Human PDAC ascites tumor cell strain and application thereof

Abstract

The invention provides a human-derived PDAC ascites tumor cell strain, and belongs to the technical field of cells, the cell strain is cytokeratin pan-CK positive and mucoprotein MUC4 expression positive, the human-derived PDAC ascites tumor cell strain is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO: 46044. The human PDAC ascites tumor cell strain provided by the invention is huge in number, can be stably passed, and is convenient to store and amplify. The blank that there is no circulating tumor cell line for pancreatic ductal adenocarcinoma is filled, and the cell line provides a powerful tool for deeply researching the natural generation process of circulating tumor cells and searching new circulating tumor cell markers and drugs.

Description

Technical Field

[0001] The invention belongs to the field of cell technology, and specifically relates to a human pancreatic ductal adenocarcinoma ascites tumor cell line and application thereof. Background Art

[0002] Pancreatic duct adenocarcinoma (PDAC) is the most common malignant tumor of the pancreas. This cancer usually has no obvious symptoms in the early stages, resulting in most patients being in advanced or late stages when diagnosed.

[0003] PDAC is highly heterogeneous and is caused by a variety of different gene mutations and molecular changes, including mutations in genes such as KRAS, TP53, CDKN2A, and SMAD4. Since PDAC does not respond well to traditional chemotherapy and radiotherapy, its treatment mainly relies on surgical resection. However, due to its highly invasive and early metastatic characteristics, many patients no longer have the opportunity for surgery at the time of diagnosis. Therefore, especially in unresectable advanced cases, systemic treatment, such as FOLFIRINOX or gemcitabine combined with albumin-bound paclitaxel chemotherapy, is also an important treatment option.

[0004] Stable and reliable PDAC cell lines are an important basis for scientific research. Although there are many human PDAC cell lines on the market, such as PANC-1, MIA PaCa-2, and BxPC-3 cell lines from in situ PDAC, and Capan-1, HS 766T, and SW 1990 cell lines from solid metastatic tumors. However, there are currently no reports of PDAC cell lines derived from ascites. The only AsPC-1 cell line comes from cancerous ascites of human pancreatic cancer nude mice xenografts, which is not representative enough. Therefore, the development of a stable circulating tumor cell line and the establishment of an in vitro culture method will greatly help promote the research on tumor metastasis and treatment. Summary of the invention

[0005] In order to solve at least one of the above problems, the present disclosure provides a human pancreatic ductal adenocarcinoma (PDAC) ascites tumor cell line and application thereof.

[0006] According to one aspect of the present disclosure, a human pancreatic ductal adenocarcinoma (PDAC) ascites tumor cell line is provided, wherein the cell line is positive for cytokeratin pan-CK and / or positive for mucin 4 (MUC4).

[0007] In some embodiments, the cell line is CD45 negative.

[0008] In some embodiments, the cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 28, 2024, with the deposit number CGMCC NO:46044, and the recommended classification name is human PDAC ascites tumor cell line.

[0009] According to another aspect of the present disclosure, provided is the use of the cell line in constructing an animal model or a cell model of human pancreatic ductal adenocarcinoma.

[0010] According to another aspect of the present disclosure, a method for constructing an animal model of human pancreatic ductal adenocarcinoma is provided, wherein the method comprises the following steps: inoculating the cell line into an animal.

[0011] In some embodiments, the animal comprises a mammal.

[0012] In some embodiments, the animal includes mice, rats, dogs, horses, rabbits, sheep, pigs, cows, monkeys, etc.

[0013] According to another aspect of the present disclosure, provided is the use of the cell line or the animal model obtained by the method in developing drug targets for pancreatic ductal adenocarcinoma.

[0014] According to another aspect of the present disclosure, provided is the use of the cell line or the animal model obtained by the method in screening drugs for preventing or treating human pancreatic ductal adenocarcinoma.

[0015] According to another aspect of the present disclosure, provided is the use of the cell line or the animal model obtained by the method in screening agents for diagnosing or detecting human pancreatic ductal adenocarcinoma.

[0016] According to another aspect of the present disclosure, provided is the application of the cell line or the animal model obtained by the method in a research platform for markers, etiology, metastasis mechanism, related signaling pathways, tumor occurrence, development or metastasis, drug resistance mechanism and clinical intervention of pancreatic ductal adenocarcinoma.

[0017] The human PDAC ascites tumor cell lines provided by the present disclosure are huge in number, can be stably propagated, and are easy to store and expand. This fills the gap that there is no circulating tumor cell line for pancreatic ductal adenocarcinoma. The cell line provides a powerful tool for in-depth research on the natural occurrence of circulating tumor cells and finding new circulating tumor cell markers and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The cell growth curve of the PDAC ascites tumor cell line cultured in Example 1 is shown, wherein the abscissa is the cell growth generation, and the ordinate is the cell count number.

[0019] Figure 2 The figure shows the microscopic observation of the PDAC ascites tumor cell line at the third passage of cell culture.

[0020] Figure 3 The staining observation diagram of the PDAC ascites tumor cell line in Example 3 is shown, wherein the red color is pan-CK staining, and the blue color is DAPI staining.

[0021] Figure 4 The staining observation diagram of other cells filtered out in Example 3 is shown, wherein green is CD45 staining, and blue is DAPI staining.

[0022] Figure 5 The diagram shows the observation results of the detection of the pancreatic tumor cell-specific marker MUC4 on the RB011 cell line in Example 3. The yellow color represents MUC4 (pancreatic tumor cell-specific marker) staining, and the blue color represents DAPI staining. DETAILED DESCRIPTION

[0023] The present invention collects ascites or peritoneal lavage fluid from PDAC patients, uses a microfluidics system to enrich and separate tumor cells, and cultures them in vitro to establish a stable human PDAC ascites tumor cell line. The cell line can further advance the research on this refractory tumor.

[0024] The present invention discloses that human PDAC ascites tumor cells RB011 capable of stable propagation were obtained from clinical patients with pancreatic ductal adenocarcinoma by isolation and culture, and were classified and named as human PDAC ascites tumor cell line, which was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Committee on August 28, 2024, with the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the deposit number is CGMCC NO:46044.

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail in some embodiments in conjunction with the following embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0026] definition Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0027] As used herein, the term "circulating tumor cell" or "CTC" refers to any circulating cancer cell found in a biological sample. Typically, CTCs are shed from solid tumors. Thus, CTCs are typically epithelial cells shed from solid tumors that are present in the circulation of patients with cancer at very low concentrations.

[0028] When used in this article, the term "biological sample" refers to any sample including CTC. The source of the sample includes whole blood, bone marrow, pleural fluid, peritoneal fluid, central spinal fluid, metastatic tumors, fresh biopsy samples, urine, saliva and bronchial washings. Specifically, the sample is a blood sample or a peritoneal effusion sample, and the blood sample includes, for example, whole blood or any fraction or component thereof. Blood samples suitable for the present disclosure can be extracted from any known source containing blood cells or their components, such as venous blood, arterial blood, peripheral blood, tissue, etc. For example, the sample can be obtained and processed using a well-known and conventional clinical method (e.g., a procedure for extracting and processing whole blood). In a specific embodiment, the sample can be peripheral blood extracted from a subject suffering from cancer.

[0029] In this article, the term "Cytokeratin (broad spectrum)" is also called pan-CK (Cytokeratin Pan), which is a mixture of two antibodies that can mark all epithelial cells, including simple epithelium, squamous epithelium, and benign and malignant tumors of urothelial origin.

[0030] In this article, the term "mucin 4 (MUC4)" refers to a large membrane-anchored glycoprotein of the mucin family that is expressed by epithelial cells in a variety of normal tissues, including the lung, bronchus, stomach, colon, and cervix. MUC4 is not usually present in the normal pancreas, but is expressed in the vast majority of pancreatic tumors, such as pancreatic ductal adenocarcinoma.

[0031] In this article, the term "short tandem repeats (STR)", also known as microsatellite DNA, is usually a DNA repeat sequence consisting of 1 to 6 base units in the genome. Since the number of core unit repeats is highly variable between individuals and the number is abundant, it constitutes the genetic polymorphism of the STR locus. Cell STR identification is to establish the genetic characteristics of the cell line through STR information.

[0032] In this article, the term "DAPI (Diamidino-phenyl-indole)" refers to 4',6-diamidino-2-phenylindole, a fluorescent dye that strongly binds to DNA and is commonly used in fluorescence microscopy. Because DAPI can penetrate intact cell membranes, it can be used to stain both living and fixed cells.

[0033] In this article, the term "CD45" is also called the common leukocyte antigen (CLA), which is a single-chain transmembrane protein widely present on the surface of leukocytes. It contains three domains: the cytoplasmic C-terminal, the transmembrane region, and the extracellular glycosylated amino-terminal region. CD45 antibodies labeled with fluorescent dyes can specifically bind to the CD45 antigen on the cell surface, so that the cells carry fluorescent dyes and can be used for fluorescence microscopy observation.

[0034] In this disclosure, the terms "around", "about" or "approximately" generally refer to within 20%, within 10%, within 5%, 4%, 3%, 2% or 1% of a given value or range. If not explicitly stated, the quantities given are approximate, meaning that the terms "around", "about" or "approximately" can be inferred.

[0035] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail in some embodiments in conjunction with the following embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications. Example Experimental Materials

[0037] 1) Preparation of screening medium: Add 50 mL FBS solution (Sijiqing), 1.1 mg bFGF (Sino Biological), 1.1 mg EGF (Sino Biological), 60.5 mg sodium pyruvate (ThermoFisher Scientific), 80.38 mg L-glutamine (Solarbio), and 11 mL penicillin-streptomycin mixture (Gibco) (5000 U / mL penicillin, 5000 ug / mL streptomycin) to 500 mL H-DMEM (Solarbio) to a total volume of 500 mL. After mixing, filter using a 0.22 μm syringe filter.

[0038] 2) Maintenance medium preparation: Add 50 mL KSR solution (Thermo Fisher Scientific), 1.1 mg bFGF (Sino Biological), 1.1 mg EGF (Sino Biological), 60.5 mg sodium pyruvate (Thermo Fisher Scientific), and 80.38 mg L-glutamine (Solarbio) to Knockout-DMEM (Thermo Fisher Scientific) to a total volume of 500 mL. After mixing, filter using a 0.22 μm syringe filter. Example 1. Methods and steps for isolating and culturing PDAC ascites tumor cells

[0039] 1) Sample collection: Ascites samples were obtained from clinical patients with pancreatic ductal adenocarcinoma. 25-50 mL of ascites was collected.

[0040] 2) Sample pretreatment: The samples were treated with human peripheral blood lymphocyte separation medium (Solebo) (800g / 30min / 25°C, ACC: 9, DEC: 0) to obtain a nucleated cell layer (or PBMC layer) containing circulating tumor cells (CTCs).

[0041] 3) Add an equal volume of sterile PBS (Sanggong) to the PBMC solution in a 1:1 ratio, mix well, and centrifuge again (120g / 10min / 25℃).

[0042] 4) Remove the supernatant to obtain the cell pellet, and add 10 mL of sterile PBS (Shanghai Biotechnology Co., Ltd.) to resuspend the cell pellet.

[0043] 5) Use a pipette to add 10 mL of sample into the sample slot of a disposable cell separation and enrichment collector (Laborith Biotechnology Co., Ltd., LABYRINTH-K01), and place the collector into the slide compartment of a cell sorter (Laborith Biotechnology Co., Ltd., LABYRINTH-CE01). Run for 15 minutes. After the machine is finished running, open the slide compartment, take out the collector, and then take out the detachable cell collection tube.

[0044] 6) After enrichment and sorting, the sample was resuspended in screening medium after centrifugation, and the cells were counted and inoculated into a six-well plate for culture. The six-well plate was placed in an incubator at 37°C and 5% CO2 for 24 hours. At this time, the cells were at the P1 generation.

[0045] 7) After 24 hours of culture, remove the waste liquid from the six-well plate and continue culturing using maintenance medium.

[0046] 8) After 48 hours of culture, subculture according to cell density. Digest the cells with 0.25% trypsin (Solabo), resuspend the cells with screening medium, and count the cells. At this time, the cells are at the P2 generation.

[0047] 9) After 24 hours of culture in a T25 flask, use maintenance medium to replace the waste liquid in the flask to accelerate cell proliferation.

[0048] 10) After 24 hours, harvest the cells, which are now at the P3 generation. Count the cells and calculate the proliferation multiples. Dilute the cells appropriately and inoculate them into multiple T75 culture flasks. Refer to steps 8) to 9) for the passaging method.

[0049] 11) After 24 hours of culture in T75 flasks, use maintenance medium to replace the waste liquid in the flask to accelerate cell proliferation.

[0050] 12) After 24 hours, harvest the cells, which are now P4. Count the cells and calculate the proliferation multiples. Dilute the cells appropriately and inoculate them into multiple T75 culture flasks. Refer to steps 8) to 9) for the passaging method.

[0051] 13) After 24 hours of culture in a T75 flask, use maintenance medium to replace the waste liquid in the flask to accelerate cell proliferation.

[0052] 14) After 24 hours, harvest the cells, which are now P5. Count the cells and calculate the proliferation multiples. Dilute the cells appropriately and inoculate them into multiple T75 culture flasks. Refer to steps 8) to 9) for the passaging method.

[0053] Take the 1st to 5th generation (P1~P5) cells and count the cells. The experimental results are shown in Tables 1~2, and the growth curve is shown in Figure 1 .

[0054]

[0055]

[0056] Cell cryopreservation: After cell culture, observe under a microscope. When the cells have grown to 2 / 3 of the bottle, use trypsin-EDTA (0.25%) to digest, centrifuge (120g / 25℃ / 5min), discard the supernatant, and add an appropriate amount of CryoStor ® The CS10 cell freezing solution was dispensed into 1 mL cryotubes, which were placed in a gradient cooling box and stored in liquid nitrogen after being stored in a -80°C refrigerator overnight.

[0057] Cell recovery: Take out the cryovials stored in liquid nitrogen, thaw them in a 37°C water bath, add 1 mL of the first culture medium to the cryovials, centrifuge (120g / 25°C / 5min), discard the supernatant, resuspend the cell solution, and add it to the culture flask for continued culture. The experimental results show that the obtained cells can be passaged and frozen normally, and can be stably passaged for more than 21 generations. Example 2. Morphological observation of PDAC ascites tumor cells

[0058] The PDAC ascites tumor cells cultured to the third generation in Example 1 were taken for microscopic observation. The circulating tumor cells and subcultured cells in the ascites of patients with pancreatic ductal adenocarcinoma were epithelial-like. The results of cell observation under the microscope were shown in Figure 2. Figure 2 . Example 3. Identification of PDAC ascites tumor cells

[0059] This example verifies the expression of pan-CK, a pan-cancer tumor cell marker, and MUC4, a pancreatic tumor cell-specific marker.

[0060] The PDAC ascites tumor cells collected in Example 1 were fixed with 4% PFA (Thermo Fisher Scientific) and diluted to a reasonable concentration. At the same time, the cells filtered out in Example 1 were diluted to a reasonable concentration as a control. Use an immunohistochemistry pen to draw a box on a glass slide (Shitai), and the diluted cell suspension was dripped into the box respectively, dried at 39°C, and placed in a wet box to prepare for the experiment. Add 200 μL of 0.2% Triton x-100 (Merck) to the slide, discard the excess solution after reacting for 3 minutes, and use a pipette to carefully drip PBST (Solebo) to wash the slide three times, each time standing for 3 minutes. Then carefully add 200 μL of 10% goat serum (Sijiqing) along the edge of the slide, and absorb the serum after blocking at room temperature for 30 minutes. Add 25 μL of diluted CD45 primary antibody (Bio-RAD) and pan-CK primary antibody (Thermo Fisher Scientific) to the target area of ​​the slide, and the dilution ratio of both is 1:100. After incubation at room temperature for 1 hour, wash three times with PBST. Add 25 μL of secondary antibodies Goat anti-mouse IgG2a Cross-Adsorbed, Alexa Fluor® 488 (Thermo Fisher Scientific) and Goat anti-mouse IgG1 Cross-Adsorbed, Alexa Fluor 546 (Thermo Fisher Scientific) to the square area of ​​the slide again, with a dilution ratio of 1:100, and incubate at room temperature in the dark for 45 minutes. After incubation, use a pipette to add 200 μL PBST to wash the slide three times. After washing, add a drop of mounting medium solution containing DAPI dye (Abcam, abcam), carefully cover the cover glass, and there should be no bubbles at the sample position. Fix the cover glass with nail polish, let it stand in a refrigerator at 4°C overnight, and observe it under a fluorescence microscope the next day.

[0061] The fluorescence identification results of RB011 cells are as follows Figure 3 As shown, the fluorescence identification results of the cells filtered out in Example 1 are as follows Figure 4 As shown. Figure 3The results shown are that RB011 cells are pan-CK positive (+), DAPI positive (+), and CD45 negative (-). Figure 4 As shown in the results, the filtered cells were pan-CK negative (-), DAPI positive (+), and CD45 positive (+). These test results further confirmed that RB011 cells were tumor cells.

[0062] RB011 cells were tested for pancreatic tumor cell-specific marker MUC4 using the same procedure as above. The primary antibody was mucin MUC4 (Thermo Fisher Scientific) and the secondary antibody was goat anti-mouse IgG (H+L) Cross-Adsorbed, AlexaFluor™ 647 (Thermo Fisher Scientific) to identify the origin of the RB011 cell line. The results of the identification of pancreatic tumor cell-specific marker MUC4 are shown in Figure 2. Figure 5 As shown, according to Figure 5 Results shown, RB011 cells are MUC4(+).

[0063] The identification results showed that the pan-CK and MUC4 staining of RB011 cells were both positive, indicating that the cells were human PDAC ascites tumor cells. Example 4. STR identification of PDAC ascites tumor cells

[0064] In this example, 20 STR loci and 1 sex locus of RB011 cells and human pancreatic ductal adenocarcinoma cell line PANC-1 (Cellosaurus) were amplified using a multiplex PCR composite amplification system (CELLSTRID®); the PCR amplification products were analyzed using an ABI 3130xl DNA analyzer (Applied Biosystems, ABI); the test results were analyzed using GeneMapperID-X v1.5 (Applied Biosystems, ABI) software. The cell identity was confirmed by comparing with the cell database (Cellosaurous database) of the Swiss Institute of Bioinformatics. According to the cell STR identification standards established by the International Committee on Cell Authentication (ICLAC), when the matching degree of cell lines is ≥80%, they are considered to be related, that is, derived from a common ancestral cell; when the matching degree is between 55% and 80%, the correlation needs to be further verified; less than 55%, indicating that the two are not related.

[0065] The STR identification results are shown in Table 3.

[0066]

[0067] The identification results showed that the match rate between RB011 and PANC-1 was 30.77%. In addition, no human pancreatic ductal adenocarcinoma cells with a cell typing match greater than 80.00% were found in the Cellosaurus database for RB011.

[0068] Based on the above results, it can be determined that the cell RB011 isolated in the present disclosure is a new PDAC ascites tumor cell line. The cell was named tumor cell RB011 and deposited in the General Microbiology Center of China Microbiological Culture Collection (CGMCC) on August 28, 2024, with the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the deposit number is CGMCC NO:46044.

[0069] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A human pancreatic ductal adenocarcinoma ascites tumor cell line, characterized in that: The cell line is positive for cytokeratin pan-CK and mucin 4 expression.

2. The cell line according to claim 1, characterized in that The cell lines described are CD45 negative.

3. The cell line according to claim 1, characterized in that The cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 28, 2024, with the deposit number CGMCC NO:46044.

4. Use of the cell line according to any one of claims 1 to 3 in constructing an animal model or a cell model of human pancreatic ductal adenocarcinoma.

5. A method for constructing an animal model of human pancreatic ductal adenocarcinoma, characterized in that: The method comprises the following steps: inoculating the cell line according to any one of claims 1 to 3 into an animal.

6. The method according to claim 5, characterized in that The animals include mammals.

7. The method according to claim 5, characterized in that The animals include mice, rats, dogs, horses, rabbits, sheep, pigs, cows or monkeys.

8. Use of the cell line according to any one of claims 1 to 3 or the animal model obtained by the method according to any one of claims 5 to 7 in developing drug targets for pancreatic ductal adenocarcinoma.

9. Use of the cell line according to any one of claims 1 to 3 or the animal model obtained by the method according to any one of claims 5 to 7 in screening drugs for preventing or treating human pancreatic ductal adenocarcinoma and / or screening agents for diagnosing or detecting human pancreatic ductal adenocarcinoma.

10. Use of the cell line according to any one of claims 1 to 3 or the animal model obtained by the method according to any one of claims 5 to 7 in the study of markers, etiology, metastasis mechanism, related signaling pathways, tumor occurrence, development or metastasis, drug resistance mechanism and clinical intervention of pancreatic ductal adenocarcinoma.

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