Malignant mucosal melanoma cancer related fibroblast line as well as construction method and application thereof
By isolating and culturing fibroblasts associated with mucosal malignant melanoma, and constructing and passageing stable CAFs cell lines, the problem of difficulty in establishing effective CAFs cell lines in the prior art is solved, and a stable cell line for scientific research, drug preparation and immunotherapy is achieved.
Patent Information
- Application Number
- CN202411892463.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-25
AI Technical Summary
The prior art is difficult to effectively establish and maintain cancer-related fibroblast cell lines associated with mucosal malignant melanoma, resulting in the lack of effective preclinical models for conducting MM studies.
By isolating and culturing mucosal malignant melanoma-associated fibroblasts, using DMEM high-glycemic medium and a specific proportion of fetal bovine serum and antibiotic solution, combined with trypsin digestion and differential adherence method, a stable CAFs cell line was successfully constructed and passaged.
The establishment and passage of CAFs cell lines with stable growth state have been achieved, ensuring the stability of cell morphology and proliferation rate, making it possible to be used in scientific research, drug preparation and immunotherapy exploration related to mucosal malignant melanoma.
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Figure CN119685249A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to a mucosal malignant melanoma cancer-associated fibroblast cell line and a construction method and application thereof. Background Art
[0002] Mucosal melanoma (MM) is a subtype of malignant melanoma with high invasiveness, unknown pathogenesis, high incidence in East Asian populations, lack of therapeutic targets, and extremely poor prognosis, with a 5-year survival rate of less than 25%. Currently, there is no systematic clinical treatment recommendation for patients with recurrent and metastatic MM, and the clinical effect of first-line / second-line chemotherapy is not ideal. Most patients in the middle and late stages often have no drugs available.
[0003] In recent years, although conventional treatment methods (such as surgery, radiotherapy, interferon, immunotherapy, etc.) and comprehensive treatment strategies have made certain progress in the clinical treatment of MM, a standardized clinical diagnosis and treatment plan or guideline has not yet been formed, which seriously affects the treatment and prognosis of MM patients. One of the main reasons is the lack of effective clinical trials and preclinical models for conducting MM research, especially disease models that can reflect the MM tumor microenvironment.
[0004] A large number of different types of tumors have confirmed the presence of cancer-associated fibroblasts (CAFs) in the tumor microenvironment. Since the sample size of MM tumors is relatively small compared to common malignant tumors, and the success rate of CAFs extraction and passaging is low, it is difficult to establish a CAFs cell line for MM. Therefore, the research progress of MM has been hindered to a certain extent. Summary of the invention
[0005] In view of the defects of the prior art, the object of the present invention is to provide a mucosal malignant melanoma cancer-associated fibroblast cell line and a construction method and application thereof.
[0006] The technical solution of the present invention is:
[0007] The first aspect of the present invention provides a mucosal malignant melanoma cancer-associated fibroblast cell line, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024383 and a deposit date of November 13, 2024.
[0008] The mucosal malignant melanoma cancer-associated fibroblast cell line provided by the present invention has at least the following applications:
[0009] a. Application in in vitro research;
[0010] b. Application in the preparation of drugs for treating mucosal malignant melanoma;
[0011] c. Application in exploring immunotherapy of mucosal malignant melanoma.
[0012] The second aspect of the present invention provides a method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line, comprising the following steps:
[0013] Step 1: Isolation and culture of mucosal malignant melanoma cancer-associated fibroblasts;
[0014] Step 2: Subculture of mucosal malignant melanoma cancer-associated fibroblasts.
[0015] Furthermore, the steps of isolating and culturing mucosal malignant melanoma cancer-associated fibroblasts are as follows:
[0016] S1. Cut the obtained MM tissue into pieces in a culture dish on a clean bench, wash the cut tissue pieces in DMEM culture medium 2-3 times, transfer the tissue pieces into a T25 culture bottle, and add 2 ml of culture medium 1 for culture;
[0017] S2, place the culture bottle upside down in the incubator and culture for 6-12 hours, then turn the culture bottle over and continue to culture for 6-12 hours;
[0018] S3, add 3-5 ml of culture medium 1 and continue culturing for 36h-72h;
[0019] S4, discard medium 1, wash the cells twice with PBS, add 3-6 ml medium 2, and culture;
[0020] S5. After the MM cells and CAFs cells have crawled out, the epithelial cells and CAFs cells are separated repeatedly by the differential adhesion method to finally obtain stably passaged CAFs cells.
[0021] More preferably, the culture medium 1 is based on DMEM high-glucose culture medium, to which 10% fetal bovine serum and 1% penicillin-streptomycin solution are added.
[0022] More preferably, the culture medium 2 is based on DMEM high-glucose culture medium, to which 20% fetal bovine serum and 1% penicillin-streptomycin solution are added.
[0023] Furthermore, the steps of subculturing the mucosal malignant melanoma cancer-associated fibroblasts are as follows:
[0024] Use trypsin to digest CAFs cells and tap the wall of the culture dish to terminate the digestion.
[0025] More preferably, the time for digesting CAFs cells with trypsin is 1-4 min.
[0026] More preferably, the subculture ratio is 1:4-1:2.
[0027] More preferably, the passage ratio is 1:2.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[0029] By using the method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line provided by the present invention, CAFs with a stable growth state are obtained, and the CAFs are subcultured to establish a cell line. After multiple subcultures, the related fibroblasts still maintain a stable cell morphology and proliferation rate, so that they can be applied to scientific research related to mucosal malignant melanoma cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:
[0031] Figure 1 A schematic diagram of the subculture results of mucosal malignant melanoma cancer-associated fibroblasts provided in an embodiment of the present invention (P1 refers to the first generation of CAFs, and P40 refers to the 40th generation of CAFs);
[0032] Figure 2 A schematic diagram of the immunofluorescence detection and identification results of mucosal malignant melanoma cancer-associated fibroblasts provided in an embodiment of the present invention;
[0033] Figure 3 A schematic diagram of flow cytometry identification results of mucosal malignant melanoma cancer-associated fibroblasts provided in an embodiment of the present invention. DETAILED DESCRIPTION
[0034] The present invention is described in detail below with reference to specific embodiments.
[0035] The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.
[0036] Example 1
[0037] Tumor specimen: obtained from a 72-year-old male who underwent surgical resection in the Department of Oral and Maxillofacial-Head and Neck Tumors of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Pathological diagnosis was primary mucosal malignant melanoma. This was a first-time patient with the primary lesion located in the lower lip. The patient signed an informed consent form.
[0038] Tumor sample collection: Tumor specimens were collected during surgery with an ex vivo time of less than 30 minutes. Typical areas of the tumor lesions were selected, avoiding areas of liquefaction and necrosis. The tumor tissue was cut into several small pieces of approximately 3 mm × 3 mm × 3 mm in size. 2-3 small pieces were retained for model construction and placed in a centrifuge tube containing PBS solution (with 500 IU / mL penicillin and 500 mg / mL streptomycin), stored at 4°C, and sent to the laboratory for experiments within 1 hour.
[0039] This embodiment provides a method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line, comprising the following steps:
[0040] Step 1: Isolation and culture of mucosal melanoma cancer-associated fibroblasts:
[0041] S1. Culture by tissue block method. Cut the obtained MM tissue into pieces in a culture dish on a clean bench, wash the cut tissue pieces in DMEM culture medium 2-3 times, transfer the tissue pieces into a T25 culture bottle, add 2 ml of culture medium 1 for culture;
[0042] S2, place the culture bottle upside down in the incubator and culture for 6-12 hours, then turn the culture bottle over and continue to culture for 6-12 hours;
[0043] S3, add 3-5 ml of culture medium 1 and continue culturing for 36h-72h;
[0044] S4, discard medium 1, wash the cells twice with PBS, add 3-6 ml medium 2, and culture;
[0045] S5. After the MM cells and CAFs cells have crawled out, the epithelial cells and CAFs cells are separated by the differential adhesion method, which is repeated several times to finally obtain stably passaged CAFs cells.
[0046] Culture medium 1 is based on DMEM high-glucose culture medium, with 10% fetal bovine serum and 1% penicillin-streptomycin solution added.
[0047] Culture medium 2 is based on DMEM high-glucose culture medium, with 20% fetal bovine serum and 1% penicillin-streptomycin solution added.
[0048] Step 2: Subculture of mucosal malignant melanoma cancer-associated fibroblasts.
[0049] Use trypsin to digest CAFs cells for 1-4 minutes, tap the wall of the culture dish to terminate the digestion. The subculture ratio is 1:4-1:2.
[0050] In this example, a subculture ratio of 1:2 was used for subsequent subculture, and a mucosal malignant melanoma cancer-associated fibroblast cell line was finally obtained. The mucosal malignant melanoma cancer-associated fibroblast cell line was deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024383 and a deposit date of November 13, 2024.
[0051] The mucosal malignant melanoma cancer-associated fibroblast cell line obtained by the construction method of this embodiment has at least the following applications:
[0052] a. Application in in vitro research;
[0053] b. Application in the preparation of drugs for treating mucosal malignant melanoma;
[0054] c. Application in exploring immunotherapy of mucosal malignant melanoma.
[0055] Experimental example
[0056] The following experimental examples not only verified the existence of CAFs with MM, but also proved that the CAFs obtained by the construction method provided in Example 1 can maintain a stable cell morphology and proliferation rate.
[0057] The test methods and results used in the experimental examples of the present invention are described in detail as follows:
[0058] 1) Observation experiment of CAFs cell morphology
[0059] In the process of constructing the mucosal malignant melanoma cancer-associated fibroblast cell line in Example 1, the gross morphology of the subcultured mucosal malignant melanoma-associated fibroblast cells at different growth densities at various stages was observed using a stereomicroscope and photographed for record.
[0060] Morphological observation results of CAFs:
[0061] Long spindle-shaped, containing a small number of irregular polygonal cells (probably MM cells). The cell proliferation rate of cells that maintain a stable cell morphology after continuous passage is shown in Figure 1 (P1 refers to the first generation of CAFs, and P40 refers to the 40th generation of CAFs).
[0062] Through the subculture of CAFs, a mucosal malignant melanoma-associated fibroblast cell line was established and deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: C2024383 and the deposit date: November 13, 2024.
[0063] 2) Immunofluorescence (IF) detection and analysis:
[0064] The second generation CAFs were selected and removed after 48 hours of climbing, washed twice with PBS, and then fixed with 4% neutral buffered formaldehyde at room temperature for 30 minutes, and permeabilized with membrane permeabilization solution for 15 minutes. Then, CAFs were blocked with blocking buffer (PBS containing 5% skim milk powder) for 1 hour, and antibodies (ACTA2, FAP, CKPan) were added and incubated overnight at 4°C. After washing with PBS, Anti-mouse IgG (H+L), F(ab')2Fragment (Alexa Fluor 500) were added at room temperature in the dark. The cells were treated with secondary antibody #4408 (488Conjugate) for 30 min, and washed with PBS three times. DAPI mounting medium containing antifade agent was added dropwise to cover the cells and observed under a fluorescence microscope.
[0065] Immunofluorescence detection of the expression of CAFs markers ACTA2, FAP and CKPan showed that CAFs cells were positive in antibodies ACTA2 and FAP, but negative in antibody CKPan. Figure 2 .
[0066] 3) Flow cytometry detection:
[0067] A PBS solution containing 0.5% BSA was prepared as a buffer for blocking and antibody incubation; the second generation CAFs were digested and resuspended, and then blocked on ice for half an hour; flow cytometry antibodies (CD45, CD31, EPCAM and PDPN) were prepared with PBS solution and stained on ice for one hour; the stained CAFs were washed with PBS solution and detected by flow cytometer.
[0068] The results of cell flow cytometry showed that CD45, CD31 and EPCAM were negatively expressed, and the PDPN positive rate was 6.23%. Figure 3 .
[0069] 4) Short Tandem Repeat (STR) Identification:
[0070] The third generation CAFs were selected for STR detection. The CAFs adherent cells were digested with trypsin, washed with PBS after centrifugation, and resuspended in 0.5-1 ml PBS to 1×10 5 The above cells were placed in a 1.5 ml EP tube, sealed with sealing film, and sent to the company for testing at room temperature.
[0071] The results of short tandem repeat sequences showed that the STR test results of the third generation CAFs showed that the CAFs were human cells that were different from any data results in the ExPASy STR database, and no other known cell lines were found to be mixed. See Table 1.
[0072] Table 1 STR test results
[0073]
[0074] 5) Mycoplasma infection detection:
[0075] The third generation CAFs were selected for mycoplasma infection detection. CAFs adherent cells were digested with trypsin (Suzhou Xinsaimei Company, 0.25% Trypsin-EDTA, 1×), washed with PBS after centrifugation, and the digested CAFs cells were resuspended with 0.5-1 ml PBS and sent to the detection unit for detection at room temperature.
[0076] The results of mycoplasma infection test showed that the results of mycoplasma infection test of the third generation CAFs cells were negative, which indicated that the cell culture process was not contaminated by mycoplasma and the cell state was relatively healthy. See Table 2.
[0077] Table 2 Mycoplasma infection detection results
[0078]
[0079] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.
Claims
1. A mucosal malignant melanoma cancer-associated fibroblast cell line, characterized in that: The mucosal malignant melanoma cancer-associated fibroblast cell line is deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC NO: C2024383 and the deposit date being November 13, 2024.
2. The mucosal malignant melanoma cancer-associated fibroblast cell line according to claim 1 has at least the following uses: a. Application in in vitro research; b. Application in the preparation of drugs for treating mucosal malignant melanoma; c. Application in exploring immunotherapy of mucosal malignant melanoma.
3. A method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line, characterized in that: The following steps are involved: Step 1: Isolation and culture of mucosal malignant melanoma cancer-associated fibroblasts; Step 2: Subculture of mucosal malignant melanoma cancer-associated fibroblasts.
4. A method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line according to claim 3, characterized in that: The steps of isolating and culturing mucosal malignant melanoma cancer-associated fibroblasts are as follows: S1. Cut the obtained MM tissue into pieces in a culture dish on a clean bench, wash the cut tissue pieces in DMEM culture medium 2-3 times, transfer the tissue pieces into a T25 culture bottle, and add 2 ml of culture medium 1 for culture; S2, place the culture bottle upside down in the incubator and culture for 6-12 hours, then turn the culture bottle over and continue to culture for 6-12 hours; S3, add 3-5 ml of culture medium 1 and continue culturing for 36h-72h; S4, discard medium 1, wash the cells twice with PBS, add 3-6 ml medium 2, and culture; S5. After the MM cells and CAFs cells have crawled out, the epithelial cells and CAFs cells are separated repeatedly by the differential adhesion method to finally obtain stably passaged CAFs cells.
5. A method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line according to claim 4, characterized in that: The culture medium 1 is based on DMEM high-glucose culture medium, with 10% fetal bovine serum and 1% penicillin-streptomycin solution added.
6. A method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line according to claim 4, characterized in that: The culture medium 2 is based on DMEM high-glucose culture medium, and 20% fetal bovine serum and 1% penicillin-streptomycin solution are added.
7. A method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line according to claim 3, characterized in that: The steps of subculturing the mucosal malignant melanoma cancer-associated fibroblasts are as follows: Use trypsin to digest CAFs cells and tap the wall of the culture dish to terminate the digestion.
8. The method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line according to claim 7, characterized in that: The time for trypsin digestion of CAFs cells is 1-4 minutes.
9. The method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line according to claim 7, characterized in that: The passaging ratio is 1:4-1:
2.
10. The method for constructing a mucosal malignant melanoma cancer-associated fibroblast cell line according to claim 9, characterized in that: The subculture ratio was 1:2.