Cancer-associated fibroblast cell line, method for constructing the same and use thereof

Abstract

This invention relates to the interdisciplinary field of cell biology, experimental oncology, and translational medicine, specifically to a cancer-associated fibroblast cell line, its construction method, and its applications. This invention fills the research gap in mouse-derived oral squamous cell carcinoma-associated fibroblast cell line models and provides a method for constructing such a cell line. Through the inverted adherent culture and enzymatic digestion methods of this invention, a mouse-derived oral squamous cell carcinoma-associated fibroblast cell line with a clear origin, high purity, and long-term phenotypic and functional stability can be obtained. This allows for the establishment of a cryopreservable and resuscitable mouse oral squamous cell carcinoma-associated fibroblast cell bank, providing technical support for research related to the tumor microenvironment and immunotherapy.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of cell biology, experimental oncology, and translational medicine, specifically to a cancer-associated fibroblast cell line, its construction method, and its uses. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, characterized by high invasiveness, high recurrence rate, and poor prognosis. In recent years, the role of the tumor microenvironment (TME) in tumorigenesis and development has received widespread attention. Cancer-associated fibroblasts (CAFs) are one of the main stromal cell types in the TME, playing a crucial role in regulating immunosuppression, promoting tumor cell migration, angiogenesis, and extracellular matrix remodeling.

[0003] Numerous studies have shown that cancer cells (CAFs) are closely related to tumor immune escape, invasion and metastasis, and treatment resistance in OSCC, and are important non-tumor cellular factors affecting tumor malignant progression. CAFs participate in the modification of the immune microenvironment through mechanisms such as secreting cytokines (e.g., TGF-β, IL-6, CXCL12), expressing immune checkpoint molecules (e.g., PD-L1), and participating in antigen presentation, and are potential synergistic targets for immunotherapy.

[0004] Currently, human CAF cell lines have been established in some tumors (such as breast cancer and pancreatic cancer) and applied to drug screening and functional studies. However, in the field of OSCC, especially the systematic construction of mouse-derived CAFs, remains very limited. Since mice are currently the most widely used immune intact animal model, the lack of CAF cell lines in mice restricts the in-depth advancement of tumor immunity-related research.

[0005] Existing studies largely rely on human CAFs, which suffer from problems such as cell instability, poor reproducibility, and lack of immune background validation. To further investigate the biological functions, drug sensitivity, and treatment tolerance mechanisms of CAFs in OSCC, there is an urgent need for an easily obtainable, reproducible, cryopreservable, phenotypically stable, purified mouse-derived OSCC-CAF cell model. This would fill the gap in model research and support in-depth exploration of immunotherapy, combination therapies, and mechanisms. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides a cancer-associated fibroblast cell line, its construction method, and its uses.

[0007] A cancer-associated fibroblast cell line, wherein the cancer-associated fibroblast cell line is derived from mouse oral squamous cell carcinoma and is constructed by inverted adherent culture and enzymatic digestion of mouse oral squamous cell carcinoma tumor cells.

[0008] The above-mentioned method for constructing cancer-associated fibroblast cell lines includes the following steps: Step 1: Invert the mouse oral squamous cell carcinoma tumor cells for adherent culture; Step 2: Perform enzymatic digestion on the cells obtained in Step 1.

[0009] Preferably, the procedure also includes steps such as constructing a mouse oral squamous cell carcinoma tumor model, collecting mouse tongue tumor tissue for mechanical decomposition, and passage purification.

[0010] Preferably, in step 1, the inverted adherent culture time is 3–5 h.

[0011] Preferably, the inverted adherent culture time is 4 hours.

[0012] Preferably, in step 2, a 0.125%-0.25% concentration of trypsin is used for digestion, and the digestion time is 1-2 minutes.

[0013] Preferably, the concentration is 0.25% and the digestion time is 1 minute.

[0014] The cancer-associated fibroblast cell lines described above can be used for tumor immunology, drug screening, microenvironment research, or organoid modeling of oral squamous cell carcinoma based on mouse models.

[0015] By adopting the technical solution of the present invention, the following beneficial effects can be obtained: This invention fills the research gap in mouse-derived OSCC-CAFs cell models and provides a method for constructing mouse-derived OSCC-CAFs cell lines. Through the inverted adherent culture and enzymatic digestion methods of this invention, OSCC-CAFs cell lines with clear origin, high purity, and long-term phenotypic and functional stability can be obtained. This allows for the establishment of cryopreservable and resuscitable mouse OSCC-CAFs cell banks to support research related to the tumor microenvironment and immunotherapy.

[0016] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0017] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0018] Figure 1 A schematic diagram of the process for constructing mouse-derived OSCC-CAFs cell lines.

[0019] Figure 2 The expression level of FAP in mouse-derived OSCC-CAFs extracted by three different methods was detected using Western blotting (WB).

[0020] Figure 3 This is an image of FAP immunofluorescence staining of mouse-derived OSCC-CAFs.

[0021] Figure 4 This is a bright-field microscopic illustration of mouse-derived OSCC-CAFs cell lines. The left image shows the P1 generation fibroblast population, and the right image shows the P10 generation fibroblast population.

[0022] Figure 5 The image shows a bright-field microscopic view of the mouse-derived OSCC-CAFs cell line extracted according to the method in Comparative Example 1. The left image shows the P1 generation fibroblast population, and the right image shows the P3 generation fibroblast population.

[0023] Figure 6 The image shows the FAP immunofluorescence staining of mouse-derived OSCC-CAFs cell line extracted according to the method in Comparative Example 1.

[0024] Figure 7 The image shows a bright-field microscopic view of the mouse-derived OSCC-CAFs cell line extracted according to the method in Comparative Example 2. The left image shows the P1 generation fibroblast population, and the right image shows the P3 generation fibroblast population.

[0025] Figure 8 The image shows the FAP immunofluorescence staining of mouse-derived OSCC-CAFs cell line extracted according to the method in Comparative Example 2. Detailed Implementation

[0026] Example 1: Construction of mouse-derived OSCC-CAFs cell line Mouse-derived OSCC-CAFs cell lines were constructed according to the established procedure. Figure 1 Specifically, it includes the following steps: (1) Establishment of a mouse tongue OSCC tumor model: Six-week-old C57BL / 6 mice were randomly divided into groups and housed in an SPF-grade animal facility. They underwent one week of acclimatization feeding before the experiment (N=6 per group). The experimental group was given sterile water (100 μg / mL) containing 4-nitroquinoline-N-oxide (4NQO) for 16 consecutive weeks, and then switched to ordinary drinking water. The control group drank ordinary sterile water. The mice were continued to be housed until the 24th week, when they were sacrificed for tissue collection.

[0027] (2) Collection of tongue tumor tissue and mechanical decomposition: Tongue tumor tissue was excised under aseptic conditions, thoroughly washed with PBS containing 10% penicillin and streptomycin, and then transferred to serum-free DMEM containing an equal proportion of penicillin and streptomycin. The tissue was shaken on a shaker at 37°C for 1 hour to reduce contamination. Subsequently, the tissue was cut into small pieces of about 3–4 mm in a clean bench with sterile scissors for later use. Enzyme (K2 ONCOLOGY, K20-EMC-20) was added and digested on a shaker at 37°C for 30 min.

[0028] (3) Short-term inverted adhesion enrichment of fibroblasts: The above-mentioned tumor tissue blocks were evenly spread in pre-moistened complete culture medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibody). After removing an appropriate amount of excess culture medium, the culture dish was inverted for 4 h to allow the tissue blocks to fully adhere to the bottom of the dish and facilitate preferential adhesion and growth of fibroblasts. Then the culture dish was inverted again and 5 mL of complete culture medium was added. The culture was then continued in a constant temperature incubator at 37 ℃ and 5% CO2. The culture medium was replenished to 10 mL the next day, and half of the culture medium was replaced every 3 days.

[0029] (4) Mild enzymatic digestion and purification of fibroblasts: When a large number of fibroblast-like cells (referred to as P0 generation) crawl out from the edge of the tissue block and cover about 60-70% of the culture area, discard the culture medium and wash thoroughly with PBS to remove some non-adherent cells. Digest with 0.25% trypsin for 1 minute, collect the adherent cells and remove some epithelial cell clumps to obtain a primary cell population mainly composed of fibroblast-like cells.

[0030] (5) Continuous passage selection from P0 to P3: The above cells were seeded at the conventional ratio and passaged continuously from P0 to P3. Each passage was performed when the cell density was about 70-80% to gradually remove non-fibroblast cell populations with slow growth or poor adhesion. After continuous selection from P0 to P3, a fibroblast cell population with uniform morphology, long spindle shape, good adhesion and not easy to detach was obtained, which was named musCAFs.

[0031] The following experimental examples illustrate the beneficial effects of the mouse-derived OSCC-CAFs cell line construction method of the present invention: Experimental Example 1: Identification of the mouse-derived OSCC-CAFs cell line of the present invention The mouse-derived OSCC-CAFs cell line (musCAFs) constructed in Example 1 was subjected to morphological identification and the expression of the CAFs-specific marker FAP was detected. (1) Morphological identification: musCAFs were observed under a 200x bright field microscope to observe cell morphology.

[0032] (2) Immunofluorescence staining: One day before the experiment, healthy mouse CAFs were seeded onto cell slides, allowing them to reach approximately 50–60% confluence by the next day. After discarding the culture medium, the cells were washed three times with pre-cooled PBS, and then fixed with 4% paraformaldehyde for 15–30 min. Subsequently, the cells were gently rinsed three times with PBS under shaking conditions, and treated with 0.3% Triton X-100 for 5–10 min to achieve permeabilization. Then, 5% BSA was added for blocking for 1 h, followed by the addition of primary antibody FAP and incubation at 4°C overnight. The next day, the cells were slowly washed three times with TBST, followed by the addition of secondary antibody—Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L)—while simultaneously staining the cytoskeleton F-actin. The cells were washed three times again with TBST, and finally mounted with DAPI-containing mounting medium for fluorescence microscopy observation. FAP + Cell proportions were assessed by cell counting analysis following immunofluorescence staining. Specifically, in multiple randomly selected fields of view, the total number of DAPI-labeled cells and the number of FAP-positive cells were counted, and the proportion of FAP-positive cells was calculated.

[0033] (3) Western blot detection: Take cultured cells or tissue samples, add pre-chilled RIPA lysis buffer (containing protease and phosphatase inhibitors) for complete lysis, and incubate on ice for 30 min. Centrifuge at 12,000×g for 15 min, collect the supernatant and determine the protein concentration using the BCA method. Add an equal volume of protein (20–40 µg) to 5×SDS loading buffer and denature at 95 °C for 5 min. Separate the samples on a 10%–12% SDS-PAGE gel and transfer them to a PVDF membrane. Block the membrane with 5% skim milk powder or BSA at room temperature for 1 h, and add primary antibodies (anti-FAP and anti-Tubulin) (incubate overnight at 4 °C). The next day, wash three times with TBST for 10 min each time, and then add HRP-labeled secondary antibody and incubate at room temperature for 1 h. After washing with TBST again, develop the gel using ECL chemiluminescence, record the bands using a gel imaging system, and perform quantitative analysis.

[0034] Results: Western blot ( Figure 2 ) and FAP immunofluorescence ( Figure 3Table 1) indicates that the FAP of the musCAFs of the present invention + Cell proportion > 92.3 ± 2.0%, uniform morphology, elongated spindle shape, good adhesion and not easy to detach, and continued to maintain the elongated spindle-shaped fibrous morphology even after continuous passage to P10 generation, with no obvious senescence or phenotypic drift observed. Figure 4 ).

[0035] Table 1 The advantages of the mouse-derived OSCC-CAFs cell line constructed using the method of this invention in terms of CAFs purity, phenotypic stability, and contamination control are further verified below through comparative examples.

[0036] Comparative Example 1: Traditional adherent culture method After the tongue tumor tissue induced by 4NQO was mechanically minced, it was directly placed in a complete culture medium without inverting it for adhesion or performing enzyme digestion. The rest of the method was the same as in Example 1, and the primary cell population was obtained by changing the medium and passage multiple times.

[0037] The obtained cell population was subjected to morphological identification, immunofluorescence, and Western blot identification using the cell identification method for mouse-derived OSCC-CAFs cell lines in Experiment Example 1.

[0038] The results showed that, morphologically, a large number of epithelioid cells, immune cells, and a small number of fibroblast-like cells coexisted. Figure 5 ); FAP immunofluorescence and WB findings, FAP + The cell percentage was approximately 50.0 ± 10.0% ( Figure 2 , Figure 6 (Table 2); After continuous passage to P3, the cell composition remained relatively complex, proliferation slowed down, and the overall model stability was poor. Figure 5 ).

[0039] Table 2 Comparative Example 2: Rapid Digestion Method Using High-Concentration Enzymes Tumor tissue was rapidly digested with 0.5% trypsin, followed by short-term adherent culture, and the remaining methods were the same as in Example 1 to obtain fibroblast-like cells.

[0040] The obtained cell population was subjected to morphological identification, immunofluorescence, and Western blot identification using the cell identification method for mouse-derived OSCC-CAFs cell lines in Experiment Example 1.

[0041] The results showed that although the proportion of fibroblast-like cells in the primary adherent cells was higher than that in control group 1, the composition was still mixed. Figure 7FAP protein expression assays showed that the expression levels of the target biomarker were unstable. + The cell percentage was approximately 66.73 ± 3.20% ( Figure 2 , Figure 8 (Table 3); when passaged to P3, the cell composition remained complex ( Figure 7 Based on the combined results of the method of this invention and the cell line identification of Comparative Examples 1 and 2, the mouse-derived OSCC-CAFs cell line constructed using the method of this invention exhibits the best performance in terms of purity, phenotypic stability, and passage stability (Table 4).

[0042] Table 3 Table 4 In summary, the method for constructing mouse OSCC-CAFs cell lines of the present invention has the advantages of simple operation and good reproducibility. The mouse OSCC-CAFs cell lines constructed by the method of the present invention have the advantages of high purity and long-term stability of phenotype and function. Therefore, the method for constructing mouse OSCC-CAFs cell lines of the present invention has great application value in research related to tumor microenvironment and immunotherapy.

Claims

1. A cancer-associated fibroblast cell line, characterized in that, The cancer-associated fibroblast cell line is derived from mouse oral squamous cell carcinoma and is constructed from mouse oral squamous cell carcinoma tumor cells through inverted adherent culture and enzymatic digestion.

2. The method for constructing the cancer-associated fibroblast cell line according to claim 1, characterized in that, Includes the following steps: Step 1: Invert the mouse oral squamous cell carcinoma tumor cells for adherent culture; Step 2: Perform enzymatic digestion on the cells obtained in Step 1.

3. The construction method according to claim 2, characterized in that, It also includes steps such as constructing a mouse oral squamous cell carcinoma tumor model, collecting mouse tongue tumor tissue for mechanical decomposition, and passage purification.

4. The construction method according to claim 2, characterized in that, In step 1, the inverted adherent culture time is 3–5 h.

5. The construction method according to claim 4, characterized in that, The inverted adherent culture time was 4 hours.

6. The construction method according to claim 2, characterized in that, In step 2, trypsin at a concentration of 0.125%-0.25% is used for digestion, and the digestion time is 1-2 minutes.

7. The construction method according to claim 6, characterized in that, The concentration is 0.25%, and the digestion time is 1 minute.

8. The use of the cancer-associated fibroblast cell line according to claim 1, characterized in that, The cancer-associated fibroblast cell lines can be used for tumor immunology, drug screening, microenvironment research, or organoid modeling of oral squamous cell carcinoma based on mouse models.

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