Drug-resistant breast cancer cells and their applications

Abstract

This invention relates to drug-resistant breast cancer cells and their applications, belonging to the field of tumor cell technology. The drug-resistant breast cancer cells of this invention are human mammary ductal carcinoma cells T47DR and / or human breast cancer cells MCF7R. The human mammary ductal carcinoma cells T47DR were deposited at the Guangdong Provincial Microbial Culture Collection Center on October 25, 2024, with accession number GDMCC No: 65348; the human breast cancer cells MCF7R were deposited at the same center on October 25, 2024, with accession number GDMCC No: 65347. This invention demonstrates that the clonogenic ability, migration ability, anti-apoptotic ability, and spheroidization ability of drug-resistant breast cancer cells are significantly enhanced. They can form tumors independently of estrogen and metastasize throughout the body in a short period of time. The tumor stemness of the drug-resistant cells is enhanced, and they exhibit resistance to apelexifen and / or tamoxifen.

Description

Technical Field

[0001] This invention relates to the field of tumor cell technology, and more particularly to drug-resistant breast cancer cells and their applications. Background Technology

[0002] Breast cancer is the uncontrolled proliferation of mammary epithelial cells and is the most common malignant tumor in women. Conventional treatment strategies for breast cancer include surgical resection, endocrine therapy, radiotherapy and chemotherapy, targeted therapy, and immunotherapy. However, approximately 20% of patients develop metastasis after treatment, and the 5-year survival rate is less than 30%.

[0003] Based on molecular and histological characteristics, breast cancer can be classified into three types: estrogen receptor (ER+) positive or progesterone receptor (PR+) positive, human epidermal receptor 2 (HER2+) positive, and triple-negative (TNBC) (ER- / PR- / HER2-). Hormone receptor-positive breast cancer is the most common, accounting for 70% of all breast cancer patients. The cancer cells in this type of patient exhibit strong estrogen dependence; therefore, endocrine therapy is the primary treatment for these patients, including estrogen antagonists such as tamoxifen and aromatase inhibitors that inhibit estrogen production. However, more than 30% of early-stage ER+ breast cancer patients relapse within 15 years after receiving adjuvant tamoxifen therapy, and approximately 20% of patients receiving aromatase inhibitor therapy relapse within 9 years. About 35% of patients develop endocrine therapy resistance within 15 years. Therefore, overcoming endocrine therapy resistance and prolonging patient survival has become a pressing issue in the clinical diagnosis and treatment of ER+ breast cancer patients.

[0004] Alpelisib (BYL719) is a selective intracellular phosphatidylinositol kinase (PI3K) inhibitor, approved by the FDA in 2019 for the treatment of patients with luminal advanced breast cancer harboring phosphatidylinositol 3-kinase α (PIK3CA) mutations. However, due to the continuous evolution of tumor cells, drug resistance is inevitable. PIK3CA gene mutations frequently occur in cancer, especially in ER-positive breast cancer patients with a mutation rate as high as 40%. Clinical trials have shown that apelisib, whether used as monotherapy or in combination with other drugs, achieves good efficacy in patients with PIK3CA-mutated solid tumors. However, many factors, such as the deletion of the phosphatase and tensin homologous gene (PTEN), persistent activation of upstream receptor tyrosine kinases, systemic glucose-insulin feedback, and upregulation of downstream PI3K and mitogen-activated protein kinase (MAPK) signaling pathways, may limit the therapeutic effect of apelisib. Therefore, in-depth exploration of the molecular mechanisms of apelips resistance in breast cancer and screening for key genes related to apelips resistance can help overcome the current situation of apelips resistance in breast cancer patients, improve treatment outcomes, and prolong patient survival.

[0005] Studies have shown that cancer stem cells (CSCs) are a subset of cells with strong self-renewal and differentiation capabilities found in various solid tumors, including breast cancer. They are significantly associated with tumorigenesis and recurrence, and are a major cause of metastasis, recurrence, and drug resistance in breast cancer. Because the specific mechanisms regulating CSC generation and proliferation remain unclear, there are currently no effective methods to eradicate CSCs. Whether resistance to the PI3K inhibitor apelelis is related to the stemness of breast cancer CSCs is also unclear. Therefore, in-depth investigation into the crucial role of CSCs in apelelis resistance in breast cancer is essential for elucidating the molecular mechanisms of apelelis resistance and developing new strategies for the prevention and treatment of breast cancer drug resistance.

[0006] However, there is currently a lack of breast cancer cell lines resistant to apelelis, making it difficult to construct relevant resistance models to further elucidate the molecular mechanisms of breast cancer drug resistance and to find treatment strategies to reverse apelelis resistance. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide drug-resistant breast cancer cells resistant to apelelis and / or tamoxifen and their applications.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a drug-resistant breast cancer cell, wherein the drug-resistant breast cancer cell is a human mammary duct carcinoma cell T47DR and / or a human breast cancer cell MCF7R. The human mammary duct carcinoma cell T47DR was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2024, with accession number GDMCC No: 65348; and the human breast cancer cell MCF7R was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2024, with accession number GDMCC No: 65347.

[0010] The drug-resistant breast cancer cells constructed in this invention can be further developed into cell and animal models to provide a reliable model for understanding the molecular mechanisms of breast cancer resistance to apelips and / or tamoxifen and for finding therapeutic strategies to reverse resistance.

[0011] Secondly, the present invention provides the application of the drug-resistant breast cancer cells in the preparation of drug-resistant breast cancer tumor models.

[0012] Furthermore, the drug-resistant breast cancer tumor model includes a drug-resistant breast cancer mouse model and / or a drug-resistant breast cancer in vitro cell model.

[0013] In a specific embodiment of the present invention, the drug-resistant breast cancer mouse model includes a drug-resistant in situ tumorigenesis mouse model and / or a drug-resistant tumor metastasis mouse model.

[0014] Thirdly, the present invention provides a reagent for preparing a drug-resistant breast cancer tumor model, wherein the reagent contains the drug-resistant breast cancer cells.

[0015] Fourthly, the present invention provides a method for preparing a drug-resistant breast cancer mouse model, wherein the drug-resistant breast cancer cells or the reagent are injected into the mouse to induce tumor formation.

[0016] In a specific embodiment of the present invention, the drug-resistant breast cancer cells or the reagent are injected into the breast pads of mice to obtain a mouse drug-resistant in situ tumorigenesis model.

[0017] In a specific embodiment of the present invention, the drug-resistant breast cancer cells or the reagent are injected into the atrium or tail vein of a mouse to obtain a mouse model of drug-resistant tumor metastasis.

[0018] Furthermore, each mouse was injected with 10 5 ~5×10 5 One drug-resistant breast cancer cell.

[0019] In a specific embodiment of the present invention, 5 × 10⁵ mg / mL was injected into each mouse. 5 Using a sample of drug-resistant breast cancer cells as an example, a corresponding mouse model of drug-resistant tumor metastasis can be obtained. The drug-resistant breast cancer cells can be resuspended in PBS and injected.

[0020] In a specific embodiment of the present invention, the mouse breed can be a balbc / nu mouse.

[0021] Fifthly, the present invention provides a method for preparing an in vitro cell model of drug-resistant breast cancer, wherein the drug-resistant breast cancer cells are passaged and cultured.

[0022] Furthermore, the culture medium used can be a complete culture medium containing serum.

[0023] Furthermore, the serum used was fetal bovine serum, and the culture medium used was DMEM medium.

[0024] Furthermore, the drug-resistant breast cancer cells were cultured at 36.3–37.3 °C.

[0025] Furthermore, the fetal bovine serum was subcultured in DMEM medium containing 8–15% (v / v) fetal bovine serum in an incubator at 37°C, 5% CO2, and 95% humidity, with the fetal bovine serum content preferably being 10% (v / v).

[0026] Furthermore, the resistance is to apelis and / or tamoxifen.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] To elucidate the molecular mechanism of apelelis resistance in breast cancer, this invention established a drug-resistant breast cancer cell model and demonstrated that the clonogenic ability, migration ability, anti-apoptotic ability, and spheroidization ability of drug-resistant breast cancer cells were significantly enhanced. Simultaneously, an in vivo mouse tumor model was obtained, demonstrating that drug-resistant cells can form tumors independently of estrogen and metastasize throughout the body in a short period. Molecular experimental results indicate enhanced tumor stemness in drug-resistant cells. Furthermore, the drug-resistant cells of this invention exhibit resistance to apelelis and / or tamoxifen. The drug-resistant cells and established model of this invention can provide new theoretical basis for elucidating apelelis and / or tamoxifen resistance in breast cancer and provide potential therapeutic targets for reversing apelelis and / or tamoxifen resistance.

[0029] In addition, this invention found that serum glucocorticoid kinase 3 (SGK3) is upregulated in apeleliximab-resistant breast cancer cells, and confirmed that SGK3 regulates breast cancer stemness through the GSK3β / β-catenin signaling pathway, thereby promoting cell resistance to apeleliximab. Attached Figure Description

[0030] Figure 1To construct apelelix (BYL719) resistant breast cancer cells and identify related features. A shows the effect of BYL719 on the AKT signaling pathway in T47D and MCF7 cells; B shows a morphological comparison between parental and resistant cells; C shows the IC50 of parental MCF7 and MCF7R cells against BYL719. 50 Value; D represents the IC50 value of parental T47D cells and T47DR cells against BYL719. 50 value.

[0031] Figure 2 This study aimed to analyze the biological characteristics of apeleliximab-resistant breast cancer cells in vitro. The assay included: A) plate colony formation assay; B) Transwell cell migration assay; C) Annexin V-FITC / PI double staining assay; and D) 3D tumor stem cell spheroidization assay.

[0032] Figure 3 This study aimed to analyze the biological characteristics of apelexilis-resistant breast cancer cells in vivo. A shows the mouse mammary pad in situ tumorigenesis model protocol; B shows tumor images of the mouse mammary pad in situ tumorigenesis model without additional estrogen supplementation; C shows in vivo imaging of a mouse ventricular injection hematogenous tumor metastasis model established using parental MCF7 cells and resistant MCF7R cells; D shows in vivo imaging of a mouse ventricular injection hematogenous tumor metastasis model established using parental T47D cells and resistant T47DR cells; E shows survival analysis of a mouse ventricular injection hematogenous tumor metastasis model established using parental MCF7 cells and resistant MCF7R cells; and F shows survival analysis of a mouse ventricular injection hematogenous tumor metastasis model established using parental T47D cells and resistant T47DR cells.

[0033] Figure 4 This section presents the expression of SGK3 in apelexilis-resistant breast cancer cells. A shows the RNA map of upregulated SGK3 expression in resistant cells; B shows the Kaplan-Meier curves depicting intraluminal breast cancer patients with high and low SGK3 scores; C shows the RNA expression levels of SGK3 and tumor stemness-related markers in parental and resistant cells; D shows the proportion of CD24- / CD44+ breast stem cells in parental and resistant cells; E shows the tumor spheroid formation ability of parental and resistant cells; F shows the protein levels of CD44 and c-Myc detected by Western blot; G shows the GO enrichment analysis of differentiation-expressing genes in intraluminal breast cancer patients with high and low SGK3 expression; H shows the detection of total phosphorylated SGK3, GSK3β, and β-catenin by Western blot; I shows the protein levels of β-catenin in the cytoplasm and nucleus by Western blot; and J shows the localization of β-catenin detected by immunofluorescence staining. Unpaired t-test; *p<0.05, **p<0.01, ***p<0.001.

[0034] Figure 5 This shows the dose-response curves of tamoxifen in parental and resistant cells. A represents MCF7R cells; B represents T47DR cells. Detailed Implementation

[0035] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0036] Example 1: Construction and Identification of BYL719 Drug-Resistant Breast Cancer Cells

[0037] I. Experimental Methods

[0038] 1. Drug resistance culture: T47D (PIK3CA gene H1047R mutation, Allele Registry ID: CA123326, alias: HIS1047ARG, RS121913279) and MCF7 (PIK3CA gene E545K mutation, Allele Registry ID: CA123334, alias: GLU545LYS, RS104886003) human breast cancer cell lines with PIK3CA hotspot mutations were used to culture BYL719 for drug resistance.

[0039] T47D and MCF7 cells were cultured in DMEM medium (containing various amino acids and glucose, developed based on MEM medium) with 10% (v / v) FBS (fetal bovine serum) added. The initial concentration of BYL719 was 0.8 μM. The cells were cultured in an incubator at 37°C, 5% CO2, and 95% humidity. After the cells reached stable growth at this concentration, they were cultured until the cell density reached over 90%. The cells were passaged and cryopreserved once. When the cells reached 70-90% confluence, medium containing a high concentration of apelips was added, and the cells were cultured for another 3-4 days until the cells reached stable growth at this concentration. The above steps were repeated until the apelips concentration reached 10 μM, resulting in a drug-resistant breast cancer cell line, i.e., a luminal-type drug-resistant breast cancer cell line induced by BYL719.

[0040] 2. Identification

[0041] (1) Changes in downstream genes of PI3K after treatment with BYL719: Different concentrations of BYL719 (0, 0.082, 0.741, 0.247, 2.222, 6.227, 20 and 60 μM) were added to parental cells (T47D and MCF7 cells). After culturing for 4 h, the protein was collected, and the phosphorylation levels of PI3K downstream phosphorylated activated ribosomal S6 protein (S6) and protein kinase B (AKT) were detected by Western blotting (WB).

[0042] (2) Cell morphology observation

[0043] Parental cells (T47D and MCF7 cells) and BYL719 resistant cells (T47DR and MCF7R cells) were observed under an electron microscope, photographed under 10x magnification, and their morphology was recorded.

[0044] (3) Detection of IC50 of apeleli in drug-resistant cells and parental cells. 50 Parental cells (T47D and MCF7 cells) and BYL719-resistant cells (T47DR and MCF7R cells) were digested into single-cell suspensions and evenly seeded in 96-well plates at a concentration of 2000 cells / 100 μL / well. After cell adhesion, different concentrations of BYL719 (0, 0.082, 0.741, 0.247, 2.222, 6.227, 20, and 60 μM) were added. The culture medium was DMEM containing 10% (v / v) FBS. The cells were cultured in an incubator at 37°C, 5% CO2, and 95% humidity for 48 h. Then, CCK-8 reagent was added, and the cells were incubated in a cell culture incubator for 2 h. The absorbance was measured using a microplate reader, and dose-response curves were plotted in Graphpad software. The IC50 was calculated. 50 value.

[0045] II. Experimental Results

[0046] 1. With increasing BYL719 concentration, the inhibitory effect of BYL719 on phosphorylated pS6 and AKT became stronger, and the expression of apoptosis markers also increased with BYL719 concentration, indicating that BYL719 significantly inhibited the PI3K / AKT signaling pathway in breast cancer cells and promoted apoptosis. Figure 1 A).

[0047] 2. When breast cancer develops resistance to apelelis, its cell morphology undergoes significant epithelial-mesenchymal transition, with reduced adhesion between resistant cells and a decrease in cell aggregation. Figure 1 B).

[0048] 3. CCK-8 assay confirmed the successful establishment of apeleliximab-resistant breast cancer cells, among which MCF7R showed an IC50 response to apeleliximab. 50The IC of T47DR reaches 19.35μM. 50 Reaching 16.66 μM, which is higher than the parental cell IC50. 50 Increased by more than 10 times ( Figure 1 C and Figure 1 D).

[0049] The above results indicate that BYL179 can significantly inhibit the PI3K / AKT signaling pathway in breast cancer cells and induce apoptosis; this invention successfully constructed apelexix-resistant breast cancer cells T47DR and MCF7R.

[0050] The obtained apelexilis-resistant breast cancer cell line T47DR was named human breast duct carcinoma cell line T47DR and deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2024, with accession number GDMCCNo: 65348. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0051] The obtained apelexilis-resistant breast cancer cells MCF7R were named human breast cancer cells MCF7R and deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2024, with accession number GDMCCNo: 65347. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0052] Example 2: In vitro experimental analysis of the biological characteristics of apelexilis-resistant breast cancer cells

[0053] I. Experimental Methods

[0054] 1. Plate colony formation experiment

[0055] The parental cells and drug-resistant cells from Example 1 were digested into single-cell suspensions and evenly spread in 6-well plates at a concentration of 1000 cells / 2 mL / well. The culture medium was changed every 2 days, and the culture conditions were the same as in Example 1. The cells were photographed after 25 days of culture.

[0056] 2. Transwell cell migration experiment

[0057] Cell migration: Parental cell suspension and drug-resistant cells were seeded at a concentration of 5000 cells / well in Transwell chambers of 24-well plates and cultured for 8 hours. After 8 hours, the cells were fixed and photographed.

[0058] 3. Annexin V-FITC / PI double staining experiment

[0059] Flow cytometry: The single-cell suspension from step 1 was evenly spread in 6-well plates and cultured until the cell density reached 90%. 1 μM BYL719 was added, and after 4 h, the cells were digested and fixed. Annexin V-FITC (Annexin V labeled with FITC as a fluorescent probe, a reagent for detecting cell apoptosis) was added, followed by PI (propidium iodide) staining, flow cytometry analysis, and data plotting using FlowJO.

[0060] 4. 3D Tumor Stem Cell Spheroidization Assay: The parental cells and drug-resistant cells from Example 1 were resuspended at a density of 5000 cells / well in 1×B27 (human leukocyte antigen B27) serum-free DMEM-F12 (Dulbeccos Modified Eagle Medium / Nutrient Mixture F-12) medium containing 20 ng / mL basic fibroblast growth factor (bFGF) and 10 ng / mL epidermal growth factor (EGF) to obtain a cell suspension. The cells were then seeded into low-adhesion 24-well plates and cultured continuously for 21 days. The size and number of tumor spheroids were photographed and counted.

[0061] II. Experimental Results

[0062] 1. Plate colony formation experiments revealed that the size and number of colonies formed by apeleliximab-resistant cells exceeded those of the parental cells. Figure 2 A).

[0063] 2. Transwell cell migration assays also confirmed that the motility and migration ability of apeleliximab-resistant cells was significantly enhanced. Figure 2 B).

[0064] 3. Annexin V-FITC / PI double staining assay confirmed that the apoptotic cell ratio of apexilase-resistant cells was significantly lower than that of parental cells. Figure 2 C).

[0065] 4. Studies have shown that tumor cell stemness may be the main culprit for targeted drug resistance. 3D tumor stem cell spheroidization experiments confirmed that the size and number of tumor spheroids formed by apeleliximab-resistant cells significantly exceeded those formed by parental cells. Figure 2 D).

[0066] The above results indicate that the clonogenic ability, migration ability, anti-apoptotic ability, and spheroidization ability of apelexilis-resistant breast cancer cells are all significantly increased.

[0067] Example 3: In vivo experimental analysis of the biological characteristics of apelexilis-resistant breast cancer cells

[0068] I. Experimental Methods

[0069] 1. Establishing a mouse model of in situ tumor formation in mammary pads

[0070] Using 5-6 week old balbc / nu mice, 5×10 6 In Example 1, the parental cells and drug-resistant cells were resuspended in 0.1 mL of sterile phosphate-buffered saline (PBS) to obtain cell suspensions. After anesthetizing mice, the cell suspensions were injected in situ into the mammary fat pads of the mice.

[0071] When the tumor volume reaches 100mm 3 Mice were then randomly divided into groups and treated with either PBS or apegliflozin. During the experiment, mouse body weight and tumor volume were measured every two days. The experiment concluded when the largest tumor in any group reached 2000 mm. 3 Afterwards, all mice were euthanized, tumor samples were surgically separated, weighed, photographed, and recorded. Tumor tissue was partially preserved at -80°C, and some was fixed in formaldehyde and embedded in paraffin for storage.

[0072] 2. Establish a mouse model of hematogenous tumor metastasis via intraventricular injection.

[0073] Using 5-6 week old balbc / nu mice, 5×10 6 In Example 1, the parental and drug-resistant cells were resuspended in 0.1 mL of sterile PBS to obtain a cell suspension. Mice were anesthetized and the cell suspension was injected into the atrium of the ventricle using an insulin injection. Starting from week 2, mice were intraperitoneally injected with D-fluorescein potassium every 3 days, and in vivo imaging was performed to detect tumor cell metastasis within the mice. During the experiment, mouse mortality was recorded promptly for survival analysis.

[0074] II. Experimental Results

[0075] 1. Mouse mammary pad in situ tumorigenesis model: In mice without estrogen implants, only drug-resistant cells could form tumors, while parental cells did not grow tumors, indicating that the drug-resistant cells constructed in Example 1 of this invention exhibited enhanced stemness. Figure 3 A and Figure 3 B).

[0076] 2. Mouse model of hematogenous tumor metastasis via ventricular injection: Only drug-resistant cells showed systemic metastasis in mice. Imaging results showed metastasis in the brain, lungs, liver, and femur of the mice. Figure 3 C and Figure 3 D). Furthermore, mice injected with drug-resistant cells into the left ventricle quickly died. Figure 3 E and Figure 3 F).

[0077] Example 4: Upregulation of SGK3 in apegliflozin-resistant breast cancer cells

[0078] I. Experimental Methods

[0079] To investigate the resistance mechanism of apelips, RNA was extracted from four groups of cells in Example 1: parental cells (MCF7 and T47D) and drug-resistant cells (MCF7R / T47DR) for RNA-seq data analysis and experimental verification.

[0080] 1. RNA-seq sequencing analysis: Volcano plots were used to display differentially expressed genes, and GO and KEGG analyses were used to identify signaling pathways significantly enriched by differentially expressed genes.

[0081] 2. Experimental verification of the expression of differentially expressed genes and signaling pathways: RNA and proteins were extracted from the four groups of cells to verify the expression of significantly altered genes and key genes in the pathways. Immunofluorescence experiments were used to verify the expression changes of SGK3 and β-catenin: Climbing slides were placed at the bottom of 12-well plates, and then the resuspension of the four groups of cells was spread on the climbing slides. After the cell density reached 90%, the cells were fixed with formaldehyde, permeabilized, blocked, and incubated overnight with primary antibody. Then, fluorescent secondary antibody was added, and the cells were incubated at room temperature for 1 hour. After DAPI staining for 10 minutes, the climbing slides were removed, flipped onto glass slides, mounted, and photographed.

[0082] II. Experimental Results

[0083] 1. To identify key regulatory factors associated with apeleliximab resistance, the RNA profiles of parental and resistant cells in Example 1 were compared. SGK3 was found to be significantly upregulated in MCF7R and T47DR cells, indicating that SGK3 plays an important role in apeleliximab resistance. Figure 4 A).

[0084] In Kaplan-Meier survival analysis, high SGK3 expression was associated with shorter disease-free survival in patients with intraluminal breast cancer compared to cases with low SGK3 expression. Figure 4 B).

[0085] In addition, stemness-related markers of genes such as CD44 (a membrane-integrated protein), CD133 (a transmembrane glycoprotein), MYC (a proto-oncogene), OCT4 (octamer-binding transcription factor 4), NANOG (a homeobox protein), and TWIST1 (a basic helical-loop-helical transcription factor) were also highly expressed in MCF7R and T47DR cells. Figure 4 C).

[0086] Previous studies have shown that the stem cell-like nature of tumor cells is closely related to drug resistance. Therefore, flow cytometry was used to evaluate breast cancer stem cells. The results showed that, compared with parental cells, MCF7R and T47DR cells had a significantly higher proportion of CD24- (differentiation antigen cluster 24) / CD44+ cells. Figure 4 D).

[0087] Tumor spheroid formation experiments also confirmed that tumor spheroids formed by MCF7R and T47DR were significantly larger and more numerous than those formed by parental cells. Figure 4 E).

[0088] In drug-resistant cells, the levels of CD44 and c-Myc proteins were also significantly upregulated. Figure 4 F).

[0089] Therefore, the increased stem phenotype helps enhance the resistance of breast cancer cells to apelips.

[0090] 2. In order to find potential downstream signaling pathways regulated by SGK3, the RNA profiles of breast cancer patients with high and low SGK3 expression were compared.

[0091] Graphene oxide enrichment analysis showed that β-catenin binding was positively correlated with SGK3. Figure 4 G).

[0092] GSK3β / β-catenin is one of the known downstream pathways of SGK3 and plays a key role in mediating cancer development. Therefore, we wanted to verify whether the GSK3β / β-catenin pathway is activated in anti-alpelisib cells.

[0093] As expected, phosphorylated GSK3β (S9) and β-catenin (S552 and S675) were significantly increased in resistant cells. Figure 4 H).

[0094] Simultaneously, nuclear / cytoplasmic separation and immunofluorescence staining showed that the subcellular localization of β-catenin in drug-resistant cells shifted from the cell membrane to the cytoplasm and nucleus. Figure 4 I~J).

[0095] In summary, enhanced stemness and activated SGK3 / GSK3β / β-catenin pathway were found in the drug-resistant cells of Example 1.

[0096] Example 5: Resistance of Apelis-resistant breast cancer cells to tamoxifen.

[0097] I. Experimental Methods

[0098] The IC50 of tamoxifen in resistant cells and parental cells was detected according to the method in Example 1. 50 The concentrations of tamoxifen were 0, 0.082, 0.741, 0.247, 2.222, 6.227, 20 and 60 μM, respectively.

[0099] II. Experimental Methods

[0100] like Figure 5 A and Figure 5 As shown in B, MCF7R is his moxifen IC 50 The IC of T47DR reaches 24.67μM. 50 Reaching 26.26 μM, it was higher than the IC50 of the parental cells. 50 It has increased by more than 5 times.

[0101] Example 6: Preparation of a drug-resistant breast cancer tumor model

[0102] 1. Mouse model of drug-resistant in situ tumor formation

[0103] 5×10 6 The drug-resistant cells (T47DR and MCF7R cells) from Example 1 were resuspended in 0.1 mL of sterile PBS to obtain a cell resuspension. After anesthetizing the mice, the cells were injected in situ into the mammary fat pads of 5-6 week old Balbc / nu mice.

[0104] 2. Mouse model of drug-resistant tumor metastasis

[0105] 5×10 6 The drug-resistant cells (T47DR and MCF7R cells) from Example 1 were resuspended in 0.1 mL of sterile PBS to obtain a cell resuspension. After anesthetizing mice, the cells were injected in situ into the atrium or tail vein of 5-6 week old Balbc / nu mice.

[0106] 3. In vitro cell model of drug-resistant breast cancer

[0107] The drug-resistant cells (T47DR and MCF7R cells) from Example 1 were cultured in DMEM medium containing 10% (v / v) FBS and passaged in an incubator at 37°C, 5% CO2, and 95% humidity to serve as an in vitro cell model of drug-resistant breast cancer for various routine cell experiments.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A drug-resistant breast cancer cell line resistant to apelelis or tamoxifen, characterized in that, The drug-resistant breast cancer cells are human mammary ductal carcinoma cells T47DR, which were deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2024, with accession number GDMCC No: 65348.

2. A drug-resistant breast cancer cell line resistant to apelelis or tamoxifen, characterized in that, The drug-resistant breast cancer cells are human breast cancer cells MCF7R, which were deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2024, with accession number GDMCC No: 65347.

3. The use of the drug-resistant breast cancer cells according to claim 1 or 2 in the preparation of drug-resistant breast cancer tumor models resistant to apelelix or tamoxifen; The drug-resistant breast cancer tumor model is any one of the following: a mouse drug-resistant in situ tumorigenesis model, a mouse drug-resistant tumor metastasis model, or a drug-resistant breast cancer in vitro cell model.

4. A reagent for preparing a drug-resistant breast cancer tumor model resistant to apelelix or tamoxifen, characterized in that, The reagent contains the drug-resistant breast cancer cells as described in claim 1 or 2.

5. A method for preparing a mouse model of drug-resistant in situ tumor formation resistant to apelelix or tamoxifen, characterized in that, The drug-resistant breast cancer cells of claim 1 or 2 or the reagent of claim 4 were injected into the mammary pads of mice to obtain a mouse drug-resistant in situ tumorigenesis model.

6. A method for preparing a mouse model of drug-resistant tumor metastasis resistant to apelelix or tamoxifen, characterized in that, A mouse model of drug-resistant tumor metastasis was obtained by injecting the drug-resistant breast cancer cells of claim 1 or 2 or the reagent of claim 4 into the atrium of the ventricle of a mouse.

7. A method for preparing an in vitro cell model of drug-resistant breast cancer resistant to apelelix or tamoxifen, characterized in that, The drug-resistant breast cancer cells described in claim 1 or 2 are passaged.

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