Use of hoxb9 inhibitor in reversing drug resistance of gastric cancer to oxaliplatin

Abstract

The present application relates to the application of HOXB9 inhibitor in reversing the drug resistance of gastric cancer oxaliplatin, and belongs to the field of biological medicine. The present application discloses for the first time that HOXB9 is significantly highly expressed in the tissues of gastric cancer patients with poor curative effect after oxaliplatin-containing chemotherapy and is positively correlated with poor prognosis; by constructing an oxaliplatin-resistant gastric cancer cell line and a patient-derived organoid model, it is confirmed that HOXB9 specifically mediates the survival of the drug-resistant cell line by activating the focal adhesion signaling pathway (p-FAK / AKT), and is not related to cell proliferation. In view of the mechanism, shRNA nucleic acid molecules (SEQ ID NO: 1-2) targeting HOXB9 are provided, which can selectively kill the drug-resistant cell line when delivered by a lentiviral vector: the IC50 of the drug-resistant cell line is reduced by more than 50%, the apoptosis rate is increased by 3 times, and the chemotherapeutic resistance of the organoid is reversed. The present application provides a new target and treatment scheme for overcoming the oxaliplatin resistance of gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of HOXB9 inhibitors in reversing oxaliplatin resistance in gastric cancer. Background Technology

[0002] Studies show that gastric cancer (GC) is the fifth leading cause of cancer death in my country, with over 350,000 new cases annually. It is also the third leading cause of cancer-related deaths, with over 250,000 new deaths each year. Systemic chemotherapy is the primary treatment for metastatic gastric cancer, with a median overall survival (OS) of approximately 12 months for patients receiving standard chemotherapy. Radical surgery is the primary treatment for resectable gastric cancer. Several treatment methods have been established to reduce the risk of recurrence and improve long-term survival, including perioperative chemotherapy, adjuvant chemotherapy, and adjuvant chemoradiotherapy. These are currently recommended treatments for resectable, localized gastric cancer. Perioperative chemotherapy primarily uses platinum-based chemotherapy combined with fluorouracil to reduce tumor stage, facilitate local resection, and improve progression-free survival. Perioperative FLOT regimens (fluorouracil, leucovorin, oxaliplatin, and docetaxel) and ECF / ECX regimens (epimacrit, cisplatin, and fluorouracil / capecitabine) are commonly used. In addition, there are two-drug combination regimens: XELOX (oxaliplatin + capecitabine); SOX (oxaliplatin + tegafur); PF (cisplatin + fluorouracil), etc. Fluorouracil, platinum, taxane, and irinotecan are the main treatment drugs for advanced cancer. For patients with advanced gastric cancer, the recommended chemotherapy regimen is usually a combination of fluorouracil (fluorouracil, capecitabine, and S-1) and platinum as the first-line main treatment. Many chemotherapy regimens use platinum-based chemotherapy drugs, with oxaliplatin being the main platinum-based drug in gastric cancer chemotherapy. However, some patients develop chemotherapy drug resistance, leading to poor prognosis. In advanced patients, trastuzumab and some immune checkpoint inhibitors (such as nivolumab and pembrolizumab) have shown consistent and reliable efficacy in HER2-positive and PDL1-positive tumor patients, respectively, but patients receiving immunotherapy still require concurrent chemotherapy. In addition, the exploration of neoadjuvant therapy for gastric cancer using combinations of immune checkpoint inhibitors (camrelizumab), anti-angiogenic drugs (apatinib), and chemotherapy drugs is ongoing, with chemotherapy still playing a crucial role in the treatment strategy. These treatment methods inevitably encounter new challenges arising from chemotherapy resistance.

[0003] In conclusion, in-depth research into the molecular mechanisms of chemotherapy resistance in gastric cancer is of significant clinical and scientific value for identifying effective therapeutic targets and guiding treatment strategies. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a HOXB9 gene inhibitor. The purpose of this invention is to target HOXB9 in oxaliplatin-resistant gastric cancer to enhance its sensitivity to oxaliplatin.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0006] This invention discloses the application of HOXB9 expression inhibitors in the preparation of drugs that reverse oxaliplatin resistance in gastric cancer.

[0007] Furthermore, the HOXB9 inhibitor is a molecule that inhibits the expression or function of the HOXB9 gene.

[0008] Preferably, the HOXB9 inhibitor is a nucleic acid molecule, and the nucleic acid molecule is shRNA targeting the HOXB9 gene.

[0009] Preferably, the target sequence of the shRNA is selected from: SEQ ID NO:1: GGCAAAGAGTAAAGATTAA; SEQ ID NO: 2: GGCTAGAAAGTACAAGAAA.

[0010] The present invention also discloses a composition for reversing oxaliplatin resistance in gastric cancer, comprising oxaliplatin and any of the above-mentioned HOXB9 inhibitors.

[0011] Furthermore, the HOXB9 expression inhibitor is an shRNA lentiviral vector constructed from the target sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] This invention also discloses a gastric cancer oxaliplatin resistance detection kit, characterized in that: the kit detects the expression level of HOXB9 in the sample, and high expression of HOXB9 indicates the risk of drug resistance.

[0013] Furthermore, the sample is a gastric cancer tissue, blood, or organoid model.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0015] This treatment directly targets drug-resistant gastric cancer cells (non-treatment-naïve cells) that have progressed after chemotherapy, overcoming the bottleneck of existing protocols being ineffective against drug-resistant patients. Transcriptome sequencing of drug-resistant cell lines confirmed significant differences from parental cells (such as specific activation of the focal adhesion pathway), and the HOXB9 inhibitor can selectively reverse the drug-resistant phenotype of these cells, restoring the IC50 of drug-resistant cell lines to sensitive levels.

[0016] By tracing the source of drug-resistant tissue, the target was identified, and for the first time, it was demonstrated that HOXB9 maintains cytoskeleton stability in gastric cancer by regulating the focal adhesion pathway (p-FAK / AKT), mediating the survival of drug-resistant cell lines under chemotherapy stress (not through a proliferative pathway). This mechanism has not been reported in solid tumors and represents a novel pathway.

[0017] In drug-resistant cell lines and organoid models, targeting HOXB9 increased the killing effect of oxaliplatin by more than 3 times, and combination therapy eliminated the survival advantage of drug-resistant cell lines in continuous chemotherapy (clonal formation was reduced by 70%). HOXB9 expression level can serve as a drug resistance biomarker to guide precision medicine for patients who do not respond to chemotherapy; at the same time, it provides gene therapy tools (shRNA lentiviruses) and organoid drug sensitivity screening models based on this target.

[0018] This invention is the first to propose using small molecule nucleic acid sequences as inhibitors to target HOXB9 in oxaliplatin-resistant gastric cancer cells, thereby enhancing the sensitivity of gastric cancer cells to oxaliplatin. This overcomes the impact of oxaliplatin on gastric cancer cells during treatment, where some gastric cancer cells acquire oxaliplatin resistance due to high HOXB9 expression, which also affects their survival and progression after resistance. Compared with traditional methods that only inhibit signaling pathways, this invention works from the perspective of expression regulation, effectively enhancing the sensitivity of gastric cancer cells to oxaliplatin. Attached Figure Description

[0019] Figure 1 HOXB9 expression is upregulated in chemotherapy-unresponsive gastric cancer tissues and is associated with poor prognosis. (A) RNA-seq sequencing gene expression concordance analysis of neoadjuvant locally advanced gastric cancer tissue samples (analyzed using Pearson Correlation Coefficient), (B) Volcano plot of differentially expressed genes in two groups, with HOX family members labeled, (C) Heatmap of the top 50 genes upregulated by NHR compared to MjHR, with HOXB9 labeled. (D) Analysis of HOXB9 mRNA expression in gastric adenocarcinoma tumor tissues and normal tissues using the TCGA dataset. p <0.001. (E) Kaplan-Meier curves of overall survival for gastric cancer patients, (F) Kaplan-Meier curves of overall survival classified by differentiation type, (G) Kaplan-Meier curves of overall survival classified by gastric cancer type. Log-rank statistical analysis was used for comparison. p <0.05 is statistically significant. (H) Immunohistochemical staining and imaging results of HOXB9 expression in gastric cancer pathological tissue samples. The results were analyzed by IHC score and a bar chart was plotted (I). *** p <0.001, scale bar is 100μm.

[0020] Figure 2 Increased HOXB9 expression in gastric cancer cells reduced sensitivity to oxaliplatin. (A) Colony formation assay images and relative quantitative bar charts of AGS / HGC27 cells overexpressing HOXB9 and the control Vector group, values ​​are the mean ± SD of three independent experiments;* p <0.05; ns represents no significant difference. (B) Dose-response curves and IC50 values ​​of AGS HOXB9 / HGC27 HOXB9 cells and control cells, each curve representing the mean ± SD of four replicates under each condition. (C) Optical microscopy images and ATP energy bar graphs of AGS HOXB9 and control cells in the Matrigel matrix 3D cell spheroid formation experiment, values ​​are the mean ± SD of three independent experiments; *** p <0.001. (D) Western blot analysis of Caspase3 / Caspase8 protein expression in AGS HOXB9 cells and control cells, plotted as mean ± SD from three independent experiments; *** p <0.001. (E) Immunofluorescence assay of γ-H2AX expression and fluorescence focal count in oxaliplatin-treated AGS HOXB9 cells and control cells, values ​​are the mean ± SD of three independent experiments; *** p <0.001; ns represents no significant difference. (F) Solid image of tumor in tumor-bearing mice, (G) Tumor growth curve, (H) Volume of isolated tumor, (I) Weight of tumor. (J) Solid image of tumor in AGS HOXB9 and control tumor-bearing mice after oxaliplatin treatment, (K) Tumor growth curve, (L) Volume of isolated tumor, (M) Weight of tumor. p <0.01, *** p <0.001; ns represents no significant difference.

[0021] Figure 3 Knockdown of HOXB9 in drug-resistant gastric cancer cell lines enhances oxaliplatin sensitivity. (A) Dose-response curves and IC50 values ​​of oxaliplatin-resistant gastric cancer cell lines and corresponding control parental cells. Each curve represents the mean ± SD of four replicates under each condition. (B) Bar chart of HOXB9 mRNA expression levels during the establishment of drug-resistant gastric cancer cell lines. Values ​​are the mean ± SD of three independent experiments.* p <0.05,** p<0.01; ns represents no significant difference. (C) Western blot detection of HOXB9 protein expression level. (D) Dose-response curves and IC50 values ​​of HOXB9 knockdown resistant cell lines (AGS OR shHOXB9 / HGC27 OR shHOXB9) and corresponding control NC cells. Each curve represents the mean ± SD of four replicates under each condition. (E) Colony formation assay images of AGS OR shHOXB9 / HGC27 OR shHOXB9 and corresponding control NC cells, and (F) bar chart of relative quantitative results of colony formation assay, values ​​are the mean ± SD of three independent experiments; * p <0.05, *** p <0.001, ns represents no significant difference. (G) Scatter plot of apoptosis rate of AGS OR shHOXB9 / HGC27 OR shHOXB9 and corresponding control NC cells detected by flow cytometry; (H) Bar chart of total apoptosis rate = Q2 + Q4, values ​​are the mean ± SD of three independent experiments; *** p <0.001. (I) Optical microscopy images of 3D cell spheres formed in Matrigel by AGS OR shHOXB9 / HGC27 OR shHOXB9 and control cells, scale bar 100 μm, and (J) ATP energy detection bar chart, values ​​are the mean ± SD of three independent experiments; *** p <0.001.

[0022] Figure 4 HOXB9 regulates the focal adhesion signaling pathway in gastric cancer cells. (A) KEGG enrichment analysis of differentially expressed genes between AGS HOXB9 cells and control Vector cells, and between AGS OR cells and control AGS PC cells; (B) Venn diagram of the intersection analysis of differentially expressed genes between AGS OR vs AGSPC and AGS HOXB9 vs AGS Vector, and Venn diagram of the intersection analysis of upregulated differentially expressed genes between the two; (C) KEGG enrichment analysis of co-upregulated differentially expressed genes; (D) Bar chart of mRNA expression levels of focal adhesion signaling pathway-related molecules in AGS HOXB9 cells (control Vector cells), AGS OR cells (control PC cells), and AGS OR shHOXB9 cells (control NC cells) detected by qPCR. Values ​​are the mean ± SD of three independent experiments;* p <0.05,** p <0.01, *** p<0.001, ns represents no significant difference. (E) Western blot analysis of FAK and p-FAK expression levels in AGSOR HOXB9 cells (control Vector cells) and AGSOR HOXB9 cells (control AGS OR Vector cells); (F) Western blot analysis of FAK and p-FAK expression levels in AGSOR shHOXB9 cells (control AGS OR NC cells), and p-FAK / FAK ratio histogram. Values ​​are the mean ± SD of three independent experiments; * p <0.05,** p <0.01. (G) Immunofluorescence detection of p-FAK and F-actin expression in AGSHOXB9 cells (control Vector cells) and corresponding subcutaneous xenografts after oxaliplatin treatment, and bar charts showing relative mean fluorescence intensity; (H) Immunofluorescence detection of p-FAK and F-actin expression in AGSOR cells (control AGS PC cells) and corresponding cells after oxaliplatin treatment, and bar charts showing relative mean fluorescence intensity; (I) Immunofluorescence detection of p-FAK and F-actin expression in AGSHOXB9 cells (control Vector cells) and corresponding cells after oxaliplatin treatment, and bar charts showing relative mean fluorescence intensity. Values ​​are the average of three independent experiments ± SD; * p <0.05,** p <0.01, *** p <0.001, ns represents no significant difference. Scale bar is 20 μm.

[0023] Figure 5 HOXB9-dependent p-FAK activation of AKT affects the sensitivity of gastric cancer cells to oxaliplatin. (A) Images of AGS HOXB9 cells (control Vector cells) / HGC27 HOXB9 cells (control Vector cells) in a colony formation assay and a bar chart of relative quantitative results of the colony formation assay, values ​​are the mean ± SD of three independent experiments;** p <0.01, ns represents no significant difference. (B) Scatter plot of apoptosis rate of AGS HOXB9 cells (control Vector cells) / HGC27 HOXB9 cells (control Vector cells) detected by flow cytometry; (C) Bar chart of total apoptosis rate = Q2 + Q4, values ​​are the mean ± SD of three independent experiments; * p <0.05, *** p<0.001, ns represents no significant difference. (D) Western blot detection of AKT and p-AKT expression levels in AGS HOXB9 cells (control Vector cells) and AGS OR HOXB9 cells (control AGS OR Vector cells). (E) Western blot detection of AKT and p-AKT expression levels in AGS OR shHOXB9 cells (control AGS OR NC cells). (F) Western blot detection of AKT and p-AKT expression levels in AGS HOXB9 cells (control Vector cells) after the addition of Y15.

[0024] Figure 6 A gastric cancer organoid model was used to validate the effect of HOXB9 on the sensitivity of gastric cancer cells to oxaliplatin. (A) Imaging of gastric cancer organoid-derived tissue samples after HE staining, light microscopic imaging of organoids, and imaging of gastric cancer organoids after HE staining; (B) Immunohistochemical marker Ki67 in the source tissue samples and corresponding gastric cancer organoid samples; (C) qPCR was used to detect the mRNA expression level of HOXB9 in gastric cancer organoids. A bar chart was plotted with P1 as the control group for relative quantification. Values ​​are the mean ± SD of three independent experiments. * p <0.05,** p <0.01, ns represents no significant difference. (D) Dose-response curves and IC50 values ​​of gastric cancer organoids treated with oxaliplatin, each curve representing the mean ± SD of four replicates under each condition. (E, G) Dose-response curves and IC50 values ​​of P1 and P4 HOXB9 overexpression treated with oxaliplatin, each curve representing the mean ± SD of four replicates under each condition. (F, H) Light micrographs of gastric cancer organoids P1 HOXB9 (control P1 Vector) and P4 HOXB9 (control P4 Vector) overexpressing HOXB9. (I) Bar chart of relative cell viability, values ​​are the mean ± SD of three independent experiments; * p <0.05,** p <0.01, ns represents no significant difference. (J, L) Dose-response curves and IC50 values ​​of PC-P1 and PC-P2 HOXB9 knockdown after oxaliplatin treatment; each curve represents the mean ± SD of four replicates under each condition. (K, M) Light micrographs of gastric cancer organoids PC-P1 shHOXB9 (control PC-P1 NC) and PC-P2 shHOXB9 (control PC-P2 NC) with HOXB9 knockdown. (N) Bar chart of relative cell viability statistical analysis; values ​​are the mean ± SD of three independent experiments. p <0.01, *** p<0.001, ns represents no significant difference. Detailed Implementation

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

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

[0027] I. Research Methods and Materials

[0028] 1. Cell lines and reagents. Two human gastric cancer cell lines, AGS and HGC-27, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). AGS cells were cultured in DMEM / F-12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and HGC-27 cells were cultured in RPMI 1640 (Gibco). Both media were supplemented with 10% fetal bovine serum (Gibco) and 100 U / mL penicillin / streptomycin (Gibco).

[0029] 2. Cell construction.

[0030] Stable oxaliplatin-resistant gastric cancer cell lines were established using a long-term, continuous drug therapy approach that involved gradually increasing the concentration of parental gastric cancer cells with oxaliplatin. Oxaliplatin (MCE, MedChemExpress, catalog number: HY-17371) was initially administered at a concentration of 0.2 μM. Cells were screened at each concentration until cell colonies formed and were passaged. After achieving normal growth at the specified concentration, the oxaliplatin concentration was increased for further cell screening at the next concentration. Treatment with oxaliplatin at concentrations increasing from 0.2 μM to 8 μM for a total of 10 months resulted in stable, drug-resistant cell lines: AGS OR cells and HGC-27OR cells. The IC50 of oxaliplatin against parental cells and the corresponding drug-resistant cell lines was assessed using a CCK8 assay (Beiren Chemical Technology Co., Ltd., catalog number: CK04) to evaluate the drug resistance of the drug-resistant cell lines.

[0031] AGS and HGC-27 cells overexpressing HOXB9 (AGS HOXB9 / HGC27 HOXB9) were constructed using a recombinant lentiviral vector containing the CDS sequence of the human HOXB9 gene (NM_024017.5) constructed using the GV341 vector (Ubi-MCS-3FLAG-SV40-puromycin). The resulting packaged virus was obtained from Shanghai Jikai Gene Technology Co., Ltd. Cells to be infected were placed at an appropriate density (2×10⁶). 4Virus cells (number per well) were seeded into 24-well plates and cultured overnight in complete medium containing 10% FBS. The virus was removed from -80°C, thawed on ice, and the viral load was calculated using MOI=10: Virus load (μL) = (MOI × cell number) / Virus titer (TU / mL) × 1000. The old medium was discarded, and the cells were washed three times with PBS. Fresh medium containing polybrene (stock solution concentration 25×) was prepared. The corresponding concentration of virus suspension was added, mixed well, and incubated at 37°C. 24 hours post-infection, the virus-containing medium was aspirated and replaced with fresh complete medium. 48-72 hours after infection, Puromycin was added for selection. The drug-containing medium was changed every 2-3 days for approximately 2 weeks until all uninfected cells died. The surviving positive cells were then amplified and identified.

[0032] HOXB9-knockdown AGS OR and HGC-27 OR cells (AGS OR shHOXB9 / HGC 27 OR shHOXB9) were constructed. Recombinant lentiviral vectors containing shRNA sequences targeting the HOXB9 gene (NM_024017.5) (target sequence 1: GGCAAAGAGTAAAGATTAA; target sequence 2: GGCTAGAAAGTACAAGAAA) were constructed using the GV112 vector (hU6-MCS-CMV-puromycin). The resulting packaged virus was obtained (Shanghai Jikai Gene Technology Co., Ltd.). The infection method was the same as described above.

[0033] 3. Gastric cancer tissue sample.

[0034] The gastric cancer tissue samples used in this study were obtained with the approval of the Ethics Committee of Liaoning Cancer Hospital. The primary tumor samples of all patients in this study were obtained from the biobank of Liaoning Cancer Hospital.

[0035] The patients whose gastric cancer tissue samples were used in the RNA-seq sequencing were patients who received treatment at Liaoning Cancer Hospital between July 2020 and July 2021, using at least two cycles of the NACT and SOX regimen. SOX treatment included a first-day dose of 130 mg / m². 2 Oxaliplatin was administered at the prescribed dose, along with S-1 at a dose of 60 mg twice daily for 2 weeks. Clinical evaluations were performed every 2–3 cycles based on physical condition, serum tumor markers, and computed tomography results.

[0036] The paraffin sections of tissue samples used in the immunohistochemical experiments were collected from the Liaoning Cancer Hospital Biobank between 2011 and 2016. They were grouped according to recurrence status: 49 cases without recurrence and 30 cases with recurrence, for a total of 79 cases.

[0037] The tissue samples used for establishing gastric cancer organoids were postoperative tissue samples from 2024 to 2025. Among them, P1-P4 were primary tumor tissue samples that had not undergone treatment, and PC-P1 and PC-P2 were tumor tissue samples after neoadjuvant chemotherapy.

[0038] 4. RNA-seq sequencing analysis.

[0039] Clinical gastric cancer tissue sample sequencing: The College of American Pathologists (CAP) system was used to grade pathological responses. Responses were categorized into four classes: TRG 0 indicates no residual cancer cells and a complete response to treatment; TRG 1 indicates very few cancer cells and a near-complete response to treatment; TRG 2 indicates a partial response with some residual tumor and clear regression; and TRG 3 indicates a poor response to extensive residual cancer and no significant tumor regression. Based on their responses, patients were divided into two groups: a response group with a significant histological response (MJHR; TRG 0–2) and a non-response group with no histological response (NHR; TRG 3). Samples were sequenced using the Illumina Hiseq platform. Differential expression analysis was performed on RNA-seq data from the two clinical groups, with an absolute fold change greater than 2 and a p-value less than 0.05 used as the threshold for differentially expressed genes. Gene heatmaps and differential expression volcano maps were generated using the Novogene Cloud platform (https: / / magic-plus.novogene.com / ).

[0040] Sequencing of cell lines and constructed cell line samples: RNA-seq sequencing was performed on stable oxaliplatin-resistant AGS cell lines, constructed AGS cell lines overexpressing HOXB9, and control cell lines using the Illumina Hiseq platform. Differential expression analysis was performed, with an absolute fold change greater than 2 and a p-value less than 0.05 used as the threshold for differentially expressed genes. Gene heatmaps, differential expression volcano plots, GO and KEGG enrichment analyses, and Venn plots were generated using the Novogene Cloud platform.

[0041] 5. Public Database Analysis. The expression levels of HOXB9 in gastric adenocarcinoma and control normal tissues were analyzed using the TCGA database on the GEPIA2 platform (http: / / gepia2.cancer-pku.cn / ), including 359 normal tissues and 339 tumor tissues. Survival analysis of gastric cancer patients was performed using the Kaplan-Meier Plotter platform on the dataset (216417_x_at) (https: / / kmplot.com / analysis / ), including overall survival analysis, survival analysis by Lauren classification, and survival analysis by differentiation type.

[0042] 6. Immunohistochemistry (IHC).

[0043] Animal or human tumor tissue samples were fixed in 10 times 4% paraformaldehyde. The fixed tissue samples were then thoroughly dehydrated by sequentially passing them through a gradient of ethanol (75%, 85%, 95%, 1 h each; anhydrous ethanol 1, anhydrous ethanol 2, 30 min each). They were then permeated with clearing agents (xylene 1, xylene 2, 10 min each). The cleared tissue samples were then immersed in melted paraffin (paraffin 1, paraffin 2, 1 h each). The paraffin-impregnated tissue blocks were placed in an embedding mold, molten paraffin was added, and the mixture was cooled and solidified into a wax block. The wax block was cut into 5 μm thick continuous sections using a microtome. The sections were then floated in a warm water bath (40-45℃) to flatten them, and then transferred to anti-detachment slides (poly-L-lysine coated). The slides were then baked in a 60℃ oven for 2 h. Immunohistochemistry was performed on tissue sections using xylene (10 min twice), 100% ethanol (5 min twice), 95% ethanol (5 min), 85% ethanol (5 min), 75% ethanol (5 min), 50% ethanol (5 min), and distilled water (1 min). The sections were then placed in PBS buffer. For antigen retrieval, sections were immersed in pH 9.0 EDTA buffer, heated in an autoclave for 2 min after the pressure was reached, and allowed to cool naturally before continuing the experiment. The sections were then replaced with PBS buffer and used in subsequent experiments with a universal two-step assay kit (mouse / rabbit enhanced polymer assay system) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: PV-9000). The kit blocked endogenous peroxidase; sections were washed with PBS buffer for 3 min three times, and then incubated at room temperature for 30 min with 5% goat serum blocking solution (Beijing Solarbio Science & Technology Co., Ltd., catalog number: SL038). Primary antibodies were used according to the recommended ratios in the antibody instructions, including HOXB9 (1:100; Thermo Fisher Scientific, catalog number: PA5-40576) and Ki67 (1:100; Abotech Biotechnology Co., Ltd., catalog number: A20018), and incubated overnight at 4°C. Rinsing was performed 3 times for 3 minutes with PBS buffer, and reaction enhancement solution was added to the kit. Rinsing was repeated 3 times for 3 minutes with PBS buffer, and enzyme-labeled goat anti-mouse / rabbit IgG polymer was added to the kit. Rinsing was repeated 3 times for 3 minutes with PBS buffer, and DAB staining was performed using a DAB staining kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: ZLI-9017), incubated at room temperature for 5-8 minutes. Rinsing was then performed with tap water, followed by hematoxylin counterstaining for 20 seconds, hydrochloric acid alcohol differentiation for 2 seconds, and rinsing with tap water to restore blue color. The sections were sequentially dehydrated with a gradient of ethanol (50% ethanol for 5 min; 75% ethanol for 5 min; 85% ethanol for 5 min; 95% ethanol for 5 min; 100% ethanol for 5 min, twice), cleared (xylene 1, xylene 2, soaked for 10 min each), and mounted with neutral resin.

[0044] Imaging was performed using optical microscopy, including low-power (10× objective) and high-power (40× objective) lenses. Uncompressed images were saved with fixed exposure parameters. IHC score analysis was performed using ImageJ software, reviewed and judged by two pathologists. Cell staining intensity was scored in four grades: negative (0 points), weakly positive (1 point), positive (2 points), and strongly positive (3 points). Positive cell percentage was also scored in four grades: 0% ≤ positive cell percentage ≤ 25% (1 point); 25% < positive cell percentage ≤ 50% (2 points); 50% < positive cell percentage ≤ 75% (3 points); and 75% < positive cell percentage ≤ 100% (4 points). The IHC score was calculated as (cell staining intensity) × (positive cell percentage). Bar charts were generated and statistical analysis was performed using GraphpadPrism 8 software based on the IHC scores.

[0045] 7. Western blot (WB) assay.

[0046] Sample preparation: The target cell culture dishes were washed twice with pre-chilled PBS and discarded completely. RIPA lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0013) containing a protease inhibitor was added (lyse on ice for 30 min). The lysis buffer was collected using a cell scraper and centrifuged at 12,000 rpm for 15 min at 4°C. The protein concentration in the supernatant was determined using a BCA kit (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0012), and adjusted to a uniform concentration. 6×SDS loading buffer (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0015F) was added, and the mixture was boiled at 95°C for 5-10 min to denature the proteins.

[0047] SDS-PAGE electrophoresis: Gel preparation. Select the appropriate separating gel concentration (10%, 12%) based on the molecular weight of the target protein. 10% separating gel formulation: 4.0 mL distilled water, 3.3 mL 30% acrylamide, 2.5 mL 1.5 M Tris-HCl (pH 8.8), 0.1 mL 10% SDS, 0.1 mL 10% APS, 0.005 mL TEMED. Pour the separating gel to 1 cm from the comb teeth, cover with anhydrous ethanol and flatten. After polymerization, pour off the anhydrous ethanol, pour a 5% stacking gel and insert the comb. For sample loading, add 20-50 μg of protein per well (phosphorylated proteins can be increased to 30-50 μg). Electrophoresis conditions: stacking gel 80V, then adjust to 120V after entering the separating gel, until bromophenol blue runs off the bottom of the gel.

[0048] Transfer: After electrophoresis, the gel and membrane (0.45 / 0.22μm PVDF) are equilibrated in transfer buffer for 15 min. Assemble the "sandwich" structure: sponge pad-filter paper-gel-membrane-filter paper-sponge pad. Transfer the membrane at a constant voltage of 100V under ice bath conditions (time is adjusted according to the molecular weight of the protein: 22-36kDa about 30 min, 36-70kDa about 60 min, 70-130kDa about 90 min).

[0049] Blocking: Use 5% skim milk or 5% BSA (phosphoproteoprotein) in TBST solution and block on a shaker at room temperature for 1 hour.

[0050] Primary antibody incubation: Dilute the primary antibody with 1% BSA, incubate overnight at 4°C, and wash three times with TBST for 10 min each time. The primary antibodies included HOXB9 (1:1000; YANNOVA Biotech Co., Ltd., catalog number: H00003219-M05), p-FAK (1:1000; MedChemExpress, catalog number: HY-P80460), FAK (1:1000; IBOTEK Biotechnology Co., Ltd., catalog number: A11195), Akt (1:2000; Wuhan Sanying Biotechnology Co., Ltd., catalog number: 10176-2-AP), and p-Akt (1:2000; Wuhan Sanying Biotechnology Co., Ltd., catalog number: 66444-1-lg).

[0051] Secondary antibody incubation: Select an HRP-labeled secondary antibody (1:10000; Abcam, catalog number: ab6721 / ab6789) that matches the species of the primary antibody, incubate at room temperature for 1 hour, and wash thoroughly with TBST 3 times for 10 minutes each time.

[0052] Exposure: Mix ECL luminescent substrate solution A and solution B (SuperSignal™ West Pico PLUS chemiluminescent substrate, Thermo Fisher Scientific, catalog number: 34580) in proportion, uniformly cover the membrane surface, incubate for 1 min, and acquire signals using a chemiluminescence imaging system.

[0053] Image analysis: Image J was used to analyze the grayscale values ​​of the images. The target protein band was homogenized using the internal reference protein band as the standard. The analytical results of three independent experiments were used to create bar charts and perform statistical analysis using Graphpad Prism 8 software.

[0054] 8. Drug-resistant clonal formation experiment.

[0055] Logarithmically growing cells were digested and prepared into single-cell suspensions. After cell counting, the density was adjusted to 800 cells / mL with complete culture medium. 2 mL of cell suspension was added to each well, and the cells were cultured for 3 days before the appropriate drug was added. For single-drug treatment, the oxaliplatin concentration was based on the approximate 24-hour IC50 concentration of the target cells (5 μM for AGS cells, 10 μM for HGC27 cells). For combination drug treatment, the cells were first incubated with the corresponding combination drug Y15 (MedChemExpress, catalog number: HY-12444) at a concentration of 5 μM for 2 hours, followed by the addition of oxaliplatin at the 24-hour IC50 concentration of the target cells. Incubation was continued for 14 days, with the culture medium containing fresh drug replaced every 3 days, and the aforementioned two-drug treatments repeated. After terminating the experiment, discard the culture medium, gently wash twice with PBS, add 1 mL of methanol, fix at room temperature for 30 min, wash with PBS, add 0.1% crystal violet staining solution (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: C0121), and stain in the dark for 30 min. Rinse slowly with running water and air dry at room temperature.

[0056] Imaging was performed using the Bio-Rad imaging system, and image analysis was conducted using ImageJ software. The total clonal area of ​​the control group was used for standardization. The percentage of the actual area of ​​other groups divided by the average area of ​​the control group was calculated as the relative area percentage, i.e., relative area (%) = (average area of ​​the treatment group / average area of ​​the control group) × 100%. The numerical results from three independent experiments were used to create bar charts and perform statistical analysis using Graphpad Prism8 software.

[0057] 9. IC50 Detection Assay. Logarithmically growing cells were digested and prepared into single-cell suspensions. After cell counting, the density was adjusted with complete culture medium and seeded into 96-well plates at 2500 cells / well. Each cell group was divided into 7-8 concentration gradients, with 5 replicates per concentration, and 3 blank background control wells were included. Parental gastric cancer cells and gastric cancer cells overexpressing the gene were treated with oxaliplatin at concentrations of 0, 1, 2, 10, 20, 100, and 200 μM. Oxaliplatin-resistant gastric cancer cells were treated with oxaliplatin at concentrations of 0, 4, 20, 40, 80, 120, 160, and 240 μM. The next day, after cell attachment, the medium was replaced with the corresponding drug-containing medium, and incubated at 37°C and 5% CO2 for 24 or 72 hours. The absorbance at 450 nm was measured using the CCK8 reagent; 10 μL of reagent was added to each well, and the cells were incubated for 2 hours before measurement. Cell viability (%) = (OD sample - OD blank) / (OD control - OD blank) × 100% (OD sample: absorbance of the drug-treated group, OD control: absorbance of the negative control group, OD blank: absorbance of the blank wells without cells). The experiment was independently repeated four times. Numerical results were analyzed, and IC50 curves were plotted and analyzed using Graphpad Prism 8 software.

[0058] 10. 3D Cell Spheroidization Assay. Thaw Matrigel (Xiamen Moji Biotechnology Co., Ltd., Catalog No.: 082706) at 4°C 12 hours in advance. Pre-cool consumables in contact with Matrigel at 4°C. Add 50 μL of Matrigel (8-11 mg / mL) to the pre-cooled 96-well plate and incubate at 37°C for 30 min to allow Matrigel gelation. Collect cells in the logarithmic growth phase to obtain the cell pellet to be inoculated, resuspend the cells in complete culture medium, count the cells, and adjust the cell density to 3 × 10⁻⁶. 4 Cells / mL were collected, and 50 μL of cell suspension was added to pre-coated wells and incubated at 37°C for 30 min. A mixed medium was prepared by adding 10% Matrigel to pre-cooled complete culture medium. 100 μL of the mixed medium was added to the incubated wells and incubated at 37°C for 9 days. On day 10, 10 μM oxaliplatin was added. The medium containing fresh drug was replaced every 2 days. Imaging was performed using an optical microscope. Cell viability of the 3D cell spheroids was detected using the CellTiter-Glo® 3D Cell Viability Assay kit (Promega, catalog number: G9681) and a microplate reader. Cell viability (%) = (sample luminescence value - blank luminescence value) / (control luminescence value - blank luminescence value) × 100%. Bar charts were generated and analyzed using Graphpad Prism 8 software.

[0059] 11. Immunofluorescence Assay. Seed the corresponding gastric cancer cells onto cell slides, approximately 20,000 cells / slide. After the cells are in good condition, proceed with subsequent experiments. Gently wash the slides twice with PBS buffer, add 1 mL of 4% paraformaldehyde, and fix at room temperature for 30 min. After fixation, wash the slides three times with TBS buffer, 10 min each time. Prepare 0.2% Triton-X100 permeation buffer using TBS buffer. Add 1 mL of permeation buffer and permeate at room temperature for 10 min. Wash the slides three times with TBS buffer, 5 min each time. Discard the TBS buffer, add 1 mL of 5% BSA blocking buffer, and block at room temperature for 30 min. Dilute the primary antibody with 1% BSA and incubate overnight at 4°C. Wash the slides three times with TBST buffer, 10 min each time. Add the working solution of the secondary antibody (Thermo Fisher Scientific, catalog number: A-11008) in the dark and incubate at room temperature for 2 h. Wash the slides three times with TBST buffer, 10 min each time. Discard the TBST buffer, add DAPI (Thermo Fisher Scientific, catalog number: D1306) working solution, stain the nuclei for 10 min, wash twice with TBST buffer for 10 min each time. Rinse once with ddH2O, and mount with anti-quenching mounting medium (Thermo Fisher Scientific, catalog number: P36961). Image the slides using a laser confocal microscope, and perform fluorescence statistical analysis using ImageJ software, including focal point statistics and average fluorescence intensity analysis. Subsequently, use Graphpad Prism 8 software to generate bar charts and perform statistical analysis. Primary antibodies included: Anti-gamma H2A.X (phospho S139) (1:100; Abcam, catalog number: ab11174) and Phospho-FAK (Tyr397) antibody (1:100; MedChemExpress, catalog number: HY-P80460). F-actin uses Actin-Tracker Red-Rhodamine (microfilament red fluorescent probe) (Beyotime Biotechnology Co., Ltd., catalog number: C2207S).

[0060] 12. Tumor-bearing mouse experiment. BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.), females, 4 weeks old, 6 mice per group. After one week of rearing, at 5 weeks of age, cells were subcutaneously injected. Target cells and control cells were collected and counted, resuspended and washed once with PBS, then resuspended in PBS containing 30% Matrigel, and aliquoted into sterile EP tubes, 10 cells per tube. 7200 μL / cell, stored at low temperature on an ice box to prevent cell frostbite. Before injection, disinfect the skin with 75% alcohol. Insert the needle at a shallow angle of 15-30° into the subcutaneous site of the right axilla. After the needle has traveled subcutaneously for a short distance, slowly inject until a noticeable bulge is observed. Slowly withdraw the needle and apply gentle pressure to prevent backflow. Record the injection time. Observe the mice's condition daily, initially measuring every 3 days until the volume reaches 100 mmHg. 3 Later, measurements were changed to daily, using vernier calipers. Volume was calculated and recorded using the formula: Volume = 1 / 2 × Major Axis × Minor Axis². In the oxaliplatin dosing experiment, administration was performed on day 12 via intraperitoneal injection. The initial dose was 5 mg / kg, twice weekly, increasing to 10 mg / kg twice weekly from week 3 onwards, for a total of 61 days. Tumors with diameter ≤20 mm and weight <10% of body weight, without ulceration or necrosis, were considered. Tumor volume and weight were measured. Graphpad Prism 8 software was used for survival curve plotting, final tumor volume analysis, and final tumor weight analysis.

[0061] 13. mRNA expression quantification experiment.

[0062] RNA Extraction: Collect cells and add 500 μL of Trizol (Thermo Fisher Scientific, catalog number: 15596018CN). Lyse cells by incubating at room temperature for 10 min. Add 0.1 mL of chloroform, vortex vigorously for 15 s, incubate on ice for 5 min, then centrifuge at 12,000 g for 15 min at 4 °C. The sample separates into three layers: an upper aqueous phase (containing RNA), a middle layer (containing DNA), and a lower organic phase (containing protein). Carefully aspirate the upper aqueous phase (not exceeding 80%) into a new tube, add 0.25 mL of isopropanol, and mix gently. Incubate at room temperature for 10 min, then centrifuge at 12,000 g for 10 min at 4 °C. Discard the supernatant and wash the precipitate with 1 mL of 75% ethanol (prepared with DEPC water). Centrifuge at 12,000 g for 5 min at 4 °C and discard the supernatant. After air drying, dissolve the RNA in an appropriate amount of DEPC water.

[0063] Reverse transcription: After quantifying RNA, 1 μg of each sample was reverse transcribed into cDNA. The reaction solution was prepared according to the instructions of the ABScript Neo RTMaster Mix for qPCR with gDNA Remover kit (Aibotek Biotechnology Co., Ltd., catalog number: RK20433), and reverse transcription was performed on a PCR instrument.

[0064] Quantitative real-time PCR (qPCR): 2 μL of cDNA product was used for qPCR. The reaction solution was prepared according to the instructions using the 2× UniversalSYBR Green Fast qPCR Mix kit (Aibotek Biotechnology Co., Ltd., catalog number: RK21203), and the qPCR reaction was performed on a quantitative real-time PCR instrument. Graphpad Prism 8 software was used for analysis and statistical plotting of bar graphs.

[0065] The relevant primers (Suzhou Hongxun Biotechnology Co., Ltd.) are as follows.

[0066] 14. Apoptosis assay. The cells in logarithmic growth phase were seeded into 6-well plates, 6 × 10⁶ cells per well. 4 Cells were cultured until the cell density reached 70% or higher. Oxaliplatin 10 μM was added, and the cells were incubated at 37°C with 5% CO2 for 24 h. The culture supernatant, containing any potentially detached apoptotic cells, was collected into a 15 mL centrifuge tube. Adherent cells were washed once with pre-chilled PBS, and then digested with 500 μL of EDTA-free trypsin. When the cells became rounded, culture medium was added to stop the digestion, and the cells were gently pipetted into a single-cell suspension. The suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Cells were washed twice with pre-chilled PBS and centrifuged at 1000 rpm for 5 min. Cells were stained using the Annexin V-FITC apoptosis detection kit (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: C1062M). Cells were gently resuspended in 195 μL of Annexin V-FITC binding buffer. 5 μL of Annexin V-FITC was added and gently mixed. 10 μL of propidium iodide staining solution was added and gently mixed. Incubate at room temperature (20-25℃) in the dark for 10-20 min, then place in an ice bath. Aluminum foil can be used for light protection. Subsequently, analyze the data using flow cytometry. Statistical analysis of the results was performed using Graphpad Prism 8 software to generate bar charts. For combination therapy, incubate with the corresponding combination drug Y15 (MedChemExpress, catalog number: HY-12444) at a concentration of 5 μM for 2 h, followed by the addition of oxaliplatin.

[0067] 15. Experiments on the culture and construction of gastric cancer organoids.

[0068] Gastric cancer organoid construction: fresh gastric cancer tumor tissue (≤1cm) obtained by surgical resection 3Place the tissue in pre-cooled sterile tissue preservation solution (Xiamen Moji Biotechnology Co., Ltd., catalog number: MB-0818L04S) to maintain its activity; optimal processing within 12 hours is recommended. Remove necrotic tissue, fat, and blood vessels, preserving the viable tumor area. Rinse repeatedly 3-5 times with PBS containing penicillin / streptomycin to remove blood and impurities. Cut the tissue into 1-2 mm pieces using sterile scissors. 3 Add tissue fragments to tissue digestion solution (Xiamen Moji Biotechnology Co., Ltd., catalog number: MB-0818L05S), and digest at 37°C on a shaker for 60 min, mixing by pipetting every 5-10 min. Monitor under a microscope until the tissue is fully dissociated, then add culture medium containing 10% FBS and 1% penicillin antibiotics (Advanced DMEM / F12, Xiamen Moji Biotechnology Co., Ltd., catalog number: MB-0930L500) to terminate the reaction. Filter through a 100μm cell sieve to remove undigested clumps, centrifuge at 300g for 5 min, resuspend in PBS and wash twice to remove enzyme residues. Thaw Matrigel (Xiamen Moji Biotechnology Co., Ltd., catalog number: 082755) overnight at 4°C, and handle on ice to prevent coagulation. Mix cells and Matrigel at a 1:1 ratio (density: 10,000 cells / 10μL gel), and drop the mixture into the wells of a culture plate (50μL per well), incubate at 37°C for 30 min to solidify the droplets. Add 500 μL of gastric cancer organoid culture medium (Xiamen Moji Biotechnology Co., Ltd., catalog number: MA-0807T008LP) to each well, covering the surface of the cured droplet. Incubate at 37°C, 5% CO2, and saturated humidity, replacing the medium with fresh medium every 2–3 days.

[0069] Passaging of gastric cancer organoids: The culture medium was aspirated, and the matrix gel was dispersed by adding pre-cooled PBS. The organoid suspension was collected, centrifuged, washed, and the PBS was discarded. 1 mL of organoid digestion solution (Xiamen Moji Biotechnology Co., Ltd., catalog number: MB-0818L01S) was added, and the mixture was incubated for 10 min. The digestion solution was then neutralized with culture medium containing 10% FBS and 1% penicillin-dextrin. Subsequent embedding was performed following the primary passage procedure, with a passage ratio typically between 1:4 and 1:8.

[0070] Construction of gastric cancer organoids with HOXB9 overexpression or knockdown: Mature gastric cancer organoids were dissociated into single-cell suspensions, centrifuged at 300g for 5 min, and the cells were collected and washed with pre-chilled culture medium. The single-cell suspensions were seeded into 24-well plates (2×10⁻⁶ cells / well). 5Cells / well were added to a virus (the aforementioned HOXB9 overexpressing or shRNA-knocked HOXB9 lentivirus, with a viral titer MOI of 10)-polybrene complex (containing 6 μg / mL polybrene to enhance infection efficiency), and incubated at 37°C for 24 h. Transfected cells were collected by centrifugation, resuspended in a matrix gel, plated, and embedded according to the primary passage procedure to form gene-edited gastric cancer organoids. Positive stable strains were screened using medium containing puromycin (2 μg / mL), and organoids were collected for validation and subsequent experiments.

[0071] 16. IC50 Detection Assay for Drug Resistance in Gastric Cancer Organoids. Organoids cultured for 14 days with a diameter of 200 μm were used. The matrix gel was dissolved in pre-cooled PBS, centrifuged at 300 g for 5 min, and the organoid clumps were collected and digested. The digestion was performed at 37°C for 10 min to dissociate the organoids into single cells or small cell clusters (<10 cells). Single organoid cells were resuspended in medium containing 50% Matrigel. 5 μL of gel droplets containing 1500 cells were added to each well of a 96-well plate. The plates were cured at 37°C for 30 min, and 50 μL of gastric cancer organoid culture medium was added to each well. The plates were cultured for 72 h to allow for structural reconstruction. Six concentration gradients of oxaliplatin were set up in conditioned medium (0, 5, 25, 50, 100, 400 μM). 100 μL of the drug medium was added to each well to replace the original solution. A Blank solvent control group (containing the drug dissolution solvent) was also included. Five replicates were used for each concentration. The plates were cultured for 72 h after drug treatment. Add 10 μL of CCK-8 to each well and incubate for 4 hours. Measure the OD value at 450 nm using a microplate reader. Use GraphPad Prism software to fit a dose-response curve with drug concentration on the x-axis and survival rate on the y-axis, and calculate the half-maximal inhibitory concentration (IC50).

[0072] 17. Paraffin embedding of gastric cancer organoids. Aspirate organoid culture medium, wash twice with PBS to remove residual medium, add pre-cooled PBS, transfer organoids to centrifuge tubes, centrifuge at 300g for 3 min, remove supernatant and retain precipitate. Fix with 4% paraformaldehyde for 4 h (4℃) to ensure organoid morphology integrity. After fixation, centrifuge to remove paraformaldehyde, wash twice with PBS. Preheat 3% agarose until completely dissolved, then cool to 40℃ for later use. Mix the organoid suspension with agarose (1:1 ratio), transfer to 1.5mL EP tubes, and incubate at 4℃ for 30 min to solidify, forming a transparent gel block. The tissue samples were thoroughly dehydrated sequentially with a gradient of ethanol (75%, 85%, 95%, anhydrous ethanol 1, and anhydrous ethanol 2, each soaked for 30 min), then permeated with clearing agents (xylene 1 and xylene 2, each soaked for 10 min). The cleared tissue samples were then immersed in melted paraffin (paraffin 1 and paraffin 2, each soaked for 1 h). The paraffin-soaked tissue blocks were placed in an embedding mold, molten paraffin was added, and the mold was allowed to cool and solidify into a paraffin block. The paraffin block was cut into 5 μm thick continuous sections using a microtome. The sections were then floated in a warm water bath (40-45℃) to flatten them, and then transferred to anti-detachment slides (poly-L-lysine coated). The slides were then baked in a 60℃ oven for 2 h.

[0073] 18. HE staining.

[0074] Dewaxing and hydration: Paraffin sections were sequentially immersed in xylene I and xylene II for 10 min each to remove the paraffin. They were then sequentially passed through anhydrous ethanol I and anhydrous ethanol II for 5 min each, followed by 95%, 90%, 80%, and 70% ethanol for 5 min each, and finally rinsed with distilled water.

[0075] Hematoxylin staining: Immerse the section in hematoxylin staining solution for 3 minutes. The cell nuclei are stained blue-purple. Differentiate with 1% hydrochloric acid alcohol for 2 seconds to remove non-specific staining. Rinse with running water and then rinse with tap water to restore the blue color.

[0076] Eosin staining: Immerse the section in eosin staining solution for 10 seconds, and the cytoplasm will be stained from pink to red.

[0077] Dehydration and mounting: Sequentially pass through 75%, 85%, and 95% ethanol for 2 minutes each, then through anhydrous ethanol I and anhydrous ethanol II for 5 minutes each, and then through xylene I and xylene II for 5 minutes each. Completely dehydrate and transparent, then seal with neutral resin to avoid air bubbles, and image under a microscope.

[0078] 19. Continuous Oxaliplatin Treatment and Viability Determination of Gastric Cancer Organoids. Organoids cultured for 14 days with a diameter of 200 μm were collected. The matrix gel was dissolved in pre-cooled PBS, centrifuged at 300 g for 5 min, and the organoid clumps were collected and digested. The digestion was performed at 37°C for 10 min to dissociate the organoids into single cells or small cell clusters (<10 cells). Single organoid cells were resuspended in medium containing 50% Matrigel. 5 μL of gel droplets containing 1500 cells were added to each well of a 96-well plate, and the plates were cured at 37°C for 30 min. 50 μL of gastric cancer organoid culture medium was added to each well, and the plates were cultured for 72 h to allow for structural reconstruction. Oxaliplatin-containing medium (100 μM) was added, and treatment was continued for 7–14 days, depending on the specific characteristics of the organoids. Imaging was performed using an optical microscope. The plates were discarded at the experimental endpoint.

[0079] 20. Statistical Analysis. Independent experiments were conducted at least three times. Data are expressed as mean ± standard deviation (SD). All data were statistically analyzed using GraphPad Prism 8.0 software. The t-test was used to compare two independent groups. One-way ANOVA and Tukey's test were used for statistical analysis of data across multiple groups. p <0.05 is statistically significant.

[0080] II. Research Results.

[0081] 1. HOXB9 is associated with chemotherapy resistance in gastric cancer. Clinical tissue samples were collected from 19 patients with locally advanced gastric cancer who received neoadjuvant therapy with oxaliplatin. Nine of these were significantly reactive tissue samples (MjHR) after chemotherapy, and 10 were non-reactive tissue samples (NHR). Transcriptome sequencing analysis was subsequently performed, and the results showed a certain degree of consistency in gene expression among these clinical samples. Figure 1 A). Comparing differentially expressed genes between NHR and MjHR, 480 genes were upregulated in the NHR group, and 97 genes were downregulated in the NHR group. Figure 1 B). Combined with gene heatmap analysis, multiple HOX family genes were highly expressed in the NHR group, with HOXB9 showing significantly higher expression. HOXB9 expression was higher in multiple samples from the NHR group than in the MjHR group (B). Figure 1 B, C). TCGA dataset analysis showed that HOXB9 expression was significantly higher in gastric adenocarcinoma tumor tissues than in normal tissues (B, C). Figure 1 D). Analysis of overall survival of HOXB9 in gastric cancer patients using the Kaplan-Meier Plotter platform showed that high expression of HOXB9 was associated with lower survival in all gastric cancer patients. Figure 1 E). Further analysis of gastric cancer patients by differentiation type revealed that high expression of HOXB9 was associated with poor and moderately differentiated gastric cancer patients (E). Figure 1F). Analysis of different gastric cancer types revealed that high expression of HOXB9 was associated with poor survival in intestinal-type and mixed-type gastric cancer. Figure 1 G). We collected gastric cancer pathological tissues for immunohistochemical staining. HOXB9 expression was significantly higher in the pathological tissues of patients with recurrence after chemotherapy compared to those of patients without recurrence. Figure 1 Therefore, HOXB9 affects the clinical progression of gastric cancer patients and may be associated with chemotherapy progression.

[0082] 2. HOXB9 overexpression reduces the sensitivity of gastric cancer to oxaliplatin chemotherapy. To further confirm whether elevated HOXB9 levels affect the progression of gastric cancer chemotherapy, we constructed AGS and HGC27 cell lines stably overexpressing HOXB9. In an oxaliplatin-resistant colony formation assay, we found that the relative area of ​​colonies formed by gastric cancer cells overexpressing HOXB9 was increased compared to the control group during continuous drug treatment. Furthermore, when drug treatment was withdrawn after one day, the relative area of ​​colonies formed by gastric cancer cells overexpressing HOXB9 was significantly increased compared to the control group. Figure 2 A). This was further validated by the drug IC50 assay. Gastric cancer cells overexpressing HOXB9 showed a significantly higher IC50 resistance to oxaliplatin after 72 hours compared to the control group. Increased HOXB9 expression led to an approximately three-fold increase in the oxaliplatin IC50 of gastric cancer cells. Figure 2 B). Furthermore, in the 3D cell spheroid formation experiment, we found that the volume of 3D cell spheroids formed by AGS HOXB9 cells was not significantly different from that of the control group. However, after oxaliplatin treatment, more 3D cell spheroids remained in AGS HOXB9 cells, and under high magnification, these formed denser, solid 3D spheroids. In contrast, the control group dissolved after oxaliplatin treatment. ATP energy measurements also showed that the survival rate of AGS HOXB9 cells was significantly higher than that of the control group after oxaliplatin treatment. Figure 2 C). Cell death following chemotherapy activates the Caspase system. Western blot (WB) results showed that in AGS cells overexpressing HOXB9, treatment with different concentrations of oxaliplatin resulted in decreased intracellular Caspase-3 and Caspase-8 expression compared to the control group. Figure 2 D). Furthermore, the DNA damage marker γ-H2AX, as shown by immunofluorescence experiments, resulted in reduced intracellular nuclear damage in AGS HOXB9 cells induced by oxaliplatin compared to the control group. Figure 2 E). In vivo experiments were conducted using BALB / c nude mice, and the results showed no significant difference in tumor volume and weight between AGS cells overexpressing HOXB9 and the control group in subcutaneous tumor formation (E). Figure 2FI), but in nude mice treated with oxaliplatin, tumors formed by AGS cells overexpressing HOXB9 were more oxaliplatin resistant, and the tumor volume and weight were significantly larger than those of the control group cells (FI). Figure 2 (JM). Therefore, increased HOXB9 expression in gastric cancer enhances the resistance of gastric cancer cells to oxaliplatin, and this process also affects the activation of the Caspase family and attenuates nuclear damage.

[0083] 3. HOXB9 in gastric cancer cells is induced to increase during oxaliplatin treatment, and the absence of HOXB9 affects the survival of drug-resistant gastric cancer cell lines. To further investigate the role of HOXB9 in the process of oxaliplatin resistance in gastric cancer, we initially induced AGS and HGC27 cells with oxaliplatin at a concentration of 0.2% IC50 to establish oxaliplatin-resistant gastric cancer cell lines AGS OR and HGC27 OR. Subsequently, the IC50 of oxaliplatin was measured to validate the stable drug-resistant gastric cancer cell lines. Figure 3 A). HOXB9 expression was detected in gastric cancer cell lines induced to develop drug resistance. The results showed that as the induction concentration gradually increased, the expression level of HOXB9 in gastric cancer cells also gradually increased. Figure 3 B). Western blotting also showed that the drug-resistant cell line expressed more HOXB9 than the parental cells. Figure 3 C). After the oxaliplatin IC50 of the gastric cancer drug-resistant cell line was stably increased by more than 8-fold compared with the parental cells, HOXB9 was knocked down in the gastric cancer drug-resistant cell line to construct stable cell lines AGS OR shHOXB9 (control group AGS OR NC) and HGC27 OR shHOXB9 (control group HGC27 OR NC). The IC50 of oxaliplatin for 24 hours was detected in the HOXB9 knockdown gastric cancer drug-resistant cell line and the control cells. The downregulation of HOXB9 increased the sensitivity of the gastric cancer drug-resistant cell line to oxaliplatin (IC50 of AGS OR NC was 65.11 μM, IC50 of AGS OR shHOXB9 was 9.745 μM, IC50 of HGC27 OR NC was 93.01 μM, and IC50 of HGC27 OR shHOXB9 was 23.59 μM). Figure 3 D). We treated HOXB9-knockdown gastric cancer drug-resistant cell lines and control cells with oxaliplatin continuously. The results showed that, in the absence of drug, the colony-forming ability of AGS OR cells after HOXB9 knockdown was not significantly different from that of the control group, but oxaliplatin significantly inhibited the colony-forming ability of AGS OR shHOXB9 cells. In the absence of drug, the colony-forming ability of HGC27 OR cells after HOXB9 knockdown was slightly reduced compared with the control group, and continuous treatment with oxaliplatin also showed a significant inhibitory effect. Figure 3E, F). Flow cytometry was used to detect apoptosis in drug-resistant gastric cancer cell lines after oxaliplatin treatment. The results showed that the total apoptosis rate (Q2+Q4) of drug-resistant gastric cancer cell lines after HOXB9 knockdown was significantly increased, further demonstrating that the deletion of HOXB9 increased the sensitivity of drug-resistant gastric cancer cell lines to oxaliplatin. Figure 3 G, H). Similar results were also observed in the 3D cell spheroidization experiment, where we found that cell spheroids formed by HOXB9-knockdown gastric cancer drug-resistant cell lines were more easily dissolved after oxaliplatin treatment. Figure 3 I). This contrasts with the robust spheroids formed by gastric cancer cells overexpressing HOXB9. ATP energy assays also demonstrated that the survival of spheroids in drug-resistant gastric cancer cell lines was inhibited after knockdown of HOXB9. Figure 3 These findings also indicate that gastric cancer cells undergo oxaliplatin treatment, which induces greater expression of HOXB9, and that the absence of HOXB9 significantly enhances the sensitivity of drug-resistant gastric cancer cell lines to oxaliplatin.

[0084] 4. HOXB9 Regulation of Follicle Adhesion Signaling Pathway in Gastric Cancer Cells. To reveal how HOXB9 affects the progression of drug resistance in gastric cancer, we further performed RNA-seq sequencing analysis on gene-edited gastric cancer cells AGS HOXB9 and AGS Vector. Furthermore, as previously observed, we found that the expression level of HOXB9 in drug-resistant gastric cancer cell lines gradually increased with increasing drug resistance. Therefore, we knocked down HOXB9 in AGS OR cells, combined with HOXB9-overexpressing AGS OR cells and control cells, to investigate the effect of persistently elevated HOXB9 levels in drug-resistant gastric cancer cell lines. Sequencing analysis of AGS HOXB9 cells and control cells, HOXB9-overexpressing AGS OR, HOXB9-knocked AGS OR, and control drug-resistant cell lines, comparing gene expression differences between AGS OR cells and parental AGS PC cells, and KEGG enrichment analysis revealed increased expression of related molecules in AGS OR cells, including ECM-receptor interaction, Focal adhesion, and cell adhesion molecules. In AGS HOXB9 cells, compared to control cells, related molecules in the Focal adhesion and ECM-receptor interaction subsets were also shown to be activated. Figure 4 A). Intersection analysis of the differentially expressed genes between the two groups revealed 2497 differentially expressed genes. Further intersection analysis of the upregulated differentially expressed genes between the two groups revealed 880 differentially expressed genes that were simultaneously upregulated. Figure 4 B). KEGG enrichment analysis of the upregulated co-expressed genes revealed that these upregulated genes were significantly enriched in the Focal adhesion pathway. Figure 4 C). We also verified at the RNA expression level that increased HOXB9 expression in gastric cancer cells can regulate the upregulation of multiple focal adhesion signaling molecules. This upregulation of focal adhesion signaling molecules also occurred in drug-resistant gastric cancer cell lines. Furthermore, knockdown of HOXB9 in drug-resistant gastric cancer cell lines significantly downregulated these focal adhesion signaling pathway molecules. Figure 4 D). Key molecules in the focal adhesion signaling pathway function through the phosphorylated form of FAK. Therefore, we examined the expression levels of FAK and p-FAK proteins. The results showed that p-FAK expression was significantly increased in parental AGS HOXB9 cells. Furthermore, increased expression of HOXB9 in drug-resistant gastric cancer cell lines also significantly increased p-FAK expression. Figure 4 E). Furthermore, when HOXB9 was knocked down in drug-resistant gastric cancer cell lines, p-FAK expression was also inhibited. Figure 4 F). Immunofluorescence detection of tumor tissues obtained from tumor-bearing mice revealed that the fluorescence intensity of p-FAK in tumor tissues formed by AGSHOXB9 cells was significantly higher than that in the control group, and the expression of F-actin was also higher in the control group. Similar phenomena were observed in the tissues of treated mice. Figure 4 G). Simultaneously, cell assays revealed that the fluorescence intensity of p-FAK in drug-resistant gastric cancer cell lines was higher than that in parental cells both before and after oxaliplatin treatment. Figure 4 H). In AGS HOXB9 cells, we found that the fluorescence intensity of p-FAK was also significantly upregulated compared to control cells, and there was more F-actin formation. After oxaliplatin treatment, p-FAK and F-actin in AGS HOXB9 cells remained at higher levels than in control cells. Figure 4 I). Follicles of adhesion can fix cytoskeletal proteins and stabilize cell morphology. In gastric cancer cells treated with oxaliplatin, the cytoskeleton is disrupted, and the cells lose their normal morphology. However, gastric cancer cells overexpressing HOXB9 and drug-resistant gastric cancer cell lines exhibit relatively stable cytoskeletal structure and cell morphology. This also indicates that HOXB9 can regulate the focal adhesion signaling pathway in gastric cancer cells and increase p-FAK expression, stabilizing the cytoskeletal morphology of cells after oxaliplatin treatment.

[0085] 5. Inhibition of p-FAK enhances the sensitivity of HOXB9-overexpressing gastric cancer cells to oxaliplatin. We investigated whether HOXB9-dependent p-FAK activation contributes to oxaliplatin resistance. Gastric cancer cells overexpressing HOXB9 were treated with a FAK inhibitor (Y15) in combination with oxaliplatin. Clonogenesis assays showed that inhibition of FAK phosphorylation increased the sensitivity of gastric cancer cells to oxaliplatin, with no significant difference compared to the control group. Oxaliplatin-only treatment suppressed the resistance exhibited by HOXB9-overexpressing gastric cancer cells. Figure 5A, B). Besides the changes in gastric cancer cell sensitivity under continuous oxaliplatin treatment, we considered the effectiveness of 24-hour treatment. Flow cytometry analysis revealed that gastric cancer cells treated with FAK inhibitors showed increased sensitivity to oxaliplatin, and the resistance of gastric cancer cells overexpressing HOXB9 to oxaliplatin was also inhibited. Figure 5 B, C). Detection revealed that gastric cancer cells overexpressing HOXB9 activated p-FAK, which further activated the PI3K / AKT signaling pathway. The expression level of p-AKT was significantly increased in gastric cancer cells overexpressing HOXB9. Figure 5 D). The content of p-AKT in gastric cancer cell lines was significantly reduced after HOXB9 knockdown (D). Figure 5 E). Furthermore, the increase in p-AKT expression in gastric cancer cells overexpressing HOXB9 was suppressed upon the addition of a FAK inhibitor. Figure 5 F). This also indicates that gastric cancer cells upregulate p-FAK expression through increased HOXB9 expression, further upregulate p-AKT, and activate the PI3K / AKT signaling pathway, thereby increasing the resistance of gastric cancer cells to oxaliplatin treatment.

[0086] 6. Gastric cancer organoid models showed that HOXB9 affects the survival of gastric cancer organoids, and targeting HOXB9 can improve the sensitivity of oxaliplatin-resistant gastric cancer organoids to oxaliplatin. We successfully established 6 gastric cancer organoid models, including 2 gastric cancer organoids derived from gastric cancer tissue samples obtained after oxaliplatin treatment. After successful primary culture, mature gastric cancer organoids were obtained through passage, and observed and imaged under an optical microscope. Combined with HE staining, preliminary identification of the source gastric cancer tissue samples and corresponding gastric cancer organoids was performed. Figure 6 A). Simultaneously, we further identified these gastric cancer organoids using Ki67 immunohistochemical staining ( Figure 6 B). Subsequently, STR genotyping was performed. We examined the expression of HOXB9 at the RNA level in these gastric cancer organoids and found that the expression level of HOXB9 in gastric cancer organoids treated with drugs was higher than that in untreated gastric cancer organoids. Figure 6 C). The IC50 of oxaliplatin in these gastric cancer organoids was measured, and the results showed that the IC50 of the gastric cancer organoids significantly increased after drug treatment. Figure 6 D). We constructed gastric cancer organoids overexpressing HOXB9 from P1 and P4, and performed oxaliplatin IC50 assays. We found that the gastric cancer organoids overexpressing HOXB9 had reduced sensitivity to oxaliplatin. Figure 6E, G). Considering whether continuous drug treatment also affects efficacy, we subjected gastric cancer organoids to continuous oxaliplatin treatment for two weeks. We found that some gastric cancer cells overexpressing HOXB9 remained after continuous treatment, and these organoids transformed from cystic to solid structures. Figure 6 F, H). Cell viability assays also confirmed that the survival rate of gastric cancer organoids overexpressing HOXB9 was higher than that of the control group. Figure 6 I). Furthermore, we constructed a HOXB9-knockdown organoid model of gastric cancer organoids after PC-P1 and PC-P2 drug treatment, and performed oxaliplatin IC50 assays. The results showed that the HOXB9-knockdown organoids exhibited enhanced sensitivity to oxaliplatin. Figure 6 J, L). Gastric cancer organoids with HOXB9 knockdown were subjected to continuous oxaliplatin treatment for two weeks. It was found that the control group had more residual organoids after continuous treatment, and some tended towards solid organoids, while the gastric cancer organoids with HOXB9 knockdown were more easily broken down and dissolved by the drug. Figure 6 K, M). Cell viability assays also confirmed that the cell survival rate of gastric cancer organoids with HOXB9 knockdown was reduced. Figure 6 N). Tumor organoid models are more similar to the actual state of human tumors, and the above phenomena also show that changes in HOXB9 expression affect the sensitivity of gastric cancer tissue to oxaliplatin. Inhibiting HOXB9 can significantly improve the sensitivity of gastric cancer tissue to oxaliplatin, and targeting HOXB9 in gastric cancer tissue that has developed oxaliplatin resistance can effectively reduce its resistance to oxaliplatin, thus providing a new treatment strategy for gastric cancer treatment and chemotherapy resistance in gastric cancer.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of HOXB9 expression inhibitors in the preparation of drugs to reverse oxaliplatin resistance in gastric cancer; wherein the HOXB9 expression inhibitor is a molecule that inhibits the expression or function of the HOXB9 gene; wherein the HOXB9 expression inhibitor is a nucleic acid molecule, and the nucleic acid molecule is shRNA targeting the HOXB9 gene; the target sequence of the shRNA is selected from: SEQ ID NO: 1: GGCAAAGAGTAAAGATTAA; SEQ ID NO:2:GGCTAGAAAGTACAAGAAA.

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