Human laryngeal squamous cell carcinoma related fibroblast and application thereof

Abstract

The invention discloses human laryngeal squamous cell carcinoma related fibroblasts, which are classified and named as human laryngeal squamous cell carcinoma related fibroblasts GY-HNSCC-CAF Homo sapiens, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCC NO: C2024179, and the preservation date is July 7, 2024. The invention enriches the resource library of tumor-related fibroblasts, especially fibroblasts from head and neck squamous cell carcinoma, can be used as an effective in-vitro study cell model, and provides a new experimental model for the purposes of etiology, metastasis mechanism, drug resistance mechanism, drug screening and the like of head and neck squamous cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to human laryngeal squamous cell carcinoma-related fibroblasts and applications thereof. Background Art

[0002] According to the 2020 Global Cancer Statistics, head and neck cancer is the sixth most common form of cancer in the world, with more than 870,000 new cases and 440,000 deaths each year, accounting for 4.5% of cancer deaths. Most of these cancers are squamous cell carcinomas that originate from the mucosal surfaces of the oral cavity, larynx, oropharynx, and hypopharynx. Head and neck squamous cell carcinoma (HNSCC) is usually diagnosed in the late stage, and despite aggressive treatment, the 5-year survival rate of HNSCC is less than 50%. It is worth noting that advanced HNSCC is usually composed of up to 80% tumor-associated fibroblasts (CAFs), suggesting that CAFs play an important role in the occurrence and development of advanced HNSCC and are of extremely important research value.

[0003] Tumor-associated fibroblasts (CAFs) are a type of stromal cell found in the tumor microenvironment that play a crucial role in promoting tumor progression and metastasis. They are a heterogeneous population of activated fibroblasts that secrete a variety of factors that affect tumor occurrence, development, inflammation, drug resistance, metastasis, and recurrence. Mechanistically, CAFs establish and remodel the structure of the extracellular matrix (ECM), enabling tumor cells to invade through the TME, and promote tumor growth, invasion, metastasis, and angiogenesis by interacting with cancer cells or other stromal cells through the secretion of growth factors, cytokines, and chemokines. At the same time, CAFs can also inhibit T cell activity and promote tumor cell immune escape by secreting immunosuppressive factors. CAFs play a multifaceted role in promoting tumor progression and metastasis, regulating immune responses, and contributing to drug resistance. Therefore, there is an urgent need to enhance the study of the mechanisms by which tumor-associated fibroblasts affect tumor occurrence and development.

[0004] In the current mainstream cell banks at home and abroad, there are no more than 10 types of tumor-associated fibroblasts that can be queried, including three types from the American Type Culture Collection (ATCC): hTERT PF179T, CAFHCC2218, and Hs 895.T, and six types from the Zhejiang Meisen Cell (MeisenCTCC): human primary gastric cancer fibroblasts, human primary bladder cancer fibroblasts, human primary lung cancer fibroblasts, human primary breast cancer-related fibroblasts, and human immortalized pancreatic cancer-related fibroblasts. In addition, although there are a large number of tumor-associated fibroblasts reported in the literature, there is a large heterogeneity and lack of homogeneity due to different sample sources and extraction methods. Therefore, the current source of model cell lines for human tumor-associated fibroblasts at home and abroad is seriously insufficient, which limits the research and development of the composition and mechanism of action in the tumor microenvironment. Stable and passable cell lines are important tools for the study of molecular biology of human tumors. Under appropriate conditions, tumor-associated fibroblasts can simulate the living environment of tumor cells, which is a favorable means for studying advanced HNSCC. Therefore, the establishment of each new CAFs cell line can enrich the cell research library of the tumor microenvironment and provide more research support.

[0005] In summary, discovering new human tumor-associated fibroblast cell lines is one of the urgent issues to be solved in the field of TME research in HNSCC. Summary of the invention

[0006] The purpose of the present invention is to provide a human laryngeal squamous cell carcinoma-associated fibroblast and its application, which enriches the tumor-associated fibroblast resource library, especially the fibroblast resource library derived from head and neck squamous cell carcinoma, and can be used as an effective in vitro research cell model to provide a new experimental model for the etiology, metastasis mechanism, drug resistance mechanism, drug screening and other uses of head and neck squamous cell carcinoma.

[0007] The technical solution adopted by the present invention to solve its technical problem is: A human laryngeal squamous cell carcinoma-associated fibroblast, classified and named: human laryngeal squamous cell carcinoma-associated fibroblast GY-HNSCC-CAF Homo sapiens, preserved in: China Center for Type Culture Collection, preserved in: CCTCC NO: C2024179, preserved on: July 7, 2024.

[0008] The human laryngeal squamous cell carcinoma-associated fibroblasts are used to construct a head and neck squamous cell carcinoma cell model.

[0009] The human laryngeal squamous cell carcinoma-associated fibroblasts are used in screening anti-tumor drugs for preventing or treating head and neck squamous cell carcinoma.

[0010] The human laryngeal squamous cell carcinoma-associated fibroblasts are used to evaluate the efficacy of anti-tumor drugs for treating head and neck squamous cell carcinoma.

[0011] The human laryngeal squamous cell carcinoma-associated fibroblasts are used to construct an animal model of head and neck squamous cell carcinoma.

[0012] The beneficial effects of the present invention are as follows: the human laryngeal squamous cell carcinoma-associated fibroblasts (GY-HNSCC-CAF) established by the present invention have stable properties, can be stably passaged for multiple times, are identified as a new single cell strain by STR, have strong proliferation activity, can be amplified in large quantities and passaged in vitro, enrich the resource library of tumor-associated fibroblasts, especially fibroblasts derived from head and neck squamous cell carcinoma, can be used as an effective in vitro research cell model, and provide a new experimental model for the etiology, metastasis mechanism, drug resistance mechanism, drug screening and other uses of head and neck squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a morphological picture of human laryngeal squamous cell carcinoma-associated fibroblasts GY-HNSCC-CAF in a high-density state after isolation and purification, with a magnification of 40 times.

[0014] Figure 2 This is the protein immunoblot of FAP and β-actin of four cell lines: human laryngeal squamous cell carcinoma-associated fibroblasts GY-HNSCC-CAF, FaDu, CAL-27, and CAL-33.

[0015] Figure 3 This is an analysis chart of the cell scratch experiment results.

[0016] Figure 4 This is an analysis chart of the cell sphere formation experiment results.

[0017] Figure 5 This is the design diagram of the cell scratch experiment. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail below through specific embodiments.

[0019] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0020] Embodiment 1: This example establishes a head and neck squamous cell carcinoma-associated fibroblast cell line - a laryngeal squamous cell carcinoma fibroblast cell line. The cell line is derived from a middle-aged male laryngeal squamous cell carcinoma patient of the Han nationality in China. The application for human samples was conducted under the supervision of the Medical Ethics Committee of Zhejiang Provincial People's Hospital and followed the principles of the Declaration of Helsinki. All people signed an informed consent form. This study was approved by the Medical Ethics Committee of Zhejiang Provincial People's Hospital; Ethics Approval Number: Zhejiang People's Medical Ethics Review 2022 Research No. (124).

[0021] Fresh tissue was obtained by surgery and fresh laryngeal cancer tissue from patients was washed with pre-cooled sterile phosphate-buffered saline to remove excess blood and connective tissue. The tissue was then cut into 1 mm 3 The tissue blocks were placed in a 24-well plate with the bottom moistened with fetal bovine serum, one tissue block was placed in each well, and placed in a 37°C, 5% CO2 incubator for 2-3 hours. After the tissue blocks are fully attached to the culture dish, 500ul DME / F12 (cytiva, SH30023.01) complete growth medium was slowly added from the edge for incubation. The culture medium consists of 90% DME / F12 (cytiva, SH30023.01) and 10% fetal bovine serum (FBS) (BioChannel Biological Technology Co., Ltd.), and 1% penicillin / streptomycin (penicillin-streptomycin solution (100x), Zhejiang Senrui Biotechnology Co., Ltd.) is added. After 7-14 days of continuous culture, long spindle-shaped fibroblasts can be seen migrating out from the edge of the tissue block and continuing to proliferate. When the local cell density reaches about 90%, the supernatant and tissue block are removed, and the cells are digested with 0.25% trypsin-EDTA (Trypsin-EDTA Solution, BL512A, biosharp) and transferred to a 12-well plate and cultured for 2-3 generations to obtain purified fibroblasts (see Figure 1). Then amplify and culture. After the amplification is completed, take some cells and evenly spread them in a 6-well plate, keeping the cell density after attachment at about 30%. After the cells are attached, use immortalization virus to infect fibroblasts to construct laryngeal squamous cell carcinoma-related fibroblast cell line. Infection system: Polybrene (ST1380, Beyotime Biotechnology Co., Ltd): 2ul, Lenti-SV40T Lentivirus, Applied Biological Materials Inc. (abm): 30ul. After 24 hours of infection, use DMEM / F-12 (cytiva, SH30023.01) complete culture medium to replace the medium. When the cell density reaches about 80%, use puromycin with a final concentration of 1ug / ml to select immortalized cells. After three consecutive days of screening, the cells that have not been immortalized will be killed by puromycin. After continuous selection with puromycin for 1 week, the cells are frozen and continued to be cultured. The culture medium contained 10% fetal bovine serum (BioChannel Biological Technology Co., Ltd.) and 1% penicillin / streptomycin solution (Pen-Streptomycin Solution (100x), Zhejiang Senrui Biotechnology Co., Ltd.). The cells were cultured at 37°C and 5% CO. 2 Cell culture incubator.

[0022] Embodiment 2: This example analyzes the biological characteristics and applications of cell lines.

[0023] (1) Cell morphology The morphology of the tumor cells obtained in Example 1 was observed under an inverted microscope. Figure 1 As shown, the cells grow adherently, are evenly dispersed, have relatively uniform sizes, and are spindle-shaped.

[0024] (2) STR identification The cells were digested with trypsin, and DNA was extracted using Axygen's genomic extraction kit. The 21-STR amplification protocol was used to amplify the STR loci and the sex gene Amelogenin were detected on the ABI 3730XL genetic analyzer. The detected loci included: D5S818; D13S317; D7S820; D16S539; VWA; TH01; AMEL; TPOX; CSF1PO; D12S391; FGA; D2S1338; D21S11; D18S51; D8S1179; D3S1358; D6S1043; PENTAE; D19S433; PENTAD; D1S1656; and the results of STR analysis of GY-HNSCC-CAF cell lines are shown in Table 1.

[0025] Note: In the database of the German Collection of Microbiological Cultures DSMZ (Deutsche SammlungvonMikroorganismen und Zellkulturen), no identical STR test results were found. No cell line matching this cell was found in the database, indicating that this cell line is a new cell line; no multi-alleles were found, indicating that this cell line is a single cell without contamination by other cells. The cell was named human laryngeal squamous cell carcinoma-associated fibroblast GY-HNSCC-CAF. It is deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: C2024179.

[0026] Western blot detection of fibroblast-specific marker FAP (fibroblast activation protein) Prepare the required cells GY-HNSCC-CAF, head and neck squamous cell carcinoma cell lines: CAL-27, CAL-33, FaDu (purchased from Zhejiang Meisen Cell Technology Co., Ltd.). Use 0.1% trypsin to digest the cells and prepare the cell suspension, centrifuge at 3000rpm for 5min, obtain the cell pellet, use the cell lysis buffer RIPA (Prilai, C1053) to lyse on ice for 30min, and vortex for 30s every 10min. After the lysis is completed, place it in a 4℃ high-speed centrifuge and centrifuge it at 12000rpm for 10min, and take the supernatant. The protein concentration in the above four cell lysates was determined by the Coomassie Brilliant Blue G-250 (5X Coomassie Brilliant Blue G-250 solution, Cat#PC0015, Solarbio) method to prepare protein samples with the same protein concentration. After the sample preparation is completed, perform gel electrophoresis, transfer, milk blocking and other standard procedures, and then perform primary antibody incubation, secondary antibody incubation and development. The specific operations are as follows: 1. SDS-PAGE electrophoresis: Gel preparation: Gel was prepared using a one-step gel preparation kit (Hangzhou Fude Biotechnology Co., Ltd., FD341-100). Electrophoresis solution: Use SWE fast high-resolution electrophoresis buffer powder pack (Sevier Bio, G2081-1L) to prepare 1L electrophoresis solution. Sample loading: Load protein samples into the gel wells according to groups and sequence.

[0027] Electrophoresis: Use an electrophoresis apparatus (BIO-RAD, basic electrophoresis apparatus power supply 164-5050) to perform electrophoresis at 80 V for 30 minutes, and then continue to perform electrophoresis at 120 V for 40 minutes.

[0028] Transfer: ·Configure transfer solution: Configure the appropriate transfer solution according to the eBlotL1 instruction manual Activation and balancing of PVDF membrane: Cut the membrane to the same size as the gel, first use anhydrous ethanol to activate the membrane, then place the membrane in the matching balancing solution for use. Transfer: Use eBlot L1 fast wet transfer instrument for fast transfer.

[0029] Closure: Block the membrane with 5% skim milk powder in TBST and incubate at room temperature for 1 hour Primary antibody incubation: Primary antibody preparation: Dilute the antibody in TBST at a ratio of 1:1000 according to the instructions. · Membrane washing: Use TBST, wash three times on a horizontal shaker at 100 rpm to thoroughly wash away the blocking residual milk, 7 minutes each time · Incubation: Add diluted primary antibody, incubate with antibodies on a horizontal shaker at 60 rpm, at 4°C overnight.

[0030] Secondary antibody incubation: Primary antibody recovery: Recover and store the primary antibody overnight at 4°C Membrane washing: Use TBST, wash three times on a 100rpm horizontal shaker to thoroughly wash away the residual primary antibody, 7 minutes each time Secondary antibody incubation: Use species-matched secondary antibodies, dilute at a ratio of 1:2500, and incubate at room temperature for 1 hour development: Secondary antibody recovery: Recover secondary antibodies Membrane washing: Use TBST, wash three times on a 100rpm horizontal shaker to thoroughly wash away the residual secondary antibody, 7 minutes each time ·Developer solution: Use Fekote ultra-sensitive ECL luminescent solution (MeilunBio, MA0186-1), with a ratio of 1:1 between solution A and solution B · Visualization: The signal was captured using a chemiluminescent imaging system.

[0031] Depend on Figure 2 It can be seen that only GY-HNSCC-CAF specifically expresses FAP protein, while CAL-27, CAL-33, and FaDu cells do not express or express low levels of FAP. This further clarifies that GY-HNSCC-CAF is a fibroblast.

[0032] Antibody:

[0033] Cell scratch assay (1) Cell digestion: CAL-27\CAL-33\FaDu were digested with trypsin, centrifuged, resuspended in complete culture medium (90% DMEM, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution) and the cells were counted.

[0034] (2) Planting cells. Taking a 12-well plate as an example, each cell type is planted in three sub-wells, and the cell density is ensured to be around 70-80%.

[0035] (3) Cell scratching: When the cell fusion rate reaches 90%, in a sterile environment, remove the lid of the 12-well plate. Without removing the culture medium, use a sterile 100 μl pipette tip to draw a horizontal line vertically downward in the center of each well under the guidance of a sterile straight edge. Then wash the detached cells and use a marker to draw a vertical line perpendicular to the scratch line at the bottom of the outer side of the 12-well plate to mark the position. After marking, each well is marked according to Figure 5 The experimental design was to add HPF-1 culture supernatant (NF CM ) and GY-HNSCC-CAF culture supernatant (CAF CM ), collect images at 0h and 24h and on both sides of the position marked by the marker, and mark them.

[0036] The method for obtaining culture supernatant is as follows: HPF-1 culture supernatant (NF CM ): After continuous culture for 48 hours, the DMEM complete medium of HPF-1 (CCC-HPF-1 cells) was taken out and placed in a tube. Centrifuged at 1000 rpm for 5 min and the supernatant was taken as NF CM .

[0037] GY-HNSCC-CAF culture supernatant (CAF CM ): After continuous culture for 48 hours, the DME / F12 complete medium of GY-HNSCC-CAF was taken out, placed in a tube, centrifuged at 1000 rpm for 5 min, and the supernatant was taken, which was CAF CM .

[0038] Experimental results: Depend on Figure 3 It can be seen that using GY-HNSCC-CAF (CAF CM ) supernatant compared with HPF-1 (NF CM ) supernatant culture can significantly promote the wound healing ability of FaDu, CAL-27, and CAL-33. CM The remaining wound surface percentages of FaDu, CAL-27, and CAL-33 cells were 20%, 25%, and 13%, respectively.CM The wound surface after culture was 50%, 60%, and 30%, respectively, and the healing ability was significantly increased by 20-30% to varying degrees. CM ) cell culture supernatant can promote the migration ability of tumor cells.

[0039] Balling experiment: Experimental method: at 37°C, 5% CO 2 GY-HNSCC-CAF, HPF-1, CAL-27, CAL-33, FaDu five cell lines were cultured in an incubator. After the cell culture density reached 80-90%, the corresponding cells were collected by trypsin digestion, and the cells were counted using a count-star instrument, and the corresponding culture medium (GY-HNSCC-CAF used DME / F12 complete culture medium, and the other cells used DMEM complete culture medium) was used to prepare a cell suspension of 30000 cells / ml. Then, equal volumes of GY-HNSCC-CAF (CAF) and HPF-1 (NF) were mixed with CAL-27, CAL-33, and FaDu at a ratio of 1:1, and after mixing, 30 μL of the cell suspension was evenly spread on the inner cover of the bacterial culture dish, inverted and cultured for 3-5 days, and 20-30 microspheres were collected in a 6-well plate according to the group, and PBS was added and placed under an inverted microscope for photography.

[0040] Note: Complete culture medium consists of 90% corresponding basal culture medium (such as DMEM, DME / F12), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution.

[0041] Experimental results: Depend on Figure 4 It can be seen that using GY-HNSCC-CAF (CAF CM ) were spheroidized with FaDu, CAL-27, and CAL-33, and the diameter of the spheroids was significantly larger than that of HPF-1 (NF CM ) is a sphere formed by the co-sphere formation of FaDu, CAL-27, and CAL-33. GY-HNSCC-CAF has a stronger ability to promote the sphere formation of FaDu, CAL-27, and CAL-33 than HPF-1.

[0042] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A human laryngeal squamous cell carcinoma-associated fibroblast, characterized in that: Classification name: Human laryngeal squamous cell carcinoma-associated fibroblasts GY-HNSCC-CAF Homo sapiens, preservation unit: China Center for Type Culture Collection, preservation number: CCTCCNO: C2024179, preservation date: July 7, 2024.

2. Use of the human laryngeal squamous cell carcinoma-associated fibroblasts as described in claim 1 in constructing a head and neck squamous cell carcinoma cell model.

3. Use of the human laryngeal squamous cell carcinoma-associated fibroblasts as claimed in claim 1 in screening anti-tumor drugs for preventing or treating head and neck squamous cell carcinoma.

4. Use of the human laryngeal squamous cell carcinoma-associated fibroblasts as claimed in claim 1 in evaluating the efficacy of anti-tumor drugs for treating head and neck squamous cell carcinoma.

5. Use of the human laryngeal squamous cell carcinoma-associated fibroblasts as claimed in claim 1 in constructing an animal model of head and neck squamous cell carcinoma.

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