Human chronic myeloid leukemia cell line and use thereof

Abstract

A human chronic myeloid leukemia cell line and the use thereof. The human chronic myeloid leukemia cell line is the first cell line internationally established from chronic-phase leukemia cells of chronic myeloid leukemia, and was named human chronic myeloid leukemia cell YYXY-M6, which was deposited at the China Center for Type Culture Collection (Wuhan University, Wuhan, China) on July 24, 2023, under the deposit number of CCTCC NO: C2023219. The leukemia cell line exhibits primitive cell morphology and has three karyotypes, i.e. t(6:11)(q25:q23), del(11)(q23) and normal karyotype (46, XX); is BCR-ABL gene-negative; has good in-vitro proliferation ability; can be used as cellular material for the study of the mechanism of occurrence and development of the chronic phase of chronic myeloid leukemia, from the chronic phase thereof to the blastic phase thereof, and of BCR-ABL gene-negative chronic myeloid leukemia, and for the in-vitro study of individualized treatment; and can also be used for both in-vitro and in-vivo studies of drug screening and evaluation for the chronic phase of chronic myeloid leukemia, from the chronic phase thereof to the blastic phase thereof, and BCR-ABL gene-negative chronic myeloid leukemia, providing guidance for clinical medication.

Description

Human chronic myeloid leukemia cell line and its application Technical Field

[0001] The present invention relates to the fields of biology and oncology, and relates to a human chronic myeloid leukemia cell line and a construction method and application thereof. Background Art

[0002] Chronic myeloid leukemia (CML) is a type of leukemia characterized by excessive proliferation of myelocytes and metamyelocytes in the peripheral blood or bone marrow, which suppresses the normal hematopoietic system in the bone marrow. This chronic disease course is often accompanied by hepatomegaly and splenomegaly. CML progresses through three phases: chronic, accelerated, and blast phases. If the chronic phase is untreated, the disease progresses to the accelerated or blast phases, ultimately transforming into acute leukemia. CML has the characteristic Philadelphia chromosome and produces a specific BCR-ABL fusion gene. The use of tyrosine kinase inhibitors targeting BCR-ABL in CML has resulted in a 10-year overall survival rate exceeding 90s, making CML one of the few malignancies managed as a chronic disease.

[0003] CML generally has a good prognosis with targeted therapy using tyrosine kinase inhibitors. However, once CML enters the accelerated phase or blast crisis, the prognosis becomes extremely poor, with an overall survival of less than one year. However, the mechanisms by which CML progresses from the chronic phase to the accelerated and blast crisis phases remain unclear. CML patients treated with standard tyrosine kinase inhibitors have a long natural course, and the majority achieve long-term survival. Therefore, studying the mechanisms of CML progression from the chronic, accelerated, and blast crisis phases of a single patient is difficult. Establishing CML cell lines at different stages of disease progression is crucial for understanding the mechanisms of CML progression and drug development. The K562 cell line, first established in 1976 from leukemic cells of a patient in the blast crisis phase of chronic myeloid leukemia, was the second cell line established internationally for hematologic malignancies and is currently widely used in research on the pathogenesis of CML, targeted drugs, and animal models. The K562 cell line, positive for the BCR-ABL fusion gene, provides an excellent cellular tool for the discovery and preclinical research of tyrosine kinases. Subsequently, only 13 CML cell lines were successfully established, but all of these were derived from CML in the blast crisis phase and were BCR-ABL positive. Studies have shown that some patients with CML who enter the accelerated or blast crisis phase may lose the BCR-ABL fusion gene. These patients have a worse prognosis and often lose sensitivity to tyrosine kinase inhibitors. CML patients entering the blast crisis phase can present with acute myeloid leukemia, acute lymphoblastic leukemia, or mixed cell leukemia, with an overall poor prognosis. Therefore, key challenges currently facing CML research and treatment are: 1. Uncovering the mechanisms of CML progression, particularly in the blast crisis phase; 2. Developing drugs to prevent CML progression; and 3. Unraveling the mechanisms of CML resistance to tyrosine kinase inhibitors. Establishing cell lines at different stages of CML progression is crucial for studying the pathogenesis, progression, and resistance mechanisms of CML, as well as for drug screening. Currently, no CML cell lines have been established in China, and no cell lines established from the chronic phase of CML have been reported internationally.

[0004] Abnormal chromosomal karyotype plays an important role in the occurrence, development and drug resistance of tumors. Among them, t(6:11) translocation is considered to be a tumor-associated clone, and it has been confirmed that t(6:11)(q27:q23) translocation plays an important role in the occurrence of AML. There are also reports of t(6:11)(q27:q23) in T-cell acute lymphoblastic leukemia and chronic eosinophilic leukemia, but no reports of t(6:11)(q25:q23) translocation have been found so far. The del(11)(q23) chromosomal change has been found in myelodysplastic syndrome, Turner syndrome, primary small testicles, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, and acute myeloid leukemia. The presence of del(11)(q23) in chronic lymphocytic leukemia is associated with poor prognosis of patients. A case of chronic neutrophilic leukemia with del(11)(q23) was also reported, and the patient was unresponsive to hydroxyurea and cytarabine treatment. However, the specific mechanism of action of del(11)(q23) remains unclear. Currently, no hematologic tumor cell lines with t(6:11)(q27:q23) abnormalities or del(11)(q23) abnormalities have been established internationally, nor have hematologic tumor cell lines with both t(6:11)(q27:q23) and del(11)(q23) abnormalities been established.

[0005] Because each patient has a unique genetic background and gene expression profile, tumors at different stages exhibit distinct molecular mechanisms. Tumor cell lines retain the biological characteristics of tumors and can be continuously subcultured in vitro, enabling numerous experiments impossible in vivo. They overcome the limitations of primary cells, which are limited in number and unable to undergo long-term, continuous proliferation in vitro. They provide irreplaceable cellular tools for tumor research. Therefore, establishing CML cell lines at different stages and leveraging advanced molecular biology techniques to reveal the mechanisms of CML progression, particularly blast crisis, and using cell and animal models to screen therapeutic agents for CML blast crisis are crucial for further improving the poor prognosis after CML progression.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a human chronic myeloid leukemia cell line established from human chronic myeloid leukemia chronic phase cells and its construction method and application.

[0008] The object of the present invention is achieved through the following technical solution: A human chronic myeloid leukemia cell line, the cell line is named human chronic myeloid leukemia cell YYXY-M6, which was deposited in the China Center for Type Culture Collection on July 24, 2023, with the preservation number CCTCC NO: C2023219.

[0009] The present invention also provides progeny cells of the human chronic myeloid leukemia cell line described above.

[0010] The human chronic myeloid leukemia cell line YYXY-M6 provided by the present invention is established from chronic myeloid leukemia cells in the chronic phase, exhibits primitive cell morphology, is BCR-ABL gene negative, and is a suspension cell.

[0011] The present invention also provides a use of the human chronic myeloid leukemia cell line as described above, selected from any one or more of the following:

[0012] a. To study the mechanism of transition from chronic phase to blast crisis in chronic myeloid leukemia;

[0013] b. Used for chromosome karyotype analysis of chronic myeloid leukemia after blast crisis for complex karyotypes, specifically: t(6:11)(q25:q23), del(11)(q23) and normal karyotype (46,XX);

[0014] c. To study the mechanism of BCR-ABL negativity after blast crisis in chronic myeloid leukemia;

[0015] d. Preparation of tumor cell models or animal tumor models; tumor cell models include progeny cells established from this cell line or cells established from progeny cells of this cell line transfected with a fluorescently labeled gene. Animal tumor models include animal models of chronic myeloid leukemia established by subcutaneous tumor placement or tail vein injection.

[0016] e. In vitro screening and / or evaluation / preparation of tumor therapeutic drugs; the method for screening tumor chemotherapy drugs can include: adding different therapeutic drugs to the culture medium of human chronic myeloid leukemia cells YYXY-M6, observing the drug cytotoxicity, and obtaining preliminary effective candidate drugs. The candidate drugs are then administered to the cells, and the half-maximal inhibitory concentration (IC50) of the screened effective drugs is calculated. The drug with the lowest IC50 is selected for further application in an animal model, and the survival, tumor size, and metastasis of the animals in the untreated group are compared to screen for potential drugs for the treatment of chronic myeloid leukemia. The animal model used is immunodeficient mice.

[0017] f. Develop cancer drug targets;

[0018] g. Preparation of tumor diagnostic products;

[0019] h. In vitro screening of tumor biotherapeutic drugs / reagents.

[0020] The present invention also provides a method for constructing the human chronic myeloid leukemia chronic phase cell line, comprising the following steps: obtaining fresh blood separated by a blood cell separator from a chronic myeloid leukemia patient in the chronic phase. Take 5 ml of the separated blood and add it dropwise to a 15 ml clean sterile centrifuge tube pre-added with 5 ml of human peripheral blood lymphocyte separation fluid, and centrifuge at 2000 rpm for 20 minutes. After the centrifugation, take the white film cell layer at the junction of the lymphocyte separation fluid and the plasma to a new 15 ml sterile centrifuge tube, add 5 ml of sterile 4°C pre-cooled 1xPBS to resuspend the cells, and centrifuge at 2000 rpm for 5 minutes. Then discard the supernatant, add 5 ml of IMDM complete medium (IMDM 90s + fetal bovine serum 10h) to resuspend the cells, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, add 5 ml of IMDM complete medium, and resuspend the cells. Use an automatic cell counter to count the cell density of the cell suspension, and take 1 to 2*10 8 After adding cells to a T25 cell culture flask, add IMDM medium to 6-8ml, mix thoroughly, and culture the cells in a 37°C constant temperature and humidity incubator. After 5-7 days, replace the IMDM medium with fresh complete medium by centrifugation. Depending on the cell growth status, replace the IMDM medium with fresh complete medium every week until the cells begin to proliferate.

[0021] The beneficial effects of the present invention are as follows: the human chronic myeloid leukemia cells of the present invention are isolated from the peripheral blood of a chronic phase CML patient, and achieve stable in vitro proliferation and continuous passage without any stimulation factors in vitro, and the cell shape is stable in vitro and conforms to the biological characteristics of clinical tumors. The human chronic myeloid leukemia cell line originates from the chronic phase of CML. After establishment, the cell morphology shows a primitive cell morphology. Flow cytometry analysis shows the expression of CD13\CD38\HLA-DR\TdT\CD19\CD20\CD22. Chromosome karyotype analysis shows three karyotypes: t(6:11)(q25:q23), del(11)(q23) and normal karyotype (46,XX). RT-PCR analysis shows negative BCR-ABL fusion gene. The human chronic myeloid leukemia cells can be used to study the pathogenesis of CML, especially the mechanism of occurrence and development of CML from chronic phase to blast phase. The cells can also be used to analyze the efficacy of new anti-leukemia drugs and combination regimens, screen and evaluate leukemia drugs, and guide clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below with reference to examples and accompanying drawings;

[0023] FIG1 shows the results of Wright-Giemsa staining of the primary and established cell lines from human chronic myeloid leukemia patients;

[0024] FIG2 is a cell growth curve of the human chronic myeloid leukemia cell line at different cell densities;

[0025] FIG3 shows the expression results of some surface antigens of primary cells and established cell lines of human chronic myeloid leukemia patients; among them, AD corresponds to CD13, CD38, HLA-DR, and TdT surface antigens, respectively;

[0026] FIG4 shows another part of the surface antigen expression results of primary cells and established cell lines of human chronic myeloid leukemia patients; among them, AD corresponds to CD19, CD20, CD22, and CD4 surface antigens respectively;

[0027] FIG5 shows the results of chromosome karyotype analysis of the human chronic myeloid leukemia cell line; wherein AC corresponds to t(6:11)(q25:q23), 46,XX,del(11)(q23) and normal karyotype (46,XX), respectively;

[0028] FIG6 shows the BCR-ABL gene expression levels in primary cells and established cell lines of human chronic myeloid leukemia patients. DETAILED DESCRIPTION

[0029] The present invention is further illustrated by the following examples, but the present invention is not limited thereto. The experimental methods in the following examples, where no specific conditions are specified, are generally based on conventional conditions.

[0030] Example 1 Preparation of YYXY-M6 cell line

[0031] Primary cell culture: Leukemia cells were immediately isolated from 5 ml of peripheral blood specimens (female) obtained from CML patients diagnosed with CML chronic phase at the People's Hospital Affiliated to Ningbo University. In a biological safety cabinet, 5 ml of blood specimen was added dropwise to a 15 ml sterile centrifuge tube pre-added with 5 ml of lymphocyte separation solution, and centrifuged at 2000 rpm for 20 minutes. After centrifugation, the buffy coat cells were transferred to a new 15 ml sterile centrifuge tube, 5 ml of sterile 1xPBS was added to resuspend the cells, and centrifuged at 2000 rpm for 5 minutes. Discard the supernatant, add 5 ml of IMDM complete medium (IMDM 90% + fetal bovine serum 10%) to resuspend the cells, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, add 5 ml of IMDM complete medium, and resuspend the cells. Count the cells using a cell counting plate, and take 1*10 8 Add cells to a 25cm culture flask and add IMDM medium to 6ml, mix thoroughly, and culture the cells in a 37°C constant temperature and humidity incubator. After one week, remove cell debris by low-speed centrifugation and replace with fresh IMDM complete medium for continued culture. Change the medium weekly thereafter.

[0032] Cell subculture: After 1-2 weeks of culture, cells undergo apoptosis. The remaining non-apoptotic cells remain in a state of neither proliferation nor death, and the culture medium is replaced weekly. Three months into the culture, the cells begin to proliferate and grow in suspension. At this point, the culture medium is replaced every 48-72 hours and subculture begins. To date, the cells have been passaged for over 50 generations, representing an immortalized cell line.

[0033] In the present invention, the cells grow in a suspended state, grow as single cells or in clusters, are round or oval, and have a stable cell growth rate. They are named human chronic myeloid leukemia cells YYXY-M6 and were deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on July 24, 2023, with the deposit number CCTCC NO: C2023219.

[0034] Example 2 Biological Properties and Applications of Human Chronic Myeloid Leukemia Cell Lines

[0035] The present invention uses IMDM culture medium containing 10% fetal bovine serum to culture YYXY-M6 cells, allowing them to grow stably in vitro and be passaged stably. Microscopic observation shows that the cells grow individually or in clusters, and are round or oval in shape. Wright-Giemsa staining shows that the cells are acute leukemia primitive cells with large and darkly stained nuclei (Figure 1). Flow cytometry analysis shows the expression of CD13\CD38\HLA-DR\TdT\CD19\CD20\CD22\CD4 (Figures 3-4). Chromosome karyotype analysis shows t(6:11)(q25:q23), 46,XX,del(11)(q23) and a normal karyotype (Figure 5). RT-PCR analysis shows a negative result for the BCR-ABL fusion gene (Figure 6). This is the first cell line established internationally from a chronic myeloid leukemia patient in the chronic phase. This cell line can be used to study the pathogenesis of chronic myeloid leukemia from chronic phase to blast crisis, screen and / or evaluate / prepare tumor therapeutic drugs; develop tumor drug targets; prepare tumor diagnostic products; screen tumor biotherapeutic drugs / reagents; and develop and detect tumor-related bioengineering products. The details are as follows:

[0036] Morphological observation

[0037] Primary cells from chronic myeloid leukemia patients and cultured YYXY-M6 cell line cells were taken from 1*10 6Cells were centrifuged at 1500 rpm for 5 minutes in a 1.5 ml EP tube. The supernatant was discarded, and the cells were resuspended in 10 μl of culture medium and then smeared. After the cell smear dried, it was stained with Wright-Giemsa solution for 5 minutes, rinsed, and air-dried. Cell morphology was observed under an inverted microscope. As shown in Figure 1A, primary cells from a CML patient exhibit morphological features characteristic of chronic phase chronic myeloid leukemia, with mature granulocytes predominating. As shown in Figure 1B, cells from the YYXY-M6 cell line exhibited large, darkly stained nuclei with mononucleation, demonstrating primitive cell morphology.

[0038] In vitro proliferation ability observation

[0039] The cultured YYXY-M6 cell line cells were divided into 1, 2, 4, 8*10 5 The concentration of the cells was plated in a 96-well plate, with 100 μl plated in each well. 20 μl of cell proliferation reagent MTS was added at 0, 24, 48, 72, 96, and 120 hours, respectively. After 4 hours, the absorbance of the 96-well plate was measured at a wavelength of 492 nm using a microplate reader. The proliferation curves of the cells at different plating concentrations were drawn using GraphPad software, as shown in Figure 2. The cell line cells have good in vitro proliferation ability, and the proliferation rate is related to the cell plating density.

[0040] Flow cytometry surface antigen test

[0041] Take 1*10 6 23 aliquots were dispensed into 23 clean, sterile EP tubes. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and wash the cells once with 1xPBS. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 100 μl of 1xPBS per tube. No antibody was added to tube 1. Tubes 2 through 23 were then loaded with different surface antibodies in the following order: CD11b / CD13 / CD14 / CD33 / CD34 / CD36 / CD38 / HLA-DR / CD2 / CD3 / CD4 / CD7 / CD8 / CD10 / CD19 / CD20 / CD22 / CD117 / TdT / CD64 / CD66c / MPO. 5 μl of each antibody was added. Incubate at room temperature for 30 minutes, then add 1 ml of 1xPBS and mix thoroughly. Intracellular antigens were permeabilized with BD Permeabilization Reagent for staining. Centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in 300 μl of 1xPBS per tube and analyze them on a flow cytometer to measure cell surface antigen expression. The results, as shown in Figures 3-4, show that this cell line expresses CD13, CD38, HLA-DR, TdT, CD19, CD20, CD22, and CD4 surface antigens.

[0042] Chromosome karyotype analysis

[0043] Colchicine was added to the cultured cells to a final concentration of 0.05 g / ml, shaken, and incubated in a 37°C incubator for 2-3 hours. The cells were harvested. At least 8 ml of a 0.075 mol / L KCl solution pre-warmed to 37°C was slowly added along the tube wall and incubated at 37°C for 40 minutes. The fixative was then added to prepare a cell suspension. Cell chromosome banding was observed using the R banding method. The cell karyotype was determined. The results are shown in Figures 5A-C. The cells of this cell line had t(6:11)(q25:q23) (Figure 5A), del(11)(q23) (Figure 5B), and a normal karyotype (Figure 5C).

[0044] BCR-ABL gene testing

[0045] The leukemia cells of chronic myeloid leukemia patients and YYXY-M6 cell line cells were counted and at least 1*10 7 The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cell pellet was stored in an ultra-low temperature freezer at -80 degrees Celsius. The BCR-ABL fusion gene was subsequently detected by a third-party medical testing platform of Deshuobaken. The results are shown in Figure 6. The BCR-ABL gene expression in primary cells of chronic myeloid leukemia patients was 83.94%, while no BCR-ABL gene expression was detected in YYXY-M6 cell line cells.

[0046] Cell STR identification

[0047] Both the cultured cell line and the patient's freshly isolated cells were sent to Shanghai Yihe Biotechnology for genotyping of the cell STR loci and the amelogenin locus. The results indicated that the cell line was unique and did not match any existing international cell lines. The genotype of the cell line matched the patient's freshly isolated cells 100% perfectly at both the STR and amelogenin loci, indicating that the cells were derived from the correct culture source and there was no cross-contamination. The genotyping of the cell STR loci and the amelogenin locus is shown in Table 1.

[0048] Table 1: Genotyping results of STR loci and Amelogenin loci of cells

[0049] The above embodiments are used to explain the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A human chronic myeloid leukemia cell line, characterized in that: The cell line was named human chronic myeloid leukemia cell YYXY-M6 and was deposited in the China Center for Type Culture Collection on July 24, 2023, with the deposit number CCTCC NO: C2023219.

2. The human chronic myeloid leukemia cell line according to claim 1, characterized in that: The human chronic myeloid leukemia cell line YYXY-M6 was established from chronic phase chronic myeloid leukemia cells, exhibiting primitive cell morphology, and chromosome karyotype analysis showed a complex karyotype, specifically: t(6:11)(q25:q23), del(11)(q23) and 46,XX three karyotypes, BCR-ABL gene negative, and the cell line was a suspension cell.

3. A use of the human chronic myeloid leukemia cell line according to any one of claims 1 to 2, characterized in that: Select one or more of the following: a. To study the mechanism of transition from chronic phase to blast crisis in chronic myeloid leukemia; b. Used for chromosomal karyotype analysis of chronic myeloid leukemia after blast crisis for complex karyotypes, specifically: t(6:11)(q25:q23), del(11)(q23) and 46,XX karyotype mechanism study; c. Used for studying the mechanism of BCR-ABL negativity after blast crisis in chronic myeloid leukemia.

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