Use of sunitinib malate in the preparation of targeted therapy drugs for gastric signet ring cell carcinoma

Abstract

The application discloses application of sunitinib malate in preparation of a targeted treatment drug for gastric signet ring cell carcinoma. Sunitinib malate has obvious inhibitory effect on growth of organoids from five patients with gastric signet ring cell carcinoma, and the inhibitory effect is more obvious with the increase of the concentration, and presents a concentration-dose dependent relationship, and a half maximal inhibitory concentration (IC50) is obviously smaller than that of a positive control drug, apatinib. The application can play a targeted treatment role on gastric signet ring cell carcinoma, has small toxic and side effects, is economical and practical, and is low in cost.

Description

Technical Field

[0001] This application belongs to the biomedical field, specifically relating to the application of sunitinib malate in the preparation of a targeted therapy drug for gastric signet ring cell carcinoma. Background Technology

[0002] In recent years, although the incidence and mortality rates of gastric cancer have decreased, the incidence of gastric signet ring cell carcinoma has continued to rise, accounting for 8% to 30% of gastric cancer subtypes. The incidence of gastric signet ring cell carcinoma increased nearly tenfold between 1970 and 2000. Compared to non-signet ring cell carcinoma, gastric signet ring cell carcinoma has an earlier onset, primarily affecting women aged 55-61, about 7 years earlier than non-signet ring cell carcinoma. Currently, the treatment of gastric signet ring cell carcinoma still faces severe challenges. A lack of in-depth understanding of its pathogenesis and widespread chemotherapy resistance are the main factors limiting treatment effectiveness. Therefore, exploring cost-effective and patient-compliant targeted therapies is an urgent priority in the clinical treatment of gastric signet ring cell carcinoma.

[0003] For a long time, targeted therapy options for gastric signet ring cell carcinoma have been limited. Apart from apatinib, no other targeted drugs with a clear indication for gastric signet ring cell carcinoma have been included in first-line treatment regimens. Since 2010, personalized medication guidance based on gene mutation detection has been widely used in cancer treatment, but the analysis results for patients with gastric signet ring cell carcinoma are often unsatisfactory. In recent years, due to the continuous development of omics and organoid technologies, it has become possible to screen drug-disease correlations based on gene expression data and artificial intelligence algorithms, combined with rapid drug screening using organoid technology. Targeted therapies screened using this methodology can adjust for global gene expression abnormalities in the development and progression of gastric signet ring cell carcinoma. Simultaneously, preliminary validation using patient-derived organoids in vitro can clarify their therapeutic effects in advance, significantly reducing the risk of drug resistance or initial ineffectiveness during clinical use. This saves valuable treatment time for advanced-stage patients while improving the efficacy of targeted therapy.

[0004] Therefore, this application utilizes patient omics data from gastric signet ring cell carcinoma patients and, based on a previously developed large-scale drug repositioning model, predicts sunitinib malate as a potential targeted therapy. Sunitinib malate is a multi-target receptor tyrosine kinase inhibitor, primarily used clinically to treat adult patients with gastrointestinal stromal tumors, renal cell carcinoma, and pancreatic neuroendocrine tumors. This application subsequently found in drug sensitivity studies on gastric signet ring cell carcinoma organoids that, compared to traditional treatments for gastric signet ring cell carcinoma such as apatinib and tegafur, sunitinib malate exhibits a significant killing effect on gastric signet ring cell carcinoma. Summary of the Invention

[0005] The technical solution provided in this application is the application of sunitinib malate in the preparation of a targeted therapy drug for gastric signet ring cell carcinoma.

[0006] In the above applications, the drug may be an oral preparation or an injection, with a drug concentration of 0.1-100 μmol / L or 50-500 mg per unit.

[0007] In summary, this application has the following beneficial effects: This application provides a new targeted therapy drug for gastric signet ring cell carcinoma, and compared with traditional gastric signet ring cell carcinoma treatment drugs apatinib and tegafur, it has significant efficacy and can rapidly kill gastric signet ring cell carcinoma organoids in organoid experiments. Attached Figure Description

[0008] The present application will be further described below with reference to the accompanying drawings and embodiments.

[0009] Figure 1 Light micrographs of the postoperative pathology report of Patient 1's tissue, where A is an image of the paraffin-embedded block after surgery, and B is an image of the histopathological section stained with hematoxylin and eosin (HE).

[0010] Figure 2 Light micrograph of the postoperative pathology report of the tissue from Patient 2.

[0011] Figure 3 Light micrographs of the postoperative pathology report of patient 3, where A is the postoperative tissue image and B is the HE staining image of the histopathological section.

[0012] Figure 4 Light micrograph of the postoperative pathology report of the tissue from Patient 4.

[0013] Figure 5 Light micrographs of the postoperative pathological reports of patient 5, where A is the HE staining image of the histopathological section, B is the HE staining image of the histopathological sections of lymph node metastases in groups 5, 6, 7, 8, 9, 10, and 11, and C is the HE staining image of the histopathological sections of lymph node metastases in groups 1, 3, and 11.

[0014] Figure 6 Microscopic image of a primary cultured gastric signet ring cell carcinoma organoid from Patient 1 on day 10.

[0015] Figure 7 Microscopic image of a primary cultured gastric signet ring cell carcinoma organoid from Patient 2 on day 10.

[0016] Figure 8 Microscopic image of a primary cultured gastric signet ring cell carcinoma organoid from Patient 3 on day 10.

[0017] Figure 9 Optical microscope image of primary cultured gastric signet ring cell carcinoma organoids of patient 4 on day 10.

[0018] Figure 10Microscopic image of a primary cultured gastric signet ring cell carcinoma organoid from Patient 5 on day 10.

[0019] Figure 11 Comparison of hematoxylin-eosin staining of gastric signet ring cell carcinoma-derived tissue and organoid pathology from Patient 1.

[0020] Figure 12 Comparison of hematoxylin-eosin staining of gastric signet ring cell carcinoma-derived tissue and organoid pathology from Patient 2.

[0021] Figure 13 Comparison of hematoxylin-eosin staining of gastric signet ring cell carcinoma tissue and organoid pathology from Patient 3.

[0022] Figure 14 Comparison of hematoxylin-eosin staining of gastric signet ring cell carcinoma tissue and organoid pathology from Patient 4.

[0023] Figure 15 Comparison of hematoxylin-eosin staining of gastric signet ring cell carcinoma tissue and organoid pathology from Patient 5.

[0024] Figure 16 Comparison of Ki67 immunohistochemistry between tissues derived from gastric signet ring cell carcinoma of Patient 1 and organoid pathology.

[0025] Figure 17 Comparison of Ki67 immunohistochemistry between tissues derived from gastric signet ring cell carcinoma of patient 2 and organoid pathology.

[0026] Figure 18 Comparison of Ki67 immunohistochemistry between tissues derived from gastric signet ring cell carcinoma of patient 3 and organoid pathology.

[0027] Figure 19 Comparison of Ki67 immunohistochemistry between tissues derived from gastric signet ring cell carcinoma of patient 4 and organoid pathology.

[0028] Figure 20 Comparison of Ki67 immunohistochemistry between tissues derived from gastric signet ring cell carcinoma of patient 5 and organoid pathology.

[0029] Figure 21 Comparison of CK7 immunohistochemical staining between tissues and organoids derived from gastric signet ring cell carcinoma in Patient 1.

[0030] Figure 22 Comparison of CK7 immunohistochemistry between tissues and organoids derived from gastric signet ring cell carcinoma in Patient 2.

[0031] Figure 23 Comparison of CK7 immunohistochemistry between tissues and organoids derived from gastric signet ring cell carcinoma in patient 3.

[0032] Figure 24Comparison of CK7 immunohistochemistry between tissues and organoids derived from gastric signet ring cell carcinoma in patient 4.

[0033] Figure 25 Comparison of CK7 immunohistochemical staining between tissues and organoids derived from gastric signet ring cell carcinoma in patient 5.

[0034] Figure 26 Immunohistochemical comparison of patient 1's gastric signet ring cell carcinoma-derived tissue and organoid pathology MUC5AC.

[0035] Figure 27 Immunohistochemical comparison of patient 2's gastric signet ring cell carcinoma-derived tissue and organoid pathology MUC5AC.

[0036] Figure 28 Immunohistochemical comparison of patient 3's gastric signet ring cell carcinoma-derived tissue and organoid pathology MUC5AC.

[0037] Figure 29 Immunohistochemical comparison of patient 4's gastric signet ring cell carcinoma-derived tissue and organoid pathology MUC5AC.

[0038] Figure 30 Immunohistochemical comparison of patient 5's gastric signet ring cell carcinoma-derived tissue and organoid pathology MUC5AC.

[0039] Figure 31 Image showing the results of drug sensitivity cell viability assay (CellTiter-Glo, CTG) for gastric signet ring cell carcinoma organoids from Patient 1.

[0040] Figure 32 Image showing the results of drug sensitivity cell viability assay (CellTiter-Glo, CTG) for gastric signet ring cell carcinoma organoids from Patient 2.

[0041] Figure 33 Image showing the results of drug sensitivity cell viability assay (CellTiter-Glo, CTG) for gastric signet ring cell carcinoma organoids from Patient 3.

[0042] Figure 34 Image showing the results of drug sensitivity cell viability assay (CellTiter-Glo, CTG) for gastric signet ring cell carcinoma organoids from Patient 4.

[0043] Figure 35 Image showing the results of drug sensitivity cell viability assay (CellTiter-Glo, CTG) for gastric signet ring cell carcinoma organoids from patient 5. Detailed Implementation

[0044] To better understand this application, the following description, in conjunction with embodiments and accompanying drawings, further illustrates the application. It should be noted that the specific embodiments described herein are merely for illustrative and explanatory purposes and are not intended to limit the scope of this application. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments used without a specified manufacturer are all commercially available conventional products.

[0045] Example 1: Sensitivity test of sunitinib malate based on organoids derived from tumor tissue of 5 patients with gastric signet ring cell carcinoma.

[0046] Experimental protocol: Sunitinib malate treatment group, apatinib treatment group, tegafur treatment group, dimethyl sulfoxide (DMSO) control group, and blank control group were set up. Nine concentration gradients were set up for each group, diluted 1.5 times, with three replicates for each concentration. After 72 hours, organoid viability was measured using CellTiter-Glo (CTG) assay. Lower viability indicated stronger inhibitory effect of the drug on gastric cancer organoids, and vice versa.

[0047] The compound to be tested.

[0048]

[0049] Primary establishment of human gastric cancer organoids.

[0050] Surgery or biopsy samples of human gastric signet ring cell carcinoma tissue should be immediately placed in sterile centrifuge tubes filled with tissue preservation solution, wrapped with foam, and kept away from direct contact with ice packs. Transport the tissue back to the laboratory within 24 hours at 0–4°C (if immediate transport is not possible, preserve at 4°C for no more than 48 hours). At the time of sampling, collect information on the source tissue sample and detailed pathological results from the hospital's pathology department. The light micrograph of the source tissue from the hospital's pathology report is attached. Figure 1 -Appendix Figure 5 As shown, attached Figure 1 -Appendix Figure 5 Patients 1-5 of this application were diagnosed with gastric signet ring cell carcinoma or with signet ring cell carcinoma components in the tumor tissue. The detailed pathological diagnoses are as follows.

[0051]

[0052] After the samples arrived at the laboratory, the tissue samples were poured into new sterile 6 cm cell culture dishes. Two pieces of tumor tissue, about the size of soybeans, were cut using scissors and forceps that had been rinsed with epithelial organoid basal culture medium. White connective tissue was removed using sterile scissors, leaving the pinkish and eroded tumor tissue. The tissue was transferred to new sterile 6 cm cell culture dishes and washed with 3-4 mL of Dulbecco-phosphate buffered solution (D-PBS) containing 2%-5% triple antibodies, rinsing 10 times from top to bottom and left to right. The tissue was then transferred to new sterile 6 cm cell culture dishes and washed again. This step was repeated twice more.

[0053] Transfer the cleaned tissue to a rinsed 1.5 mL centrifuge tube. Use scissors to directly cut the tissue into small particles smaller than 2 mm in the tube. Prepare 5 mL of preheated 37°C tumor tissue digestion solution (using a 15 mL centrifuge tube). Cut off the tip of the 1250 μL pipette tip with new scissors. Resuspend the cut tissue in 1 mL of digestion solution, transfer it to 5 mL of digestion solution for resuspending, and wash the scissors in the digestion solution as well.

[0054] Digest at 37℃ and 130 rpm on a shaker for 10 minutes. Remove the cells and observe them under a microscope. Cell clumps of various shapes will appear. Continue digesting for another 5 minutes and observe under a microscope until you see small cell clumps of about 30 μm around large tissue masses. Digestion can be stopped at this point. The entire digestion process takes about 25-30 minutes. (Note: Rinse the 100 μm cell filter before digestion. The rinsing method is to place the filter in a 6 cm cell culture dish and use 1 mL of rinsing solution to rinse the filter evenly with a 1250 μL pipette tip, avoiding the formation of air bubbles.)

[0055] To terminate digestion, add 100 μL of FBS to 5 mL of tumor tissue digestion solution and mix by inverting the container 3-5 times. Pour the mixture directly into a 100 μm cell filter and gently shake the filter until all the liquid flows into the cell culture dish. Rinse the centrifuge tube once with 1 mL of epithelial organoid basal culture medium and then filter it through the cell filter. Rinse the filter again with 1 mL of epithelial organoid basal culture medium. Transfer the liquid from the cell culture dish to a new, rinsed 15 mL centrifuge tube (add the liquid gently along the tube wall to avoid disturbing the cell clusters). Centrifuge at 300-350 g for 3 min at 4°C.

[0056] Use a Bass pipette to remove the supernatant, being careful not to aspirate the precipitate; retain 50-100 μL of liquid. Add 1 mL of erythrocyte lysis buffer, gently disperse and mix the precipitate, transfer to a rinsed 1.5 mL centrifuge tube, and immediately centrifuge at 4°C, 300 g, for 3 min.

[0057] Discard the supernatant, gently resuspend the cells in 1 mL of epithelial organoid basal culture medium, and aspirate 30 μL to observe the density under a microscope. After determining the seeding density, according to the number of wells in the seed plate, aspirate an appropriate amount of suspension from the middle or lower section (at this point, the cell clumps will settle to the bottom of the centrifuge tube), and centrifuge at 300 g for 3 min at 4°C.

[0058] Discard the supernatant, add an appropriate amount of matrix gel (the gel content should not be less than 70%), gently blow and stir to mix evenly, avoiding the formation of air bubbles, and put it into an ice box.

[0059] Dispense 30 μL of gel per well (for experimental 48-well plates) into the center of the bottom of each well of a preheated cell culture plate, avoiding contact with the sidewalls. Invert the plate and incubate at 37°C in a 5% CO2 incubator for 15-20 minutes until the cell mixture solidifies. Slowly add complete human gastric cancer culture medium along the well walls. Supplement the gaps between wells with phosphate-buffered saline (PBS). Incubate the plate for primary culture of human gastric cancer organoids. Closely monitor organoid growth. Ideally, human gastric signet ring cell carcinoma organoids should form in approximately 3-5 days. Once formed, the gastric signet ring cell carcinoma organoids can be observed under a microscope; their bright-field morphology appears vacuolated. Figure 6 -Appendix Figure 10 As shown.

[0060] For successfully constructed gastric signet ring cell carcinoma organoids, hematoxylin-eosin staining and immunohistochemical staining for characteristic markers (Ki67, CK7, MUC5AC) are required to confirm their consistency with the source tissue and determine whether the construction was successful. The hematoxylin-eosin staining results of 6 organoids and their source tissues are shown in the attached table. Figure 11 -Appendix Figure 15 The gastric signet ring cell carcinoma organoids exhibited a vacuolated appearance, with nuclei showing atypia and the nuclei being pushed to one side of the cell membrane by intracellular mucus, forming a typical signet ring morphology, highly consistent with the pathological characteristics of the source tissue. Immunohistochemical staining results of 6 organoids and source tissues showed that Ki67 (with appended...) was present in both the gastric signet ring cell carcinoma organoids and the source gastric cancer tissue. Figure 16 -Appendix Figure 20 ), CK7 (with) Figure 21 -Appendix Figure 25 ), MUC5AC (with) Figure 26 -Appendix Figure 30 The expression of the proteins was strongly positive. The morphological and immunohistochemical staining results of the above gastric signet ring cell carcinoma organoids and primary gastric cancer tissues indicate that the gastric signet ring cell carcinoma organoids were successfully constructed.

[0061] Drug sensitivity testing of gastric signet ring cell carcinoma organoids.

[0062] Plate culture of human gastric signet ring cell carcinoma organoids: Discard the old culture medium and add an appropriate amount of ice-cold (4°C) epithelial organoid basal medium (catalog number: ZKBL-S004) to each well. Use a rinsed pipette tip to repeatedly pipette and scrape off the matrix gel and organoids, transferring the mixture to a pre-rinsed 5 mL centrifuge tube. Resuspend the suspension with a rinsed pipette tip to separate the organoids from the matrix gel. Centrifuge at 300 g for 3 minutes at 4°C, discarding the supernatant. Add an appropriate volume of organoid digestion solution (catalog number: ZKBL-D001), resuspend, mix well, and incubate at 37°C. During digestion, pipette every 2 minutes to aid organoid dissociation. Closely monitor the digestion process to minimize the incubation time of the organoids in the digestion solution. When the organoids disperse into cell clusters containing 10-50 cells, add 1-2 ml of epithelial organoid basal medium (containing 2% FBS) to terminate the digestion. The cells were filtered through a 70 μm filter, and an appropriate amount of epithelial organoid basal culture medium was added to wash the filter. The collected solution was centrifuged at 300 g for 3 minutes at 4°C, and the supernatant was discarded. The washed organoid pellet was resuspended in a small amount of basal culture medium containing antibiotics. A small amount of the suspension was taken for viable cell detection and counting. Extracellular matrix (>70%) was added at a density of 100,000~500,000 cells / ml and mixed on ice (note that the mixing action should be gentle to avoid generating a large number of bubbles; if mixing at room temperature, it should be controlled within 15 seconds). After mixing, it was placed on ice. Seeds were rapidly applied to the center of the bottom of each well in a preheated 96-well cell culture plate, avoiding contact with the sidewalls. The plate was incubated at 37°C in a 5% CO2 incubator for approximately 15 minutes until the extracellular matrix solidified. Then, 100 μL of complete human gastric cancer organoid culture medium (catalog number: ZKBL-S003) was slowly added along the well wall. The organoid status was recorded on day 0. The cell culture plate was then placed in an incubator for 2–3 days. Drug sensitivity testing was performed once the organoids were in good condition.

[0063] Preparation of 10x Compound Working Solution and Compound Treatment of Organoids: Preparation of 10x Compound Working Solution: Add 158 μL of complete culture medium to a V-bottom 96-well plate. Add 2 μL of the compound from an 800x compound storage plate to the 96-well plate. Add 2 μL of DMSO to the solvent control and blank control. Mix thoroughly by pipetting after adding the compound or DMSO. Add the compound: Discard the old culture medium from the cell culture plate and add 90 μL of fresh organoid complete culture medium. Add 10 μL of the 10x compound working solution to the cell culture plate. Add 10 μL of the DMSO-complete culture medium mixture to the solvent control and blank control. The final DMSO concentration is 0.125%. Incubate the 96-well cell culture plate in an incubator for 72 hours.

[0064] Organoid cell viability assay after drug administration: Observe and photograph the cells using an inverted microscope, recording the state of each organoid in each well. Add 50 μL of cell viability assay reagent to each well, gently shake to mix, and incubate in the dark for 10 min. Turn on the chemiluminescent microplate reader and place the culture plate on the sample slot for detection. Calculate the inhibition rate (IR) of the detected compound using the following formula: IR(%) = (1 - (compound group detection value - blank control group detection value) / (solvent control group detection value - blank control group detection value)) * 100%. Calculate the inhibition rate of different concentrations of the compound in a spreadsheet, then use GraphPad Prism software to plot the inhibition curve and calculate the half-maximal inhibitory concentration (IC50). The results showed that the half-maximal inhibitory concentrations (IC50) of organoids from patients 1 to 5 in response to the positive control drug apatinib were 13.58 μM, 10.21 μM, 22.10 μM, 37.23 μM, and 13.11 μM, respectively. Patient 4 did not show an IC50. Sunitinib malate showed good efficacy to organoids from patients 1 to 5, with IC50 values ​​of 7.14 μM, 26.94 μM, 13.11 μM, and 35.26 μM, respectively. Patient 5's organoids were almost completely killed within the dosing range, so no IC50 value was found. All these values ​​were significantly lower than the IC50 of the positive control drug apatinib. Detailed results are attached. Figure 31 -Appendix Figure 35 .

Claims

1. Application of sunitinib malate in the preparation of targeted therapy drugs for gastric signet ring cell carcinoma.

2. The application of sunitinib malate according to claim 1 in the preparation of a targeted therapy for gastric signet ring cell carcinoma, characterized in that, The drug is an oral preparation or an injection.

3. The application of sunitinib malate according to claim 1 in the preparation of a targeted therapy for gastric signet ring cell carcinoma, characterized in that, The concentration of sunitinib malate used is 0.1-100 μmol / L.

4. The application of sunitinib malate according to claim 1 in the preparation of a targeted therapy for gastric signet ring cell carcinoma, characterized in that, The dosage of sunitinib malate is 50-500 mg per unit.

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