Use of an agent for detecting LMTK3 protein, a combination drug, and use of a combination of a LMTK3 inhibitor and a platinum drug

Abstract

This invention provides the use of a reagent for detecting LMTK3 protein, a combination drug, and the use of LMTK3 inhibitors in combination with platinum-based drugs, belonging to the field of pharmaceutical technology. This invention is the first to discover that LMTK3 is significantly highly expressed in ovarian cancer patients compared to healthy individuals; and that LMTK3 is significantly highly expressed in cisplatin-resistant ovarian cancer patients compared to cisplatin-sensitive ovarian cancer patients. Detecting the expression level of LMTK3 in ovarian tissue can differentiate between ovarian cancer patients and healthy individuals, as well as between cisplatin-sensitive and cisplatin-resistant ovarian cancer patients. Furthermore, this invention also discovers that combining LMTK3 inhibitors with platinum-based drugs to treat platinum-resistant ovarian cancer can effectively increase the sensitivity of platinum-based drugs to drug-resistant ovarian cancer cells, improve the therapeutic efficacy of platinum-based drugs for ovarian cancer, and lay the foundation for developing new drugs to reverse drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of reagents for detecting LMTK3 protein, a combination drug, and the use of LMTK3 inhibitors in combination with platinum-based drugs. Background Technology

[0002] Ovarian cancer is one of the most common malignant tumors of the female reproductive organs, ranking third in incidence among all gynecological cancers, after cervical cancer and endometrial cancer; however, it has the highest mortality rate. High-grade serous ovarian cancer (HGSC) accounts for 70-80% of all ovarian cancer deaths, with a 5-year overall survival rate of less than 40%. The reasons for this are twofold: first, ovarian cancer often has an insidious early onset, and patients are frequently diagnosed at an advanced stage; second, while surgery combined with platinum-based chemotherapy is the standard treatment for ovarian cancer, even though 85% of patients respond to initial chemotherapy, most develop acquired chemotherapy resistance, leading to recurrence and death. Currently, there is no effective method to distinguish between pre-treatment resistant and sensitive HGSC, meaning that even if resistant patients endure the toxicity of platinum-based chemotherapy, they ultimately cannot benefit from it.

[0003] Cisplatin primarily inhibits cell division by inducing DNA damage in tumor cells, ultimately leading to apoptosis. It also promotes apoptosis by causing cellular oxidative stress, disrupting calcium homeostasis, impairing mitochondrial function, and inducing reactive oxygen species. Early-stage ovarian cancer is highly sensitive to chemotherapy, with an initial response rate of 85%. However, approximately 15% of patients do not respond to treatment and have a poor prognosis, with a median overall survival of only 12 months. Moreover, most of the 85% of patients who respond to initial chemotherapy relapse due to drug resistance. The molecular mechanisms of ovarian cancer chemoresistance (OCC) are not yet fully understood; therefore, there is an urgent need to find more effective therapeutic targets and drugs for resistant patients.

[0004] Occupational cancer cell (OCC) formation is the result of multiple genes, factors, and pathways. Reduced or inactivated intracellular drugs, abnormal target sites, abnormal apoptosis regulation, increased tolerance to DNA damage or enhanced repair function after damage, and alterations in the tumor microenvironment can all lead to increased resistance of ovarian cancer cells to chemotherapy drugs, resulting in chemotherapy resistance. Epigenetic changes are also associated with drug resistance in various cancers, including ovarian cancer.

[0005] Lemur tyrosine kinase 3 (LMTK3), an oncogenic kinase, is closely related to the occurrence and development of various malignant tumors and other diseases, such as central nervous system-related diseases. However, whether LMTK3 participates in the development and progression of platinum-resistant ovarian cancer cells has not yet been reported. Therefore, it is urgent to further elucidate the molecular mechanism by which LMTK3 regulates OCC, clarify its potential value as a tumor biomarker, and confirm the effectiveness of its inhibitors, thus laying a theoretical foundation for the clinical translational treatment of platinum-resistant ovarian cancer. Summary of the Invention

[0006] The purpose of this invention is to provide the use of a reagent for detecting LMTK3 protein, a combination drug, and the use of LMTK3 inhibitors in combination with platinum-based drugs.

[0007] This invention provides the use of reagents for detecting LMTK3 protein in the preparation of ovarian cancer screening kits.

[0008] Furthermore, the ovarian cancer screening kit is a reagent for screening ovarian cancer patients and healthy individuals, or the ovarian cancer screening kit is a reagent for screening platinum-resistant ovarian cancer patients and platinum-sensitive ovarian cancer patients.

[0009] Furthermore, the reagent for detecting LMTK3 protein is an enzyme-linked immunosorbent assay (ELISA) reagent or an ELISA assay reagent.

[0010] Furthermore, the reagent used to detect LMTK3 protein is a Western blot reagent or an IHC reagent.

[0011] Furthermore, the reagent for detecting LMTK3 protein is a reagent for detecting LMTK3 protein in ovarian tissue.

[0012] The present invention also provides a combination drug for the prevention and / or treatment of platinum-resistant ovarian cancer, wherein the combination drug comprises an LMTK3 inhibitor and a platinum-based drug in the same or different strength units for simultaneous or separate administration, and a pharmaceutically acceptable carrier.

[0013] Furthermore, the mass ratio of the LMTK3 inhibitor to the platinum-based drug is 15:1.

[0014] Furthermore, the LMTK3 inhibitor includes reagents that knock out or knock down the LMTK3 gene, reagents that inhibit LMTK3 gene expression, reagents that reduce the content of LMTK3 protein in the body, or reagents that reduce the stability of LMTK3 protein; the platinum-based drugs include cisplatin, carboplatin, or oxaliplatin.

[0015] Furthermore, the reagent for knocking down the LMTK3 gene includes shRNA1 and / or shRNA2 targeting the LMTK3 gene; the forward and reverse primer sequences of shRNA1 are shown in SEQ ID NO.1~2; the forward and reverse primer sequences of shRNA2 are shown in SEQ ID NO.3~4;

[0016] The reagent for knocking out the LMTK3 gene includes sgRNA1 and / or sgRNA2 targeting the LMTK3 gene, and the forward and reverse primer sequences of sgRNA1 are shown in SEQ ID NO.5~6; the forward and reverse primer sequences of sgRNA1 are shown in SEQ ID NO.7~8;

[0017] The reagents used to reduce LMTK3 protein levels include small molecule inhibitors C28 or C36.

[0018] The present invention also provides the use of LMTK3 inhibitors and platinum-based drugs in combination in the preparation of medicaments for the prevention and / or treatment of platinum-based drug-resistant ovarian cancer.

[0019] Furthermore, the drug can inhibit the growth of ovarian cancer cells and / or inhibit the metastasis of ovarian cancer cells.

[0020] This invention demonstrates through experiments that LMTK3 is significantly highly expressed in ovarian cancer patients compared to healthy individuals; and that LMTK3 is significantly highly expressed in cisplatin-resistant ovarian cancer patients compared to cisplatin-sensitive ovarian cancer patients. LMTK3 can promote the tolerance of ovarian cancer cells to platinum-based drugs, indicating that LMTK3 is a potential biomarker for screening ovarian cancer and predicting platinum-based drug resistance in ovarian cancer patients. It can be used to predict the sensitivity of ovarian cancer patients to platinum-based chemotherapy before treatment, thereby achieving patient stratification and precision treatment.

[0021] The key to this invention lies in determining that the content of LMTK3 protein in ovarian tissue is significantly correlated with the risk of ovarian cancer, and that the content of LMTK3 protein in ovarian tissue is significantly correlated with platinum-based drug tolerance in ovarian cancer patients. Therefore, the risk of ovarian cancer can be assessed by detecting the content of LMTK3 protein in ovarian tissue, and the sensitivity of ovarian cancer patients to platinum-based drugs can also be determined. As for the specific methods for detecting the content of LMTK3 protein in human tumor tissue, various methods disclosed in the prior art can be used. In this embodiment of the invention, Western blot and IHC are specifically used for detection, but it is not limited to these methods. Any method that can detect the content of LMTK3 protein can be used for screening platinum-resistant ovarian cancer.

[0022] This invention also verified the effectiveness of targeting the LMTK3 gene through comparative experiments involving LMTK3 gene knockdown. LMTK3 expression significantly promoted the proliferation of platinum-resistant ovarian cancer cells and increased their tolerance to platinum-based drugs, demonstrating that LMTK3 is a highly reliable therapeutic target for ovarian cancer resistance. Blocking LMTK3 function can effectively enhance the sensitivity of platinum-based drugs to ovarian cancer cells, laying the foundation for developing new drugs to reverse drug resistance. This invention also shows that LMTK3 can be downregulated through gene silencing techniques such as shRNA, siRNA, antisense oligonucleotides, CRISPR / Cas9 gene editing, or epigenetic modification drugs, thereby achieving drug resistance reversal. This lays a solid foundation for the subsequent development of drugs using different technical routes.

[0023] Furthermore, this invention demonstrates that inhibiting LMTK3 expression promotes platinum resistance by inhibiting the pyroptosis pathway, which provides a theoretical basis for research on drug resistance treatment of ovarian cancer and also means that drug design targeting pyroptosis may become an effective strategy to overcome drug resistance.

[0024] Simultaneously, this invention also demonstrates that combining LMTK3 inhibitors with platinum-based anti-ovarian cancer drugs can significantly enhance their anti-tumor effects, showing significant effectiveness in treating drug-resistant ovarian cancer. It proposes a highly effective treatment strategy to reverse platinum resistance, offering great promise for improving chemotherapy response rates and survival in patients with advanced or recurrent ovarian cancer. This also implies that LMTK3 inhibitors can be combined with other anti-ovarian cancer drugs to enhance their anti-tumor effects.

[0025] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0026] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0027] Figure 1 Figure A shows the results of Western blot analysis of LMTK3 expression in ovarian cancer cell lines A2780, COC1 and their corresponding platinum-resistant cell lines A2780CP and COC1 / DDP. Figure 1 B~D are microarray images of the results of cisplatin-resistant and cisplatin-sensitive patient tissue analysis. Figure 1 E is a graph showing the relationship between LMTK3 expression and survival prognosis in ovarian cancer patients; Figure 1F is the ROC curve used to distinguish between cisplatin-sensitive and cisplatin-resistant ovarian cancer patients by the expression level of LMTK3 in ovarian tissue. Figure 1 G is the ROC curve used to distinguish between ovarian cancer patients and healthy individuals by the expression level of LMTK3 in ovarian tissue.

[0028] Figure 2 The following figures show the results of Western blotting experiments on sensitive and drug-resistant ovarian cancer cells: A shows the expression of LMTK3 in cells treated with cisplatin; B shows the expression of LMTK3 in the nucleus after nucleocytoplasmic separation; C shows the expression of LMTK3 in the nucleus after cisplatin treatment.

[0029] Figure 3 A~B represent the construction status of LMTK3 knockdown cell lines as detected by q-PCR and Western blotting. Figure 3 C~E is the sensitivity of cisplatin-treated cells and COC1 / DDP cells to LMTK3 knockdown using CCK8 and colony formation assays. Figure 3 F is an experiment on cell viability with C28+ cisplatin; Figure 3 G~K is used to detect the expression of LMTK3 in cells and cell proliferation after the LMTK3-WT full-length expression vector is transfected into cells.

[0030] Figure 4 The following figures show the experimental results of LMTK3 inhibiting pyroptosis in drug-resistant ovarian cancer cells: A is a phase-contrast image of A2780CP cells before and after stable LMTK3 knockdown; B is the expression levels of PYCARD and CASPASE4 in A2780CP and COC1 / DDP cells before and after stable LMTK3 knockdown detected by q-PCR; C is the mRNA levels of IL-1β and IL-18 in A2780CP cells before and after stable LMTK3 knockdown detected by q-PCR; DE is the mRNA levels of IL-1β and IL-18 in A2780CP cells treated with the LMTK3 inhibitor C28 detected by PCR; F is the changes in live and dead cells in A2780CP cells before and after stable LMTK3 knockdown detected by immunofluorescence.

[0031] Figure 5 The following figures show the experimental results of the effect of LMTK3 on drug-resistant ovarian cancer cells in vivo: A is the flowchart of the mouse experiment; B-D show the peritoneal seeding and growth of A2780CP cells in the control group (L-glucose / PEG400) and the treatment group (C28 / PEG400); E-G show the peritoneal seeding and growth of A2780CP-Luc-Puro cells and scrambled cells with stable LMTK3 knockdown transplanted into mice.

[0032] Figure 6In the diagram, A represents the construction of ID8 Lmtk3 KO cells in protein immunoassay; B-C represent the CCK8 assay results of the control group and ID8Lmtk3 KO cells; D-E represent Lmtk3... WT -PBS, Lmtk3 WT -Cis、Lmtk3 KO -PBS, Lmtk3 KO - Peritoneal seeding and growth of Cis cells (carrying Luciferase fluorescent labeling); F shows in vivo imaging results of Luciferase substrate taken every 7 days in mice; G shows in vivo imaging results of Luciferase substrate taken at 42 days; H shows the results of Luciferase substrate imaging on Lmtk3. WT -PBS, Lmtk3 KO -Graph showing the statistical results of ascites volume collected from mice in the PBS group, and I is a graph showing the change in mouse body weight. Detailed Implementation

[0033] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0034] Experimental Example 1: LMTK3 is highly expressed in platinum-resistant ovarian cancer cells and tissues.

[0035] 1.1 Experimental Subjects

[0036] Tissue samples were collected from clinical patients with ovarian cancer (tissue microarray from Shanghai Chipover Biotechnology Co., Ltd.: HOvaC143Su01). According to clinical criteria, ovarian cancer patients with a disease-free survival (DFS) greater than 6 months were defined as cisplatin-sensitive, and those with a DFS less than or equal to 6 months were defined as cisplatin-resistant. The corresponding results are as follows: Figure 1 As shown in B~F.

[0037] LMTK3 expression data from ovarian cancer tumor samples (sourced from TCGA, project number OV) and normal ovarian samples (sourced from GTEx) were collected for comparison. A total of 379 ovarian cancer patient samples and 88 normal ovarian samples were collected for analysis. The corresponding results are as follows: Figure 1 As shown in G.

[0038] Ovarian cancer cell lines A2780 and COC1, and their corresponding platinum-resistant cell lines A2780CP and COC1 / DDP, were used; the corresponding results are as follows: Figure 1 A and Figure 2 As shown.

[0039] 1.2 Experimental Methods

[0040] Approximately 50,000 cells were seeded in each well of a 6-well plate and cultured at 37°C and 5% CO2. LMTK3 expression was then detected using Western blotting. LMTK3 expression in cisplatin-sensitive / resistant tissues was detected using an ovarian cancer tissue microarray, and changes in survival prognosis were analyzed. LMTK3 expression in cisplatin-treated cells, nuclei of cells after nucleocytoplasmic separation, and nuclei of cells treated with cisplatin were also detected using Western blotting.

[0041] 1.3 Experimental Results

[0042] (1) Differences in LMTK3 expression between ovarian cancer patients and healthy individuals

[0043] like Figure 1 As shown in Figure E, ovarian cancer patients with high LMTK3 expression had a significantly reduced survival prognosis. Figure 1 The ROC curve shown in G, which distinguishes the expression level of LMTK3 in ovarian cancer patients from that of healthy individuals, has an AUC of 0.9678, verifying that LMTK3 can specifically distinguish between ovarian cancer patients and healthy individuals.

[0044] (2) Differences in LMTK3 expression between cisplatin-resistant and cisplatin-sensitive ovarian cancer patients

[0045] Tissue microarray analysis showed that the expression level of LMTK3 in the tissues of cisplatin-resistant patients was significantly higher than that in the sensitive group, and the expression of LMTK3 in the metastasis group was also significantly upregulated. Figure 1 (B~D). For example... Figure 1 The ROC curve shown in Figure F, which differentiates cisplatin-sensitive from cisplatin-resistant ovarian cancer patients by immunohistochemical expression levels of LMTK3 protein in tissues of ovarian cancer patients, has an AUC of 0.693, verifying that LMTK3 can specifically distinguish between cisplatin-resistant and cisplatin-sensitive ovarian cancer patients.

[0046] (3) Ovarian cancer cell experiment

[0047] like Figure 1 As shown in Figure A, LMTK3 expression was significantly upregulated in platinum-resistant cells A2780CP and COC1 / DDP. Figure 2 As shown, after treatment with cisplatin, the expression of LMTK3 was upregulated in drug-resistant cells. Figure 2 A); Nuclear-cytoplasmic separation revealed that LMTK3 expression in the nucleus of drug-resistant cells was significantly higher than that in the nucleus of sensitive cells. Figure 2 B); moreover, the addition of cisplatin significantly upregulated the expression of LMTK3 in the nucleus ( Figure 2 C).

[0048] The above results indicate that LMTK3 is significantly overexpressed in ovarian cancer patients compared to healthy individuals, and ovarian cancer patients with high LMTK3 expression have a significantly lower survival prognosis. Detecting the LMTK3 expression level in ovarian tissue can help distinguish between ovarian cancer patients and healthy individuals. Furthermore, LMTK3 is significantly overexpressed in cisplatin-resistant ovarian cancer patients compared to cisplatin-sensitive ovarian cancer patients. Detecting the LMTK3 expression level in ovarian tissue can help distinguish between cisplatin-sensitive and cisplatin-resistant ovarian cancer patients.

[0049] Experimental Example 2: Decreasing LMTK3 expression significantly inhibited the proliferation of cisplatin-resistant ovarian cancer cells.

[0050] 2.1 Experimental Subjects

[0051] shRNA targeting LMTK3 was constructed using lentivirus, and then platinum-resistant cells A2780CP and COC1 / DDP were infected with the lentivirus; the corresponding results are as follows. Figure 3 As shown in A~F.

[0052] The full-length LMTK3-WT expression vector was constructed and transfected into A2780 cells using Lipo-3000 transfection reagent; the corresponding results are as follows. Figure 3 As shown in G~K.

[0053] 2.2 Experimental Methods

[0054] In the experiment of infecting platinum-resistant A2780CP cells with lentivirus, the lentiviral vector used was pLKO.1-puro; the knockdown and verification procedures are as follows:

[0055] The shLMTK3-Forward-1-cds and shLMTK3-Reverse-1-cds sequences with SEQ ID NO.1~2 were annealed to form the shRNA1 target sequence, which was then constructed into the pLKO.1-puro vector to obtain the shRNA1-LMTK3-pLKO.1-puro vector. The shRNA1-LMTK3-pLKO.1-puro vector was then used to infect A2780CP cells, followed by selection with 2.5 μg / mL puromycin. Western blotting was used to further verify the LMTK3 knockdown effect. The shLMTK3-Forward-2-utr and shLMTK3-Reverse-2-utr sequences (SEQ ID NO. 3-4) were annealed to form the shRNA2 target sequence, which was then constructed into the pLKO.1-puro vector to obtain the shRNA2-LMTK3-pLKO.1-puro vector. The shRNA2-LMTK3-pLKO.1-puro vector was then used to infect A2780CP cells, followed by selection with 2.5 μg / mL puromycin. Western blotting was used to further verify the LMTK3 knockdown effect.

[0056] Table 1 Primer Sequences

[0057]

[0058] A2780CP cells were seeded into six-well plates. When the cells reached 70% confluence, lentivirus-infected cells were added, and three groups were established: a scrambled control group, a shLMTK3-1 group, and a shLMTK3-2 group. After culturing under suitable conditions for 48 hours, the cells were digested with trypsin, counted, and then seeded into 48-well plates. After 48 hours of cell growth, different concentrations of cisplatin (2, 4, 6, 8, 10, and 12 μM) were added for 24 hours, and then the culture medium was replaced with fresh medium. After two weeks of cell growth, the cells were fixed with 4% paraformaldehyde and then stained with crystal violet. After staining, the cells were rinsed with water, photographed, and the number of clones was counted.

[0059] LMTK3 levels in the Scrambled control group, as well as the shLMTK3-1 and shLMTK3-2 groups, were detected by q-PCR and Western blotting. The sensitivity of the three groups of cells to cisplatin was detected by CCK8 and colony formation assays. Similarly, COC1 / DDP cells were infected with lentivirus to detect the sensitivity of the three groups of cells to cisplatin.

[0060] The full-length LMTK3-WT expression vector was constructed and transfected into A2780 cells using Lipo-3000 transfection reagent. The expression of LMTK3 and cell proliferation in A2780 cells of the Vehicle group and OE-LMTK3 group were detected by q-PCR, Western blotting, CCK8 assay, and colony formation assay.

[0061] 2.2 Experimental Results

[0062] like Figure 3 As shown, the shLMTK3-1 and shLMTK3-2 groups were constructed by knocking down LMTK3 in cisplatin-resistant ovarian cancer cells using shRNA targeting LMTK3. The results showed that q-PCR and Western blotting experiments confirmed successful construction of the knockdown cell lines. CCK8 assays revealed a significant increase in cisplatin sensitivity after LMTK3 knockdown. Compared to cells with LMTK3 knockdown, the control group showed higher LMTK3 expression, significantly promoted cell proliferation, and improved cisplatin tolerance. The results from the Vehicle and OE-LMTK3 groups also indicated that the OE-LMTK3 group showed higher LMTK3 expression, significantly promoting cell proliferation and improving cisplatin tolerance.

[0063] Experiments have shown that reducing LMTK3 expression can significantly inhibit the proliferation of cisplatin-resistant ovarian cancer cells, while simultaneously increasing the sensitivity of cisplatin-resistant ovarian cancer cells to cisplatin.

[0064] Experimental Example 3: LMTK3 inhibits pyroptosis in cisplatin-resistant ovarian cancer cells.

[0065] 3.1 Experimental Methods

[0066] Further experiments were conducted based on Experiment 2:

[0067] (1) Phase contrast images show the morphology of A2780CP cells in the Scrambled control group and the LMTK3 knockdown groups (shLMTK3-1 and shLMTK3-2); the corresponding results are as follows: Figure 4 As shown in Figure A.

[0068] (2) The expression levels of PYCARD, LMTK3, and other related proteins in A2780CP and COC1 / DDP cells before and after stable LMTK3 knockdown were detected by q-PCR experiment; the corresponding results are as follows: Figure 4 As shown in B.

[0069] (3) The mRNA levels of IL-1β and IL-18 in A2780CP cells before and after stable knockdown of LMTK3 were detected by q-PCR; the mRNA levels of IL-1β and IL-18 in A2780CP cells treated with the LMTK3 inhibitor C28 were detected by q-PCR; the corresponding results are as follows: Figure 4 As shown in C~E.

[0070] (4) Immunofluorescence assay was used to detect changes in live and dead cells in A2780CP cells before and after stable LMTK3 knockdown; Calcein-AM / EthD-I dye was used to label live and dead cells respectively; the corresponding results are as follows: Figure 4 As shown in F.

[0071] 3.2 Experimental Results

[0072] Morphology of A2780CP cells in the control group and LMTK3 knockdown group was observed using a conventional phase-contrast microscope. The knockdown group cells showed significant swelling, exhibiting a "fried egg" appearance. Figure 4 A).

[0073] CASPASE-4, a key regulator of pyroptosis, can cleave GSDMD, releasing the activated GSDMD-N domain, causing membrane perforation, releasing IL-1β and IL-18, and triggering pyroptosis. In this experiment, q-PCR analysis revealed that knockdown of LMTK3 in platinum-resistant A2780CP cells significantly promoted the expression levels of CASPASE-4 and PYCARD. Figure 4 B).

[0074] Furthermore, q-PCR analysis revealed that both LMTK3 knockdown and C28 treatment significantly upregulated the levels of IL-1β and IL-18. Figure 4 C~E). Immunofluorescence assays were used to detect changes in viable and dead cells in A2780CP cells before and after stable LMTK3 knockdown. The results showed that LMTK3 knockdown significantly promoted cell death in drug-resistant cells. Figure 4 F).

[0075] The above experimental results demonstrate that knocking down LMTK3 and reducing LMTK3 protein levels can both promote pyroptosis in cisplatin-resistant ovarian cancer cells.

[0076] Experiment Example 4, Animal Experiment

[0077] 4.1 Experimental Methods

[0078] (1) Select 4-6 week old female BALB / c-nu nude mice, inject each nude mouse with a mixture of 2 million A2780CP-Luc-puro cells intraperitoneally, monitor and record the growth of the tumor and the weight of the mice; on the 7th day after tumor implantation, or when the tumor volume reaches a certain size, each group of mice is randomly divided into four groups of 6 mice each, namely L-glucose / PEG400, Cisplatin, C28 / PEG400 and Cisplatin (2mg / kg / week) + C28 (30mg / kg / day), and intraperitoneal drug injection combined with gavage, six times a week for 2-3 weeks. Before the end of the treatment or before the tumor volume of the control group reaches the endpoint, the mice are sacrificed, and the tumor tissue is embedded and fixed for HE and IHC staining;

[0079] Formulation preparation: First, C28 is dissolved in the required volume of PEG400, vortexed for 1 min, then sonicated at 4℃ for 30 min, and then 5% L-glucose (5% glucose:PEG400=1:1, volume ratio) is added to the formulation under vigorous vortexing.

[0080] (2) Using the method of knocking down LMTK3 in Experiment Example 2 to construct a stable knockdown of LMTK3 in the peritoneal xenograft of A2780CP: The mice were randomly divided into groups of 7 female BALB / c-nu nude mice aged 4-6 weeks. 8 million A2780CP-Luc-puro cells (including the scrambled control group, shLMTK3-1 and shLMTK3-2 stable knockdown groups) were suspended in 200 μL of DMEM medium suspension and injected intraperitoneally. Subsequently, cisplatin (2 mg / kg / week) or the corresponding volume of PBS was injected intraperitoneally at predetermined times. All nude mice with tumor implantation were tracked regularly. In vivo imaging was performed using 15 mg / mL of D-fluorescein potassium salt dissolved in PBS to monitor and record the tumor growth and weight changes of nude mice until the control group nude mice reached the endpoint. The mice were sacrificed by overdose anesthesia, and the tumor tissue was removed, embedded and fixed for HE and IHC staining.

[0081] (3) Wild-type C57BL / 6 mice were intraperitoneally injected with 1 × 10⁻⁶ PBS suspended in 200 μL of sterile PBS. 7 Mice were injected with ID8-NGL cells. Thirty days later, they were randomly divided into the following treatment groups: control group (Lmtk3). WT -PBS, cisplatin control group Lmtk3 WT -Cis, Knock out Lmtk3 group Lmtk3 KO -PBS, Lmtk3 knockout + cisplatin group Lmtk3 KO-Cis; cisplatin, 2 mg / kg, once weekly; no signs of drug toxicity were observed in any of the treated mice, and tumor progression was monitored by body weight and abdominal circumference measurements; at sacrifice, ascites was aspirated and its volume was measured using a subcutaneous syringe, and tumor implantation in the peritoneal wall and mesentery was measured, then harvested and quick-frozen or formalin fixed for further analysis.

[0082] Lmtk3 knockout experiment: Lentiviral sgRNAs targeting Lmtk3 were constructed and then mice were infected with the lentivirus. This experiment used the LentiCRISPR V2 viral vector to simultaneously amplify and transcribe two sgRNAs targeting the Lmtk3 site for knockout. The knockout and validation procedures are as follows:

[0083] The Lm3-sg1-PCR-F and Lm3-sg1-PCR-R sequences (SEQ ID NO. 5-6) were annealed to form the sgRNA1 target sequence, which was then constructed into the LentiCRISPR V2 vector to obtain the sgRNA1-Lmtk3-LentiCRISPR V2 vector. The Lm3-sg2-PCR-F and Lm3-sg2-PCR-R sequences (SEQ ID NO. 7-8) were annealed to form the sgRNA2 target sequence, which was then constructed into the LentiCRISPR V2 vector to obtain the sgRNA2-Lmtk3-LentiCRISPR V2 vector. The sgRNA1-Lmtk3-LentiCRISPR V2 vector and the sgRNA2-Lmtk3-LentiCRISPR V2 vector were then combined. The V2 vectors were transfected into ID8 cells at a molar ratio of 1:1. Single-clone screening and sequencing were then performed, and Western blotting was used to further verify the Lmtk3 knockout effect. Finally, the cells were preserved at -80℃.

[0084] Table 2 Primer Sequences

[0085]

[0086] 4.2 Experimental Results

[0087] A mouse model was established by intraperitoneal injection of A2780CP-Luc-Puro cells. After approximately 12 days of normal feeding, the colonization of A2780CP-Luc-Puro cells in the peritoneal cavity was assessed using in vivo imaging. Subsequently, mice in both the L-glucose / PEG400 and C28 / PEG400 groups were treated daily by gavage. The results showed that the peritoneal seeding and growth of A2780CP cells in the C28 / PEG400 group were significantly lower than those in the L-glucose / PEG400 group. Importantly, post-mortem examination confirmed that metastatic nodules in the viscera, peritoneal wall, and omentum of mice in the C28 / PEG400 group were significantly reduced in size. Figure 5 B~D).

[0088] Depend on Figure 3 Experimental results on cell survival rates of A2780CP-Luc-puro cells treated with the combination of LMTK3 inhibitor C28 and cisplatin showed that the combination therapy significantly reduced the survival rate of A2780CP-Luc-puro cells and effectively killed cancer cells, demonstrating the effectiveness of LMTK3 inhibitor combined with cisplatin in the treatment of cisplatin-resistant ovarian cancer.

[0089] To further clarify the effect of LMTK3 on cisplatin-resistant ovarian cancer cells in vivo, scrambled cells and stably knocked-down LMTK3 A2780CP-Luc-Puro cells (shLMTK3-1 and shLMTK3-2) were transplanted into the peritoneum of mice. The results showed that the combination of LMTK3 knockdown and cisplatin significantly inhibited the seeding and growth of A2780CP cells in the peritoneum of mice; post-mortem examination also confirmed that metastatic nodules in the viscera, peritoneal wall, and omentum of mice in the LMTK3 knockdown group were significantly reduced in size. Figure 5 E~G).

[0090] Furthermore, Western blotting experiments demonstrated that ID8 Lmtk3 in this experimental case... KO Successfully built ( Figure 6 A), and at the same time, the results of the CCK8 experiment show that ID8 Lmtk3 KO Cells showed increased sensitivity to cisplatin (Figures B-C); in a mouse peritoneal xenograft model, Lmtk3... KO -PBS, Lmtk3 KO -Cis cells showed significantly lower peritoneal seeding and growth compared to the control group Lmtk3. WT -PBS Figure 6 D); importantly, the final autopsy confirmed that Lmtk3 KO -In the Cis treatment group, metastatic nodules in the viscera, peritoneal wall, and omentum of mice were significantly reduced ( Figure 6 E). By Figure 6 The FI results show that knocking out Lmtk3 has a positive therapeutic effect on mice and can further enhance the therapeutic effect of cisplatin.

[0091] Animal experiments have shown that the combination of small molecule inhibitor C28, LMTK3 knockdown or knockout, and cisplatin has a significant inhibitory effect on the growth and metastasis of cisplatin-resistant ovarian cancer cells.

[0092] In summary, the experimental results demonstrate that detecting LMTK3 expression levels in ovarian tissue can differentiate between ovarian cancer patients and healthy individuals. Furthermore, detecting LMTK3 expression levels in ovarian tissue can also differentiate between cisplatin-sensitive and cisplatin-resistant ovarian cancer patients. Simultaneously, LMTK3 knockdown significantly increased cell sensitivity to cisplatin, while LMTK3 expression promoted cell proliferation and metastasis, increasing cell tolerance to cisplatin, indicating that LMTK3 is a highly reliable therapeutic target for ovarian cancer resistance. The results also verified that both LMTK3 knockdown and treatment with the LMTK3 inhibitor C28 promoted pyroptosis in cisplatin-resistant ovarian cancer cells. In vivo experiments further showed that LMTK3 knockdown and treatment with the LMTK3 inhibitor C28 in combination with cisplatin significantly inhibited the growth and metastasis of cisplatin-resistant ovarian cancer cells in the peritoneal cavity of mice.

[0093] This invention demonstrates that LMTK3 can specifically differentiate between ovarian cancer patients and healthy individuals, as well as cisplatin-resistant and cisplatin-sensitive ovarian cancer patients. Inhibiting LMTM3 expression can increase pyroptosis and enhance the sensitivity of ovarian cancer cells to cisplatin. This invention proves the effectiveness of LMTK3 functional blockade or the combination of LMTK3 inhibitors and cisplatin in the treatment of cisplatin-resistant ovarian cancer, providing a theoretical basis for research on ovarian cancer drug resistance.

Claims

1. The use of reagents for detecting LMTK3 protein in the preparation of cisplatin-resistant ovarian cancer screening kits.

2. The use according to claim 1, characterized in that: The cisplatin-resistant ovarian cancer screening kit is a kit for screening patients with cisplatin-resistant ovarian cancer and patients with cisplatin-sensitive ovarian cancer.

3. The use according to claim 1, characterized in that: The reagents used to detect LMTK3 protein are enzyme-linked immunosorbent assay (ELISA) reagents, Western blot reagents, or IHC reagents.

4. The use according to claim 1, characterized in that: The reagent used to detect LMTK3 protein is an enzyme-linked immunosorbent assay (ELISA) reagent.

5. The use according to any one of claims 1 to 4, characterized in that: The reagent for detecting LMTK3 protein is a reagent for detecting LMTK3 protein in ovarian tissue.

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