Human chronic myeloid leukemia blast crisis cell line, its use, method of preparation and kit

Abstract

The present application relates to a kind of human chronic myeloid leukemia acceleration phase cell strain and its application, preparation method and kit, cell strain is named human chronic myeloid leukemia acceleration phase cell YYXY-M10, on October 12, 2024, be preserved in China typical culture preservation center, and the preservation number is as follows: CCTCC NO: C2024346.The present application is isolated from the bone marrow or peripheral blood sample of chronic myeloid leukemia acceleration phase patient, can reach stable proliferation in vitro without adding any cell growth factor and continuous passage, cell in vitro shape is stable, can be tumorigenic in vivo immunodeficient animal, resistant to three generations tyrosine kinase inhibitor.It can be used for the pathogenesis of chronic myeloid leukemia, the development mechanism from acceleration phase to acute phase, drug resistance mechanism research, has wide application prospect and practical value in the basic research of human chronic myeloid leukemia, drug screening and evaluation, clinical drug guidance and the like.

Description

Technical Field

[0001] This invention relates to a human chronic myeloid leukemia accelerated phase cell line, its application, preparation method, and reagent kit, and is applicable to the field of biotechnology. Background Technology

[0002] Chronic myeloid leukemia (CML) is a type of leukemia caused by the clonal proliferation of hematopoietic stem cells in the bone marrow. According to international consensus classification, CML is divided into chronic phase, accelerated phase, and blast crisis phase. These different phases of CML exhibit significant differences and vastly different prognoses. In the chronic phase, treatment with tyrosine kinase inhibitors targeting BCR-ABL can achieve a 10-year survival rate of 90%. However, in the accelerated and blast crisis phases, even with tyrosine kinase inhibitors, the prognosis deteriorates significantly, especially in the blast crisis phase, where overall survival is less than one year. Currently, the specific mechanisms by which CML progresses from the chronic phase to the accelerated and blast crisis phases remain unclear in CML research. Furthermore, because CML patients using tyrosine kinase inhibitors typically have a longer disease course and most achieve long-term survival, it is difficult to study the disease process directly. Therefore, constructing cell lines for different stages of CML is of great significance in understanding the development mechanism of CML and in drug development.

[0003] The first CML cell line internationally was the K562 cell line, established in 1976 from leukemia cells of patients in the blast crisis phase of chronic myeloid leukemia. Subsequently, several other cell lines were successfully established, such as NALM-1, BV173, CML-C-1, CML-T1, KBM7, and KCL-22. However, most existing cell lines are derived from cells in the blast crisis phase, and there is currently a lack of CML cell lines established from the accelerated phase. Therefore, it is necessary to establish corresponding cell lines for the accelerated phase of CML to provide strong support for elucidating the development and drug resistance mechanisms of the accelerated phase of CML and screening therapeutic drugs for the accelerated phase of CML. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention proposes a human chronic myeloid leukemia accelerated phase cell line, its application, preparation method, and reagent kit.

[0005] The technical solution adopted in this invention is as follows: Firstly, this invention provides a human chronic myeloid leukemia accelerated phase cell line, named human chronic myeloid leukemia accelerated phase cell line YYXY-M10, which was deposited on October 12, 2024, at the China Center for Type Culture Collection (CCTCC) in Wuhan, with accession number CCTCC NO: C2024346. The cell line is classified as human chronic myeloid leukemia accelerated phase cell line, with the Latin scientific name *Homo sapiens*.

[0006] Furthermore, this cell line was derived from primary cells of patients with accelerated phase chronic myeloid leukemia who had been treated with first-, second-, and third-generation tyrosine kinase inhibitors. It exhibited resistance to third-generation tyrosine kinase inhibitors, specifically olaribatibin.

[0007] Furthermore, the cell line exhibits a normal karyotype with 46 chromosomes, including XY sex chromosomes, and displays a primitive cell morphology, exhibiting suspension growth during culture. This cell line is capable of forming tumors in immunodeficient animals and possesses the ability to proliferate stably and continuously.

[0008] On the other hand, the present invention also provides the application of the above-mentioned human chronic myeloid leukemia accelerated phase cell line in the preparation of drug models for screening treatment of chronic myeloid leukemia accelerated phase. Specifically, the drug models here include, but are not limited to, in vitro cell-level screening models, in vitro molecular-level screening models, in vivo animal tumorigenesis and leukemia infiltration models, organoid models, etc.

[0009] On the other hand, this invention also provides the application of the aforementioned human chronic myeloid leukemia accelerated phase cell line in the preparation of drug models for screening and reversing resistance to tyrosine kinase inhibitors. Specifically, the drug models here include, but are not limited to, in vitro drug-resistant cell line models, in vitro combined drug synergistic screening models, signaling pathway molecular-level screening models, in vivo xenograft drug-resistant leukemia animal models, 3D tumor sphere and bone marrow microenvironment co-culture models, and primary clinical drug-resistant cell models.

[0010] On the other hand, the present invention also provides the application of the above-mentioned human chronic myeloid leukemia accelerated phase cell line in the preparation of products for evaluating the therapeutic effect of tyrosine kinase inhibitors on accelerated phase chronic myeloid leukemia. Specifically, the products herein include, but are not limited to, drugs, test kits, related reagents and consumables, animal models and related products, molecular mechanism detection related products, data analysis and detection instruments and equipment, etc.

[0011] On the other hand, the present invention also provides a method for preparing the above-mentioned human chronic myeloid leukemia accelerated phase cell line, comprising: S1. Collect peripheral blood or bone marrow samples from patients with accelerated phase of chronic myeloid leukemia and separate mononuclear cells using gradient centrifugation. Specifically, take 3 mL of sample and add it to a sterile centrifuge tube containing 5 mL of Ficoll-Paque. Centrifuge at 2000 rpm for 60 min at 4°C. Then, take the white membrane cell layer at the junction of Ficoll-Paque and plasma and place it in a new sterile centrifuge tube. Add 5 mL of 1×PBS pre-cooled at 4°C to resuspend the cells and centrifuge at 2000 rpm for 5 min. Collect the cell pellet. S2. Place the product obtained in step S1 into IMDM basal medium containing fetal bovine serum, and then place the medium in a culture flask for primary culture. The volume fraction of fetal bovine serum is 10-15%, and the density of mononuclear cells in the culture flask is 1-2 × 10⁻⁶. 8 Cells / mL; specifically: add 5-15% fetal bovine serum to IMDM medium and count cells using an automated cell counter at a rate of 1-2 × 10⁻⁶. 8 The product obtained in step S1 was added at a cell density of cells / mL. Then the culture medium was placed in a T25 cell culture flask and placed in a constant temperature and humidity incubator for primary culture. The environment in the incubator was 37℃ and 5% CO2. S3. Continuously observe the cell status in the culture flask, replace the IMDM medium weekly, and replace the culture flask when adherent cells appear, until the cells have stably proliferated and passaged for more than 40 generations. Specifically: observe the cell status using an optical microscope, and replace the IMDM complete medium weekly. When adherent cells appear during the culture process, replace the culture flask promptly, retaining only suspension cells. If the cells are in obvious apoptosis, centrifuge at 600-800 rpm for 5 minutes and replace the medium to allow the cells to continue to proliferate until the cells have stably proliferated and passaged for more than 40 generations. Then, perform cell proliferation capacity, tumorigenicity, and STR identification, and test the in vitro growth rate, doubling time, and viability stability of the cell line to verify its ability to stably passage and malignantly proliferate, and screen to obtain the desired human chronic myeloid leukemia accelerated phase cell line.

[0012] On the other hand, the present invention also provides a kit for screening drugs against chronic myeloid leukemia, comprising the above-mentioned human chronic myeloid leukemia accelerated phase cell line.

[0013] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: This invention relates to a human chronic myeloid leukemia (CML) accelerated phase cell line, its application, preparation method, and reagent kit. The cell line is isolated from bone marrow or peripheral blood samples from patients with CML accelerated phase. Without the addition of any cell growth factors, it can achieve stable in vitro proliferation and continuous passage. The cells exhibit stable in vitro morphology, can form tumors in immunodeficient animals, and are resistant to third-generation tyrosine kinase inhibitors. It can be used to study the pathogenesis of CML, the progression from accelerated phase to blast crisis, and drug resistance mechanisms. It has broad application prospects and practical value in basic research, drug screening and evaluation, and clinical medication guidance for human CML. Attached Figure Description

[0014] The following sections will describe some specific embodiments of the invention in a detailed manner, by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar components or parts. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings: Figure 1 This is a staining image of donor bone marrow primitive cells in Embodiment 1 of the present invention; Figure 2 yes Figure 1 The diagram shows the proportion of donor bone marrow blast cells in the illustrated embodiment; Figure 3 This is a staining image of YYXY-M10 cells in Example 2 of the present invention; Figure 4 yes Figure 3 The growth curve of YYXY-M10 cells in the illustrated embodiment is shown. Figure 5 yes Figure 3 The karyotype diagram of YYXY-M10 cells in the illustrated embodiment; Figure 6 yes Figure 3 Flow cytometry plot of mouse bone marrow infiltration in the illustrated embodiment; Figure 7 yes Figure 3 The mouse survival curves in the illustrated embodiment; Figure 8 yes Figure 3 The illustrated embodiment shows the inhibition curves of olaribatitinib on different cells; Figure 9 yes Figure 3 The illustrated example shows the drug resistance index of YYXY-M10 cells and patient primary cells compared to K562. Detailed Implementation

[0015] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0017] Example 1: Preparation of the human chronic myeloid leukemia accelerated phase cell line YYXY-M10 Sample Collection: Bone marrow samples were obtained from the Affiliated People's Hospital of Ningbo University. The patient is a 76-year-old male who has been diagnosed with chronic myeloid leukemia for 6 years. During his illness, he was treated with first-generation tyrosine kinase inhibitors imatinib, second-generation tyrosine kinase inhibitors flumatinib and dasatinib, and third-generation tyrosine kinase inhibitor olaribatinib. Routine bone marrow examinations were performed upon admission, as shown in the attached sample. Figure 1 , 2 As shown, Wright-Gymsa staining of the bone marrow indicated the presence of primitive cells in the bone marrow smear. Counting revealed that the proportion of primitive cells was 10%, which meets the diagnostic criteria for accelerated phase of CML according to the 2022 International Consensus Classification (ICC).

[0018] Primary culture: Take 3 mL of the patient's bone marrow sample and add it to a sterile centrifuge tube containing 5 mL of Ficoll-Paque. Centrifuge at 2000 rpm for 60 min at 4°C. Then, take the white membrane cell layer at the junction of Ficoll-Paque and plasma and place it in a new sterile centrifuge tube. Add 5 mL of sterile 1×PBS pre-cooled at 4°C to resuspend the cells and centrifuge at 2000 rpm for 5 min. Collect the cell pellet.

[0019] Prepare IMDM medium containing 10% fetal bovine serum and count cells using an automated cell counter at a concentration of 1×10⁶ cells / mL. 8 Add the cell pellet to the culture medium at a cell density of cells / mL and resuspend the cells; then take 6 mL of culture medium and place it in a T25 cell culture flask and place the flask in a constant temperature and humidity incubator containing 5% CO2 at 37°C for culture; after 1 week, centrifuge the culture flask at 600 rpm for 5 min to remove cell debris, and then replace it with new IMDM complete culture medium, and change the culture medium once a week thereafter.

[0020] Passaging: Continuously observe the cell status in the culture flask. Apoptosis occurs after 2 weeks of cell culture, and non-apoptotic cells are in a state of neither proliferation nor death. Change the culture medium weekly. After 4 weeks of cell culture, cells begin to proliferate and adhere to the culture vessel. Replace with a new cell culture flask and continue to change the culture medium weekly. After 8 weeks of cell culture, cells grow in suspension and no obvious apoptosis or adherent cells are observed. At this time, change the culture medium every 48 hours and start passage until the cells proliferate stably and passage for more than 40 generations, forming an immortalized cell line. The cells grow at a stable rate, are round, and grow in suspension, either as single cells or in clusters.

[0021] The resulting cell line was named YYXY-M10, a human chronic myeloid leukemia accelerated phase cell line, and was deposited on October 12, 2024, at the China Center for Type Culture Collection (CCTCC) in Wuhan, with accession number CCTCC NO: C2024346.

[0022] Example 2: Biological characteristics and applications of human chronic myeloid leukemia accelerated phase cell lines YYXY-M10 cells were cultured in IMDM medium containing 10% fetal bovine serum to ensure stable growth and passage in vitro.

[0023] Morphological observation: Take 1×10 6 One YYXY-M10 cell line was placed in a 1.5 mL EP tube and centrifuged at 1500 rpm for 5 min. After discarding the supernatant, 10 μL of IMDM medium was added to resuspend the cells. Then, 2-3 μL of the cell suspension was transferred to a glass slide and spread evenly. After the cell smear dried, it was stained with Wright-Giemsa stain for 5 min, rinsed, and air-dried. The cell smear was then observed under a microscope to observe the cell state and photographed under an oil immersion microscope. (See attached image) Figure 3 As shown, the YYXY-M10 cell line exhibits a primitive cell morphology with large, deeply stained, and mononuclear nuclei, consistent with the morphological characteristics of primitive malignant cells in the accelerated phase of chronic myeloid leukemia.

[0024] Observation of in vitro proliferation capacity: YYXY-M10 cells in logarithmic growth phase were harvested and divided into 1×10⁻⁶ cells. 5 cells / mL, 2×10 5 cells / mL, 4×10 5 cells / mL, 8×10 5 Cells were seeded at a density of 100 μL / mL in 96-well cells culture plates. Then, at the same time on days 0, 1, 2, 3, and 4, 20 μL of MTS cell proliferation reagent was added to each well. After incubation for 4 hours, the absorbance of each well was measured at 492 nm using a microplate reader. Cell proliferation curves at different initial seeding densities were then plotted using software. (See attached image) Figure 4As shown, YYXY-M10 cells have good in vitro proliferation ability, and the cell proliferation rate is significantly correlated with the initial seeding density.

[0025] Chromosome karyotype analysis: YYXY-M10 cell lines in stable culture were treated with colchicine to a final concentration of 0.05 g / mL. After thorough mixing, the cells were incubated at 37°C for 3 hours, and then collected. The collected cells were placed in centrifuge tubes, and preheated 0.075 mol / L KCl solution (37°C) was slowly added along the tube wall to a final volume of at least 8 mL. The tubes were then incubated at 37°C for 40 minutes. After incubation, cell fixation solution was added, and a cell suspension was prepared. Chromosome banding was performed using the R-banding technique, and the results were observed under a microscope, as shown in the attached figure. Figure 5 As shown, the YYXY-M10 cells have a normal karyotype and a chromosome number of 46, XY.

[0026] Tumorigenicity analysis in immunodeficient mice: NCG immunodeficient mice bred by Jicui Pharmaceutical Co., Ltd., strain name: NOD / ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22 / Gpt, were used. Six-week-old female mice were placed in an SPF-grade animal facility for one week of acclimatization. Then, YYXY-M10 cells in the logarithmic growth phase were collected and processed at a rate of 2 × 10⁻⁶ cells / mL. 6 Cells were injected into mice via tail vein injection at a rate of 100 μL / cell. Mice were continuously monitored, and their survival time was recorded until the experimental endpoint (weight loss ≥25%, limb paralysis, or death). Flow cytometry was then used to detect bone marrow tumor cell infiltration at the experimental endpoint: The femur of the mouse hind leg was isolated and placed in 1×PBS buffer. The femoral bone marrow cavity was thoroughly rinsed with 1×PBS using a 1 mL syringe. The rinsing fluid was collected into a 15 mL centrifuge tube, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 500 μL of 1×PBS and filtered through a 70 μm cell sieve. The filtered cells were divided into two aliquots: one aliquot was treated with isotype IgG antibody as a negative control, and the other with APC-labeled human CD45 antibody as a positive control. Both aliquots were incubated at room temperature in the dark for 20 min. After incubation, 1 mL of 1×PBS was added to each aliquot, mixed thoroughly, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. Finally, the cells were resuspended in 500 μL of 1×PBS. Beckman flow cytometry was used for detection. First, the detection threshold was determined based on the negative control, then the positive control samples were analyzed, and the proportion of CD45-positive cells was recorded, as shown in the attached figure. Figure 6 As shown, significant YYXY-M10 cell infiltration was observed in the bone marrow of mice at the experimental endpoint. Survival data were analyzed using the life table method, and survival curves were plotted, as shown in the attached figure. Figure 7As shown, the median survival time in mice was 37.25 days. These results indicate that YYXY-M10 cells possess good in vivo tumorigenesis ability.

[0027] Tyrosine kinase inhibitor sensitivity analysis: Orabatinib final concentration gradients were set at 0, 0.1, 0.5, 1, 5, 10, 25, and 50 nM. Logarithmic growth phase CML cell lines K562 and YYXY-M10, as well as freshly isolated primary bone marrow cells from the patient in Example 1, were selected. After cell counting, the cells were divided into groups of 2 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL in 24-well culture plates, followed by the addition of olaribinib at the aforementioned concentration. After 48 hours of treatment, cells were transferred from the 24-well plates to 96-well plates, with 100 μL of cell suspension transferred to each well. Then, 20 μL of MTS reagent was added to each well, and the plates were incubated at 37°C for 4 hours. The absorbance of each well was measured at 492 nm using a microplate reader, and the half-maximal inhibitory concentration (IC50) of the drug for each group was calculated using software. (See attached figure.) Figure 8 , 9 As shown, the IC50 value of olaribatitinib against K562 cells was 1.74 nM, against primary bone marrow cells from patients was 18.43 nM, and against the YYXY-M10 cell line was 16.11 nM. The experimental results indicate that both primary cells from patients and the YYXY-M10 cell line exhibited significant drug resistance characteristics compared to K562 cells. Furthermore, after successful cell line construction, the cell resistance did not significantly decrease. Further calculation of the resistance index revealed that the resistance indices of primary cells from patients and the YYXY-M10 cell line relative to K562 cells were 10.59 and 9.26, respectively, confirming that both cells exhibited significant resistance to olaribatitinib.

[0028] In summary, the YYXY-M10 cell line is proven to be an ideal experimental cell material, suitable for studies on tyrosine kinase inhibitor resistance mechanisms, olabratinib resistance reversal, and screening of novel drug candidates for drug resistance.

[0029] STR identification of cells: YYXY-M10 cell line cultured in vitro and freshly isolated primary cells from the patient were sent to Shanghai Yihe Biotechnology Co., Ltd. for STR locus and Amelogenin sex typing. Genomic DNA was extracted from the cells using the Axygen genomic DNA extraction kit, and locus amplification was performed using the 21-STR amplification system. STR locus and Amelogenin sex gene detection were completed using an ABI 3730XL genetic analyzer. All experimental procedures were strictly performed according to standard experimental protocols. The results showed that the STR typing data of the YYXY-M10 cell line did not match any existing cell lines in international public databases, indicating it was a newly established cell line. Furthermore, the STR locus and Amelogenin typing results of this cell line were completely homologous to the primary cells from the donor patient, confirming accurate cell origin and no cross-contamination from other cells during in vitro passage culture, indicating a pure and stable cellular genetic background. The STR locus and Amelogenin genotyping results for each cell line are detailed in the table below.

[0030]

[0031] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: This invention relates to a human chronic myeloid leukemia (CML) accelerated phase cell line, its application, preparation method, and reagent kit. The cell line is isolated from bone marrow or peripheral blood samples from patients with CML accelerated phase. Without the addition of any cell growth factors, it can achieve stable in vitro proliferation and continuous passage. The cells exhibit stable in vitro morphology, can form tumors in immunodeficient animals, and are resistant to third-generation tyrosine kinase inhibitors. It can be used to study the pathogenesis of CML, the progression from accelerated phase to blast crisis, and drug resistance mechanisms. It has broad application prospects and practical value in basic research, drug screening and evaluation, and clinical medication guidance for human CML.

[0032] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A human chronic myeloid leukemia blast crisis cell line, characterized in that: The cell line was named human chronic myeloid leukemia accelerated phase cell line YYXY-M10 and was deposited at the China Center for Type Culture Collection on October 12, 2024, with accession number CCTCC NO: C2024346.

2. The human chronic myeloid leukemia blast crisis cell line according to claim 1, characterized in that: The cell line was derived from primary cells from patients with accelerated phase chronic myeloid leukemia who had been treated with first-, second-, and third-generation tyrosine kinase inhibitors.

3. The use of any one of the human chronic myeloid leukemia accelerated phase cell lines according to claims 1 to 2 in the preparation of a drug model for screening treatment of chronic myeloid leukemia accelerated phase.

4. A kit for screening an anti-chronic myeloid leukemia drug, characterized by: The kit contains the human chronic myeloid leukemia accelerated phase cell line as described in any one of claims 1 to 3.

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