Lobaplatin drug-resistant cell strain for triple negative breast cancer as well as construction method and application thereof

Abstract

The invention relates to the technical field of cell engineering, in particular to a lobaplatin drug-resistant cell strain for triple negative breast cancer, the cell strain is named as the lobaplatin drug-resistant cell strain SUM-159PT-LBP for triple negative breast cancer, the cell strain is preserved in China Center for Type Culture Collection (CCTCC) on May 7, 2025, and the preservation number is CCTCC NO: C2025150; the drug-resistant cell strain provided by the invention is constructed by adopting a lobaplatin concentration gradual increase method for induction and intermittent maintenance culture, and finally can stably grow and pass in a lobaplatin culture system with the action concentration of 5 mu M, and the drug-resistant index (RI) is 5.72. Besides, the drug-resistant cell strain can also be used for analyzing the biological phenotype and the drug-resistant mechanism of the triple negative breast cancer after drug resistance, screening drug-resistant markers and finding potential antitumor drugs, and has a scientific research transformation effect and a clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering, and in particular relates to a triple-negative breast cancer lobaplatin-resistant cell line and a construction method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is one of the most common and aggressive breast cancer subtypes, accounting for 10% to 15% of all cases. TNBC lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), resulting in high tumor heterogeneity and the potential for recurrence and drug resistance. The disease mechanism is complex and the prognosis is generally poor.

[0003] Currently, platinum-based chemotherapy remains the standard treatment for patients with TNBC. Our research team has conducted a series of prospective studies focusing on the neoadjuvant treatment of stage I-III TNBC. For example, the "docetaxel (T) + epirubicin (E) + lobaplatin (L)" (TEL) combined neoadjuvant chemotherapy study demonstrated that compared with the TE regimen, lobaplatin significantly improved the short-term efficacy of neoadjuvant therapy in TNBC patients, achieving an overall response rate of 93.5% (lobaplatin group) versus 73% (non-lobaplatin group). Subsequently, the combination of a taxane and lobaplatin was found to be more effective than a taxane combined with an anthracycline (41.2% versus 21.2%). Further studies have shown that the anti-angiogenic drug anlotinib combined with taxane and lobaplatin exhibited excellent antitumor activity, with a pathological complete response rate (pCR) of 57.8%. These studies demonstrate that lobaplatin offers a new option for the neoadjuvant treatment of TNBC.

[0004] However, in recent years, our research team has conducted retrospective analyses and found that nearly 23.5% of TNBC patients treated with neoadjuvant chemotherapy with lobaplatin developed drug resistance or even died, leading to treatment failure. Therefore, establishing lobaplatin-resistant cell lines is essential for studying the mechanisms of drug resistance.

[0005] So far, there has been no report on the establishment of lobaplatin-resistant human breast cancer cell lines using the human breast cancer cell line SUM-159PT as the induction object. Summary of the Invention

[0006] To this end, the main purpose of the present invention is to provide a triple-negative breast cancer lobaplatin-resistant cell line and its construction method and application.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A triple-negative breast cancer lobaplatin-resistant cell line, classified and named triple-negative breast cancer lobaplatin-resistant cell line SUM-159PT-LBP, was deposited in the China Center for Type Culture Collection (CCTCC) on May 7, 2025, with the deposit number CCTCC NO: C2025150, and the deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072.

[0009] The present invention also provides a method for constructing a triple-negative breast cancer lobaplatin-resistant cell line. The human breast cancer cell line SUM-159PT is used as the induction object, and SUM-159PT-LBP is constructed by inducing with a stepwise increase in lobaplatin concentration and intermittent maintenance culture.

[0010] In certain specific embodiments, the method for constructing the triple-negative breast cancer lobaplatin-resistant cell line as described above comprises the following steps:

[0011] S1: Lobaplatin induction, specifically, the induction method is as follows: add lobaplatin to the initial culture medium of the human breast cancer cell line SUM-159PT containing premium serum, wherein the initial concentration of lobaplatin is 0.05-0.25 μM, continue culturing, and maintain this drug concentration until the cells can grow stably. After the cell density reaches 80%-90%, perform 1:2 passage, and then use culture medium without lobaplatin to maintain the culture intermittently;

[0012] S2: After the cell density to be passaged reaches 80%-90%, the induction method described in step S1) is repeated, and the lobaplatin concentration is gradually increased until the cells can stably grow and be passaged in the culture medium containing 3-5 μM lobaplatin, thereby obtaining the target cell line;

[0013] S3: The target cell line is repeatedly cultured in a culture medium containing 2-5 μM lobaplatin, followed by at least two freezing and thawing treatments to ultimately obtain a stable cell line, which is a lobaplatin-resistant triple-negative breast cancer cell line;

[0014] Preferably, the method further comprises rinsing and centrifuging the cells before and after passage using PBS containing double antibodies.

[0015] Furthermore, the rinsing and centrifugation treatment is specifically as follows: rinsing twice with PBS containing 1% double antibody, adding 1 ml of 0.25% trypsin cell digestion solution (containing EDTA) to digest the cells for 2 minutes, stopping the digestion with 2 ml of culture medium, collecting the cell suspension in a 15 ml centrifuge tube, centrifuging at 800 rpm for 3 minutes, discarding the liquid, and then adding fresh culture medium to resuspend and place in a new T25 culture flask for subculture.

[0016] Preferably, the culture medium in step S1 contains 5-10% special grade fetal bovine serum.

[0017] Preferably, the culture conditions in step S1 are: culture at 37° C., 5% CO 2 , and 95% humidity;

[0018] Preferably, the gradually increasing initial concentrations of lobaplatin in step S2 are 0.5 μM, 0.75 μM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 2.75 μM, 3 μM, 3.25 μM, 3.5 μM, 3.75 μM, 4 μM, 4.25 μM, 4.5 μM, 4.75 μM, and 5 μM, respectively.

[0019] Preferably, the specific operations of the freezing and thawing treatment in step S3 are: freezing the target cell line in liquid nitrogen, then thawing and continuing to culture in a culture medium containing 2.5 μM lobaplatin for stable passage.

[0020] During the cell freezing and recovery process, adding 5ml of the specific nutrient L-Gluamine (100X, 2mM) to 500ml of incomplete culture medium can significantly increase the amino acid components required for cell growth, ensure that the newly recovered cells maintain the drug resistance concentration before freezing as much as possible, and enhance drug resistance stability.

[0021] During the construction of SUM-159PT-LBP, the SUM-159PT-LBP was obtained by inducing with a gradually increasing concentration of lobaplatin and maintaining the culture intermittently.

[0022] Specifically, the use of special-grade fetal bovine serum is very important for the stability of cell status and resistance to drug resistance, so as to avoid the weakening or disappearance of drug resistance in subsequent subculture.

[0023] Use PBS containing double antibodies to rinse to ensure that impurities and metabolic waste on the cell surface are washed away, apoptotic cells are removed in time, and the risk of contamination is reduced. The rinsing action should be gentle to avoid damaging fragile drug-resistant cell lines, and the number of times should be no less than 2.

[0024] The construction method described in the present invention is also applicable to the parental cells MDA-MB-231 or MDA-MB-453.

[0025] The present invention also provides an application of a triple-negative breast cancer lobaplatin-resistant cell line, which adopts the following technical solution:

[0026] As mentioned above, triple-negative breast cancer lopinavir-resistant cell lines are used in the study of the lopinavir resistance mechanism.

[0027] As described above, the triple-negative breast cancer lobaplatin-resistant cell line is used to prepare a drug-resistant model.

[0028] As described above, the triple-negative breast cancer lobaplatin-resistant cell line is used in screening drugs that reverse tumor resistance or anti-tumor drugs.

[0029] Compared with the prior art, the present invention has at least the following advantages:

[0030] 1) The SUM-159PT-LBP constructed by the present invention can stably grow and be passaged in a culture system with an effective concentration of 2.5 μM lobaplatin, with a resistance index (RI) of 5.72. This provides a basis for the study of the mechanism of lobaplatin resistance and the development of related targets. It also provides a drug-resistant cell model for studying the mechanism of lobaplatin resistance in human breast cancer cells, finding effective treatments to overcome human breast cancer resistance, preparing anti-tumor drugs, screening tumor resistance-reversing drugs, and guiding clinical drug use. The model plays an important role and has good application prospects.

[0031] 2) In the process of constructing SUM-159PT-LBP of the present invention, the human breast cancer cell line SUM-159PT is used as the induction object, and the induction is constructed by gradually increasing the concentration of lobaplatin and intermittently maintaining the culture. Specifically, a small-dose increasing culture method of lobaplatin is adopted to induce the human breast cancer cell line SUM-159PT to grow and pass through from a low concentration to a high concentration of lobaplatin; the cell state is restored in a culture medium without lobaplatin, and the induction cycle is repeated until the cells can be stably passaged and grown in a drug-containing culture medium to obtain a drug-resistant cell line; the cells before and after passaging are rinsed and centrifuged with PBS containing double antibodies, so that the cells can better adapt to the pressure during the drug resistance induction process, reduce contamination, promptly process cell debris and eliminate apoptotic cells, and improve the overall survival rate and stability of the drug-resistant cell line; the cell line constructed in the present application has good drug resistance to the target drug, and can stably maintain its drug resistance characteristics during long-term passage culture, and is not prone to loss of drug resistance.

[0032] 3) This application verifies the drug-resistant cell line by in situ transplantation into the breast cancer fat pad of mice to form an in vivo drug-resistant model, which can more realistically simulate clinical drug resistance and provide scientific ideas for studying the mechanism of lobaplatin resistance and reversing lobaplatin resistance. At the same time, the morphological and biological characteristics of resistant and sensitive tumor cells and the action patterns of lobaplatin in different TNBC are compared to broaden clinical diagnosis and treatment ideas and improve translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present invention, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art.

[0034] Figure 1 The morphology of SUM-159PT and SUM-159PT-LBP in a culture medium containing 3 μM effective concentration of lobaplatin was observed under a microscope (100×);

[0035] Figure 2 The growth pictures of SUM-159PT and SUM-159PT-LBP are shown below.

[0036] Figure 3 Growth curves of SUM-159PT and SUM-159PT-LBP;

[0037] Figure 4 Growth and invasion results of SUM-159PT and SUM-159PT-LBP (20×);

[0038] Figure 5 Cell apoptosis results of SUM-159PT and SUM-159PT-LBP (20×);

[0039] Figure 6 The results of the cell spheroidization ability of SUM-159PT and SUM-159PT-LBP are shown;

[0040] Figure 7 IC50 curves of SUM-159PT and SUM-159PT-LBP;

[0041] Figure 8 The results of the cell transplant tumor model of SUM-159PT and SUM-159PT-LBP are shown. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and embodiments, but it should not be understood as limiting the scope of protection of the present invention. Any formal equivalent transformation based on the concept of the present invention should be considered as the scope of the present invention.

[0043] This document provides general and / or specific descriptions of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods. All reagents and instruments used, unless the manufacturer is specified, are commercially available, conventional products and were prepared or used using conventional methods.

[0044] In the following examples, the chemical structural formula of lobaplatin used is:

[0045]

[0046] Lobaplatin, also known as lobaplatin, has the chemical name cis-(trans-1,2-cyclobutanebis(methylamine)-N,N′)-((2S)-lactic acid-O1,O2)-platinum (II), and the chemical structural formula is cis-((trans-1,2-cyclobutanebis(methylamine))((S)-lactic acid-O',O')platinum trihydrate, with the molecular formula C9H 18 N2O3Pt·3H2O, molecular weight is 451.38.

[0047] Structurally, lobaplatin is centered on the platinum ion and coordinated with the oxygen atoms of trans-1,2-cyclobutanedimethylamine and (S)-lactic acid to form a stable complex structure. This structure gives lobaplatin its unique anti-tumor activity.

[0048] The lobaplatin used in the following examples is a 2.5 mM lobaplatin solution (i.e., 1 mg / ml) prepared with sterile water for injection.

[0049] The DMEM incomplete culture medium, premium fetal bovine serum, PBS, 0.25% trypsin cell digestion solution (containing EDTA), double-streptomycin antibodies (penicillin and streptomycin), sterile water for injection, and mycoplasma preventive agent used; as well as the liquid nitrogen tank, fluorescence microscope, and clean bench used, can all be purchased through commercial channels.

[0050] The culture medium is as follows: 500 ml of incomplete DMEM medium is added with 5 ml of double antibiotics [penicillin (100 U / ml) / streptomycin (0.1 mg / ml)], 5 ml of L-Glutamine (100X, 2 mM), and 250 μl of mycoplasma preventive agent (2 mg / ml).

[0051] Example 1: Construction of SUM-159PT-LBP cell line

[0052] This embodiment provides a method for constructing a triple-negative breast cancer lobaplatin-resistant cell line, which comprises the following steps:

[0053] 1. Initial Culture: Human breast cancer cell line (abbreviated as SUM-159PT) was inoculated into a culture medium containing 10% premium fetal bovine serum in a T25 culture flask. Initial culture was performed in an incubator at 37°C, 5% CO2, and 95% humidity. After a period of incubation, when the cells were in good condition and had adapted to the culture environment, the initial culture medium was obtained.

[0054] 2. Lobaplatin induction

[0055] S1) Inducing the human breast cancer cell line SUM-159PT with lobaplatin: adding lobaplatin to an initial culture medium containing 10% premium serum (Gibco) at an initial concentration of 0.25 μM, culturing the cells and maintaining this concentration until the cells can grow stably. When the cell density reaches 80%, performing a 1:2 passage, and then continuing culturing in a culture medium without lobaplatin for one week, closely observing the cell growth status;

[0056] Before and after subculturing, cells were rinsed twice with PBS and digested with 1 ml of 0.25% trypsin cell digestion solution (containing EDTA) for 2 min. Digestion was terminated with 2 ml of complete culture medium. The cell suspension was collected in a 15 ml centrifuge tube, centrifuged at 800 rpm for 3 min, discarded, and resuspended in fresh complete culture medium. The cells were then placed in a new T25 culture flask for subculturing.

[0057] S2) After the cell density to be passaged reaches 80%, the induction method described in step S1) is repeated to induce, and the initial concentration of lobaplatin is changed to 0.5 μM, 0.75 μM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 2.75 μM, 3 μM, 3.25 μM, 3.5 μM, 3.75 μM, 4 μM, 4.25 μM, 4.5 μM, 4.75 μM, 5 μM, 6 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57 5 μM (i.e., after the induction with 0.25 μM lobaplatin, the cells obtained by induction with 0.25 μM lobaplatin are induced with 0.5 μM lobaplatin; after the induction with 0.5 μM lobaplatin, the cells obtained by induction with 0.5 μM lobaplatin are induced with 0.75 μM lobaplatin, and so on, until the concentration of lobaplatin reaches 5 μM), and the induction, medium replacement, and passaging are repeated until the cells can eventually grow and be passaged in a medium containing 5 μM lobaplatin to obtain the target cell line;

[0058] 3. The obtained target cell line was repeatedly cultured in a culture medium containing 5 μM lobaplatin, and then subjected to two freezing and thawing treatments. The specific operations of the freezing and thawing treatments were as follows: the target cell line was frozen in liquid nitrogen, then revived and continued to be cultured in a culture medium containing 5 μM lobaplatin for one week, and stably passaged. The cell line obtained at this time was a triple-negative breast cancer lobaplatin-resistant cell line; it was classified and named SUM-159PT-LBP, and was deposited in the China Center for Type Culture Collection (CCTCC) on May 7, 2025, with the preservation number CCTCC NO: C2025150.

[0059] Test Example 1: Morphological Observation of SUM-159PT-LBP Cell Line

[0060] Materials and test methods:

[0061] Place the cell culture flask on the stage of an inverted microscope and observe with objective lenses of different magnifications; first use a low-magnification (4×) and then a high-magnification (10×) objective lens (total magnification is 100) to observe the cells and take photos for record.

[0062] Results and Discussion

[0063] The results are as follows Figure 1 As shown, from Figure 1 As can be seen, SUM-159PT cells are stretched, uniform in size, and have a uniform intracellular structure; whereas, SUM-159PT-LBP cells are larger and irregular in shape. This is because drug-resistant cells have a strong ability to excrete drugs, a high number of transport proteins on the cell membrane, and may alter the expression and distribution of cytoskeletal proteins.

[0064] Test Example 2: Cell Growth Curve Assay (EDU Method)

[0065] Materials and test methods:

[0066] According to the instructions of EdU Apollo 567 in vitro cell proliferation imaging kit (CA1170, Solarbio), 1×10 5 Units of SUM-159PT and SUM-159PT-LBP were seeded in 6-well culture plates for 48 hours, and then the cells were incorporated with 5-ethynyl-2′-deoxyuridine (EdU) solution for 6 hours; the cells were fixed with 4% paraformaldehyde and then washed three times with 3% BSA; the cells were incubated with 0.3% TritonX-100 solution at room temperature for 15 minutes, and the Click reaction was performed using the Click-iT cell reaction buffer kit (C10269, Thermo Fisher Scientific Inc.); finally, Hoechst 33,342 staining dye (ab228551, Abcam) was diluted 1:1000 with 1× PBS, and 1× Hoechst33,342 was added to each well and incubated in the dark at room temperature for 10 minutes to stain the cell nuclei; after removing the wash solution, the plate was observed for fluorescence imaging.

[0067] Results and Discussion

[0068] The results are as follows Figure 2 and Figure 3 As shown, from Figure 2 and Figure 3 It can be seen that SUM-159PT cells grow relatively slowly, while SUM-159PT-LBP cells grow faster. This is because drug-resistant cells may undergo metabolic remodeling, increase nutrient utilization, activate signaling pathways related to cell proliferation, inhibit cell apoptosis, and improve cell survival and growth.

[0069] Test Example 3: Cell invasion assay

[0070] Materials and test methods:

[0071] SUM-159PT and SUM-159PT-LBP were cultured in serum-free medium and transferred to the apical chamber of a Transwell insert pre-coated with Matrigel. Medium containing 10% FBS was then added to the basolateral chamber. After 24 hours, the insert was carefully removed, and the cells in the basolateral chamber were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Finally, randomly selected fields of view were observed and recorded microscopically.

[0072] Results and Discussion

[0073] The results are as follows Figure 4 As shown, from Figure 4 It can be seen that SUM-159PT has weak invasive ability, while SUM-159PT-LBP has stronger cell invasiveness. This is because the resistant cells may activate epithelial-mesenchymal transition (EMT), remodel the extracellular matrix, and expose some signaling molecules that promote cell migration, further enhancing the invasive ability of the resistant cells.

[0074] Test Example 4: Cell apoptosis assay

[0075] Materials and test methods:

[0076] Lentivirus-infected SUM-159PT and SUM-159PT-LBP were fixed with paraformaldehyde for 25 minutes at 4°C. TNBC cell apoptosis was assessed by incubating cells with TUNEL detection reagent solution for 1 hour at 37°C according to the protocol provided by the Click-iTPlus TUNEL detection kit (C10617, Thermo Fisher Scientific Inc.).

[0077] Results and Discussion

[0078] The results are as follows Figure 5 As shown, from Figure 5 It can be seen that SUM-159PT cells are more susceptible to apoptosis after Loper treatment, while SUM-159PT-LBP cells are less susceptible to apoptosis. This is because drug-resistant cells may upregulate the expression of anti-apoptotic proteins and enhance their antioxidant capacity.

[0079] Test Example 5: Cell Spheroidization Experiment

[0080] Materials and test methods:

[0081] SUM-159PT and SUM-159PT-LBP were first seeded in spheroid culture medium at approximately 10,000 cells per well in Costar multiwell cell culture plates (3471, Corning Glass Works, Corning, NY, USA). The cells were incubated at 37°C with 5% CO2 to allow for the formation of tumor spheroids. After 10 days, the cells were imaged under an inverted microscope, and the spheroid formation efficiency (SFE) was calculated. SFE = number of spheroids with a diameter greater than 75 μm per well / total number of cells originally seeded per well.

[0082] Results and Discussion

[0083] The results are as follows Figure 6 As shown, from Figure 6 As can be seen, SUM-159PT forms fewer spheres, while SUM-159PT-LBP has enhanced sphere-forming ability. This is because the enhanced sphere-forming ability of drug-resistant cells is related to factors such as the maintenance of cell stemness, altered intercellular interactions, and the influence of the tumor microenvironment. These factors work together to give drug-resistant cells stronger sphere-forming ability, which helps them aggregate and form tumor spheres in suspension culture.

[0084] Test Example 6: Cell Drug Sensitivity Test

[0085] Materials and test methods:

[0086] The IC50 values of SUM-159PT and SUM-159PT-LBP were determined. Different concentrations (0, 1 nm, 2 nm, 3 nm, and 4 nm) of lobaplatin culture medium were added to establish a negative control group (containing the corresponding culture medium, cells, and CCK8), a blank control group (containing only the corresponding culture medium and CCK8, without cells), a PBS control group (primarily used to reduce edge effects and maintain a stable plate environment), and an experimental group (containing the corresponding culture medium, cells, CCK8, and lobaplatin). Cells were inoculated into 96-well plates, with five replicates and two time points. At each time point, the culture plates were removed, the stock solution was removed, and 100 μl of the prepared culture medium containing 10% CCK8 was added. The plates were incubated at 37°C for 2 h, and the absorbance at 450 nm was measured. The OD values were read and entered into Graphpad Prism software to draw fitting curves. The parental and resistant IC50 values were then read. A resistant IC50 / parental IC50 > 5 was considered an indicator of successful construction of a resistant cell line. (IC50 is shown in the table below)

[0087] Results and Discussion

[0088] The results are as follows Figure 7 As shown in Table 1:

[0089] Table 1 IC50 values of parental cells and drug-resistant cell lines

[0090]

[0091] As shown in the table, SUM-159PT is more sensitive to lobaplatin, with a lower IC50. The IC50 of SUM-159PT-LBP is five times greater than that of SUM-159PT. Because resistant cells are less sensitive to lobaplatin than their parental counterparts, a higher concentration of lobaplatin is required to induce killing in order to achieve the IC50.

[0092] Test Example 7: In vivo verification experiment

[0093] Materials and test methods:

[0094] (1) Mouse source: BALB / C-NSG mice. Experimental animals were housed under standard light-dark conditions, with a 12-h / d lighting cycle and a room temperature of 26°C. They were fed with standardized feed and purified drinking water.

[0095] (2) Methods for establishing an orthotopic xenograft TNBC drug-resistant mouse model:

[0096] A. Adjust the SUM-159PT and SUM-159PT-LBP density to the appropriate range (usually 3×10 6 Use a sterile syringe to draw an appropriate amount of cell suspension. Under a microscope or dissecting microscope, slowly inject the cell suspension into the fourth pair of mammary fat pads of the mice, taking care to avoid damaging surrounding tissues. Observe the mice daily after injection and record their body weight, food intake, and water intake.

[0097] B. One week later, 10 mg / kg lobaplatin was injected intraperitoneally every 3 days. The tumor volumes of SUM-159PT and SUM-159PT-LBP were compared on days 7, 14, 21, and 28. Mice were killed 30 days later.

[0098] Results and Discussion

[0099] The results are as follows Figure 8 As shown, from Figure 8 It can be seen that animal experiments have confirmed that the tumor of SUM-159PT is small; SUM-159PT-LBP is insensitive to lobaplatin stimulation, so in the drug-resistant model constructed in vivo, SUM-159PT-LBP is resistant to lobaplatin, so under lobaplatin induction, the tumor volume is larger than that of the parent.

[0100] The construction method of the present application is not suitable for high-dose shock induction. It uses the human breast cancer cell line SUM-159PT as the induction object, adopts the method of gradually increasing the concentration of lobaplatin and intermittent maintenance culture to induce and construct a stable lobaplatin-resistant strain; based on this construction method, other TNBC cell lines with parental cells such as MDA-MB-231 and MDA-MB-453 as the induction objects can also be used to construct stable lobaplatin-resistant strains; however, based on this construction method, TNBC cell lines such as parental cells BT-549 cannot be constructed into stable resistant strains.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A triple-negative breast cancer lobaplatin-resistant cell line, characterized in that: The cell line is named as triple-negative breast cancer lobaplatin-resistant cell line SUM-159PT-LBP, and its deposit number is CCTCCNO: C2025150.

2. A method for constructing a triple-negative breast cancer lobaplatin-resistant cell line, characterized in that: The human breast cancer cell line SUM-159PT was used as the induction object. The triple-negative breast cancer lobaplatin-resistant cell line SUM-159PT-LBP was constructed by inducing with stepwise increasing lobaplatin concentration and intermittent maintenance culture.

3. The method for constructing a triple-negative breast cancer lobaplatin-resistant cell line according to claim 2, characterized in that: The steps include: S1: Lobaplatin induction, specifically, the induction method is as follows: add lobaplatin to the initial culture medium of the human breast cancer cell line SUM-159PT containing premium serum, wherein the initial concentration of lobaplatin is 0.05-0.25 μM, continue culturing, and maintain this drug concentration until the cells can grow stably. After the cell density reaches 80%-90%, perform 1:2 passage, and then use culture medium without lobaplatin to maintain the culture intermittently; S2: After the cell density to be passaged reaches 80%-90%, the induction method described in step S1) is repeated, and the lobaplatin concentration is gradually increased until the cells can stably grow and be passaged in the culture medium containing 3-5 μM lobaplatin, thereby obtaining the target cell line; S3: The obtained target cell line is repeatedly cultured in a culture medium containing 2-5 μM ropaplatin, followed by at least two freezing and thawing treatments, to ultimately obtain a stable cell line, namely, a ropaplatin-resistant triple-negative breast cancer cell line.

4. The method for constructing a triple-negative breast cancer lobaplatin-resistant cell line according to claim 3, characterized in that: It also includes rinsing and centrifuging the cells before and after passaging using PBS containing double antibodies.

5. The method for constructing a triple-negative breast cancer lobaplatin-resistant cell line according to claim 3, characterized in that: The initial concentrations of lobaplatin gradually increased in step S2 are 0.5 μM, 0.75 μM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 2.75 μM, 3 μM, 3.25 μM, 3.5 μM, 3.75 μM, 4 μM, 4.25 μM, 4.5 μM, 4.75 μM, and 5 μM, respectively.

6. The method for constructing a triple-negative breast cancer lobaplatin-resistant cell line according to claim 3, characterized in that: The specific operations of the freezing and thawing treatment in step S3 are: freezing the target cell line in liquid nitrogen, then thawing and continuing to culture in a culture medium containing 2-5 μM lobaplatin, and stably passage.

7. The method for constructing a triple-negative breast cancer lobaplatin-resistant cell line according to claim 3, characterized in that: The construction method is also applicable when the parent cell is MDA-MB-231 or MDA-MB-453.

8. Use of the triple-negative breast cancer lobaplatin-resistant cell line according to claim 1 in the study of lobaplatin resistance mechanism.

9. Use of the triple-negative breast cancer lobaplatin-resistant cell line according to claim 1 in preparing a drug-resistant cell model.

10. Use of the triple-negative breast cancer lobaplatin-resistant cell line according to claim 1 in screening drugs for reversing breast cancer resistance or anti-breast cancer drugs.

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