Drug-resistant breast cancer cell and application thereof

Abstract

The invention relates to drug-resistant breast cancer cells and application thereof, and belongs to the technical field of tumor cells. The drug-resistant breast cancer cells are human breast duct cancer cells T47DR and / or human breast cancer cells MCF7R, the human breast duct cancer cells T47DR are preserved in Guangdong Microbial Culture Collection Center on October 25, 2024, and the preservation number of the human breast duct cancer cells T47DR is GDMCC No: 65348; the human breast cancer cell MCF7R is preserved in Guangdong Microbial Culture Collection Center on October 25, 2024, and the preservation number of the human breast cancer cell MCF7R is GDMCC No: 65347. The invention proves that the clone formation ability, migration ability, anti-apoptosis ability and balling ability of the drug-resistant breast cancer cells are all remarkably enhanced, tumor formation can be independent of estrogen, and whole-body metastasis occurs in a short time. The tumor stemness of the drug-resistant cells is enhanced, and the drug-resistant effect is achieved on the asperisib and / or tamoxifen.

Description

Technical Field

[0001] The invention relates to the technical field of tumor cells, in particular to drug-resistant breast cancer cells and applications thereof. Background Art

[0002] Breast cancer is a phenomenon in which breast epithelial cells proliferate uncontrollably, and its incidence ranks first among female malignant tumors. Conventional treatment strategies for breast cancer include surgical resection, endocrine therapy, radiotherapy and chemotherapy, targeted therapy, and immunotherapy. However, about 20% of patients still experience metastasis after treatment, and the 5-year survival rate is less than 30%.

[0003] According to molecular and histological characteristics, breast cancer can be divided into three categories, including estrogen receptor (ER+) positive or progesterone receptor (PR+) positive, human epidermal receptor 2 (HER2+) positive and triple negative (TNBC) (ER- / PR- / HER2-). Among them, hormone receptor positive breast cancer is the most common, accounting for 70% of all breast cancer patients. The growth of cancer cells in this type of patients is highly dependent on estrogen. Therefore, endocrine therapy is the main treatment for this type of patients, including estrogen antagonist tamoxifen, aromatase inhibitors that inhibit estrogen production, etc. However, more than 30% of early ER+ breast cancer patients relapse within 15 years after receiving adjuvant tamoxifen treatment, and about 20% of patients treated with aromatase inhibitors relapse within 9 years. About 35% of patients will show resistance to endocrine therapy within 15 years. Therefore, how to overcome endocrine therapy resistance and prolong patient survival has become an urgent problem to be solved in the clinical diagnosis and treatment of ER+ breast cancer patients.

[0004] Alpelisib (BYL719) is a selective intracellular phosphatidylinositol kinase (PI3K) inhibitor. It was approved by the US FDA in 2019 for the treatment of patients with luminal advanced breast cancer carrying phosphatidylinositol 3-kinase α (PIK3CA) mutations. However, due to the continuous evolution of tumor cells, the re-emergence of drug resistance is inevitable. PIK3CA gene mutations often occur in cancer, especially in patients with ER-positive breast cancer, where the mutation rate is as high as 40%. Clinical trials have shown that in patients with solid tumors with PIK3CA mutations, Alpelisib can achieve good therapeutic effects both as a single agent and in combination with other drugs. However, many factors, such as the loss 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 Alpelisib. Therefore, in-depth exploration of the molecular mechanism of apellisin resistance in breast cancer and screening of key genes associated with apellisin resistance will help overcome the current status of apellisin resistance in breast cancer patients, improve treatment outcomes, and prolong patient survival.

[0005] Studies have shown that cancer stem cells (CSCs) are a type of cell subpopulation with strong self-renewal and differentiation capabilities that exist in a variety of solid tumors such as breast cancer. They are significantly associated with tumor occurrence and recurrence, and are the main cause of breast cancer metastasis, recurrence and drug resistance. Because the specific mechanism regulating the generation and expansion of CSCs is still unclear, there is currently no effective method to eradicate CSCs. It is unclear whether resistance to the PI3K inhibitor apellis is related to the stemness of breast cancer CSCs. Therefore, in-depth exploration of the key role of CSCs in apellis resistance in breast cancer is very important to clarify the molecular mechanism of apellis resistance in breast cancer and to develop new strategies for the prevention and treatment of breast cancer resistance.

[0006] However, there is currently a lack of breast cancer cell lines that are resistant to apellis, making it difficult to construct relevant resistance models to further elucidate the molecular mechanism of breast cancer resistance and to find therapeutic strategies to reverse apellis resistance. Summary of the invention

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

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

[0009] In the first aspect, the present invention provides a drug-resistant breast cancer cell, which is a human ductal breast cancer cell T47DR and / or a human breast cancer cell MCF7R. The human ductal breast cancer cell T47DR was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 25, 2024, and its preservation number is: GDMCC No: 65348; the human breast cancer cell MCF7R was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 25, 2024, and its preservation number is: GDMCC No: 65347.

[0010] The drug-resistant breast cancer cells constructed by the present invention can be further constructed into cell models and animal models to provide reliable models for the molecular mechanism of breast cancer resistance to apellis and / or tamoxifen and for finding therapeutic strategies for reversing drug resistance.

[0011] In a second aspect, the present invention provides the use of the drug-resistant breast cancer cells in preparing a drug-resistant breast cancer tumor model.

[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 mouse drug-resistant in situ tumor formation model and / or a mouse drug-resistant tumor metastasis model.

[0014] In a third aspect, 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] In a fourth aspect, 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 mice 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 mammary pad of mice to obtain a drug-resistant in situ tumor model in mice.

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

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

[0019] In a specific embodiment of the present invention, 5×10 5 The corresponding mouse drug-resistant tumor metastasis model can be obtained by taking drug-resistant breast cancer cells as an example. The drug-resistant breast cancer cells can be resuspended in PBS for injection.

[0020] In a specific embodiment of the present invention, the mouse strain may be BALB / nu mice.

[0021] In a fifth aspect, the present invention provides a method for preparing an in vitro cell model of drug-resistant breast cancer, and subculturing the drug-resistant breast cancer cells.

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

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

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

[0025] Furthermore, subculture is performed in a DMEM medium containing 8-15% (v / v) fetal bovine serum in an incubator at 37° C., 5% CO 2 , and saturated 95% humidity, preferably with a fetal bovine serum content of 10% (v / v).

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

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] In order to clarify the molecular mechanism of apellis drug resistance in breast cancer, the present invention establishes a drug-resistant breast cancer cell model, and confirms that the cloning ability, migration ability, anti-apoptosis ability and spheroidization ability of drug-resistant breast cancer cells are significantly enhanced. At the same time, a mouse tumor model is obtained by in vivo experiments, confirming that drug-resistant cells can form tumors independently of estrogen, and systemic metastasis occurs in a relatively short period of time. The molecular experimental results show that the tumor stemness of drug-resistant cells is enhanced. At the same time, the drug-resistant cells of the present invention can have a drug resistance effect on apellis and / or tamoxifen. The drug-resistant cells of the present invention and the established model can provide a new theoretical basis for clarifying the drug resistance of apellis and / or tamoxifen in breast cancer, and provide a potential therapeutic target for reversing the drug resistance of apellis and / or tamoxifen.

[0029] In addition, the present invention found that serum glucocorticoid kinase 3 (SGK3) is upregulated in apellis-resistant breast cancer cells, and confirmed that SGK3 regulates breast cancer stemness through the GSK3β / β-catenin signaling pathway and thus promotes cell resistance to apellis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1To construct apellis (BYL719)-resistant breast cancer cells and related identification. A is the effect of BYL719 on the AKT signaling pathway of T47D and MCF7 cells; B is the morphological comparison of parental cells and resistant cells; C is the IC of parental MCF7 cells and MCF7R cells to BYL719 50 D is the IC value of parental T47D cells and T47DR cells to BYL719 50 value.

[0031] Figure 2 In vitro experiments were performed to analyze the biological characteristics of apellis-resistant breast cancer cells. A is a plate clone formation assay; B is a Transwell chamber migration assay; C is an Annexin V-FITC / PI double staining assay; and D is a 3D tumor stem cell sphere formation assay.

[0032] Figure 3 In vivo experiments were performed to analyze the biological characteristics of apellis-resistant breast cancer cells. A is a scheme for the mouse mammary pad orthotopic tumor model; B is a tumor photo of the mouse mammary pad orthotopic tumor model without additional estrogen supplementation; C is the in vivo imaging of the mouse ventricular injection hematogenous tumor metastasis model established by parental MCF7 cells and resistant MCF7R cells; D is the in vivo imaging of the mouse ventricular injection hematogenous tumor metastasis model established by parental T47D cells and resistant T47DR cells; E is the survival analysis of the mouse ventricular injection hematogenous tumor metastasis model established by parental MCF7 cells and resistant MCF7R cells; F is the survival analysis of the mouse ventricular injection hematogenous tumor metastasis model established by parental T47D cells and resistant T47DR cells.

[0033] Figure 4 Figure 2 is the expression of SGK3 in apellis-resistant breast cancer cells. A is the RNA profile of upregulated SGK3 expression in resistant cells; B is the Kaplan-Meier curve depicting patients with luminal breast cancer with high and low SGK3 scores; C is the RNA expression level of SGK3 and tumor stemness-related markers in parental cells and resistant cells; D is the ratio of CD24- / CD44+ breast stem cells in parental cells and resistant cells; E is the tumor sphere formation ability of parental cells and resistant cells; F is the Western blot detection of CD44 and c-Myc protein levels; G is the GO enrichment analysis of differentiation-expressed genes in patients with luminal breast cancer with high and low SGK3 expression; H is the Western blot detection of total phosphorylated SGK3, GSK3β and β-catenin; I is the Western blot showing the protein level of β-catenin in the cytoplasm and nucleus; J is the immunofluorescence staining to detect the localization of β-catenin. Unpaired t-test; *p<0.05, **p<0.01, ***p<0.001.

[0034] Figure 5 The dose-response curves of tamoxifen in parental cells and drug-resistant cells. A is MCF7R cells; B is T47DR cells. DETAILED DESCRIPTION

[0035] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0036] Example 1 Construction of BYL719 drug-resistant breast cancer cells and related identification

[0037] 1. Experimental Methods

[0038] 1. Drug-resistant culture Human breast cancer cell lines 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) with PIK3CA hotspot mutations were used for BYL719 resistance culture.

[0039] T47D and MCF7 cells were cultured with a medium supplemented with BYL719, and the medium was a DMEM medium (a medium containing various amino acids and glucose, developed on the basis of MEM medium) containing 10% (v / v) FBS (fetal bovine serum). The initial drug concentration of BYL719 was 0.8 μM, and the cells were cultured in an incubator at 37°C, 5% CO2, and 95% humidity. After the cells grew stably under this concentration of drug, they were cultured until the cell density was above 90%, and then passaged and frozen once. When the cells grew to 70-90%, a medium containing a high concentration of apellis was added, and the cells were cultured for 3-4 days until the cells grew stably under this concentration of drug. The above steps were repeated until the concentration of apellis reached 10 μM, and a breast cancer resistant cell line was obtained, that is, a luminal breast cancer resistant cell line induced by BYL719 was obtained.

[0040] 2. Identification

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

[0042] (2) Cell morphology observation

[0043] The parental cells (T47D and MCF7 cells) and BYL719 resistant cells (T47DR and MCF7R cells) were observed under an electron microscope, photographed under a 10x microscope, and their morphologies were recorded.

[0044] (3) Detection of IC of resistant cells and parental cells to apellis 50 :Parental cells (T47D and MCF7 cells) and BYL719-resistant cells (T47DR and MCF7R cells) were digested into single cell suspensions, respectively, and evenly plated in a 96-well plate at a concentration of 2000 cells / 100 μL / well. After the cells adhered, 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 medium containing 10% (v / v) FBS. The cells were cultured in a 37°C, 5% CO2, 95% humidity incubator for 48 h, and then CCK-8 reagent was added. The cells were incubated in a cell culture incubator for 2 h. The absorbance was detected by a microplate reader. The dose-effect curve was drawn in Graphpad software, and the IC 50 value.

[0045] 2. Experimental Results

[0046] 1. With the increase of BYL719 concentration, BYL719 has a stronger inhibitory effect on phosphorylated pS6 and AKT, and the expression of apoptosis markers also increases with the concentration of BYL719, indicating that BYL719 significantly inhibits the PI3K / AKT signaling pathway of breast cancer cells and promotes cell apoptosis ( Figure 1 A).

[0047] 2. When breast cancer becomes resistant to apellis, its cell morphology undergoes obvious epithelial-mesenchymal transition, the adhesion between resistant cells decreases, and the cells no longer grow in clusters ( Figure 1 B).

[0048] 3. CCK-8 confirmed that apellis-resistant breast cancer cells had been successfully established, and the IC of MCF7R to apellis 50The IC of T47DR reached 19.35 μM. 50 reached 16.66μM, which was higher than the parent cell IC 50 Improved by more than 10 times ( Figure 1 C and Figure 1 D).

[0049] The above results show that BYL179 can significantly inhibit the PI3K / AKT signaling pathway of breast cancer cells and induce cell apoptosis; the present invention successfully constructed apellis-resistant breast cancer cells T47DR and MCF7R.

[0050] The obtained apellisin-resistant breast cancer cells T47DR were named human breast ductal carcinoma cells T47DR, and were deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 25, 2024. The deposit number is: GDMCCNo: 65348, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

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

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

[0053] 1. Experimental Methods

[0054] 1. Plate colony formation experiment

[0055] The parental cells and drug-resistant cells in Example 1 were digested into single cell suspensions, respectively, and evenly plated in a 6-well plate at a concentration of 1000 cells / 2 mL / well. The culture medium was replaced every 2 days. The culture conditions were the same as in Example 1, and photos were taken after 25 days of culture.

[0056] 2. Transwell chamber migration assay

[0057] Cell migration: The parental cell suspension and drug-resistant cells were plated at a concentration of 5000 cells / well on the Transwell chamber of a 24-well plate and cultured. 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 a 6-well plate and cultured until the cell density reached 90%. 1 μM BYL719 was added, and the cells were digested and fixed after 4 hours. Annexin V-FITC (Annexin V labeled with FITC was used as a fluorescent probe, a reagent for detecting cell apoptosis) was added, and PI (propidium iodide) staining was performed. The cells were tested on the machine and the data were analyzed and plotted using flowjo.

[0060] 4. 3D tumor stem cell sphere experiment: The parental cells and drug-resistant cells in 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) culture medium containing 20ng / mL basic fibroblast growth factor (bFGF) and 10ng / mL epidermal growth factor (EGF) to obtain a cell suspension, and the cells were inoculated in a low-adhesion 24-well plate with the cell suspension and cultured for 21 consecutive days. Take pictures and count the size and number of tumor spheres.

[0061] 2. Experimental Results

[0062] 1. Through the plate cloning experiment, it was found that the clone size and number formed by the apellis-resistant cells exceeded that of the parental cells ( Figure 2 A).

[0063] 2. Transwell chamber migration assay also confirmed that the migration and motility of apellis-resistant cells was significantly enhanced ( Figure 2 B).

[0064] 3. Annexin V-FITC / PI double staining experiments confirmed that the apoptotic cell ratio of apellis-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 culprit for targeted drug resistance. The 3D tumor stem cell spheroid experiment confirmed that the size and number of tumor spheroids formed by apellis-resistant cells were significantly greater than those of parental cells ( Figure 2 D).

[0066] The above results show that the clone formation ability, migration ability, anti-apoptosis ability and sphere formation ability of apellisin-resistant breast cancer cells are significantly increased.

[0067] Example 3 In vivo experiments to analyze the biological characteristics of apellis-resistant breast cancer cells

[0068] 1. Experimental Methods

[0069] 1. Establishment of an in situ tumor model in mouse mammary pad

[0070] 5-6 week old BALB / nu mice were used and 5×10 6 The parental cells and drug-resistant cells in Example 1 were resuspended in 0.1 mL of sterile phosphate buffered saline (PBS) to obtain a cell resuspension, which was then injected in situ into the mammary fat pad of mice after anesthetizing the mice.

[0071] When the tumor volume reaches 100mm 3 Afterwards, the mice were randomly divided into groups and injected with PBS or apellis. During the experiment, the weight and tumor volume of the mice were measured every 2 days. 3 Afterwards, all mice were killed, tumor samples were surgically isolated, weighed, photographed and recorded. Some tumor tissues were stored at -80°C, and some were fixed with formaldehyde and embedded in paraffin for storage.

[0072] 2. Establishment of a mouse model of hematogenous tumor metastasis by intraventricular injection

[0073] 5-6 week old BALB / nu mice were used and 5×10 6 The parental cells and drug-resistant cells in Example 1 were resuspended in 0.1 mL of sterile PBS to obtain a cell resuspension, and the mouse was anesthetized and the cell resuspension was injected into the ventricle of the mouse with an insulin needle. From the second week, the mouse was intraperitoneally injected with D-luciferin potassium salt every 3 days and live imaging was performed to detect the metastasis of tumor cells in the mouse. During the experiment, the death of the mouse needs to be recorded in time for survival analysis.

[0074] 2. Experimental Results

[0075] 1. Mouse mammary pad in situ tumor model: In the absence of estrogen rods in mice, only resistant cells can form tumors, and parental cells do not grow tumors, indicating that the stemness of the resistant cells constructed in Example 1 of the present invention is enhanced ( Figure 3 A and Figure 3 B).

[0076] 2. Mouse ventricular injection hematogenous tumor metastasis model: Only drug-resistant cells metastasized throughout the body of the mouse. From the imaging results, it can be seen that metastasis occurred in the brain, lungs, liver, and femur of the mouse ( Figure 3 C and Figure 3 D) In ​​addition, mice injected with resistant cells in the left ventricle soon died ( Figure 3 E and Figure 3 F).

[0077] Example 4 SGK3 is upregulated in apellis-resistant breast cancer cells

[0078] 1. Experimental Methods

[0079] In order to study the drug resistance mechanism of Apellis, RNA was extracted from four groups of cells, namely, parental cells (MCF7 and T47D) and drug-resistant cells (MCF7R / T47DR) in Example 1, for RNA-seq data analysis and experimental verification.

[0080] 1. RNA-seq sequencing analysis: Use volcano plot to display differentially expressed genes, and GO and KEGG analysis to identify signal pathways where differentially expressed genes are significantly enriched.

[0081] 2. Experimental verification of the expression of differentially expressed genes and signal pathways: RNA and protein of the four groups of cells were extracted to verify the expression of significantly changed genes and key genes in the pathways. Immunofluorescence experiments were used to verify the expression changes of SGK3 and β-catenin: The slides were placed on the bottom of the 12-well plate, and then the resuspensions of the four groups of cells were spread on the slides. After the cell density reached 90%, the cells were fixed with formaldehyde, permeabilized, blocked, and the primary antibody was added overnight, followed by the fluorescent secondary antibody. After incubation at room temperature for 1 hour, DAPI staining was performed for 10 minutes, and the slides were taken out and spread on a glass slide. After sealing, the slides were photographed.

[0082] 2. Experimental Results

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

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

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

[0086] Previous studies have shown that the stemness of tumor cells is closely related to drug resistance. Therefore, breast cancer stem cells were evaluated by flow cytometry, and the results showed that the ratio of CD24- (differentiation antigen cluster 24) / CD44+ cells was significantly higher in MCF7R and T47DR cells compared with parental cells ( Figure 4 D).

[0087] Tumor sphere formation experiments also confirmed that the tumor spheres 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, CD44 and c-Myc protein levels were also significantly upregulated ( Figure 4 F).

[0089] Thus, the increased stemness phenotype contributes to the enhanced resistance of breast cancer cells to apellis.

[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 the occurrence of cancer. Therefore, we verified whether the GSK3β / β-catenin pathway was activated in alpelisib-resistant cells.

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

[0094] At the same time, nuclear / cytoplasmic separation and immunofluorescence staining showed that the subcellular localization of β-catenin in drug-resistant cells changed from cell membrane to 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 Apellis-resistant Breast Cancer Cells to Tamoxifen

[0097] 1. Experimental Methods

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

[0099] 2. Experimental Methods

[0100] like Figure 5 A and Figure 5 B shows that MCF7R has tamoxifen IC 50 The IC of T47DR reached 24.67 μM. 50 reached 26.26μM, which is higher than the IC 50 Increased by more than 5 times.

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

[0102] 1. Mouse drug-resistant orthotopic tumor model

[0103] 5×10 6 The drug-resistant cells (T47DR and MCF7R cells) in Example 1 were resuspended in 0.1 mL of sterile PBS to obtain a cell resuspension, which was then injected orally into the mammary fat pad of 5- to 6-week-old balbc / nu mice after anesthetizing the mice.

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

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

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

[0107] The drug-resistant cells (T47DR and MCF7R cells) in Example 1 were cultured separately with DMEM medium containing 10% (v / v) FBS, and subcultured 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 conventional cell experiments.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A drug-resistant breast cancer cell, characterized in that: The drug-resistant breast cancer cells are human ductal breast cancer cells T47DR and / or human breast cancer cells MCF7R. The human ductal breast cancer cells T47DR were deposited in Guangdong Provincial Microbiological Culture Collection Center on October 25, 2024, and their deposit number is: GDMCC No: 65348; the human breast cancer cells MCF7R were deposited in Guangdong Provincial Microbiological Culture Collection Center on October 25, 2024, and their deposit number is: GDMCC No: 65347.

2. Use of the drug-resistant breast cancer cells according to claim 1 in preparing a drug-resistant breast cancer tumor model.

3. The use according to claim 2, characterized in that 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.

4. The use according to claim 3, characterized in that The drug-resistant breast cancer mouse model includes a mouse drug-resistant in situ tumor formation model and / or a mouse drug-resistant tumor metastasis model.

5. A reagent for preparing a drug-resistant breast cancer tumor model, characterized in that: The reagent contains the drug-resistant breast cancer cells according to claim 1.

6. A method for preparing a drug-resistant breast cancer mouse model, characterized in that: The drug-resistant breast cancer cells according to claim 1 or the reagent according to claim 5 are injected into mice to induce tumor formation.

7. The method according to claim 6, characterized in that The drug-resistant breast cancer cells of claim 1 or the reagent of claim 5 are injected into the mammary pad of mice to obtain a drug-resistant in situ tumor model in mice.

8. The method according to claim 6, characterized in that The drug-resistant breast cancer cells according to claim 1 or the reagent according to claim 5 are injected into the ventricle of mice to obtain a mouse drug-resistant tumor metastasis model.

9. A method for preparing an in vitro cell model of drug-resistant breast cancer, characterized in that: The drug-resistant breast cancer cells according to claim 1 are subcultured.

10. The drug-resistant breast cancer cell according to claim 1, the use according to any one of claims 2 to 4, the reagent according to claim 5, and the method according to any one of claims 6 to 9, characterized in that: The drug resistance is resistance to apellis and / or tamoxifen.

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