A human dedifferentiated liposarcoma cell line and its application

Abstract

The present invention provides a human dedifferentiated liposarcoma cell line and application, belonging to the technical field of functional cell lines. The present invention is the first to successfully extract a dedifferentiated liposarcoma cell line, named RP-LPS01, and the preservation number is CGMCC No.46013. The cell line RP-LPS01 can be stably propagated for 50 generations and still maintain amplification without obvious changes, and can be directly used for the construction of a dedifferentiated fat animal model. Compared with the liposarcoma cell lines currently available in the American Type Culture Collection (ATCC), the cell line RP-LPS01 has more obvious molecular and biological characteristics of dedifferentiated liposarcoma, and a fast tumor formation rate in vivo. The cell line RP-LPS01 fills the gap in the in vitro and in vivo liposarcoma model of dedifferentiated liposarcoma, and provides a basis for studying the molecular mechanism of retroperitoneal liposarcoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional cell lines, and in particular relates to a human dedifferentiated liposarcoma cell line and application thereof. Background Art

[0002] Based on histomorphological and genetic characteristics, the WHO classified liposarcoma into five pathological subtypes in 2020, namely well-differentiated liposarcoma (WDLPS), dedifferentiated liposarcoma (DDLPS), myxoid liposarcoma (MLPS), pleomorphic liposarcoma (PLPS) and myxoidpleomorphic liposarcoma. Different liposarcoma subtypes have specific genetic changes, reflecting unique biological characteristics, clinical features and treatment sensitivity.

[0003] DDLPS accounts for 15% to 20% of liposarcoma. It is typically characterized by a sudden or gradual transition from WDLPS to non-lipogenic sarcoma tissue composed of high-grade undifferentiated cells. It is most common in the retroperitoneal region, followed by the limbs, trunk, and mediastinum. DDLPS can be primary or develop from WDLPS. It has strong invasiveness and metastatic potential, with a local recurrence rate of 40% to 80% and a distant metastasis rate of 15% to 20%. DDLPS has a poor prognosis, with a 5-year survival rate of 44%, lower than the 93% of WDLPS. The most important prognostic factor for DDLPS is the anatomical location, among which retroperitoneal DDLPS has the strongest malignancy and the worst prognosis.

[0004] DDLPS research models mainly include in vitro cell line models and in vivo animal models. Establishing mature cell lines is crucial for understanding the biological characteristics of DDLPS and developing new treatment strategies. Currently, there are only three mature commercial liposarcoma cell lines, namely 93T449, 94T778 and Sw872. Among them, 93T449 and 94T778 cell lines are cell lines of the same well-differentiated liposarcoma patient at the time of initial treatment and relapse. They are less malignant and cannot form tumors in mice, so in vivo studies cannot be performed. The Sw872 cell line is regarded as a liposarcoma cell line in some studies, but it lacks the typical 12q13-15 amplification and cannot represent the DDLPS case subtype. Since tumor cell lines are important models for studying tumor biological behavior, they need to correctly reflect the molecular biological characteristics of different pathological types. Therefore, DDLPS cell lines with typical oncological characteristics and rapid in vivo tumor formation are urgently needed. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a human dedifferentiated liposarcoma cell line RP-LPS01, which has the molecular and biological characteristics of dedifferentiated liposarcoma and has the characteristics of fast in vivo modeling.

[0006] The invention provides a human dedifferentiated liposarcoma cell line (Homo sapiens) RP-LPS01, whose deposit number is CGMCC No.46013.

[0007] The present invention provides the use of the human dedifferentiated liposarcoma cell line RP-LPS01 in preparing a dedifferentiated liposarcoma animal model.

[0008] Preferably, the type of animal comprises an immunodeficient mouse.

[0009] The present invention provides the use of the human dedifferentiated liposarcoma cell line RP-LPS01 as an in vitro dedifferentiated liposarcoma cell model.

[0010] The present invention provides a human dedifferentiated liposarcoma cell line RP-LPS01, with a deposit number of CGMCC No. 46013. The cell line RP-LPS01 has good genetic stability, can be stably propagated in conventional DMEM / F12 culture medium, and can still maintain amplification without obvious changes after 50 generations of stable propagation. Compared with the liposarcoma cell lines currently available on ATCC, the cell line RP-LPS01 has more obvious dedifferentiated liposarcoma molecular characteristics and biological characteristics. Experiments show that the cell line RP-LPS01 exhibits adhesion and proliferation characteristics without fat differentiation. At the same time, the cell line RP-LPS01 has chromosome 12q13-q15 amplification and RP-LPS01 cells overexpress CDK4 and MDM2 genes. In addition, the cell line RP-LPS01 can quickly form tumors in immunodeficient animals, and the histological morphology of the derived xenografts is similar to its parental tumor tissue, indicating that the cell line RP-LPS01 retains the DDLPS characteristics. Therefore, the cell line RP-LPS01 provided by the present invention has 12q13–q15 amplification characteristics relative to Sw872, has tumorigenicity in animals relative to 93T449 and 94T778, and is more stable and more convenient to transfect than the patient-derived tumor xenograft (PDX) model. It can provide a convenient and accurate in vivo molecular oncology model of liposarcoma, laying a foundation for studying the molecular mechanism of post-coated liposarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1This is a typical well-digested DDLPS tissue block observed under a phase contrast microscope, scale bar: 500 μm;

[0012] Figure 2 Representative phase contrast microscopy images of the DDLPS cell line RP-LPS01, where Figure A is a typical image at low confluence; Figure B is a typical image at high confluence, scale bar: 500 μm;

[0013] Figure 3 Representative phase contrast microscopy images of DDLPS cell line (RP-LPS01) at different generations under high magnification, among which Figure A is a typical image of RP-LPS01 at the 12th generation; Figure B is a typical image of RP-LPS01 at the 50th generation; scale bar: 200 μm;

[0014] Figure 4 This is the STR profile result of RP-LPS01 cell line;

[0015] Figure 5 The results of FISH probe detection of DDLPS cell line RP-LPS01, red: MDM2 probe, green: Cen12 control probe; scale bar 50μm;

[0016] Figure 6 The Western blot results of DDLPS cell line RP-LPS01 and control cell line are shown;

[0017] Figure 7 The growth curve of subcutaneous xenograft tumor (Figure A) and the result of xenograft tumor formed by RP-LPS01 cells in NOD / SCID mice (Figure B), where the bulge indicated by the arrow is the tumor mass of DDLPS;

[0018] Figure 8 Typical HE staining images of patient surgical specimens and RP-LPS01 xenografts; Figure A is a patient surgical specimen; Figure B is a RP-LPS01 tumor specimen; Scale bar: 100 μm;

[0019] Fig. 9 Representative IHC staining images of patient surgical specimens, RP-LPS01 xenografts, and Sw872 xenografts; scale bar 50 μm in the MDM2 detection group; scale bar 100 μm in the CDK4 detection group.

[0020] Biomaterial Deposit Information

[0021] A human dedifferentiated liposarcoma cell line (Homo sapiens) was deposited on July 3, 2024 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the biological deposit number CGMCC No.46013 and the cell line number RP-LPS01. DETAILED DESCRIPTION

[0022] The invention provides a human dedifferentiated liposarcoma cell line RP-LPS01, whose deposit number is CGMCC No.46013.

[0023] In the present invention, the donor source of the cell line RP-LPS01 is preferably retroperitoneal dedifferentiated liposarcoma tissue of a clinical dedifferentiated liposarcoma patient. The type of protease used for separation preferably includes collagenase IV or collagenase IV combined with at least one of the following: dispase and DNase I enzyme. In one embodiment of the present invention, the isolated cell line RP-LPS01 was detected using short tandem repeats (STRs), and the results showed that none of the gene loci of the cell line RP-LPS01 had multiple alleles, indicating that the isolated cell line did not contain cross-contamination of human cells; at the same time, the American Type Culture Collection (ATCC), the German Collection of Microorganisms and Cell Cultures (DSMZ), and the Japanese Collection of Research Bioresources (JCRB) database were compared, and no cells matching the typing of the cell line RP-LPS01 were found. The cell line RP-LPS01 was confirmed to be an unreported human-derived cell line and was not contaminated by other cell lines.

[0024] In the present invention, the cell line RP-LPS01 preferably has the molecular characteristics and biological characteristics of dedifferentiated liposarcoma. The molecular characteristics of dedifferentiated liposarcoma preferably include chromosome 12q13-q15 specific amplification and overexpression of MDM2 and CDK4 proteins. The biological characteristics of dedifferentiated liposarcoma preferably include the adhesion and proliferation characteristics of dedifferentiated liposarcoma and / or the phenomenon of no fat differentiation. In one embodiment of the present invention, the cell line RP-LPS01 is cultured in DMEM / F12 medium containing 15% FBS, and the cells grow adherently, the cell morphology is irregular polygonal, and no lipid component accumulation is observed under the microscope. In another embodiment of the present invention, a fluorescently labeled MDM2 probe (JLB301002, Jinlu Bio) is used to mark the 12q13-15 segment in the cell line RP-LPS01. The results show that the cell line RP-LPS01 has a cluster-like 12q13-15 amplification, indicating that the cell line RP-LPS01 maintains the molecular pathological characteristics of DDLPS. In another embodiment of the present invention, the cell line RP-LPS01 was detected by Western Bolt technology, and the results showed that the cell line RP-LPS01 had the characteristics of mesenchymal origin, which was manifested by the positive expression of vimentin and N-Cadherin. At the same time, the cell line RP-LPS01 was accompanied by high expression of CDK4, which is a significant feature of DDLPS. On the other hand, the cell line RP-LPS01 lacked the expression of epithelial marker E-cadherin and tumor-associated fibroblast marker α-SMA, indicating that the cell line RP-LPS01 did not contain epithelial cells and matrix fibroblasts. PPAR-γ, a typical marker of adipose differentiation, was not expressed in the cell line RP-LPS01, indicating that the cell line RP-LPS01 had a low adipose differentiation potential, which further supported that the cell line RP-LPS01 had dedifferentiation characteristics, which was consistent with the biological manifestations of DDLPS.

[0025] In the present invention, the cell line RP-LPS01 has good genetic stability. The number of generations of the cell line RP-LPS01 preferably includes 2 to 50 generations, and more preferably 4 generations, 8 generations, 10 generations, 12 generations, 16 generations, 20 generations, 24 generations, 28 generations, 32 generations, 35 generations, 38 generations, 40 generations, 43 generations, 45 generations, 48 ​​generations and 50 generations. The digestive enzyme for the cell line RP-LPS01 is preferably pancreatic enzyme. The cell line is preferably subcultured at a cell ratio of 1:2, and is subcultured every 4 to 5 days. Under this condition, the cell line can be subcultured to the 50th generation, and the proliferation rate and growth state do not change significantly at the 50th generation.

[0026] In the present invention, the cell line RP-LPS01 preferably has the ability to form tumors in immunodeficient animals. The histological morphology of the transplanted cells of the cell line RP-LPS01 is preferably highly similar to the parent tumor tissue. In one embodiment of the present invention, the concentration of 4×10 7 The cell line RP-LPS01 with a concentration of 100 μl / ml was injected into the back subcutaneous tissue of 5-week-old female immunodeficient mice NOD / SCID. Each injection point was injected with 100 μl. The length and width of the subcutaneous transplanted tumor were measured and recorded with a vernier caliper at least once a week. The results showed that the cell line RP-LPS01 could form tumors in immunodeficient mice, while the control cell lines 93T449 and 94T778 could not form tumors. The mouse tumor mass was further separated, and the HE staining results showed that the cell line RP-LPS01 and the cell line-derived xenografts were extremely similar in histology, indicating that the RP-LPS01 cells retained the biological characteristics of the parent DDLPS to a great extent, and the RP-LPS01 xenografts and their parent tumor tissues had typical high expression of CDK4 and MDM2, which further indicated that the RP-LPS01 cell line tumor formation could maintain the tumor molecular biological characteristics of dedifferentiated liposarcoma. The control cell line Sw872 had only very low expression of CDK4 and MDM2 and could not serve as a reasonable model of DDLPS.

[0027] In view of the fact that the human dedifferentiated liposarcoma cell line RP-LPS01 has good differentiated liposarcoma tumor characteristics and tumorigenicity in animals, the present invention provides the use of the human dedifferentiated liposarcoma cell line RP-LPS01 in preparing a dedifferentiated liposarcoma animal model.

[0028] In the present invention, the type of the animal preferably includes immunodeficient mice. The preparation of the dedifferentiated liposarcoma animal model is of great significance in scientific research. It is not only an effective laboratory means to study tumor cell biology, but also provides an important experimental platform and tool for drug screening and research on the mechanism of tumor occurrence and development, which helps to deeply reveal the characteristics of tumors and provide new targets for the diagnosis and treatment of tumors.

[0029] In view of the fact that the human dedifferentiated liposarcoma cell line RP-LPS01 has good differentiated liposarcoma tumor characteristics, the present invention provides the use of the human dedifferentiated liposarcoma cell line RP-LPS01 as a dedifferentiated liposarcoma cell model.

[0030] The present invention provides a detailed description of a human dedifferentiated liposarcoma cell line and its application, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1

[0032] Cell line extraction protocols

[0033] Fresh surgical specimens from patients with dedifferentiated liposarcoma are rinsed with PBS or serum-free medium to remove red blood cells and other impurities. The tissue is soaked in a serum-free medium containing 2% to 3% double antibody, and cut into small pieces with small scissors. Take part of the tissue block (about 0.5 to 1g) and place it in a new culture dish. Use ophthalmic scissors to finely chop it until each piece is small enough to be sucked up with a Pasteur pipette. Add collagenase IV and 0.5mg / ml Dispase at a final concentration of 0.5mg / ml to about 0.5g of tissue fragments, and digest at 37°C for 30min. If conditions permit, shake every 10min and blow it with a Pasteur pipette to prevent DNA release and cause viscosity, and observe. Under a phase contrast microscope, well-digested tissue should be able to see a large number of cells scattered in the culture medium, and the tissue block becomes a hard paste with cells attached to it ( Figure 1 ). Add about 5 mL of 1640 culture medium containing 10% FBS to the culture dish after digestion, and blow with a Pasteur pipette to separate as many cells as possible from the tissue blocks. Transfer the above tissue blocks and digestion solution to a 50 ml centrifuge tube, add PBS containing 2% FBS to about 40 ml, shake well and let stand for about 1 minute, and larger tissue blocks will sink to the bottom. Aspirate the cell suspension with a Pasteur pipette and filter (70μm filter) into a new 50 ml centrifuge tube. Centrifuge the cell suspension at 2000rpm for 5 to 10 minutes. Resuspend the cells in complete culture medium and culture them normally in a culture dish. Observe the cell adhesion the next day and change the medium as appropriate. Add mycoplasma inhibitors until the culture is stable and successfully passaged, and the resulting cell line is named RP-LPS01.

[0034] Example 2

[0035] In vitro culture of cell line RP-LPS01

[0036] The cell line RP-LPS01 was inoculated into DMEM / F12 medium supplemented with 15% FBS and cultured in a humidified incubator with a 5% CO2 environment. The cell culture was kept at 37°C. The cell culture cells adhered to the wall and grew in an irregular polygonal shape. No lipid components were observed under the microscope. Figure 2 ).

[0037] Example 3

[0038] Genetic stability test of cell line RP-LPS01

[0039] The cell line RP-LPS01 was treated with trypsin at a working concentration of 0.25%. (The cells were digested with phenol red (Gibco, brand: 25200072), and passaged in DMEM / F12 complete medium containing 15% FBS at a ratio of 1:2, every 4 to 5 days, until the 50th passage under this condition.

[0040] There was no significant change in proliferation rate and growth state at the 50th generation ( Figure 3 ).

[0041] Example 4

[0042] Short tandem repeats (STRs) detection in cell line RP-LPS01

[0043] Take 5×10 6 DNA was extracted from the RP-LPS01 cell line using Chelex100 (BIO-RAD) according to the instructions of the kit and then analyzed using a Microreader TM The 21ID System (Beijing Read Micro Gene Technology Co., Ltd.) kit was used to amplify 20 STR loci and sex identification loci, and the PCR products were detected using an ABI 3130xl genetic analyzer. The test results were analyzed using GeneMapper IDX software (Applied Biosystems). A blank control group was set up during the test.

[0044] The experiment showed that no PCR products were detected in the blank control group. The results of the cell line RP-LPS01 showed that no multi-allelic phenomenon occurred in each locus, and no human cell cross-contamination was found in the cells (Table 1 and Figure 4 ). No cell line matching the cell type of cell line RP-LPS01 was found by comparing with ATCC, DSMZ, and JCRB databases. RP-LPS01 was proven to be an unreported human cell line and was not contaminated by other cell lines.

[0045] Table 1 STR detection results of RP-LPS01 cell line

[0046]

[0047]

[0048] Example 5

[0049] 12q13-15 region amplification test

[0050] The cell line RP-LPS01 cultured on a cell slide was fixed with paraformaldehyde and then a fluorescently labeled MDM2 probe (JLB301002, Jinlu Bio) was used to label the 12q13-15 region in the cell line RP-LPS01. After elution according to the instructions, the slide was sealed and photographed under a high-power fluorescence microscope.

[0051] Figure 5 The FISH probe of the DDLPS cell line RP-LPS01 showed MDM2 amplification, and the amplification rate could reach 100%, indicating that the cell line RP-LPS01 isolated by the present invention had clustered 12q13-15 amplification, and the RP-LPS01 cell line maintained the molecular pathological characteristics of DDLPS.

[0052] Example 6

[0053] To further confirm that the extracted cell line RP-LPS01 is a cell line of DDLPS, further examination at the protein expression level was performed using western bolt. The experiment also included control cells QGP-1, CAF, 93T449, 94t778, and Sw872, among which QGP-1 is a pancreatic tumor cell line representing a negative control for the epithelial cell spectrum. CAF is a tumor-associated fibroblast extracted from liposarcoma, representing potential confounding cells during the extraction process. Each cell line was lysed using RIPA during the logarithmic growth phase. After the lysate was centrifuged, the protein concentration in the supernatant was determined by the BCA method. After standard SDS-PAGE and transfer, the PVDF membrane was blocked with 5% skim milk and probed with antibodies (CDK4[12790T], PPAR-γ[2435S], E-cadherin[3195S], N-cadherin[13116P], vimentin[5741P], GAPDH[5174S], α-SMA[ab7819]). Afterwards, the washed membrane was visualized by chemiluminescence.

[0054] The results are as follows Figure 6As shown. The cell line RP-LPS01 has the characteristics of mesenchymal origin, which is manifested by the positive expression of vimentin and N-Cadherin. At the same time, these cells are accompanied by high expression of CDK4, which is a prominent feature of DDLPS. On the other hand, the cell line RP-LPS01 lacks the expression of the epithelial marker E-cadherin and the tumor-associated fibroblast marker α-SMA, indicating that the RP-LPS01 isolated by the present invention does not contain epithelial cells and stromal fibroblasts. In particular, PPAR-γ, a typical marker of adipose differentiation, is not expressed in DDLPS cells, indicating that these cells have low adipose differentiation potential, which is consistent with the biological manifestations of DDLPS. In summary, these characteristics of DDLPS cells together constitute their unique molecular biological manifestations.

[0055] Example 7

[0056] DDLPS cell line tumorigenesis experiment and tumor detection

[0057] Digest RP-LPS01 cells, resuspend in PBS and count, and adjust the cell concentration to 4 × 10 7 RP-LPS01 cells were injected subcutaneously into the back of 5-week-old female immunodeficient NOD / SCID mice. 100 μl was injected at each injection point. The length and width of the subcutaneous transplanted tumor were measured and recorded with a vernier caliper at least once a week. The tumor volume was calculated according to formula I and the tumor growth curve was drawn.

[0058] Tumor volume = (tumor long diameter × tumor short diameter 2 ) / 2Formula I.

[0059] like Figure 7 As shown in the figure, 93T449 and 94T778 cells cannot form tumors, while RP-LPS01 cells and Sw872 cells can form tumors. After the mice were killed, the tumors were removed for further HE staining analysis. The results showed that the parent tumor of RP-LPS01 and the xenografts derived from the cell line RP-LPS01 were highly similar in histology ( Figure 8 ), indicating that RP-LPS01 cells retain the biological characteristics of the parent DDLPS to a great extent.

[0060] Furthermore, this example also carried out tissue immunochemistry (IHC) to detect the expression of CDK4 and MDM2 in the cell line RP-LPS01. The results showed that in the RP-LPS01 xenografts and their parents, there were typical high expressions of CDK4 and MDM2 ( Fig. 9). This indicates that the RP-LPS01 cell line can maintain the tumor molecular biological characteristics of DDLPS. However, Sw872 has only very low CDK4 and MDM2 expression and cannot be used as a reasonable model of DDLPS.

[0061] It can be seen from the results of the above examples that the cell line RP-LPS01 isolated by the present invention belongs to DDLPS cells, and is more flexible than PDX and can be used for molecular biology research. Compared with 94T778 and 93T449, it can be used for tumor formation operations. Compared with Sw872, it has more accurate DDLPS molecular oncology characteristics, as shown in Table 2.

[0062] Table 2 Comparison results of different sarcoma cell lines

[0063]

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A human dedifferentiated liposarcoma cell line ( Homo sapiens ) RP-LPS01, characterized by: The deposit number is CGMCC No.46013.

2. Use of the human dedifferentiated liposarcoma cell line RP-LPS01 according to claim 1 in preparing a dedifferentiated liposarcoma animal model, wherein the animal is an immunodeficient mouse.

3. Use of the human dedifferentiated liposarcoma cell line RP-LPS01 according to claim 1 as an in vitro dedifferentiated liposarcoma cell model.

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