for tail vein injection, with 100,000 tumor cells injected into each mouse. Since the relatively thick hair of C57BL / 6 mice affects the observation of in vivo imaging in the later stage, before constructing the model in mice, the abdomen of the mice needs to be depilated with a razor or depilatory cream. Every 3 days, in vivo imaging is used to detect the metastasis of tumor cells in the mice. After taking the last in vivo imaging at 15 days, the mice are immediately sacrificed quickly, and their organs are taken out for in vivo imaging. The whole process is kept within 10 minutes to prevent the loss of fluorescence signals. From the developed images of in vivo imaging, the fluorescence intensity of the mice in the CMT-shscr group is strong and the range is wide, while after knocking down CPS1, the fluorescence intensity becomes weaker and the fluorescence range becomes smaller ( Figure 11 A). After taking the ROI of the fluorescence area of the mice with in vivo imaging software, the fluorescence intensity is quantified and then statistically analyzed ( Figure 11 B). The data results show that the fluorescence intensity of the mice in the shscr group is significantly stronger than that in the shCPS1 group. The fluorescence development of each organ shows ( Figure 11 C) that stronger fluorescence intensity appears in multiple organs of the shscr control group of mice, especially in the lungs, heart, liver, kidneys, and legs. After knocking down CPS1, the fluorescence intensity of the lungs, heart, and liver is weaker. This shows that the tail vein injection model can indeed cause systemic metastasis of tumors, especially most obvious in the lungs. This is mainly because the tail vein first enters the pulmonary circulation and reaches the pulmonary capillary network, and the diameter of the pulmonary capillaries is relatively small, and some tumor cells are easily retained in the lungs and form metastatic foci, and the other part of the tumor cells then reach other organs or tissues through the circulation. After knocking down CPS1, it can effectively inhibit the systemic metastasis of tumors.

[0059] 3.4 Knockdown of CPS1 effectively inhibits the growth of lung cancer cells in a mouse metastasis model by tibia injection

[0060] In previous animal experiments, we deeply explored the potential role of CPS1 in lung cancer metastasis by constructing two tumor metastasis models: left ventricular injection and tail vein injection. The common metastatic sites of lung cancer include the brain, bones, liver, etc. To deeply study the role of CPS1 in lung cancer metastasis, we selected bone metastasis as the research object and used the in-situ tibia injection model to construct a tumorigenesis experiment for lung cancer bone metastases. Male C57BL / 6 mice were randomly divided into two groups, and CMTshscr-luc cells and CMTshCPS1-luc cells were injected into the mice through intra-articular injection at a dose of 10^ 5 cells per mouse. Subsequently, the growth of tibia tumors in mice was monitored every 3 days by in vivo imaging.

[0061] In vivo imaging development ( Figure 12 A) and fluorescence analysis results ( Figure 12 B) showed that in the lung cancer bone metastasis tumorigenesis model constructed by injecting lung cancer cells into the tibia of mice, after knocking down CPS1, the growth rate of tumor cells was significantly slowed down, manifested as a reduction in tumor volume, a significant decrease in the fluorescence range, and a significant decrease in fluorescence intensity.

[0062] Example 4 Chemical inhibition of CPS1 can inhibit the migration and metastasis of lung cancer cells

[0063] 4.1 The CPS1 inhibitor H3B-120 can effectively inhibit the proliferation of metastatic lung cancer cells

[0064] In this example, the half-maximal inhibitory concentration (IC50) of H3B-120 in L2 and L6 cells was first determined. The results showed that the IC50 value of H3B-120 in L2 cells was 58.5 μM, while in L6 cells it was 42.8 μM ( Figure 13 A). Based on this, we treated L2 and L6 cells with concentrations of 1 / 2 IC50, IC50, and 2-fold IC50, respectively. Subsequently, a CCK-8 kit was used to detect the proliferation ability of L2 and L6 cells under different concentration treatments, and the absorbance (OD value) was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. Finally, the change in cell proliferation ability was statistically analyzed based on the difference in OD values. The results showed that the cell proliferation rate was the fastest in the control group, while the cell proliferation rate was significantly slowed down under the 2-fold IC50 condition. Figure 13B). The colony formation assay of cells can not only reflect the cell proliferation ability, but also better evaluate the tumorigenic ability of tumor cells. To further explore the effect of H3B-120 on cell proliferation, we detected the colony formation ability of L2 and L6 cells treated with different concentrations of H3B-120. The results showed that the number of cell colonies formed in the control group was the largest and the proliferation rate was the fastest; while under the treatment of H3B-120 at 2-fold IC50 concentration, the number of cells forming colonies decreased significantly and the proliferation rate was significantly inhibited ( Figure 13 C, D). This result further confirmed that the CPS1 specific inhibitor H3B-120 has a significant inhibitory effect on cell proliferation, indicating that inhibiting CPS1 can effectively inhibit the cell proliferation ability.

[0065] 4.2 CPS1 inhibitor H3B-120 promotes apoptosis of metastatic lung cancer cells

[0066] Apoptosis is a programmed cell death process regulated by genes. To explore the effects of the CPS1 inhibitor H3B-120 on cell proliferation and apoptosis, we first observed that the cell proliferation rate of cells treated with H3B-120 decreased significantly. Based on this phenomenon, we speculated that this inhibitor might inhibit cell proliferation by inducing apoptosis. To verify this hypothesis, we quantitatively detected the apoptosis level of cells by flow cytometry. The results of flow cytometry showed that in the untreated control group, the apoptosis rate of L2 cells remained at a low level, with the early apoptosis rate being 0.57% and the late apoptosis rate being 2.2%, and almost no obvious apoptosis phenomenon was observed. However, after treatment with the CPS1 inhibitor H3B-120, the apoptosis rate of the experimental group cells increased in a dose-dependent manner, with the early apoptosis rate being 2.26% and the late apoptosis rate being 9.44%, showing a statistically significant difference compared with the control group (p < 0.01). It is worth noting that the increase in the late apoptosis rate (4.3-fold) was significantly higher than that in the early apoptosis rate (4.0-fold). The L6 cell line also showed similar apoptosis characteristics under the same treatment conditions ( Figure 14 ). Based on the above results, we inferred that the CPS1 inhibitor H3B-120 might inhibit cell proliferation by inducing apoptosis, especially promoting the late apoptosis process.

[0067] 4.3 CPS1 inhibitor H3B-120 inhibits the migration of metastatic lung cancer cells

[0068] Based on the previous experimental data, in cells with overexpression of CPS1, the migration ability of the cells becomes stronger, suggesting that CPS1 plays an important role in regulating cell migration. Based on this finding, we proposed a scientific hypothesis: inhibiting the activity of CPS1 may reverse its pro-migration effect. To verify this hypothesis, we used a specific CPS1 inhibitor, H3B-120, and evaluated the effect of the CPS1 inhibitor on cell migration ability through a Trans-well experimental system.

[0069] The Trans-well results showed that in the control group of L2 and L6 cells, the cell migration ability was the strongest. However, as cells were treated with different concentrations of the CPS1 inhibitor H3B-120, the cell migration ability showed a significant dose-dependent inhibition ( Figure 15 ).

[0070] 4.4 The CPS1 inhibitor H3B-120 effectively inhibits lung cancer cell metastasis in a left ventricular injection mouse model

[0071] To verify the inhibitory effect of the CPS1 inhibitor H3B-120 on tumor metastasis in vivo, we established a mouse tumor metastasis model induced by left ventricular injection. This experiment aimed to explore whether H3B-120 could reproduce the effect of inhibiting tumor cell growth and metastasis observed in its in vitro experiments in animals. We used BALB / c-nude mice to establish a tumor metastasis model and inoculated L6-luc cells by left ventricular injection. Subsequently, a small animal in vivo imaging system was used to detect the fluorescence signal intensity of the mice. Mice with similar fluorescence intensities were screened and randomly divided into three groups (n = 8 / group): high-dose group (80 mg / kg H3B-120), low-dose group (40 mg / kg H3B-120), and solvent control group (equal volume of DMSO). Seven days after tumor cell inoculation, intraperitoneal injection was started daily for continuous treatment for 21 days to systematically evaluate the inhibitory effect of H3B-120 on tumor metastasis.

[0072] The results of small animal in vivo imaging showed that on the 29th day of the experiment, the control group mice showed extensive tumor metastasis characteristics, and the fluorescence signals were mainly distributed in parts such as the brain, thoracic vertebrae, and lower limbs, with an average fluorescence intensity reaching 10^ 5 or so, indicating that the tumor had metastasized systemically. Although multiple metastatic foci could still be detected in the mice treated with 40 mg / kg H3B-120, their average fluorescence intensity was significantly reduced to 10 3 or so (p < 0.01), and the number of metastatic foci was significantly reduced compared with the control group. Notably, no obvious metastatic foci were detected in the mice treated with 80 mg / kg H3B-120, and the average fluorescence intensity remained at the baseline level ( Figure 16 A and Figure 16B). These results indicate that H3B-120 can significantly inhibit the metastasis of lung cancer cells in vivo in a dose-dependent manner (p<0.001).

[0073] To further verify the therapeutic effect of H3B-120, we repeated the above experiment and systematically evaluated the survival time of mice in each group. Kaplan-Meier survival analysis showed that the median survival time of mice in the control group was 39 days. Compared with the control group, the median survival time of mice in the 40 mg / kg H3B-120 treatment group was significantly extended to 53 days, while that in the 80 mg / kg treatment group was further extended to 71 days ( Figure 16 C). The dose-dependent extension of survival time was significantly positively correlated with the tumor metastasis inhibition effect. These results fully confirmed that the CPS1 inhibitor H3B-120 could effectively inhibit the tumor metastasis process in mice with high CPS1 expression and significantly improve the survival prognosis of tumor-bearing mice.

[0074] 4.5 The CPS1 inhibitor H3B-120 effectively inhibits lung cancer cell metastasis in the left ventricular injection mouse model

[0075] At the end of the experiment, we euthanized the mice according to the requirements of animal ethics. Subsequently, the main metastatic tissue of the three groups of mice (including the brain, spine, lower limbs, etc.) was systematically collected and immediately placed in 4% paraformaldehyde (PFA) fixative and fixed at 4°C for 48 hours to ensure the complete preservation of tissue morphology. To quantitatively evaluate the degree of bone tissue damage caused by tumor metastasis, we selected the spine as a representative sample and performed three-dimensional scanning using a high-resolution Micro-CT system (model: Bruker, resolution: 9μm). The scanning data was reconstructed and quantitatively analyzed using professional analysis software, focusing on evaluating bone microstructure parameters such as bone mineral density (BMD), trabecular bone number (Tb.N), and bone volume fraction (BV / TV) to systematically evaluate the inhibitory effect of different doses of H3B-120 on tumor bone metastasis. Through three-dimensional reconstruction and two-dimensional cross-sectional analysis of the Micro-CT data, we found that significant bone destruction characteristics appeared in the spines of mice in the control group (treated with DMSO). The three-dimensional reconstruction images showed typical worm-eaten-like changes on the bone surface, accompanied by extensive destruction of the bone cortex and fracture of the trabecular bone ( Figure 17A). In contrast, the bone microstructure of mice in the 40mg / kg H3B-120 treatment group remained relatively intact, with only local slight erosion on the bone surface. The 80mg / kg treatment group showed the best protective effect, with intact trabecular structure and smooth and continuous bone surface, which was not significantly different from normal bone tissue. These morphological analysis results were mutually confirmed by the three-dimensional reconstruction images, confirming that H3B-120 can effectively inhibit tumor-related bone destruction. The analysis software (CT analysis) provided by Micro-CT was used to systematically and quantitatively evaluate the microstructure of mouse spinal trabeculae. The three-dimensional reconstruction images showed that the bone density of mice in the control group (DMSO treatment) was significantly lower than that in the treatment group, and the trabecular structure showed obvious sparse characteristics. The quantitative analysis results showed that the bone density of mice in the 40mg / kg H3B-120 treatment group was significantly higher than that in the control group, and the trabecular network structure was significantly improved; while there was no significant difference between the 80mg / kg treatment group and the 40mg / kg treatment group. Further analysis showed that the bone volume fraction (BV / TV) and trabecular number (Tb.N) of the control group were significantly lower than those of the two treatment groups ( Figure 17 B). These results collectively indicate that in CPS1-overexpressing tumor cells, H3B-120 treatment can inhibit overexpressed CPS1 and thus inhibit lung cancer metastasis, and effectively improve tumor-related bone destruction by maintaining the integrity of trabecular structure, providing an important experimental basis for the clinical treatment of distant metastasis of CPS1-overexpressing tumors.

[0076] 4.6 CPS1 inhibitor H3B-120 effectively inhibits lung cancer cell metastasis in the tail vein injection mouse metastasis model

[0077] In addition to the left ventricular injection model, the tail vein injection model is also one of the classic animal models for studying tumor metastasis. By injecting tumor cells via the tail vein, this model can simulate the process of tumor cell dissemination through the blood circulation and is suitable for studying the systemic metastasis of tumors. To comprehensively evaluate the anti-metastatic effect of CPS1 inhibitors, we established a tail vein injection model: a suspension of murine lung cancer cells CMT-luc was injected into C57BL / 6 mice via the tail vein (cell number: 10^5 / mouse). Twenty-four hours after inoculation, mice with similar fluorescence signal intensities were screened using a small animal in vivo imaging system and randomly divided into an experimental group and a control group (n = 6). Three days after model establishment, the experimental group was treated with 80 mg / kg of H3B-120, and the control group was given an equal volume of DMSO, intraperitoneally injected once daily for 12 days, and the fluorescence intensity was quantitatively analyzed. On the 15th day of the experiment, after collecting the fluorescence signals using a small animal in vivo imaging system, the mice were immediately euthanized by cervical dislocation. Subsequently, the main organ tissues (including the lung, heart, liver, spleen, and kidney) and bone samples (femur and tibia) were systematically collected. Each tissue sample was placed on an imaging plate, and ex vivo tissue fluorescence imaging was performed using a small animal in vivo imaging system to accurately locate tumor metastases and evaluate the degree of metastasis in each organ. The imaging conditions were kept consistent with the in vivo imaging parameters to ensure data comparability.

[0078] The results of small animal in vivo imaging showed that obvious fluorescence signals were detected in the thoracic region and lower limb bone sites of the control group mice. In contrast, only weak fluorescence signals were detected in the thoracic region of the treatment group mice ( Figure 18 A). Through the dynamic monitoring and quantitative analysis of the fluorescence signals during the experimental period, it was found that the fluorescence intensity of the treatment group remained at a low level all the time, showing a significant statistical difference compared with the control group (p < 0.001) ( Figure 18 B).

[0079] Considering that the hair regrowth of C57BL / 6 mice in the later stage of the experiment might affect the accuracy of in vivo imaging, we used ex vivo tissue fluorescence imaging technology to localize the metastases in the main organs. The imaging results showed that obvious fluorescence signals were detected in multiple organs of the control group (DMSO-treated group) mice, and the fluorescence intensity in the lungs was the most significant. This was because the diameter of the pulmonary capillaries is small, and some tumor cells are easily retained and colonized in the lungs to form metastases, which is consistent with the pathophysiological process that tumor cells first pass through the pulmonary circulation after tail vein injection. In addition, different degrees of fluorescence signals were also detected in the heart, liver, spleen, kidney, and lower limb bones of the control group mice, indicating that systemic metastasis of tumor cells had occurred. In contrast, only weak fluorescence signals were detected in the lungs and heart of the treatment group mice, and no obvious fluorescence was observed in the remaining organs. Figure 18C). Through this model, we further explored the inhibitory effect of CPS1 inhibitors on the lung metastasis of lung cancer cells and its potential mechanism. These results further confirmed that H3B-120 could effectively inhibit the systemic metastasis of tumor cells.

[0080] 4.7 In the tibia injection mouse metastasis model, the CPS1 inhibitor H3B-120 effectively inhibits the growth of lung cancer cells

[0081] The left ventricular injection model and the tail vein injection model in mice are two classic animal models for studying tumor metastasis. In previous experiments, we have preliminarily explored the anti-tumor metastasis effect of the CPS1 inhibitor H3B-120 through these two models. To further investigate the specific mechanism of action of the CPS1 inhibitor H3B-120 in lung cancer metastasis, this example selected lung cancer bone metastasis as the key research direction and used the tibia in-situ injection model to construct lung cancer bone metastases to establish an animal model closer to clinical pathological characteristics.

[0082] Eight-week-old male C57BL / 6 mice were selected as the research subjects. Before the experiment, the right hind limb of the mice was depilated to facilitate subsequent tumor cell injection and observation. CMT-luc cells expressing luciferase were injected into the knee joint cavity of the mice at a cell dose of 1×10^ 5 / mouse. The luciferase substrate was injected, and 5 minutes later, the fluorescence signal intensity in the mice was detected using a small animal in vivo imaging system (IVIS). Mice with similar fluorescence intensities were selected and randomly divided into two groups (n = 6 / group). Three days after cell inoculation, the CPS1 inhibitor H3B-120 (80 mg / kg) was administered by intraperitoneal injection for treatment, once every 3 days. At the same time, the in vivo imaging system was used to regularly monitor the tumor growth, and the changes in fluorescence signal intensity were recorded to evaluate tumor progression.

[0083] The in vivo imaging results on the 15th day showed ( Figure 19 A), in the DMSO control group of mice, significant fluorescence signals were presented in the tibia region, with relatively high fluorescence intensity and a wide distribution range. In contrast, the fluorescence signal intensity in the mice treated with the CPS1 inhibitor was significantly reduced, and the fluorescence distribution area was significantly reduced. The quantitative analysis results showed ( Figure 19 B), the fluorescence intensity in the mice treated with the CPS1 inhibitor was significantly lower than that in the control group (p < 0.05) and remained at a low level, indicating that the CPS1 inhibitor effectively inhibited the growth and metastasis of tumors.

[0084] 4.8 In the lung cancer bone metastasis organoid model, the CPS1 inhibitor H3B-120 effectively inhibits the growth of lung cancer metastatic cells

[0085] Patient-derived organoids (PDO) are one of the most promising research models for evaluating drug efficacy at present. Compared with traditional two-dimensional cell culture and animal xenograft tumor models, the PDO model has significant advantages: First, PDO is directly derived from patient tumor tissues, retaining the organizational structure and molecular characteristics of the primary tumor, ensuring a high degree of consistency between the model and clinical samples; Second, PDO contains tumor stem cells with self-renewal and multi-directional differentiation potential, capable of reproducing the cellular heterogeneity of the primary tumor; More importantly, PDO completely retains the genomic characteristics, epigenetic characteristics and tumor microenvironment of the tumor, and is highly similar to the original tumor in terms of morphological and pathophysiological characteristics. Therefore, it can more accurately predict clinical drug responses and provide a reliable basis for the formulation of individualized treatment plans. So we collected fresh surgical samples from the bone metastasis sites of lung cancer patients with bone metastasis and constructed lung cancer bone metastasis organoids. Under aseptic conditions, the tumor tissues were mechanically separated and enzymatically digested, and then cultured with Matrigel using a three-dimensional culture system. To verify the reliability of the organoid model, we conducted the following systematic identification: First, the morphological characteristics of the organoids were evaluated by hematoxylin-eosin (H&E) staining, and the results showed that the cultured organoids maintained the organizational structure and cell morphological characteristics of the original tumor; Second, immunohistochemical staining (IHC) was used to detect the expression of lung squamous cell carcinoma-specific markers p40 and CK5 / 6, confirming that the organoids retained the phenotypic characteristics of the primary tumor; In addition, we also detected the expression of CPS1, and the results showed that CPS1 was stably expressed in the organoids, consistent with the data obtained from previous experiments. These results indicate that we have successfully established a lung cancer bone metastasis organoid model with high biological fidelity, providing a reliable experimental platform for subsequent drug screening and mechanism research. Figure 20 A).

[0086] Subsequently, drug sensitivity tests were conducted. The cells of the organoids were inoculated into 96-well plates. When they grew to a size of 30 - 100 μm, they were treated with the CPS1 inhibitor H3B-120. Samples were collected 96 hours after adding the drug. Calcein-AM / PI fluorescence staining was used, and the lung cancer bone metastasis organoids were photographed through an optical microscope. Calcein-AM is a lipophilic non-fluorescent dye that can penetrate intact cell membranes and enter the cell interior. Inside the cell, Calcein-AM is hydrolyzed by intracellular esterases into highly green fluorescent Calcein. Only living cells can hydrolyze Calcein-AM into Calcein. Therefore, a high green fluorescence signal indicates good cell metabolic activity and cell membrane integrity. Propidium iodide (PI) is a nucleic acid dye that can enter the cell only when the cell membrane is damaged, bind to DNA, and emit red fluorescence. Therefore, when the cell membrane is damaged, PI enters the cell and binds to DNA, emitting a red fluorescence signal. By simultaneously observing the green fluorescence of Calcein and the red fluorescence of PI, three cell states can be distinguished: living cells (only green fluorescence), dead cells (only red fluorescence), and damaged but still surviving cells (both green and red fluorescence). According to the intensity and distribution of the fluorescence signals, the cell viability and toxicity levels under different conditions can be quantified and analyzed, thereby evaluating the toxicity of the drug, the quality of cell culture, and other related biological research questions. The experimental results showed that the CPS1 inhibitor had a concentration-dependent cytotoxic effect on lung cancer bone metastasis organoids. When treated with 50 μM CPS1 inhibitor, approximately 50% of the organoid cells died; when the concentration was increased to 100 μM, the cell death rate increased significantly ( Figure 20 B).

[0087] To further verify this result, we used the luminescence method (CTG) to quantitatively detect cell viability. The CTG test results were consistent with the Calcein-AM / PI double staining results: the organoids in the control group showed the strongest cell viability, followed by the 50 μM treatment group, while the cell viability in the 100 μM treatment group was significantly reduced (p < 0.05) ( Figure 20 C). These results indicate that the CPS1 inhibitor can inhibit the cell viability of lung cancer bone metastasis organoids in a dose-dependent manner.

[0088] The above is only a preferred embodiment of the present invention, and thus does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be realized that all equivalent replacements and obvious changes made by using the content of the present invention's specification and drawings should be included in the protection scope of the present invention.

Claims

1. Use of a CPS1 inhibitor in the preparation of a medicament for treating lung cancer metastasis, characterized in that, The Gene ID of CPS1 is 1373, and the CPS1 inhibitor refers to a molecule that targets CPS1 and has an inhibitory effect on CPS1.

2. The application according to claim 1, wherein The CPS1 inhibitor is an shRNA inhibitor, an siRNA inhibitor, or a small molecule inhibitor.

3. The application according to claim 2, wherein The CPS1 inhibitor is an shRNA inhibitor, and the sequences of the shRNA are shown in SEQ ID NO.1-2.

4. The application according to claim 2, characterized in that, The CPS1 inhibitor is a small molecule inhibitor, and the small molecule inhibitor is H3B-120, with a molecular formula of C 19 H 24 N4O2S.

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