MDS-AML azacitidine drug-resistant strain and preparation method thereof

Abstract

The invention discloses an MDS-AML azacitidine drug-resistant strain, and belongs to the field of drug-resistant strains, the drug-resistant strain is preserved in the China Center for Type Culture Collection, the preservation name is SKM / Aza, and the preservation number is C2024248; the invention also discloses a construction method of the MDS-to-AML azacitidine drug-resistant strain, and the construction method is characterized by comprising the following steps: S1, subculturing an initial SKM1 cell in a cell culture device; s2, adding azacitidine with the drug concentration of 0.1 mu M into the cell culture device, treating for 24 hours, centrifuging at a low speed, changing the liquid and removing the drug to remove dead cells, treating by using the drug with the concentration of 0.1 mu M after the cell state is recovered, observing that the number of dead cells is large, namely removing the drug to recover, and repeating the operation of adding the drug and removing the drug until the cells stably grow under the concentration, so as to obtain the azacitidine. Entering the next stage of drug concentration treatment; and S3, according to the drug treatment concentration, selecting a specific drug concentration for progressive increase treatment.

Description

Technical Field

[0001] The present invention relates to the field of preparation of azacitidine-resistant strains. Background Art

[0002] Myelodysplastic syndrome (MDS) is a group of heterogeneous, malignant clonal disorders originating from hematopoietic stem cells / myeloid progenitor cells. It is characterized by ineffective clonal hematopoiesis, resulting in abnormal bone marrow cell development, peripheral blood cytopenias, and progressive bone marrow failure. Approximately 30% of patients progress to acute myeloid leukemia (AML). Secondary AML accounts for approximately 20-30% of AML cases and is primarily a transformation of MDS. Once MDS transforms into AML, patient response to treatment is poor, and survival is significantly shortened. The incidence of leukemia in my country is approximately 3.4 per 100,000 patients, with 75,000 new cases and 53,000 deaths annually, of which AML accounts for 70%. It is one of the top ten most common malignant tumors in my country, posing a serious threat to the health and lives of its citizens and increasing the healthcare burden. The current gold standard for the clinical treatment of AML is standard chemotherapy combined with hematopoietic stem cell transplantation, but the efficacy of cytotoxic chemotherapy drugs remains suboptimal in clinical practice.

[0003] Azacitidine (Aza) is currently one of the most important hypomethylating drugs. In 2004, the US Food and Drug Administration approved Azacitidine for use in hematological malignancies such as MDS and AML. Currently, Azacitidine is primarily used clinically for patients with MDS, as well as patients with AML whose blasts are 20-30%, or elderly patients with blasts >30% who are unable to tolerate conventional chemotherapy. Azacitidine, a pyrimidine analog, is phosphorylated into Aza triphosphate by uridine cytidine kinase after entry into cells. 80-90% of Azacitidine binds to RNA, interfering with nucleic acid and protein synthesis, while 10-20% of Azacitidine is incorporated into DNA during DNA replication and covalently binds to DNMTs, inhibiting DNMT activity, reducing methylation of tumor suppressor genes, and restoring transcription of tumor suppressor genes, thereby inducing anti-leukemic effects. However, a growing number of clinical studies have found that a significant proportion of MDS patients develop primary or secondary resistance to Azacitidine, meaning that some patients do not respond to Azacitidine treatment or develop resistance after responding. Drug-resistant patients have poor clinical outcomes and poor prognosis, and therefore, drug resistance is the main clinical obstacle to the successful treatment of leukemia with azacitidine compared with relatively sensitive patients.

[0004] Establishing drug-resistant cell lines in vitro is an important method for studying the mechanisms of tumor resistance. Currently, mainstream methods include high-dose drug shock, increasing drug concentration, and transgenic combined with drug screening. During drug treatment, cells that cannot tolerate the drug die. While adherent tumor cells can be depleted by changing the medium, hematologic tumor cells are often suspended, making depletion difficult. Therefore, establishing drug-resistant suspension tumor cell lines is challenging, and detailed documentation is lacking. Summary of the Invention

[0005] The purpose of the present invention is to address the problems existing in the prior art and disclose an azacitidine-resistant strain of MDS-to-AML. The resistant strain is deposited in the China Center for Type Culture Collection with the deposit name: SKM / Aza and the deposit number: C2024248.

[0006] The present invention also discloses a method for constructing the MDS-to-AML azacitidine-resistant strain, which comprises the following steps:

[0007] S1: Subculturing the initial SKM1 cells in a cell culture device;

[0008] S2: Add 0.1 μM azacitidine to the cell culture apparatus and treat for 24 hours. Centrifuge at low speed to replace the medium and withdraw the drug to remove dead cells. After the cells recover, treat with 0.1 μM drug again. If more dead cells are observed, withdraw the drug and the cells recover. Repeat the above "drug addition-drug withdrawal" operation until the cells grow stably at this concentration and enter the next drug concentration treatment stage.

[0009] S3: Based on the drug treatment concentration, select a specific drug concentration for incremental treatment; the details are as follows:

[0010] When the drug treatment concentration was ≤1 μM, the concentration was increased by 0.1, 0.2, 0.4, 0.8, and 1.2 μM;

[0011] When the drug treatment concentration was >1 μM, the concentration was increased in steps of 2.4, 3.6, 4.8, 6.4, 7.2, and 12 μM;

[0012] When the drug treatment concentration is ≥12 μM, the concentration is increased by 1 μM each time, and the drug treatment is continued without withdrawal;

[0013] When the drug treatment concentration is ≥18 μM, the concentration is increased by 2 μM each time, and the drug treatment is continued without withdrawal;

[0014] When the drug treatment concentration is ≥25 μM, the concentration is increased by 5 μM each time, and the drug treatment is continued without withdrawal;

[0015] S4: After 12 months of induction, the cells were able to stably survive at 70 μM of the drug, and the MDS-to-AML azacitidine-resistant strain was obtained.

[0016] In a preferred embodiment, the cell culture device is a cell culture flask.

[0017] In a preferred embodiment, the centrifugation condition of the low-speed centrifugation in step S2 is 500 rpm and 4° C. for 5 min.

[0018] The present invention intends to construct an azacitidine-resistant SKM1 cell line through the concentration-increasing intermittent induction method, fill the gap in AML azacitidine-resistant strains, and provide a cell model for azacitidine resistance research.

[0019] The azacitidine-resistant cell line constructed by the present invention provides an important cell model for studying the mechanism of azacitidine resistance. The high-throughput data provides expression profile changes of resistant cells relative to parental cells. It can be used for in vitro and in vivo experiments on the azacitidine resistance mechanism of MDS and AML, evaluating the anti-tumor activity of drugs, and developing target drugs to reverse drug resistance. It has high scientific research and production application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the result of SKM1 cell detection;

[0021] Figure 2 It is the inhibitory concentration detection graph;

[0022] Figure 3 It is the cytological characteristics of drug-resistant strains;

[0023] Figure 4 The results at different concentrations are shown in Figure 2.

[0024] Figure 5 It is a picture of cell apoptosis detection;

[0025] Figure 6 This is the transcriptome level detection diagram of drug-resistant strains;

[0026] Figure 7 It is a schematic diagram of dead cells;

[0027] Figure 8 It is a microscopic picture of cells;

[0028] Figure 9 It is a microscopic picture of cells. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below with reference to the accompanying drawings.

[0030] 1. Construction of azacitidine-resistant MDS-transfected AML cell line

[0031] 1. Materials

[0032] The human MDS-AML transformed cell line SKM1 was introduced and preserved by the Department of Hematology of the People's Hospital Affiliated to Jiangsu University and included in the cell bank of the German National Culture Collection (DSMZ) with the code ACC 547. It was correctly identified by STR of Wuhan Punosai Life Science Co., Ltd. (such as Figure 1 SKM1 cells are suspension cells routinely cultured in 25 cm2 breathable cell culture flasks in RPMI-1640 medium (Vicente, 350-006-CL) supplemented with 10% fetal bovine serum and 1% double-antibody. The cells were cultured in a 37°C, 5% CO2 incubator, maintaining a cell density of no more than 80%. Medium changes and subcultures were performed as needed. Conventional centrifugation conditions were 1000 rpm at 4°C for 5 minutes (Eppendorf refrigerated centrifuge, model Centrifuge 5810R).

[0033] 2. Determination of the initial sensitivity of SKM1 cells to azacitidine

[0034] Cells in good logarithmic growth phase were prepared into a suspension and 30,000 cells were seeded per well of a 96-well plate in a volume of 100 μl per well. Treatments were added to the wells, including a zero-adjustment well, a blank control group, and treatments with varying concentrations of azacitidine (0.025, 0.1, 0.5, 1, 5, and 10 μM azacitidine) (Selleck, S1782), with three replicates per group. To minimize error, sterile PBS was added to the outermost circle of wells on the culture plate. After treatment, a CCK8 assay was performed by adding 10 μl of CCK8 solution (TargetMol, C0005) to each well. After incubation in the incubator for another 2 hours, the absorbance (A) of each well was measured at a wavelength of 450 nm using a microplate reader, ensuring that the blank control reading was between 0.8 and 1.2. The calculation formula is: cell survival rate = (A value of experimental group - A value of zero adjustment group) / (A value of control group - A value of zero adjustment group) × 100%, and then calculate the half inhibitory concentration (IC50). GraphPad Prism was used for data analysis and graphing. The experimental results are shown in Figure 2 As shown, the initial 24h IC50 of SKM1 for azacitidine is 0.58 μM.

[0035] 3. Construction of drug-resistant strains by concentration-increasing intermittent induction method

[0036] The initial SKM1 cells were passaged into two cell culture flasks, one as a "parental control group" and the other as a "drug resistance induction group". The two groups of cells were cultured synchronously except for the difference between the addition and absence of drug treatment.

[0037] The above experiment obtained that the 24h IC50 of SKM1 for azacitidine was 0.58μM. A drug concentration of 1 / 6 of the initial IC50 (i.e. 0.1μM) was used as the starting drug concentration. After adding the "drug resistance induction group" for treatment for 1 day, the cell state was observed and the solution was changed by low-speed centrifugation to withdraw the drug to remove dead cells. SKM1 is a suspension cell. Dead cells cannot be removed by changing the solution of adherent cells. Low-speed centrifugation is required to remove dead cells. The centrifugation conditions are 500rpm and 4℃ for 5min. After the cell state of the "drug resistance induction group" recovered, it was treated with the same concentration of drug again. If more dead cells were observed, the drug was withdrawn and the cells recovered. Repeat the above "drug addition-drug withdrawal" operation until the cells grow stably at this concentration, indicating that they can tolerate this concentration and can enter the next higher drug concentration shock. The drug increment is as follows:

[0038] ① When the drug treatment concentration is ≤1 μM, increase the concentration by 0.1, 0.2, 0.4, 0.8, and 1.2 μM.

[0039] ② When the drug treatment concentration is greater than 1 μM, the concentration is increased in increments of 2.4, 3.6, 4.8, 6.4, 7.2, and 12 μM.

[0040] ③ When the drug treatment concentration is ≥12μM, increase it by 1μM each time, and continue the drug treatment without withdrawal.

[0041] ④ When the drug treatment concentration is ≥18 μM, increase it by 2 μM each time, and continue the drug treatment without withdrawal.

[0042] ⑤ When the drug treatment concentration is ≥25μM, increase it by 5μM each time and continue the drug treatment without withdrawal.

[0043] After 12 months of induction, the “drug-resistance induced group” could survive stably in the presence of 70 μM of the drug, indicating that the “drug-resistance induced group” could resist the proliferation inhibition caused by azacitidine treatment.

[0044] The data show that after one day of treatment with a drug at a concentration of 1 / 6 (i.e. 0.1 μM), the proportion of dead cells is 8.3%. If the solution is immediately replaced and the drug is withdrawn, and the cell state recovers after 1-2 days, the drug is treated with a concentration of 1 / 6 again, and this operation is repeated until the proportion of dead cells after treatment with this concentration is low, close to the proportion of dead cells in normal cells, that is, the above-mentioned 2.8% value, and then the next concentration (0.2 μM) is used for treatment, the proportion of dead cells is 8.9%. If the "drug addition-withdrawal" method is not adopted, the proportion of dead cells after treatment with a drug at a concentration of 1 / 6 (i.e. 0.1 μM) for 1 day, 2 days, 3 days, and 7 days is 8.3%, 11.9%, 65.3%, and 94.3%, respectively. No surviving cells can be obtained for subsequent construction. The experimental results are as follows. Figure 8Therefore, the effect of using the "drug addition-withdrawal" operation is better than that without the "drug addition-withdrawal" operation. It can ensure that during the construction of drug-resistant strains, cell activity can be maintained while drug resistance can be gradually increased.

[0045] (4) The obtained “parent control group” was named parent strain “SKM / Par”, and the “resistance-induced group” was named resistant strain “SKM / Aza”, and they were deposited in China Center for Type Culture Collection with the deposited name: SKM / Aza and the deposited number: C2024248.

[0046] 2. Morphological observation of parental strain and drug-resistant strain.

[0047] The morphological characteristics of the parental strain and the drug-resistant strain were observed using an inverted optical microscope (Olympus, model CKX53), and the changes in the nucleus and cytoplasm of the cells were observed using modified Wright Giemsa staining. The cells in the logarithmic growth phase were evenly smeared on the histochemical pen circle of the high-adhesion slide of the Setai, fixed with 70% ethanol for 10 minutes, and an appropriate amount of the prepared modified Giemsa staining working solution (1×) (Biyuntian, C0131) was added and stained for 20 minutes. The cells were thoroughly washed from one side with a small stream of tap water, and observed and photographed under a microscope after drying. SKM / Aza showed typical cytological characteristics of drug-resistant strains, including irregular morphology, increased volume, increased nuclear-cytoplasmic ratio, and increased intracellular particles. The experimental results are as follows. Figure 3 shown.

[0048] 3. Detection of proliferation and apoptosis levels of drug-resistant strains

[0049] 1. Cell proliferation detection: During the drug resistance induction process, the 48h IC50 of the "parental control group" and the "drug resistance induced group" were tested multiple times to monitor the changes in the drug resistance index (RI = IC50 of the resistant strain / IC50 of the parent strain) and the degree of drug resistance. Compared with the experimental operation of the initial 24h IC50 of SKM1 cells to azacitidine, the experiment of detecting 48h IC50 has two adjustments: first, the number of cells plated is adjusted to 20,000 cells per well of a 96-well plate to obtain the best CCK8 absorbance value; second, as the drug tolerance of the "resistance-induced group" increases, in order to obtain an accurate IC50 value, the azacitidine concentration gradient for detecting IC50 should be adjusted accordingly. The concentration gradient is set to: 0.05, 0.1, 0.5, 1, 5, 10, 20μM, or 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2μM, or 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100μM. The experimental results are as follows Figure 4As shown in the figure, during the process of drug resistance induction, the resistance index of the "resistance induced group" to azacitidine gradually increased, experiencing 5.3 times, 18.3 times, and 34.5 times. The current resistance multiple is 65.7 times, which is a highly resistant strain (RI>15 is highly resistant).

[0050] 2. Cell apoptosis detection: The collected cell suspension was washed once with pre-cooled PBS, resuspended in 100μl 1×Binding buffer and the concentration was adjusted to 1×106 / ml, 5μl PE Annexin V and 5μl 7-AAD (Novozymes, A213) were added, and the mixture was gently blown evenly. Incubated at room temperature in the dark for 10 minutes, and 400μl 1×Binding buffer was added to each well. The detection was completed within 1 hour. A blank control group, PE Annexin V and 7-AAD single staining group and double staining group were set up. The results were analyzed using FlowJo software, and the data of three independent repetitions were combined and statistically drawn. The calculation formula is: Apoptosis rate (%) = early apoptosis rate (Annexin V+ / PE-) + late apoptosis rate (Annexin V+ / PE+). The experimental results are as follows Figure 5 As shown in the data, treatment with a certain concentration of azacitidine for 48 hours can significantly increase the apoptosis level of the SKM / Par parental strain, but the same concentration of the drug cannot induce apoptosis in the SKM / Aza-resistant strain, indicating that the resistant strain can resist the apoptosis induction of azacitidine.

[0051] 4. Transcriptome-level detection of drug-resistant strains

[0052] 1×106 SKM / Par and SKM / Aza cells in good growth were placed in 15ml centrifuge tubes and centrifuged at 1000 rpm at 4°C for 5 minutes. The supernatant was discarded, the cells were resuspended in 1ml of pre-chilled sterile PBS, transferred to a 1.5ml EP tube, and washed once by centrifugation at 1000 rpm at 4°C for 5 minutes. The cell pellet was snap-frozen in liquid nitrogen for 2 minutes, stored and shipped at -80°C, and then sequenced with the help of Beijing Tiangen Biochemical Technology Co., Ltd., which provided library preparation, quality control, sequencing, and data analysis. Sequencing was performed using the Illumina sequencing platform in PE150 mode, generating 150bp paired-end reads. Each sample generated 6GB of raw data, with two biological replicates. After the raw sequencing data passed quality inspection, they were referenced to the human genome, mapped to the sequencing reads, and differential analysis was performed on the read count data using DESeq2. The differential gene screening criteria were |log2(Fold Change)|>1&FDR<0.05 (Fold Change, fold difference; FDR, false discovery rate, i.e., corrected P value). A total of 1226 significantly differentially expressed genes were identified between SKM / Aza and SKM / Par, including 958 upregulated genes and 268 downregulated genes. The experimental results are shown in Figure 2. Figure 6 These differentially expressed genes may provide molecular targets for further exploring the mechanism of secondary azacitidine resistance and overcoming resistance.

[0053] Although the drug concentration increment method is the most commonly used method for constructing drug-resistant strains, there are few existing data that provide detailed descriptions of the characteristics of drug concentration increments, and the details of the methods vary. In the early stages of construction, attempts were made to treat cells for one day with drug concentrations of 1 / 10, 1 / 6, and 1 / 2 of the initial IC50. The proportion of dead cells in the group without drug treatment was 2.8%, 3.0% at 1 / 10 concentration (i.e., 0.06 μM), 8.3% at 1 / 6 concentration (i.e., 0.1 μM), and 14.0% at 1 / 2 concentration (i.e., 0.29 μM). Considering that after drug-induced drug treatment of drug-resistant strains, it is necessary to induce dead cells to indicate that the drug is effective, but the proportion of dead cells cannot be too high, otherwise it means that the drug concentration is too high (experimental results are shown in Figure 2). Figure 7 As shown, red circles in the figure represent dead cells stained with trypan blue, and uncircled cells represent live cells not stained with trypan blue. Therefore, the present invention proposes that a drug concentration of 1 / 6 of the initial IC50 is a more appropriate starting drug concentration, and emphasizes the need to repeat the "drug addition-withdrawal" operation of a certain drug concentration until the cells can tolerate this concentration before proceeding to the next higher drug concentration challenge.

[0054] (3) Existing reports have proposed increasing the drug concentration by 2-fold each time. During the implementation of the present invention, it was found that when the concentration was low in the early stage, the concentration could be increased by 2-fold; however, when the concentration was high in the later stage, the 2-fold increase in concentration was too radical, which would make the cell state of the "drug resistance induced group" more difficult to control and result in too many dead cells. Therefore, it is necessary to flexibly adjust the drug dosage according to the cell state and gradually increase the drug dosage to obtain a drug-resistant strain with high tolerance and stability.

[0055] (3) Tumor cells are mostly adherent growth characteristics, while blood tumor cells are mostly suspended growth. It is impossible to remove dead cells by simply discarding the culture supernatant. This is a key technical problem that needs to be solved in the construction of blood tumor resistant strains. The present invention has repeatedly explored the centrifugation conditions, and the experimental results are as follows Figure 9 As shown, the dead cell percentage before centrifugation was 54.4%, but after centrifugation at 300 rpm for 5 minutes, it decreased to 49.6%, showing almost no dead cell removal. After centrifugation at 500 rpm, the percentage decreased to 17.2%, achieving both good dead cell removal and viable cell retention. After centrifugation at 800 and 1000 rpm, the percentages increased to 13.9% and 15.2%, respectively, showing no significant improvement in dead cell removal and a significant loss of viable cells. Therefore, within the 300-1000 rpm range, we found that "500 rpm, 4°C for 5 minutes" was the optimal centrifugation condition for both dead cell removal and viable cell retention in the SKM1 cell line.

Claims

1. A MDS-to-AML azacitidine-resistant strain, characterized in that: The drug-resistant strain is deposited in China Center for Type Culture Collection with the deposit name: SKM / Aza and the deposit number: C2024248.

2. A method for constructing an MDS-to-AML azacitidine-resistant strain as claimed in claim 1, characterized in that: Here are the steps: S1: Subculturing the initial SKM1 cells in a cell culture device; S2: Add 0.1 μM azacitidine to the cell culture device, treat for 24 hours, centrifuge at low speed to replace the solution and withdraw the drug to remove dead cells. After the cell state recovers, treat with 0.1 μM drug again. If more dead cells are observed, withdraw the drug and recover. Repeat the above "add drug-withdraw drug" operation until the cells grow stably at this concentration and enter the next stage of drug concentration treatment. S3: According to the drug treatment concentration, a specific drug concentration is selected for incremental treatment; the details are as follows: When the drug treatment concentration was ≤1 μM, the concentration was increased by 0.1, 0.2, 0.4, 0.8, and 1.2 μM; When the drug treatment concentration was >1 μM, the concentration was increased according to 2.4, 3.6, 4.8, 6.4, 7.2, and 12 μM; When the drug treatment concentration was ≥12 μM, the concentration was increased by 1 μM each time, and the drug treatment was continued without withdrawal; When the drug treatment concentration was ≥18 μM, the concentration was increased by 2 μM each time, and the drug treatment was continued without withdrawal; When the drug treatment concentration was ≥25 μM, the concentration was increased by 5 μM each time, and the drug treatment was continued without withdrawal; S4: After 12 months of induction, the cells were able to stably survive in 70 μM of the drug, and the MDS-to-AML azacitidine-resistant strain was obtained.

3. The method for constructing an MDS-to-AML azacitidine-resistant strain according to claim 2, characterized in that: The cell culture device is a cell culture flask.

4. The method for constructing an MDS-to-AML azacitidine-resistant strain according to claim 3, characterized in that: The centrifugation conditions of step S2 low speed centrifugation are 500 rpm and 4° C. for 5 min.

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