CN117100747A - Use of FOXO protein inhibitors in the preparation of tumor drugs for the treatment of gastric adenocarcinoma - Google Patents

Abstract

The application belongs to the technical fields of molecular biology and biological medicine, and particularly relates to application of a FOXO protein inhibitor in preparation of a medicine for treating gastric adenocarcinoma tumor. The application discovers CCKBR (Cholecystokinin B Receptor) positive stomach Dou Gan cell-like tumor cells in gastric adenocarcinoma patients for the first time, specifically exists FOXO (Forkheadbox O) family (FOXO 1, FOXO3 and FOXO 4) high expression in CCKBR+ gastric adenocarcinoma tumor patients, and the high expression of the FOXO is closely related to clinical prognosis. The application provides a novel treatment target of gastric adenocarcinoma of CCKBR+, and the FOXO protein inhibitor AS1842856 can be effectively used for the selection and prognosis evaluation of a treatment scheme of gastric adenocarcinoma, can inhibit the growth of CCKBR positive poorly differentiated gastric Dou Gan cell-like tumor cells and provides a novel possibility for the subsequent targeted treatment of gastric adenocarcinoma.

Description

Use of FOXO protein inhibitors in the preparation of tumor drugs for the treatment of gastric adenocarcinoma

Technical field

The invention belongs to the technical fields of molecular biology and biomedicine, and specifically relates to the use of FOXO protein inhibitors in preparing drugs for treating gastric adenocarcinoma tumors.

Background technique

Gastric adenocarcinoma is a type of gastric cancer, accounting for 95% of all gastric cancers. It is caused by the malignant transformation of gastric gland cells, so it is called gastric adenocarcinoma. Gastric adenocarcinoma still has problems of refractory treatment and easy recurrence. The main reason is that there is no effective therapeutic target for targeted killing. However, due to the extremely heterogeneous clinical gastric adenocarcinoma, it is impossible to choose a broad-spectrum and effective treatment. Therapeutic targets, therefore, it is crucial to select effective therapeutic targets for gastric adenocarcinoma subgroups, especially poorly differentiated gastric adenocarcinomas with higher malignancy.

Early studies have confirmed that stem cells can undergo malignant transformation to form stem cell-type gastric adenocarcinomas. For example, LGR5- and AQP5-positive stem cell-like gastric adenocarcinomas have been clinically confirmed and have different tumor cell characteristics (NatCell Biol. 2021Dec; 23(12) :1299-1313., J Exp Clin Cancer Res. 2022Nov 14; 41(1):322., Cell Physiol Biochem. 2015; 36(6):2447-55.). CCKBR (Cholecystokinin BReceptor)-positive gastric antrum cells belong to +4 stem cells and are considered to be tumor-initiating cells in mice (Gut. 2015Apr; 64(4):544-53., Cell Stem Cell. 2020May7; 26(5) ):739-754.e8.), however it has not been reported in clinical patients. Our analysis of the expression of CCKBR in clinical gastric adenocarcinoma shows that CCKBR+ tumors in gastric adenocarcinoma are tumors formed by malignant transformation of stem cells and have extremely poor survival rates, suggesting that CCKBR+ patients are patients with a higher degree of malignancy. Such tumors are often It is characterized by poor differentiation and refractory treatment.

The FOXO family is a type of transcription factor encoded by the FOXO gene. It mainly includes three protein family members: FOXO1, FOXO3 and FOXO4. They have highly conserved DNA-binding domains and therefore have similar functions. The activity of FOXO proteins plays an important role in cell homeostasis. It is strictly regulated and plays a key role in the human body. FOXO proteins are often considered lifespan genes (especially FOXO3), which are mainly related to aging, autophagy and stem cell maintenance.

At present, conventional treatment methods for gastric adenocarcinoma are still used when treating CCKBR+ gastric adenocarcinoma tumors, which lack specificity. This makes the treatment of CCKBR+ gastric adenocarcinoma tumors difficult to treat and prone to recurrence. Therefore, the treatment of CCKBR+ tumors is an urgent problem that needs to be solved in this field. It is of great clinical significance and practical value to study an effective therapeutic target for CCKBR+ gastric adenocarcinoma tumors and perform targeted killing. Currently, there are no products that regulate CCKBR+ gastric adenocarcinoma tumors through the regulation of FOXO proteins.

Contents of the invention

In order to solve the problem in the prior art that there is no product for treating CCKBR+ gastric adenocarcinoma tumors through the regulation of FOXO protein, the present invention found that AS1842856 can significantly inhibit the growth of CCKBR+ gastric adenocarcinoma tumor cells by inhibiting FOXO protein, providing a Use of FOXO protein inhibitors in the preparation of tumor drugs for the treatment of gastric adenocarcinoma.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The invention provides the use of a FOXO protein inhibitor in preparing a drug for treating gastric adenocarcinoma tumors.

Preferably, the gastric adenocarcinoma tumor is a CCKBR+ gastric adenocarcinoma tumor.

Preferably, the FOXO protein inhibitor is AS1842856, which targets the function of inhibiting the FOXO protein; the structure of AS1842856 is as follows:

Preferably, the AS1842856 inhibits the growth of CCKBR+ gastric adenocarcinoma tumor cells, and the tumor cells are CCKBR+ gastric antrum stem cell-like tumor cells.

Preferably, the AS1842856 targets the FOXO protein and inhibits the transcriptional activity of the FOXO protein.

Preferably, the degree of infiltration of the CCKBR+ gastric adenocarcinoma tumor shows a clear positive correlation with the expression of FOXO, and the clinical prognosis of the CCKBR+ gastric adenocarcinoma tumor shows a clear positive correlation with the expression of FOXO.

Preferably, the expression of FOXO includes the expression of FOXO1, FOXO3 and FOXO4 in the FOXO family.

The present invention also provides a drug for preventing and/or treating CCKBR+ gastric adenocarcinoma tumors.

Preferably, the medicament includes AS1842856 according to claim 3.

Preferably, the drug is a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, isotopically labeled derivative, stereoisomer or prodrug of AS1842856.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides the use of FOXO protein inhibitors in preparing drugs for treating gastric adenocarcinoma tumors. The present invention obtained for the first time a single-cell transcriptome library of CCKBR+ gastric adenocarcinoma tumor patients, and found that such tumors showed poor differentiation and refractory properties, and had no effective therapeutic targets. In order to identify genes related to the pathogenesis of CCKBR+ gastric adenocarcinoma tumors and discover some new therapeutic targets, the present invention screened out through single-cell sequencing technology and in vitro and in vivo experiments that FOXO is a poorly differentiated tumor formed by the malignant transformation of CCKBR+ gastric antrum stem cells. potential therapeutic targets. The present invention provides a therapeutic target for CCKBR+ gastric adenocarcinoma tumors. At the same time, it is found that the FOXO protein inhibitor AS1842856 is a potential therapeutic target for gastric adenocarcinoma transformed by CCKBR+ gastric antrum stem cells. AS1842856 can target the FOXO protein. Inhibiting its transcriptional activity and inhibiting CCKBR+ gastric adenocarcinoma tumors can provide a basis for subsequent targeted therapy of gastric adenocarcinoma.

Description of drawings

Figure 1 shows the establishment and identification of a stem cell-transformed CCKBR+ gastric adenocarcinoma tumor single cell transcriptome library in Example 1 of the present invention. A is a marker used to identify CCKBR as an epithelial cell Epi8 subpopulation using single-cell sequencing; B is a marker used to identify CCKBR+. Gastric adenocarcinoma tumors widely express gastric antrum mucus gland cell markers, proving that such tumor cells are formed by malignant transformation of gastric antrum basal cells; C is to identify such cells expressing gastric antrum basal stem cell markers LGR5, AQP5, LRIG1 and A4GNT; D is the identification of cellular molecular characteristics of CCKBR+ gastric adenocarcinoma tumor cells; E is the enrichment of transcription factors related to this type of cells, including FOXO3 and FOXO4; F is the pseudo-chronological identification of CCKBR+ gastric adenocarcinoma tumors as tumors originating from the base of the gastric antrum ; G shows that the FOXO signaling pathway is significantly upregulated in CCKBR+ gastric adenocarcinoma tumors; H shows that the exogenous single cell library GSE183904 was used to also identify CCKBR+ tumor phenotypes with higher FOXO activation levels.

Figure 2 shows that in Example 1 of the present invention, it was found that CCKBR+ gastric adenocarcinoma tumors have poor prognosis and survival. A is the expression and occupancy of CCKBR in tumors detected by immunohistochemistry; B is the statistics of CCKBR+ tumors. Clinical tumor proportion, tumor location and differentiation; C is immunofluorescence results detecting the co-expression of CCKBR, CD133 and CD44 in gastric adenocarcinoma; D is CCKBR+ gastric adenocarcinoma tumors have a poor prognosis in gastric adenocarcinoma; E is CCKBR+ stem-like tumor cells exhibit poor prognostic characteristics;

Figure 3 shows that CCKBR+ gastric adenocarcinoma tumors in Example 1 of the present invention express very high levels of FOXO1, FOXO3 and FOXO4, where A is RT-qPCR (Quantitative Real-time PCR) detection of FOXO family expression levels in CCKBR-positive gastric adenocarcinoma tumors. ; B is the change in the expression level of FOXO in CCKBR-positive and negative tumors; C is the change in the expression level of FOXO in CCKBR-positive tumors relative to adjacent tissues; D is the correlation analysis of the expression of CCKBR and FOXO in tumors;

Figure 4 shows that tumors with high CCKBR expression in the TCGA database in Example 1 of the present invention express very high FOXO1, FOXO3 and FOXO4, and FOXOs are related to worse prognosis of gastric adenocarcinoma patients. A is the detection of high CCKBR expression in the TCGA database. The change in the expression level of FOXOs relative to patients with low expression; B is the prognostic relationship of FOXOs in patients with gastric adenocarcinoma;

Figure 5 shows that the FOXO protein inhibitor AS1842856 in Example 1 of the present invention can inhibit the stemness of cancer stem cells and inhibit the formation of tumor spheres. Among them, A shows that when gastric adenocarcinoma cell lines are cultured in tumor spheres to enrich stem cells, RT-qPCR is used to detect the increased expression of CCKBR, FOXO1, FOXO3 and FOXO4; B shows that the increased expression of CCKBR and FOXO is correlated; C shows that FOXO is used When the protein inhibitor AS1842856 treats tumor cell spheroids, different concentrations of AS1842856 inhibit the growth of tumor spheroids. Bright field pictures; D is a statistical chart showing the changes in tumor spheroids diameter when AS1842856 treats tumor spheroids; E is immunofluorescence showing when AS1842856 treats tumor spheroids. Changes in the expression levels of CCKBR and CD133 in tumor spheroids; F shows immunofluorescence changes in the expression levels of CCKBR and KI67 in tumor spheroids when AS1842856 is treated with tumor spheroids.

Figure 6 shows the selective killing effect of FOXO protein inhibitor AS1842856 on stem cell-like CCKBR+ gastric adenocarcinoma tumors in Example 1 of the present invention, where A is the HE morphological staining of gastric adenocarcinoma organoids and paired tumor tissues; B is the organoids Immunohistochemistry staining of paired tumor tissues; C is a bright field picture after organoids were treated with AS1842856; D is the change in cell viability after organoids were treated with AS1842856; E is severe immune load in MKN45 cell line (CCKBR positive) treated with AS1842856 Photos of mouse tumors after 17 days of deficient mice; F is the statistical results of mouse tumors 17 days after AS1842856 was used to treat MKN45-loaded severe immunodeficient mice; G is the statistical tumor growth curve of AS1842856 used to treat MKN45-loaded severe immunodeficient mice.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but should not be understood as limitations of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources, unless otherwise specified.

Example 1

1. Establish and characterize stem cell-transformed CCKBR+ gastric adenocarcinoma single-cell transcriptome libraries

Five fresh surgically resected gastric adenocarcinoma tumor samples were placed in sterile ribonuclease (RNase)-free petri dishes containing calcium- and magnesium-free 1×PBS (NaCl8 per liter of distilled water). .0g; KCl 0.2g; Na ₂ HPO4 1.44g; KH ₂ PO ₄ 0.24g). Transfer the tumor tissue to a culture dish, use ophthalmic scissors and a scalpel to cut the tumor tissue into 0.5mm-sized tissue pieces, wash with 1×PBS, and remove excess tissue such as blood clots, connective tissue, and fat layer. Then use tissue separation solution (add 0.35g collagenase IV, 200mg papain, 12000 units DNase I per 100mL of PBS) to incubate the tumor tissue, shake it at 100rpm/min for 20 minutes in a water bath at 37°C, and obtain a single cell suspension. Digestion of the single cell suspension was stopped with 1×PBS and filtered through a 70-pore size strainer. Resuspend the filtered suspension in 100 μl of Dead Cell Removal Microparticles (MAC 130-090-101) and use Dead Cell Removal Kit (MAC 130-090-101) removes dead cells. 10X Genomics Chromium Single-Cell 3'kit (V3) was used to capture 13,000 cells for each sample and perform cDNA synthesis and single-cell library construction. The steps for cDNA synthesis and single-cell library construction are as follows:

cDNA synthesis:

Prepare Master Mix on ice. The composition of Master Mix is shown in Table 1.

Table 1 Composition of Master Mix

Components 1x(μl) 8x(μl) RT Reagent B 18.8 165.0 Template Switch Oligo 2.4 20.8 Reducing Agent B 2.0 17.3 RT Enzyme C 8.7 76.8 Total 31.8 279.8

Dispense 31.8μl Master Mix into eight rows for later use (keep on ice). Mix the Master Mix and the cells obtained by the above dissociation into 70 μl and add it to the sample well. Shake the Gel Beads (take out at -80°C and leave at room temperature for 30 minutes), attach a 10× Gasket seal, and run the 10× machine.

Pipette 100 μl of water-in-oil into a new pre-cooled eight-row tube for PCR reaction. The reaction procedure is shown in Table 2:

Table 2 Single-cell transcriptome PCR reaction procedures

Lid Temperature Reaction Volume Run Time 53℃ 125μl 55min Step Temperature Time 1 53℃ 45min 2 85℃ 5min 3 4℃ Hold

Add 125 μl of Recovery Agent to the reaction solution (do not pipet to mix), let stand at room temperature for 2 minutes, and discard 125 μl of pink Recovery Agent/Partitioning Oil.

Prepare Dynabeads Cleanup Mix according to Table 3; prepare Elution BufferI according to Table 4; add 200 μl Dynabeads Cleanup Mix to the reaction solution obtained above, mix well, and let stand at room temperature for 10 minutes. Place it on a magnetic stand until it is clear. Discard the supernatant and add 300 μl to freshly prepare it. of 80% ethanol at room temperature for 30 s and discard the supernatant. Add 200 μl of freshly prepared 80% ethanol and leave at room temperature for 30 s. Discard the supernatant, centrifuge lightly, place a magnetic stand, remove the remaining alcohol, and air-dry for 1 min. Then add 35.5 μl Elution Buffer I, mix well, keep at room temperature for 1 min, place on a magnetic stand until clear, and draw 35 μl of supernatant into a new tube.

Table 3 Ingredients of Dynabeads Cleanup Mix

Dynabeads Cleanup Mix 1x(μl) 8x(μl) Clearup Buffer 182 1602 Dynabeads MyOne SLANE 8 70 Reducing Agent B 5 44 Nulear-free Water 5 44

Table 4 Composition of Elution Buffer I

Configure cDNA Amplification Reaction Mix for PCR reaction: Mix 50 μl AmpMix obtained in the previous step and 15 μl cDNA Primers to obtain cDNA Amplification Reaction Mix. Mix cDNAAmplification Reaction Mix and 35μl purified GEM RT, and perform PCR reaction. The reaction procedure is shown in Table 5:

Table 5 cDNA amplification PCR reaction procedure

Lid Temperature Reaction Volume Run Time 105℃ 100μl 45min Step Temperature Time 1 98℃ 3min 2 98℃ 15sec 3 63℃ 20sec 4 72℃ 1min 5 Go to step 2 for11 cycles / 6 42℃ 1min 7 4℃ Hold

Next, perform cDNA quality control and quantification: add 60 μl SPRIselect Reagent to the product after the reaction, mix well, let it stand at room temperature for 5 minutes, place it on a magnetic stand until it is clear, and discard the supernatant. Add 200 μl of freshly prepared 80% ethanol at room temperature for 30 s, discard the supernatant, repeat once, and dry the pellet. Add 40.5 μl Buffer EB, mix well, leave at room temperature for 1 min, place on a magnetic stand until clear, aspirate 40 μl of supernatant into a new tube, use Qubit quality inspection and Agilent 2100 quality inspection, and record the concentration.

Next, perform single cell library construction:

Prepare Fragmentation Mix (the entire process of preparing reagents and adding samples must be on ice): Add 5 μl Fragmentation Buffer and 10 μl Fragmentation Enzyme to each sample, and mix evenly.

For each sample, take 10 μl of the cDNA obtained above, add 25 μl of Buffer EB, 15 μl of Fragmentation Mix, and mix evenly. Next, the PCR reaction is performed. The reaction procedure is shown in Table 6:

Table 6 cDNA fragmentation PCR reaction program

Lid Temperature Reaction Volume Run Time 65℃ 50μl 35min Step Temperature Time Pre-cool block 4℃ Hold Fragmentation 32℃ 5min End Repair and A tailing 65℃ 30min Hold 4℃ Hold

Then add 30 μl SPRIselect Reagent, mix well, let stand at room temperature for 5 minutes, and place on a magnetic stand until clear. Take 75 μl of the supernatant in a new tube, add 10 μl of SPRIselect Reagent, mix well, let stand at room temperature for 5 minutes, place on a magnetic stand until clear, and remove the supernatant. Add 125 μl of 80% ethanol and leave at room temperature for 30 s. Discard the supernatant and repeat once to dry the sample. Add 50.5 μl Buffer EB and mix well, wait for 1 min at room temperature, wait until clear on a magnetic stand, and pipet 50 μl supernatant into a new tube.

Prepare Adapter Ligation Mix according to Table 7, mix 50 μl Adaptor Ligation Mix and 50 μl product obtained from the above reaction, and incubate at 20°C for 15 minutes.

Add 80 μl SPRIselect Reagent, mix well, let stand at room temperature for 5 minutes, place on a magnetic stand until clear, and discard the supernatant. Then add 200 μl of freshly prepared 80% ethanol and incubate at room temperature for 30 s. Discard the supernatant, repeat once, and air-dry. Add 30.5 μl Buffer EB, mix well, and wait at room temperature for 2 minutes. Place the magnetic stand until the solution is clear. Aspirate 30 μl of the supernatant into a new tube. Add 50 μl Amp Mix, 10 μl SIPrimer, 60 μl Sample Index PCR Mix, and 10 μl individual Chromium i7Sample Index to the tube. Mix thoroughly and then perform the PCR reaction. The reaction steps are shown in Table 8.

Table 7 Components of Adapter Ligation Mix

Adapter Ligation Mix 1x(μl) 8x(μl) Ligation Buffer 20 176 DNA Ligase 10 88 Adapter Oligos 20 176 Total 50 440

Table 8 Library construction PCR reaction steps

Lid Temperature Reaction Volume Run Time 105℃ 100μl 40min Step Temperature Time 1 98℃ 45sec 2 98℃ 20sec 3 54℃ 30sec 4 72℃ 20sec 5 Go to step 2 for 16 cycles 6 72℃ 1min 7 4℃ Hold

Add 60 μl of SPRIselect Reagent to the reaction product, mix thoroughly, let stand at room temperature for 5 minutes, and place on a magnetic stand until clear. Take 150 μl of supernatant in a new tube, add 20 μl of SPRIselect Reagent, mix thoroughly, let stand at room temperature for 5 minutes, place on a magnetic stand until the solution is clear, and then remove the supernatant. Add 200 μl of 80% ethanol and leave at room temperature for 30 s. Discard the supernatant and repeat once to fully dry the pellet. Add 35.5 μl Buffer EB, mix thoroughly, leave at room temperature for 2 minutes, place a magnetic stand until the solution is clear, and pipet 35 μl supernatant into a new tube. The constructed single cell library was subjected to high-throughput sequencing using Illumina Nova 6000PE150.

The above sequencing data underwent standardized procedures for data format conversion, reference genome tagging and quality control (Cell Ranger (vision 5.0.0), Seurat (version 3.1.1), DoubletFinder package (version 2.0.2)). All cells were divided into 12 cell subpopulations through dimensionality reduction clustering, and markers of each cell subpopulation were detected to find that the CCKBR+ gastric adenocarcinoma tumor cell population was epithelial cell subpopulation 8 (Epi8). Detection and analysis of gastric mucus neck cell and basal stem cell markers in Epi8 revealed that Epi8 originates from the base of the gastric antrum. The top 2000 characteristic protein-coding genes were selected for cell group ontology analysis, and a high geometric distribution test was used to calculate the p-value representing whether the GO (GeneOntology) functional set was significantly enriched in the list of differential protein-coding genes. Then, the P value was subjected to Benjamini & Hochberg multiple tests for Q value significance analysis.

Regarding TF (Transcription Factor) enrichment, we input multiple gene lists (https://metascape.org/) and determined gene list enrichment in the ontology TRRUST script. All genes in the genome were used as enrichment background. p-values <0.01, with a minimum count of 3 and an enrichment factor >1.5 (the enrichment factor is the ratio between observed counts and counts expected by chance) were collected and grouped into clusters based on the similarity of their members. The algorithm is the same as that used for pathway and process enrichment analysis. For GSE183904, in addition to sample collection and library construction, the same operations were performed for the rest of the analysis, and finally the activation status ranking of related transcription factors and signaling pathways was obtained. In both test results, it was found that FOXO showed abnormal activation.

The results are shown in Figure 1. The present invention established and identified a gastric adenocarcinoma single cell library formed by malignant transformation of CCKBR+ stem cells, and analyzed that FOXO is a key transcription factor and signaling pathway that maintains CCKBR+ gastric adenocarcinoma tumors.

2. Identification of CCKBR+ gastric adenocarcinoma tumors associated with poorly differentiated types and poor patient prognosis

We use immunohistochemical techniques to identify and analyze the expression of CCKBR in gastric adenocarcinoma. The specific studies are as follows:

The paraffin sections were immersed in xylene I for 20 min and xylene II for 20 min respectively. Then immerse in absolute ethanol I for 10 min, absolute ethanol II for 10 min, 95% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then rinse twice with double-distilled water, 2 min each time. Then put the paraffin tissue slices into the repair box, and then add an appropriate amount of sodium citrate buffer (Zhongshan Jinqiao, ZLI-9064). The liquid level should be immersed in the tissue. Put the repair box into the microwave oven, microwave on high fire for 8 minutes, and stop for 2 minutes. , and then high heat for 2 minutes. Do not let the tissue dry out during this process. Take the repair box out of the microwave oven and let it cool down naturally. When the repair solution reaches room temperature, take out the slide and rinse it with PBS 3 times for 3 minutes each time. Add the prepared hydrogen peroxide (dilute the hydrogen peroxide with double distilled water to a concentration of 3%) dropwise on the sliced tissue to block endogenous catalase to reduce background staining, incubate at room temperature for 15 minutes, and wash three times with PBS. , 3 minutes each time. Drop about 50 μl of goat serum onto the slide and incubate at 37°C for one hour to reduce non-specific staining. Wipe the liquid around the slide with absorbent paper, draw a circle around the tissue with an immunohistochemistry pen, and then add 100 μl of diluted primary antibody (CCKBR antibody was purchased from Boster, diluted at a ratio of 1:300, CD133, KI67 and CD44 antibodies were purchased from Biolegend and diluted 1:100). After adding the primary antibody, place the sections in a humidified box at 4°C overnight (≥12h).

After taking the slices out of the refrigerator, they need to be incubated at room temperature for 15 minutes and rewarmed. Wash the slices three times with PBS, 3 minutes each time. Dry the slices with absorbent paper and add biotin-labeled secondary antibody (Nakasugi Jinqiao, SAP-9100), 37 Incubate at ℃ for 30 minutes. Wash the sections with PBS three times, 3 minutes each time, add horseradish-labeled streptavidin working solution (Zhongshan Jinqiao, SAP-9100) dropwise, and incubate at room temperature for 15 minutes. Rinse the sections with PBS 3 times, 3 minutes each time. After drying the PBS, wipe the sections with absorbent paper. Add 100 μl of freshly prepared DAB chromogenic solution (Zhongshan Jinqiao, ZLI-9017) to each section. After staining for 3 minutes, rinse with tap water. Color development. Then counterstain with 100 μl hematoxylin (Nakasugi Jinqiao, BSBA-4021) for 10 seconds, wash with water, differentiate with 1% hydrochloric acid ethanol for 1 minute, and then rinse with tap water for 15 minutes to return to blue. Then place the slices in order: 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, absolute ethanol I, absolute ethanol II, xylene I, xylene II for dehydration and transparency, and leave each reagent for a few seconds. , and finally air-dry the slices in a fume hood. Seal the slides with neutral gum and observe the dried sections under a microscope.

Finally, statistical analysis was performed on 59 clinical sections (the samples taken from the 59 clinical sections were gastric adenocarcinoma pathological tissue sections from the First Affiliated Hospital of Anhui Medical University, and the preparation method was as described above), and the statistical method used the chi-square test. Among survival-related indicators, Kaplan-Meier plots were analyzed using the Kaplan-Meier Plotter website, and violin plots were drawn using Cloupe software.

The results are shown in Figure 2. Immunohistochemistry results found that CCKBR+ gastric adenocarcinoma tumors showed low differentiation and high malignancy. These tumor cells showed more co-localization of CD44 and CD133 and showed more characteristics of cancer stem cells. And Kaplan-Meier plots and violin plots demonstrated that this type of tumor was associated with a worse prognosis for patients.

3. Verify that CCKBR+ gastric adenocarcinoma tumors express more FOXO proteins

The present invention uses RT-qPCR to detect whether clinical CCKBR+ gastric adenocarcinoma tumors also highly express FOXO. Among them, RNA was extracted using QIAGEN's RNeasy Mini Kit (74104). Specific steps are as follows:

Add an appropriate amount of BufferRLT (500 μl) to fresh gastric adenocarcinoma tumor tissue, grind the tissue for 5 minutes to destroy the tissue and release RNA. Then add 500 μl of 70% ethanol, mix by pipetting, transfer it to RNeasy spincolumn, put it into a centrifuge and centrifuge at 12000 rpm for 15 seconds. Discard the lower liquid after centrifugation. Then add 700 μl of BufferRW1, close the lid, and centrifuge at 12,000 rpm for 15 s to wash the spin column membrane. Discard the lower liquid after centrifugation. Then add 500 μl of Buffer RPE to the RNeasy spin column. Place into centrifuge and centrifuge at 12000 rpm for 15 seconds. Discard the lower liquid after centrifugation. Then add 500 μl of Buffer RPE to the upper layer, centrifuge at 12,000 rpm for 2 minutes, and discard the lower tube. Place the RNeasy spin column into a new 2ml collection tube, centrifuge for 3 minutes and place the RNeasy spincolumn into a new 1.5ml collection tube. Add 30 μl RNase-free ddH ₂ O. Close the lid and centrifuge at 12,000 rpm for 1 minute to elute RNA. Finally, the tissue RNA solution is obtained.

The extracted RNA was reverse transcribed using TOLOBIO's ToloScript ALL-in-one RT EasyMix for qPCR (22107) to obtain the corresponding cDNA. Detailed operations are as follows:

First, melt each reagent component on ice and configure the reaction system according to Table 9:

Table 9 Reaction system of reverse transcription

The reaction program is: 50°C, 15min, then 85°C, 5sec.

After the reaction, the obtained cDNA was diluted 5-fold with RNase-free ddH ₂ O. Then perform qPCR reaction. qPCR was performed using TOLOBIO's 2X Q3 SYBR Qpcr Master Mix (22204) kit. The reaction system is: Mix 10μl, ddH2O 3μl, Primer Forward 1μl, Primer Reverse 1μl, Template 5μl. Reactions were performed in a Bio-Rad CFX96 ^TM Touch machine. The reaction procedure is shown in Table 10:

Table 10 RT-qPCR reaction program

The primer sequences used in the above reaction are:

FOXO1 FORWARD 5’-AAACACCAGTTTGAATTCACCC-3’; REVERSE 5’-TCGACTTATTGTCCTGAAGTGT-3’;

FOXO3 FORWARD 5’-AGCCGAGGAAATGTTCGTC3’; REVERSE 5’-CCTTATCCTTGAAGTAGGGCAC-3’;

FOXO4 FORWARD 5’-CAAGAAGAAACCATCTGTGCTG’; REVERSE 5’-ATATCGGCTTCTCTCACGGTTTC-3’.

The results are shown in Figure 3. Through immunohistochemistry and RT-qPCR tests, the present invention proves that the FOXO family (FOXO1, FOXO3 and FOXO4) is highly expressed specifically in CCKBR+ gastric adenocarcinoma tumor patients, while in CCKBR-negative patients There is almost no expression of the FOXO family in gastric adenocarcinoma patients. Comparing CCKBR+ gastric adenocarcinoma patients with their corresponding para-cancerous tissues also shows that FOXO has extremely high expression in CCKBR+ gastric adenocarcinoma tumors, suggesting that FOXO is promising. As a therapeutic target, it kills tumor cells in a targeted manner.

4. TCGA database verifies the relative expression and clinical prognosis of CCKBR and FOXO

The present invention also detects the expression of CCKBR and FOXO from the TCGA STAD database, obtains the original data of TCGA STAD through the https://portal.gdc.cancer.gov/ website, and distinguishes high and low CCKBR expression through the expression of CCKBR. Express tumors and detect FOXO-related expression. Kaplan-Meier plots were analyzed using the Kaplan-MeierPlotter website.

The results are shown in Figure 4. The verification of the present invention also shows similar results in the TCGA STAD database, that is, gastric adenocarcinoma tumor tissues with high CCKBR expression express higher FOXO than gastric adenocarcinoma tumors with low CCKBR expression, and FOXO has high expression. associated with poorer prognosis in patients with gastric adenocarcinoma.

5. FOXO protein inhibitor AS1842856 inhibits the stemness and cell proliferation of CCKBR-positive cancer stem cells

Since gastric adenocarcinoma tumors with high CCKBR expression show high FOXO expression, in order to detect whether inhibiting FOXO can inhibit the growth of tumors with high CCKBR expression, and to detect whether FOXO is related to tumor stemness, the present invention uses the cancer stem cell spheroidization test. Whether the self-renewal ability of cancer stem cells is related to FOXO, the specific research is as follows:

Six gastric adenocarcinoma cell lines (MKN45, AGS, MKN7, HGC27, MKN28, HGC7901) were cultured in ultra-low adhesion culture dishes in DMEM/F12 (containing 2% B27, 20ng/mL EGF, 10ng/mLbFGF ), the culture environment is 37°C constant temperature, 5% CO ₂ concentration, the medium is changed every two days, (MCE, HY-100596) is added on the third day, and the final concentration of 1uM and 10uM drugs (AS1842856) are used for 4 days. Observe the cell status, and the results are shown in Figure 5.

As can be seen from Figure 5, the expression of CCKBR, FOXO1, FOXO3 and FOXO4 was widely increased in the tumor stem cells cultured into spheres, and the expression of CCKBR and the expression of FOXO family members after sphere formation were still positively correlated, proving that CCKBR+ gastric adenocarcinoma tumors and FOXO is related, and inhibiting FOXO activity inhibits tumor sphere growth, suggesting that FOXO is related to the stemness of CCKBR+ gastric adenocarcinoma tumor cells.

6. Establish a gastric adenocarcinoma organoid model and verify the growth inhibitory effect of AS1842856 on CCKBR-positive gastric adenocarcinoma organoids

In order to clinically verify the correlation between FOXO and CCKBR+ in gastric adenocarcinoma tumors, the present invention uses gastric adenocarcinoma organoids as experimental carriers for verification. Organoids have been widely proven to be able to simulate tumor characteristics in vivo, and are sensitive to drug sensitivity and primary tumors in vivo. Almost exactly the same. The present invention establishes and identifies CCKBR-positive gastric adenocarcinoma organoids, and conducts drug sensitivity tests on FOXO protein inhibitors on the organoids. The establishment method is as follows:

Cut the freshly removed gastric adenocarcinoma tissue samples from the gastric antrum into small tissue pieces of ^1-3mm3 with ophthalmic scissors, and add DMEM/F12 (containing 1x P/S, 1μg/uLPrimocin, 2.5% FBS , 0.6 mg/mL collagenase 1, 20 μg/mL hyaluronidase and 10 μMY-27632), place the mixture in a constant temperature incubator, and digest the cells at 37°C and 300 rpm for 2 hours to obtain a single cell suspension. The dissociated single-cell suspension was filtered and centrifuged through a 100-mesh filter to obtain cell pellets. Mix 10 μl of cell pellet and 100 μl of Matrigel, embed it in a 12-well plate, and add 1 mL of organoid medium (DMEM/F12 medium, containing 10mM HEPES, 2mM GlutaMax, 1X B27, 1mM N-Acetylcysteine , 50ng/mL EGF, 100ng/mLNoggin, 100ng/mLWnt3a, 100ng/mL FGF10, 1μg/mLR-Spondin1, 10nM Gastrin, 500nMA-83-01, 1μg/μl Primocin, 10μMY-27632) culture, every 2-3 times during the period Change the fluid once a day. Follow-up experiments were conducted after the organoids had grown for 14 days.

After culturing the above-mentioned organoids for 14 days, the organoids were identified in this application. Select organoids grown after 14 days for embedding. The specific steps are as follows:

Use 5 mL of pre-cooled PBS to pipette the organoids in the Matrigel to depolymerize the Matrigel and expose the organoids. Place the mixture on ice and incubate it for 30 minutes, then put it into a 4°C centrifuge at 2000 rpm for 5 minutes to obtain the organoid pellet. Fix the pellet with pre-cooled 4% paraformaldehyde solution for 1 hour, and centrifuge the solution. Centrifuge at 2000 rpm for 5 minutes to obtain the fixed organoid pellet. Then add 1g agarose to 50mL of PBS and put it in the microwave on high heat for 2 minutes to make a 2% agarose solution. Mix 1mL of the agarose solution and the organoids evenly, immediately put it into a 50mL centrifuge tube and centrifuge at 1000rpm for 3 minutes to obtain the solution. Organoids embedded in agarose blocks.

Then perform routine dehydration on the organoid agarose block. The dehydration steps are: soak the agarose block in formalin solution for 2 hours, transfer to 80% ethanol solution for 3 hours, 95% ethanol solution for 2 hours, and 95% Ethanol solution for 1 hour, absolute ethanol for 1 hour, absolute ethanol for 1 hour, xylene and absolute ethanol (1:1) for 30 minutes, xylene for 30 minutes, and paraffin at 60°C for 3 hours. After dehydration, paraffin embedding was performed. The obtained sections were subjected to immunohistochemical staining to identify whether the obtained organoids were CCKBR+ or CCKBR- gastric adenocarcinoma organoids. The experimental results are shown in Figure 5A-B. We established and identified CCKBR-positive and CCKBR-negative gastric adenocarcinoma organoids. Tumors, such gastric adenocarcinoma tumors highly express CCKBR, and their CCKBR expression and pathological morphology are consistent with the primary tumors, and are highly reproducible in vivo.

In the drug sensitivity test, the organoids were first spread in a 96-well plate, with 10 μl of Matrigel per well, and 100 μl of complete culture medium containing 1 μM and 101 μM of AS1842856 for treatment. After 6 days of treatment, an equal volume of Promega was added 3D Cell Viability Assay (G9681) kit, incubate at room temperature for 30 minutes, use a microplate reader to detect the Luminescence value in the well, which means the organoid viability is detected and a line chart is drawn based on this. The experimental results are shown in Figure 5C and D. .

Through drug sensitivity testing, we found that treating CCKBR+ gastric adenocarcinoma tumor organoids with AS1842856 can significantly inhibit the growth of the organoids.

7. Verification of the growth inhibitory effect of AS1842856 on CCKBR-positive gastric adenocarcinoma tumors in vivo

Combined with the above results, this application uses in vivo treatment to observe whether AS1842856 inhibits the growth of CCKBR+ gastric adenocarcinoma tumors in mice. The allogeneic tumor formation test in immunodeficient mice was used for verification. The detailed steps are as follows:

Ten 5-week-old nude mice with severe immunodeficiency were injected into the armpits with 5X10 ⁶ MKN45 cells, and were randomly divided into two groups, namely the drug treatment group and the control group, with 5 mice in each group. Both groups were of the same gender and age and were tested in the same environment. Starting from the third day, the mice in the drug treatment group were intraperitoneally injected with 50 mg/kg of AS1842856 every day, and the mice in the control group were intraperitoneally injected with the same amount of DMSO. . During this period, the size of the mouse tumors was measured every other day, and the volume was calculated using the formula V=L*W ² /2 for conversion.

The results are shown in Figure 6. AS1842856 has a significant inhibitory effect on the growth of CCKBR+ gastric adenocarcinoma tumor organoids at a concentration of 10 ^-6 M and inhibits tumor cell proliferation. However, it has a certain effect on tumors at a concentration of 10 ^-5 M. Cytotoxic effects. It was proved that AS1842856 can indeed inhibit the proliferation of CCKBR+ gastric adenocarcinoma tumor cells. However, it had no obvious cell killing effect on CCKBR-gastric adenocarcinoma tumor organoids. At the same time, after tumorigenesis of CCKBR+ MKN45 cells in nude mice, AS1842856 was found to inhibit tumor growth after the cells were treated with AS1842856, indicating that AS1842856 is a potential therapeutic target for CCKBR+ gastric adenocarcinoma tumors.

The present invention provides the use of a FOXO protein inhibitor: AS1842856 in preparing a drug for treating CCKBR+ gastric adenocarcinoma tumors. The present invention first constructed a CCKBR+ gastric adenocarcinoma single cell library based on single-cell RNA sequencing technology, and through enrichment analysis found that FOXO is a key transcription factor that maintains the stemness of CCKBR+ gastric adenocarcinoma tumor cells. Then, through tumor spheroid testing, organoid testing and nude mouse tumor formation testing, it was found that inhibiting FOXO activity through AS1842856 can inhibit the stemness of tumor cells, thereby inhibiting tumor cell growth. Therefore, the present invention found that AS1842856 has the potential to be used in the preparation of therapeutic drugs for the treatment and/or prevention of CCKBR+ gastric adenocarcinoma tumors, can be used for the treatment of CCKBR+ gastric adenocarcinoma tumors, and provides a method for the treatment of CCKBR+ gastric adenocarcinoma tumors. New drugs or methods.

Through single-cell sequencing technology and in vitro and in vivo experiments, the present invention identified FOXO as a potential therapeutic target for poorly differentiated tumors formed by malignant transformation of CCKBR+ gastric antrum stem cells. The present invention provides a therapeutic target for CCKBR+ gastric adenocarcinoma tumors, and also finds that the FOXO protein inhibitor AS1842856 is a potential therapeutic target for CCKBR+ gastric adenocarcinoma tumors (gastric adenocarcinoma transformed by CCKBR+ gastric antrum stem cells), AS1842856 It can target the FOXO protein, inhibit its transcriptional activity, and inhibit CCKBR+ gastric adenocarcinoma tumors, which can provide a basis for subsequent targeted therapy of gastric adenocarcinoma.

It should be noted that when the claims of the present invention refer to numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. To avoid redundancy, the present invention describes the preferred Example.

Although the preferred embodiments of the present invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once the basic inventive concepts are apparent. Therefore, it is intended that the appended claims be construed to include the preferred embodiments and all changes and modifications that fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the invention. In this way, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims (9)

1. Use of foxo protein inhibitor in the preparation of a medicament for the treatment of gastric adenocarcinoma tumor.
2. 2. The use according to claim 1, wherein the gastric adenocarcinoma tumor is a cckbr+ gastric adenocarcinoma tumor.
3. 3. The use according to claim 1, wherein the FOXO protein inhibitor is AS1842856 and the AS1842856 is targeted to inhibit the function of FOXO protein;

   the structure of the AS1842856 is AS follows:
4. 4. the use according to claim 3, wherein the AS1842856 inhibits the growth of gastric adenocarcinoma tumor cells of cckbr+, which are gastric Dou Gan cell-like tumor cells of cckbr+.
5. 5. The use of claim 4, wherein the AS1842856 is targeted to bind FOXO protein, inhibiting the transcriptional activity of FOXO protein.
6. 6. The use according to claim 4, wherein the degree of infiltration of the cckbr+ gastric adenocarcinoma tumor is in clear positive correlation with FOXO expression and the clinical prognosis of the cckbr+ gastric adenocarcinoma tumor is in clear positive correlation with FOXO expression.
7. 7. The use according to claim 6, wherein the expression of FOXO comprises expression of FOXO1, FOXO3 and FOXO4 in the FOXO family.
8. 8. A medicament for the treatment of gastric adenocarcinoma tumors, which prevent and/or treat cckbr+, characterized in that it comprises AS1842856 according to claim 3.
9. 9. The medicament of claim 8, wherein the medicament is a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, isotopically labeled derivative, stereoisomer, or prodrug of AS1842856.