Canine mammary gland liposarcoma cell line and application thereof

Abstract

The invention discloses a canine mammary gland liposarcoma cell line and application thereof, the canine mammary gland liposarcoma cell line is established through primary culture of an in-vitro canine mammary gland tumor excision specimen, the canine mammary gland liposarcoma cell line is named as canine mammary gland liposarcoma cell line CMLPS-N1, the preservation number is CCTCC NO: C2024371, the cell line is a canine mammary gland liposarcoma cell line with positive MDM2, ER and PR, and the canine mammary gland liposarcoma cell line is named as canine mammary gland liposarcoma cell line. The canine mammary gland liposarcoma cells have the same protein expression as primary tumor tissues, also have chromosome abnormal karyotype, can be continuously subcultured in vitro to keep the characteristics of the canine mammary gland liposarcoma cells unchanged, have the tumor characteristics of tumor formation ability in mice, and are suitable for being used as a good model for researching and developing canine mammary gland tumor related immunotherapy, diagnosis and drugs.

Description

Technical Field

[0001] The present invention belongs to the field of microbial medicine, and more specifically, relates to a canine mammary gland liposarcoma cell line and its application. Background Art

[0002] As the most intimate companion animals of humans, pet dogs have gradually entered most families. As of 2022, the predicted number of pet dogs in China has reached 50.85 million, ranking first in the world. Canine mammary tumors seriously affect the health of dogs due to their high incidence and high malignant conversion rate. From 2017 to 2021, canine mammary tumor cases accounted for 46.7% of all canine tumor cases in the country, exceeding skin tumors and ranking first, and its malignant conversion rate has also exceeded 50%.

[0003] Canine mammary tumors can originate from epithelial tissue or mesenchymal tissue. Among malignant tumors, epithelial-derived mammary tumors are called breast cancer, and mesenchymal-derived ones are called mammary sarcoma (CMS). Most mammary tumors originate from epithelial cells and have been widely studied, but the research on CMS is relatively less. CMS is a malignant tumor originating from the mammary mesenchymal tissue, characterized by easy metastasis and poor prognosis. Common CMS includes liposarcoma, osteosarcoma, chondrosarcoma, and fibrosarcoma.

[0004] Canine mammary gland liposarcoma (CMLPS) is a liposarcoma that occurs in the canine mammary gland and is a malignant tumor formed by the mutation of lipoblasts in the canine mammary mesenchymal tissue. In the global research on canine mammary tumors, it is rare to establish a stable cell line that can maintain long-term passage, and most of them are breast cancer cell lines. Currently, there is only 1 report on the establishment of a CMS cell line. In 2006, Kawabata et al. successfully established a canine mammary gland osteosarcoma cell line MCO-Y4 in Japan, and there has been no report on a stably passaged CMLPS cell line.

[0005] In view of the fact that the establishment of tumor cell lines plays a crucial role in the basic research of tumors, however, the available canine mammary sarcoma cell lines are limited globally, and each cell line has its unique characteristics. Therefore, it is particularly important to enrich the types and quantities of cell lines, which also provides a basis for the development of translational medicine. The present invention intends to establish a canine mammary gland liposarcoma cell line to lay a foundation for further exploring the etiology, metastasis mechanism, drug resistance mechanism, and clinical intervention of CMS. Summary of the Invention

[0006] The object of the present invention is to provide a canine mammary gland liposarcoma cell line, named Canine Mammary Gland Liposarcoma Cell Line CMLPS-N1 (Canis lupus famliaris). This cell line was deposited at the China Center for Type Culture Collection on October 29, 2024, with the deposit number CCTCC NO: C2024371. CMLPS-N1 is a mammary gland liposarcoma cell line with positive expression of MDM2, ER, and PR. It can be continuously passaged in vitro while maintaining its differentiated phenotype and molecular phenotype unchanged, and has the tumor characteristics of forming tumors in mice.

[0007] The canine mammary gland liposarcoma cell line CMLPS-N1 was obtained by the following method:

[0008] 1. Specimen collection: Obtain fresh canine mammary gland tumor resection specimens in vitro. Immerse the lesion tissue in sterile 5% triple-antibody (penicillin, streptomycin, amphotericin B) PBS solution for washing, and remove connective tissue and necrotic tissue;

[0009] 2. Primary culture: Digest the tumor tissue and inoculate it on the wall of a T25 culture flask, and perform static culture. After about 24 hours, the cells adhere and grow;

[0010] 3. Subculture: When the cells cover the bottom of the flask, aspirate the culture medium, rinse with PBS buffer, digest with 0.25% trypsin, and passage at a ratio of 1:2. Currently, it has been passaged more than 80 generations.

[0011] After biological property identification, a canine mammary gland liposarcoma cell line was obtained, named CMLPS-N1, with the deposit number CCTCC NO: C2024371, and it has the following biological properties:

[0012] 1. Grow in a monolayer and adhere to the wall, losing contact inhibition. The cell morphology is spindle-shaped to polygonal, arranged in a fibroblast-like pattern, with the ability to grow in overlapping layers. The cell nucleus is relatively large, with multiple nucleoli. The cell morphology is stable after being passaged 50 generations;

[0013] 2. Grow well in vitro, passage once every 1.5 days, and can be continuously and stably passaged. Currently, it has been passaged more than 80 generations;

[0014] 3. The result of chromosome karyotype analysis shows that both the chromosome structure and number are abnormal;

[0015] 4. The results of immunofluorescence staining of CMLPS-N1 cells are consistent with the immunohistochemical staining results of mammary gland liposarcoma tissues. PCK and HER-2 are negative, while MDM2, Vimentin, ER, PR, and Ki67 are positive, confirming that the origin of this cell line is the tumor tissue from which the specimens were taken;

[0016] 5. The CMLPS-N1 cell line has successfully established xenograft tumor models in two immunodeficient mouse strains, BALB / c and BALB / c-nude, and has the characteristic of forming tumors in mice.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. The cell line established in the present invention is the first canine mammary gland liposarcoma cell line in the country and even globally that can be stably passaged more than 50 generations while maintaining its cell characteristics unchanged.

[0019] 2. The cell line established in the present invention can successfully establish xenograft tumor models in BALB / c and BALB / c-nude mice. The tumor formation rate in BALB / c-nude mice is high, reaching 100%.

[0020] 3. The cell line established in the present invention has been successfully used in the activity inhibition test of anti-cancer drugs on tumor cells and is suitable as cell material for developing chemotherapy drugs, immunotherapy, and early diagnosis related to canine mammary sarcoma. Description of the Drawings

[0021] Figure 1 : Primary canine mammary tumor tissue for primary culture of cancer cells.

[0022] Figure 2 : Hematoxylin-eosin staining diagram of primary tumor tissue.

[0023] Figure 3 : Immunohistochemical staining diagram of primary tumor tissue.

[0024] Figure 4 : Morphology of CMLPS-N1 cells under an inverted microscope.

[0025] Figure 5 : Oil red staining diagram of CMLPS-N1 cells under an inverted microscope.

[0026] Figure 6 : Morphology and structure of CMLPS-N1 cells under a transmission electron microscope.

[0027] Figure 7 : Growth curve of CMLPS-N1 cells.

[0028] Figure 8 : Karyotype analysis diagram of CMLPS-N1 cells.

[0029] Figure 9 : Immunofluorescence diagram of CMLPS-N1 cells.

[0030] Figure 10 : Subcutaneous tumor formation diagram of CMLPS-N1, a canine mammary gland liposarcoma cell line, in BALB / c-nude mice.

[0031] Figure 11 : Gross anatomy of transplanted tumor in BALB / c-nude mice.

[0032] Figure 12 : Hematoxylin-eosin staining of transplanted tumor tissue in BALB / c-nude mice.

[0033] Figure 13 : Subcutaneous tumor formation in BALB / c mice with canine mammary liposarcoma cell line CMLPS-N1.

[0034] Figure 14 : Gross anatomy of transplanted tumor in BALB / c mice.

[0035] Figure 15 : Immunohistochemical staining of transplanted tumor tissue in BALB / c-nude mice.

[0036] Figure 16 : Inhibitory curve of cisplatin on the viability of CMLPS-N1 and CMT-7364 cells.

[0037] Figure 17 : Inhibitory curve of paclitaxel on the viability of CMLPS-N1 and CMT-7364 cells.

[0038] Figure 18 : Inhibitory curve of epirubicin on the viability of CMLPS-N1 and CMT-7364 cells.

[0039] Figure 19 : Inhibitory curve of 5-fluorouracil on the viability of CMLPS-N1 and CMT-7364 cells.

[0040] Figure 20 : Mycoplasma detection of CMLPS-N1 cells. Detailed implementation

[0041] Example 1 Preparation of canine mammary liposarcoma cell line CMLPS-N1

[0042] 1. Sample collection

[0043] In July 2024, a fresh sample of excised canine mammary tumor was obtained from the Animal Hospital of Huazhong Agricultural University (as Figure 1 shown), and the pathological diagnosis was mammary liposarcoma (as Figure 2 shown). One part of the lesion tissue was taken as 1 cm 3 and immersed in sterile PBS solution containing 5% triple antibiotics (penicillin, streptomycin, amphotericin B) for cleaning, and connective tissue and necrotic tissue were removed; another part was taken as 1 cm 3 at the junction of tumor and normal tissue and immersed in formaldehyde for fixation.

[0044] 2. Primary culture

[0045] Cut the rinsed and trimmed tumor tissue blocks into small pieces about 1 mm 3 in size, put them into a 50 ml centrifuge tube, add about 5 ml of collagenase solution, seal the centrifuge tube, and digest by shaking at 37 °C until the tissue blocks are dispersed into flocs. Filter the digested tissue fluid through a 100-mesh sieve, collect the digested fluid, transfer it into a centrifuge tube, centrifuge at 1000 r / min for 5 min, remove the supernatant, rinse 1 - 2 times with sterile PBS buffer or serum-free medium, remove the supernatant, add serum-containing culture medium to make a cell suspension, inoculate it on the wall of a T25 culture flask, and culture it statically.

[0046] 3. Subculture

[0047] When the cells cover the bottom of the flask, aspirate the old culture medium in the original flask, rinse 3 times with PBS buffer, digest with 0.25% trypsin, and subculture at a ratio of 1:2. Currently, it has been subcultured more than 80 generations; a canine mammary gland liposarcoma cell line was obtained, named canine mammary gland liposarcoma cell line CMLPS-N1, which was deposited by the China Center for Type Culture Collection on October 29, 2024. The address of the depository is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO:C2024371.

[0048] The conventional in vitro culture conditions for this experiment are DMEM cell culture medium containing 10% FBS, 2% L-glutamine, and 1% double antibiotics (penicillin, streptomycin). The culture temperature is 37 °C, and the gas environment is 5% CO 2 / 95% air, and the humidity is saturated humidity.

[0049] 4. Immunohistochemical staining of canine mammary gland liposarcoma tissue

[0050] ① Baking the slides: Preheat the tissue sections in a 65 °C constant temperature air blast drying oven for 20 min;

[0051] ② Deparaffinization to water: Immerse in xylene I for 20 min, xylene II for 20 min, 100% ethanol for 2 min, twice in total, 95%, 90%, and 85% ethanol for 2 min each, and finally immerse in distilled water to remove residual ethanol;

[0052] ③ Antigen repair: Acidic microwave repair. Place the tissue sections in sodium citrate buffer with pH = 6.0, perform antigen repair with high-temperature microwave, high fire for 5 min, stop the fire for 2 min, maintain with low fire for 20 min. After the repair is completed, cool at room temperature. After about 30 min, rinse with PBS 3 times, 5 min each time; Alkaline high-pressure repair. Put the tissue sections into Tris-EDTA antigen repair solution with pH = 9.0, heat and repair for 30 min. After completion, take out and cool naturally to room temperature, wash with PBS 3 times, 5 min each time;

[0053] ④ Remove endogenous peroxidase activity: Take out the sections, use absorbent paper to wipe off the moisture around the tissue, draw a circle around the tissue with an immunohistochemistry pen about 2 mm away from the tissue, drop peroxidase blocker in the circle, act at room temperature for 15 min, wash with PBS 3 times, 5 min each time;

[0054] ⑤ Block non-specific antigens with serum or 5% BSA: Drop a certain amount of serum or BSA onto the tissue sections, incubate at room temperature for 30 min, discard the blocking solution, do not wash;

[0055] ⑥ Incubate with primary antibodies: Add different diluted primary antibodies, including PCK, Vimentin, MDM2, Ki67, HER-2, ER, PR, incubate at 4°C for 12 h;

[0056] ⑦ Incubate with secondary antibodies: Take out the wet box, discard the primary antibodies on the tissue, wash with PBS 3 times, 5 min each time, drop diluted HRP-labeled goat anti-rabbit IgG to cover the tissue, incubate at room temperature in the wet box for 30 min;

[0057] ⑧ DAB color development: Discard the secondary antibodies on the tissue, wash with PBS 3 times, 5 min each time; Dilute DAB with diluent at 1:50, drop the diluted DAB color development solution in the circle to cover the tissue, observe the color development effect under the microscope, and immediately terminate with distilled water after the staining is completed;

[0058] ⑨ Counterstain with hematoxylin: Immerse with hematoxylin for 7 min, rinse with tap water until the water is colorless, differentiate with 0.5% hydrochloric acid for 13 s, rinse with running water for 8 min;

[0059] ⑩ Dehydration and clearing: Immerse in gradient ethanol (80%, 90%, 95%, 95%, 100%, 100%), about 2 min each time, immerse in xylene I for 5 min, immerse in xylene II for 8 min. After air-drying the glass slides in the fume hood, seal with neutral gum, store at room temperature or directly interpret under the microscope.

[0060] Immunohistochemistry results of canine mammary liposarcoma tissue: The expression of two proteins, the epithelial marker PCK and the human epidermal growth factor receptor HER-2, was negative; the expression of five proteins, the liposarcoma marker MDM2, the mesenchymal marker Vimentin, the estrogen receptor ER, the progesterone receptor PR, and the tumor proliferation marker Ki67, was positive (such as Figure 3 shown).

[0061] Example 2 Biological characteristics of canine mammary liposarcoma cell line CMLPS-N1

[0062] 1. Cell morphology

[0063] ①Observation under an inverted phase contrast microscope: The cells grow in a monolayer adhering to the wall, losing contact inhibition, with a spindle-shaped to polygonal cell morphology, arranged like fibroblasts, and capable of overlapping growth. The cells are morphologically stable after 50 generations (e.g. Figure 4 shown).

[0064] ②Oil red staining to observe intracellular lipid droplets: cells are evenly inoculated in a 24-well plate, and when the cells are confluent to 50%, the culture medium in the cell culture wells is discarded, and the cells are washed twice with PBS; 200 μL of 4% paraformaldehyde is added to each well and fixed for 15 minutes at room temperature. Discard the fixative and wash twice with PBS; treat with 60% volume isopropanol for 2 minutes; stain with 0.5% oil red for 15 minutes; wash with 60% isopropanol and then wash twice with PBS, and observe under an optical microscope. It can be seen that more than 95% of the cells have lipid droplets in the cytoplasm that are stained orange with oil red dye. The number of lipid droplets is large and distributed in clusters (such as Figure 5 shown).

[0065] ③ Transmission electron microscopy observation: When the cells are confluent to 80%, discard the culture medium, rinse with PBS for 3 times, add 2.5% glutaraldehyde electron microscopy fixative, fix at room temperature for 5 minutes, gently scrape the cells with a cell scraper, collect them in a centrifuge tube for centrifugation, and you can see a mung bean-sized cell precipitate at the bottom of the tube. Add new fixative, fix for another 30 minutes at room temperature in the dark to prepare the electron microscopy fixed sample. Under the electron microscope, the cells are long spindle-shaped; the nucleus is large, the nucleolus is obvious, and the nuclear-cytoplasmic ratio is high; there are a large number of mitochondria in the cytoplasm, some of which have complete mitochondrial cristae and clear double membrane structures, and some of which have blurred mitochondrial cristae and outer membranes, and new and immature mitochondria can also be seen; there are many rough endoplasmic reticulum structures, and the endoplasmic reticulum is dilated; Golgi bodies and lysosomes can be seen; there are also many vacuoles scattered in the cytoplasm (such as Figure 6 shown).

[0066] 2. Cell Growth Curve

[0067] Take cells in logarithmic growth phase to make 10 5Cell suspension with a cell count of / mL was inoculated into a 96-well plate and placed in an incubator at 37°C and 5% CO 2 for culturing. Cell number detection was performed at 0, 1, 2, 3, 4, 5, and 6 days respectively. Using the culture time as the horizontal axis and the cell number as the vertical axis, a cell growth curve was plotted (as shown in Figure 7 ), and the calculated cell doubling time was 33.98 h.

[0068] 3. Chromosome karyotype analysis

[0069] ① Take cells in the logarithmic growth phase, treat them with 0.05 mg / ml colchicine, and continue culturing at 37°C for 5 h;

[0070] ② Digest and collect the cells with trypsin and transfer them into a 10 ml centrifuge tube;

[0071] ③ Hypotonic treatment: Add 0.075 mol / L KCl and mix well, incubate at 37°C for 30 min;

[0072] ④ Fix with fixative (methanol: glacial acetic acid = 3:1);

[0073] ⑤ Stain with Giemsa stain. Select 50 cells with well-dispersed, non-overlapping, and non-dispersed chromosomes under the microscope for observation and counting. The number of chromosomes in canine somatic cells is 78. Count the chromosomes of metaphase nuclei under the oil immersion lens. Both the chromosome structure and number show abnormalities (as shown in Figure 8 ). The results show that the number of chromosomes in this cell line ranges from 52 to 126, and the median is 63.

[0074] 4. Immunofluorescence detection of cell marker proteins

[0075] ① After trypsinizing the cells, suspend them with 3 mL of complete medium, and thoroughly pipette them into a single-cell suspension. Evenly inoculate the cells into a 24-well plate; when the cells reach 50% confluence, discard the medium in the cell culture wells and wash twice with PBS;

[0076] ② After sucking dry the PBS, add 200 μL of 4% paraformaldehyde to each well and fix at room temperature for 20 min. After fixation, wash three times with PBS, 10 min each time, and discard the PBS; add 0.2% Triton X-100 and incubate at room temperature for 15 min for cell membrane permeabilization; discard the permeabilization solution, wash three times with PBS, 10 min each time, and discard the PBS; add 5% goat serum to block for 3 h;

[0077] ③ Select CK8, Vimentin, MDM2, Ki67, HER-2, ER, PR. Dissolve different primary antibodies in PBST respectively, add 200 μL of primary antibody dilution to each well, and incubate at 4°C for 12 h;

[0078] ④ Recover the primary antibody dilution solution, wash the cells 3 times with PBST, 10 minutes each time; incubate with 594-goat anti-rabbit IgG fluorescent secondary antibody for 2-3 hours at room temperature in the dark, wash 3 times with PBS, aspirate the fluorescent secondary antibody, and wash the cells 3 times with PBST, 10 minutes each time;

[0079] ⑤ Add 200 μL of DAPI to each well to completely cover the cells, and incubate for 15 minutes at room temperature; wash the cells 3 times with PBS, 10 minutes each time.

[0080] The immunofluorescence staining results of CMLPS-N1 cells were compared with the immunohistochemical staining results of canine mammary gland liposarcoma (as Figure 3 and Figure 9 ), and the results showed that the results of the two were consistent: PCK and HER-2 were negative, and MDM2, Vimentin, ER, PR, and Ki67 were positive, confirming that this cell line retained the molecular phenotype of the original tumor cells. 5. Tumorigenic characteristics of CMLPS-N1 cells in mice

[0081] Cell suspensions were inoculated into BALB / c-nude and BALB / c mice respectively, and their tumorigenic abilities were observed:

[0082] ① After disinfecting the fourth left mammary pad of 3 4-week-old female BALB / c-nude mice numbered 1, 2, and 3 with alcohol cotton, inject the canine mammary gland liposarcoma CMLPS-N1 cell suspension, 3×10 6 cells per mouse.

[0083] ② Five days after inoculation, white protrusions appeared at the cell injection sites in all 3 mice; on the 21st day, the longest diameter of the tumors was 7-11 mm, and the tumorigenesis rate was 100% (as Figure 10 shown). After 36 days of inoculation, the 3 mice were euthanized and dissected, and the tumor tissues were taken out (as Figure 11 shown), fixed in formaldehyde, and the pathological sections of the tissues were stained with HE. It was found that there were many blood vessels in the tumor tissues, a large number of adipocytes of different sizes were distributed in sheets, and mitotic figures were relatively common (as Figure 12 shown), which was in line with the tissue characteristics of liposarcoma.

[0084] ③ After disinfecting the armpits near the fourth left mammary pad of 3 4-week-old female BALB / c mice numbered 1, 2, and 3 with alcohol cotton, inject the canine mammary gland liposarcoma CMLPS-N1 cell suspension, 6×10 6 cells per mouse.

[0085] ④ Ten days after inoculation, white protrusions appeared at the cell injection sites in all 3 mice (as Figure 13 shown); on the 36th day, the longest diameter of the tumors was 8-12 mm, and the tumorigenesis rate was 100% (asFigure 14 as shown

[0086] 6. Immunohistochemical staining of tumor tissues in BALB / c-nude xenograft tumor mice

[0087] Immunohistochemical staining was performed on the tumor tissues collected after inoculating CMLPS-N1 cells into BALB / c-nude mice. Compared with the immunohistochemical staining results of canine mammary liposarcoma tissues and the results of cellular immunofluorescence, the results showed that the results of the three were consistent: negative for PCK and HER-2, and positive for MDM2, Vimentin, ER, PR, and Ki67 (as Figure 15 shown), confirming that the mouse xenograft tumors established from CMLPS-N1 cells retained the differentiation phenotype and molecular phenotype of the original tumor cells, and their sources were unified.

[0088] 7. Inhibition test of anticancer drugs on cell viability

[0089] CMLPS-N1 cells were seeded into 96-well plates at a density of 10 4 cells / well and cultured in a cell incubator at 37°C and 5% CO 2 . After 12 h of culture, docetaxel, cisplatin, epirubicin, and 5-fluorouracil reagents diluted in gradients were added respectively. After the cells were treated with the drugs for 24 h and 48 h (docetaxel, cisplatin, epirubicin) or 48 h and 72 h (5-fluorouracil), the cell viability was detected using a CCK8 kit, and the drug IC50 was calculated. Using the canine breast cancer cell line CMT-7364 as a reference, drug inhibition tests were performed on CMT-7364 and CMLPS-N1 cells respectively. The results showed (as Figures 16 - 19 shown): Cisplatin and epirubicin had similar inhibitory effects on both CMLPS-N1 and CMT-7363 cell lines. After 48 h of treatment, their IC50 values were both around 15 μM (cisplatin) and 100 nM (epirubicin); while 5-fluorouracil had a significantly higher inhibitory effect on CMT-7364 (48 h, IC50: 526.2 nM) than on CMLPS-N1 (48 h, IC50: 1710 nM); paclitaxel had a significantly higher inhibitory effect on CMLPS-N1 (48 h, IC50: 4.27 nM) than on CMT-7364 (48 h, IC50: 21.47 nM). The CMLPS-N1 cell line can be used for the inhibition test of anticancer drugs on the viability of tumor cells and is suitable as a cell material for the development of chemotherapy drugs related to canine mammary sarcoma.

[0090] 8. Mycoplasma examination of cells

[0091] The cells to be examined were seeded into sterile 6-well cell culture plates at an inoculation density of 1×10 4Meanwhile, inoculate the same type of cells that are normal and mycoplasma-free as a negative control. After 5 days, first aspirate the culture medium, then add 1 mL of fixative and let it stand for 20 min. Aspirate the fixative, air dry, and add 1 mL of Hoechst 33258 working solution to each well, ensuring that the working solution completely covers the cells to be tested. Incubate in the dark at 37 °C for 15 min. Aspirate the working solution and add 2 mL of sterile ultrapure water to wash three times. Observe under a fluorescence microscope using ultraviolet excitation light to check if there are small blue fluorescent dots or beaded small blue fluorescent dots around the cells. No small blue fluorescent dots or beaded small blue fluorescent dots are observed around the cells under the fluorescence microscope, indicating that the cells are clean and free of mycoplasma contamination (as Figure 20 shown).

Claims

1. Canine mammary liposarcoma cell line CMLPS-N1, characterized in that Its deposit number is CCTCC NO: C2024371.

2. Use of the cell line CMLPS-N1 according to claim 1 in preparing an animal model of liposarcoma.

3. The use according to claim 2, characterized in that: Application in preparing mouse liposarcoma animal model.

4. Use of the cell line CMLPS-N1 according to claim 1 as a cell material in the development of drugs for canine mammary liposarcoma.

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