A human PDAC ascites tumor cell line and its application

Abstract

The present disclosure provides a human PDAC ascites tumor cell line, belonging to the field of cell technology. The cell line is positive for cytokeratin pan-CK and mucin MUC4 expression. The human PDAC ascites tumor cell line is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC NO: 46044. The human PDAC ascites tumor cell line provided by the present disclosure has a large quantity, can be stably passaged, and is convenient for preservation and amplification. It fills the blank that there is no circulating tumor cell line for pancreatic ductal adenocarcinoma. This cell line provides a powerful tool for in-depth study of the natural occurrence process of circulating tumor cells, searching for new circulating tumor cell markers and drugs.

Description

Technical Field

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

[0002] Pancreatic ductal adenocarcinoma (PDAC) is the most common malignant tumor of the pancreas. This cancer usually has no obvious symptoms in the early stage, resulting in most patients being in the advanced or late stage at the time of diagnosis.

[0003] PDAC is highly heterogeneous, 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 regimens, its treatment mainly relies on surgical resection. However, due to its highly invasive and early metastatic characteristics, many patients do not have the opportunity for surgery at the time of diagnosis. Therefore, especially in inoperable advanced cases, systemic treatments, such as the FOLFIRINOX regimen or chemotherapy regimens combining gemcitabine with albumin-bound paclitaxel, are also important treatment means.

[0004] A stable and reliable PDAC cell line is an important basis for scientific research. Although there are already a variety of human PDAC cell lines on the market, such as the PANC-1, MIA PaCa-2, and BxPC-3 cell lines from in situ PDAC, and the Capan-1, HS 766T, and SW 1990 cell lines from solid metastases. However, there is currently no report on PDAC cell lines derived from ascites. The only AsPC-1 cell line is from the cancerous ascites of human pancreatic cancer xenografts in nude mice, and its representativeness is insufficient. Therefore, the development of a stable circulating tumor cell line and the establishment of an in vitro culture method will be of great help in promoting 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 (immature teratoma, PDAC) ascites tumor cell line and its application.

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

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

[0008] In some embodiments, the cell line was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 28, 2024, with the deposit number CGMCC NO: 46044, and the proposed taxonomic name is a human PDAC ascites tumor cell line.

[0009] According to another aspect of the present disclosure, there is provided 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, there is provided a method for constructing an animal model of human pancreatic ductal adenocarcinoma, the method comprising the following step: inoculating the cell line into an animal.

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

[0012] In some embodiments, the animal includes a mouse, a rat, a dog, a horse, a rabbit, a sheep, a pig, a cow, a monkey, etc.

[0013] According to another aspect of the present disclosure, there is provided 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, there is provided 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, there is provided the use of the cell line or the animal model obtained by the method in screening reagents for diagnosing or detecting human pancreatic ductal adenocarcinoma.

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

[0017] The human PDAC ascites tumor cell line provided by the present disclosure has a large quantity, can be stably passaged, and is convenient for storage and amplification. It fills the gap that there is no circulating tumor cell line for pancreatic ductal adenocarcinoma, and this cell line provides a powerful tool for in-depth study of the natural occurrence process of circulating tumor cells, searching for new circulating tumor cell markers and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the cell growth curve of the PDAC ascites tumor cell line cultured in Example 1. Where the abscissa is the cell growth passage number, and the ordinate is the cell count number.

[0019] Figure 2 The microscopic observation images of the 3rd passage of PDAC ascites tumor cell line in cell culture are shown.

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

[0021] Figure 4 The staining observation images of other cells filtered out in Example 3 are shown. Among them, the green color is CD45 staining, and the blue color is DAPI staining.

[0022] Figure 5 The detection result observation images of the pancreatic tumor cell-specific marker MUC4 in the RB011 cell line in Example 3 are shown. Among them, the yellow color is MUC4 (pancreatic tumor cell-specific marker) staining, and the blue color is DAPI staining. Detailed implementation manners

[0023] In the present disclosure, ascites or peritoneal lavage fluid of PDAC patients is collected, and tumor cells are enriched, separated and cultured in vitro by using a microfluidic system to establish a human-derived PDAC ascites tumor cell line with stable passage. This cell line can further promote the research on this refractory tumor.

[0024] In the present disclosure, a human-derived PDAC ascites tumor cell RB011 capable of stable passage is obtained by separation and culture from clinical patients with pancreatic ductal adenocarcinoma, and is classified and named as a human-derived PDAC ascites tumor cell line. It has been deposited in the China General Microbiological Culture Collection Center (CGMCC) on August 28, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, and the deposit number is CGMCC NO: 46044.

[0025] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be described in detail below in combination with embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0026] Definitions

[0027] Unless otherwise defined, all technical terms and scientific and technical terms used in the present disclosure have the same meanings as those commonly used in the field to which the present disclosure belongs. For the purpose of explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0028] In this text, the term "Circulating Tumor Cell" or "CTC" refers to any circulating cancer cell found in a biological sample. Generally, CTCs shed from solid tumors. Thus, CTCs are typically epithelial cells shed from solid tumors and are present in the circulation of cancer patients at very low concentrations.

[0029] When used in this text, the term "biological sample" refers to any sample that includes CTCs. Sources of the sample include 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 an ascites sample, and the blood sample includes, for example, whole blood or any fraction or component thereof. A blood sample 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 well-known and conventional clinical methods (such as procedures for drawing and processing whole blood). In a specific embodiment, the sample can be peripheral blood drawn from a subject with cancer.

[0030] In this text, the term "Cytokeratin (broad spectrum)", also known as pan-CK (Cytokeratin Pan), is a mixture of two antibodies that can label all epithelial cells, including benign and malignant tumors of monolayer epithelium, squamous epithelium, and urothelial origin.

[0031] In this text, the term "Mucin 4 (MUC4)" is a large membrane-anchored glycoprotein of the mucin family, which is expressed by epithelial cells in various normal tissues such as the lung, bronchus, stomach, colon, and cervix. MUC4 is usually not present in normal pancreas but is expressed in the vast majority of pancreatic tumors, such as pancreatic ductal adenocarcinoma.

[0032] In this text, the term "short tandem repeats (STR)", also known as microsatellite DNA, is generally a segment of DNA repeat sequence composed of 1 - 6 base units in the genome. Due to the high variability of the number of core unit repeats among individuals and their abundance, it constitutes the genetic polymorphism of STR loci. Cell STR identification is to establish the genetic characteristics of a cell line through STR information.

[0033] In this text, the term "DAPI (Diamidino-phenyl-indole)" refers to 4',6-diamidino-2-phenylindole, which is a fluorescent dye that can strongly bind to DNA and is commonly used for fluorescence microscopy observation. Because DAPI can penetrate intact cell membranes, it can be used for staining of both live cells and fixed cells.

[0034] In this text, the term "CD45", also known as the common leukocyte antigen (CLA), is a single-chain transmembrane protein widely present on the surface of leukocytes, containing three domains: a cytoplasmic C-terminus, a transmembrane region, and an extracellular glycosylated amino-terminal region. The CD45 antibody labeled with a fluorescent dye can specifically bind to the CD45 antigen on the cell surface, thereby making the cell carry a fluorophore and can be used for fluorescence microscopy observation.

[0035] In the present disclosure, the terms "about", "around" or "approximately" generally refer to within 20%, within 10%, within 5%, within 4%, within 3%, within 2% or within 1% of a given value or range. If not otherwise specified, the given quantity is an approximation, meaning that the terms "about", "around" or "approximately" can be inferred.

[0036] Unless the context clearly indicates otherwise, the expressions "a" and "an" as used herein include plural referents.

[0037] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following describes the present invention in detail in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications. Examples

[0038] Experimental materials

[0039] 1) Preparation of screening medium: Add 50 mL of FBS solution (Four Seasons Green), 1.1 mg of bFGF (Sino Biological), 1.1 mg of EGF (Sino Biological), 60.5 mg of sodium pyruvate (ThermoFisher Scientific), 80.38 mg of L-glutamine (Solarbio), and 11 mL of penicillin-streptomycin mixture (Gibco) (5000 U / mL penicillin, 5000 μg / mL streptomycin) to 500 mL of H-DMEM (Solarbio) respectively. The total volume is 500 mL. After mixing, filter using a 0.22 μm syringe filter.

[0040] 2) Preparation of maintenance medium: Add 50 mL of KSR solution (Thermo Fisher Scientific), 1.1 mg of bFGF (Sino Biological), 1.1 mg of EGF (Sino Biological), 60.5 mg of sodium pyruvate (Thermo Fisher Scientific), and 80.38 mg of L-glutamine (Solarbio) to Knockout-DMEM (Thermo Fisher Scientific) respectively. The total volume is 500 mL. After mixing, filter through a 0.22 μm syringe filter.

[0041] Example 1. Method and steps for isolation and culture of PDAC ascites tumor cells

[0042] 1) Sample collection: Obtain ascites samples from clinical patients with pancreatic ductal adenocarcinoma, and collect 25 - 50 mL of ascites.

[0043] 2) Sample pretreatment: Treat the sample with human peripheral blood lymphocyte separation solution (Solarbio) (800 g / 30 min / 25 °C, ACC: 9, DEC: 0) to obtain a nucleated cell layer containing circulating tumor cells (CTC) (or called PBMC layer).

[0044] 3) Add an equal volume of sterile PBS (Sangon Biotech) to the PBMC solution according to a 1:1 ratio, mix well and centrifuge again (120 g / 10 min / 25 °C).

[0045] 4) Discard the supernatant to obtain cell pellet, and resuspend it with 10 mL of sterile PBS (Sangon Biotech).

[0046] 5) Use a pipette to add 10 mL of the sample to the sample slot of a disposable cell separation and enrichment collector (LABYRINTH-K01, LABYRINTH Biotechnology Co., Ltd.), and place the collector in the slide chamber of a cell sorter (LABYRINTH-CE01, LABYRINTH Biotechnology Co., Ltd.). Run for 15 min. After the machine operation is completed, open the slide chamber, take out the collector, and take out the detachable cell collection tube.

[0047] 6) Enrich and sort to obtain the sample. After centrifugation, resuspend it with the screening medium, and count the cells. Inoculate the cells into a six-well plate and culture them in an incubator at 37 °C and 5% CO2 for 24 h. At this time, the cells are P1 generation.

[0048] 7) After culturing for 24 h, discard the waste liquid in the six-well plate and continue culturing with the maintenance medium.

[0049] 8) After culturing for 48 h, passage the cells according to the cell density. Digest the cells with 0.25% trypsin (Solarbio), resuspend the cells with the screening medium, and count the cells. At this time, the cells are P2 generation.

[0050] 9) After subculturing into a T25 culture flask and culturing for 24 h, replace the waste liquid in the culture flask with maintenance medium to accelerate the cell proliferation rate.

[0051] 10) After 24 h, harvest the cells. At this time, the cells are at passage P3. Count the cells and calculate the proliferation multiple. Appropriately dilute the cells and inoculate them into multiple T75 culture flasks. The subculture method refers to steps 8) - 9).

[0052] 11) After subculturing into a T75 culture flask and culturing for 24 h, replace the waste liquid in the culture flask with maintenance medium to accelerate the cell proliferation rate.

[0053] 12) After 24 h, harvest the cells. At this time, the cells are at passage P4. Count the cells and calculate the proliferation multiple. Appropriately dilute the cells and inoculate them into multiple T75 culture flasks. The subculture method refers to steps 8) - 9).

[0054] 13) After subculturing into a T75 culture flask and culturing for 24 h, replace the waste liquid in the culture flask with maintenance medium to accelerate the cell proliferation rate.

[0055] 14) After 24 h, harvest the cells. At this time, the cells are at passage P5. Count the cells and calculate the proliferation multiple. Appropriately dilute the cells and inoculate them into multiple T75 culture flasks. The subculture method refers to steps 8) - 9).

[0056] Take the cells of passages 1 - 5 (P1 - P5) for cell counting. The experimental results are shown in Tables 1 - 2, and the growth curve is shown in Figure 1 .

[0057]

[0058]

[0059] Cell cryopreservation: After cell culture, observe under a microscope. After the cells cover 2 / 3 of the flask, digest them with trypsin - EDTA (0.25%). After centrifugation (120 g / 25 °C / 5 min), discard the supernatant, add an appropriate amount of CryoStor ® CS10 cell cryopreservation solution, aliquot into 1 - mL cryotubes. Place the cryotubes in a gradient cooling box, leave them in an - 80 °C refrigerator overnight, and then store them in liquid nitrogen.

[0060] Cell resuscitation: Take out the cryotubes stored in liquid nitrogen, thaw them in a 37 °C water bath, add 1 mL of the first medium to the cryotubes. After centrifugation (120 g / 25 °C / 5 min), discard the supernatant, resuspend the cell suspension, and add it to a culture flask for continued culture. The experimental results show that the obtained cells can be subcultured and cryopreserved normally, and can be stably subcultured for more than 21 passages.

[0061] Example 2. Morphological observation of PDAC ascites tumor cells

[0062] Take the PDAC ascites tumor cells cultured to the 3rd generation in Example 1 and conduct microscopic observation. The circulating tumor cells and passaged cultured cells derived from the ascites of pancreatic ductal adenocarcinoma patients are epithelial-like. The microscopic observation results of the cells are shown in Figure 2 .

[0063] Example 3. Identification of PDAC Ascites Tumor Cells

[0064] This example verified the expression of the pan-cancer tumor cell marker pan-CK and the expression of the pancreatic tumor cell-specific marker MUC4.

[0065] Fix the PDAC ascites tumor cells collected in Example 1 with 4% PFA (Thermo Fisher Scientific) and dilute to a reasonable concentration. At the same time, dilute the cells filtered out in Example 1 to a reasonable concentration as a control. Use an immunohistochemistry pen to draw a square on the glass slide (Shitai), and drop the diluted cell suspension into the square respectively. Dry it at 39 °C and place it in a wet box to prepare for the experiment. Add 200 μL of 0.2% Triton X-100 (Merck) to the glass slide. After reacting for 3 min, discard the excess solution, and carefully drop and wash the glass slide three times with PBST (Solarbio) using a pipette, standing for 3 min each time. Then carefully add 200 μL of 10% goat serum (Sijiqing) along the edge of the slide. After sealing at room temperature for 30 min, aspirate the serum. Add 25 μL each of the 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 incubating at room temperature for 1 h, wash three times with PBST. Again, add 25 μL each of the 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) against the CD45 and pan-CK antibodies to the square area of the slide, and the dilution ratio of both is 1:100. Incubate in the dark at room temperature for 45 min. After incubation, use a pipette to drop 200 μL of PBST to wash the slide three times. After washing, drop a drop of mounting medium solution containing DAPI dye (Abcam) and carefully cover the cover slip. There should be no bubbles at the sample position. Fix the cover slip with nail polish and let it stand overnight in a 4 °C refrigerator. Observe it under a fluorescence microscope the next day.

[0066] The fluorescence identification results of RB011 cells are as shown in Figure 3 shown, and the fluorescence identification results of the cells filtered out in Example 1 are as shown in Figure 4 shown. According to Figure 3The results showed that RB011 cells were positive for pan-CK (+), DAPI (+), and negative for CD45 (-). According to Figure 4 the results shown, the filtered cells were negative for pan-CK (-), positive for DAPI (+), and positive for CD45 (+). These test results further confirmed that RB011 cells were tumor cells.

[0067] For the detection of the pancreatic tumor cell-specific marker MUC4 in RB011 cells, the steps were the same as above. The primary antibody was the mucin MUC4 primary antibody (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 identification results of the pancreatic tumor cell-specific marker MUC4 were as Figure 5 shown. According to Figure 5 the results shown, RB011 cells were positive for MUC4 (+).

[0068] The identification results indicated that both the pan-CK and MUC4 staining of RB011 cells were positive, suggesting that the cells were human PDAC ascites tumor cells.

[0069] Example 4. STR Identification of PDAC Ascites Tumor Cells

[0070] In this example, a multiplex PCR amplification system (CELLSTRID®) was used to amplify 20 STR loci and 1 gender locus of RB011 cells and the human pancreatic duct adenocarcinoma cell line PANC-1 (Cellosaurus); the PCR amplification products were analyzed using an ABI 3130xl DNA analyzer (Applied Biosystems, ABI, USA); the test results were analyzed using GeneMapperID-X v1.5 software (Applied Biosystems, ABI, USA). The cell identity was confirmed by comparison with the cell database of the Swiss Institute of Bioinformatics (Cellosaurous database). According to the cell STR identification standard formulated by the International Cell Line Authentication Committee (ICLAC), when the match degree of the cell line is ≥80%, they are considered to be related, that is, derived from a common ancestral cell; when the match degree is between 55% and 80%, the correlation needs to be further verified; less than 55% indicates that the two are not related.

[0071] The STR identification results are shown in Table 3.

[0072]

[0073] The identification results show that the matching rate of RB011 and PANC-1 is 30.77%. In addition, no human pancreatic duct adenocarcinoma cells with a cell typing matching degree greater than 80.00% with RB011 were found in the Cellosaurus database.

[0074] 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. This cell was named tumor cell RB011 and was deposited on August 28, 2024 at the China General Microbiological Culture Collection Center (CGMCC), deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Zip Code 100101, deposit number CGMCC NO: 46044.

[0075] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls 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 pan-cytokeratin (pan-CK) and positive for mucin 4 expression. The cell line was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on August 28, 2024, with the deposit number CGMCC NO: 46044.

2. The cell line according to claim 1, characterized in that, The cell line is negative for CD45.

3. Use of the cell line according to claim 1 or 2 in the construction of a cell model of human pancreatic ductal adenocarcinoma.

4. Use of the cell line according to claim 1 or 2 in screening for drugs for preventing or treating human pancreatic ductal adenocarcinoma.

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