A murine anaplastic thyroid cancer cell line and its use

Abstract

This invention discloses a murine undifferentiated thyroid cancer cell line and its applications. The invention provides a murine undifferentiated thyroid cancer cell line, MATC (Mus musculus), deposited on October 10, 2024, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: C2024159. The MATC murine undifferentiated thyroid cancer cell line of this invention exhibits good adherence and stable cell characteristics, allowing for stable multiple passages. STR identification confirms it as a novel single cell line, enriching the library of undifferentiated thyroid cancer cell lines. It provides new experimental materials with biological characteristics closely resembling clinical tumors for research on undifferentiated thyroid cancer, serving as an effective target cell line for target research. It possesses very strong in vitro tumorigenic ability and can successfully prepare animal models of undifferentiated thyroid cancer for basic research and drug screening.

Description

Technical Field

[0001] This invention relates to the field of cell line technology, specifically to a murine undifferentiated thyroid cancer cell line and its applications. Background Technology

[0002] Anaplastic thyroid cancer (ATC) is the most malignant and has the worst prognosis subtype of thyroid cancer. It is a rare but highly lethal malignant tumor of the endocrine system, accounting for only 1%–2% of all thyroid cancers, yet responsible for more than 50% of thyroid cancer-related deaths. The median survival is only 3–6 months, and the 1-year survival rate is less than 15%, posing a significant challenge to clinical diagnosis and treatment. This tumor is characterized by rapid growth, high invasiveness, and a high tendency for early distant metastasis (commonly to the lungs, bones, and brain). It is also highly insensitive to traditional treatments—radioactive iodine therapy is ineffective because cancer cells lose the iodine uptake function of thyroid cells, and chemotherapy and radiotherapy can only temporarily relieve symptoms and are unlikely to reverse disease progression. Although emerging therapies such as targeted therapy and immunotherapy offer hope to some patients, they are still in the exploratory stage, effective only for a small number of patients, and exhibit significant drug resistance issues, with no standardized treatment regimen yet established.

[0003] The malignant progression mechanism of undifferentiated thyroid carcinoma is complex. It is currently known that its pathogenesis is closely related to mutations in genes such as TP53, BRAF, RAS and PIK3CA, as well as RET gene rearrangement. It exhibits significant genomic instability and tumor heterogeneity. However, the specific oncogenic driving mechanism, invasion and metastasis regulatory pathways, and molecular basis of treatment resistance have not been fully elucidated, which greatly limits the development of new diagnostic technologies, targeted drugs, and immunotherapy strategies.

[0004] In vitro cell models are core tools for basic tumor research, drug screening, and mechanism exploration. Their stability and specificity directly determine the reliability of research results and the feasibility of clinical translation. Currently, in vitro studies of undifferentiated thyroid carcinoma mainly rely on human undifferentiated thyroid carcinoma cell lines, such as 8505C and CAL-62 cells. Although these cell lines have been used in some studies, they have significant limitations: First, human cell lines are mostly derived from tumor tissues of clinical patients, and their genetic background and biological behavior differ from those of primary human tumors. They are also susceptible to genetic variations due to long-term in vitro passages, leading to biased research results. Second, human cell lines cannot easily simulate the complete pathogenesis and tumor microenvironment interactions of undifferentiated thyroid carcinoma in vivo, especially in studying the early mechanisms of tumor development, host immune responses, and in vivo drug efficacy evaluation, where they have inherent limitations.

[0005] Compared to human cell lines, mouse cell lines have unique advantages such as a clear genetic background, standardized culture, and easy integration with mouse in vivo models (e.g., xenograft tumor models, transgenic models). These advantages allow them to better simulate the in vivo pathogenesis of undifferentiated thyroid carcinoma, bridging basic research and clinical translation. However, mouse-derived undifferentiated thyroid cancer cell lines are extremely rare in the domestic and international markets. Most existing thyroid-related mouse cell lines are differentiated thyroid cancer cell lines or are initially constructed through gene editing. Their biological characteristics differ from those of natural undifferentiated thyroid cancer cells, making it difficult to accurately reflect the malignant phenotype of undifferentiated thyroid carcinoma (such as high invasiveness, high metastasis, and treatment resistance), thus failing to meet the needs of related research.

[0006] Furthermore, existing murine undifferentiated thyroid cell models, which are few in number, often suffer from low induction efficiency, poor passage stability, and atypical malignant phenotypes. After continuous in vitro passage, they are prone to differentiation reversal or decreased proliferation, making them unsuitable for long-term use in subsequent mechanistic studies, drug screening, and the construction of drug resistance models. Therefore, developing a murine undifferentiated thyroid cancer cell line with a stable genetic background, well-defined biological characteristics, and the ability to consistently exhibit the typical malignant phenotype of undifferentiated thyroid cancer (high proliferation, high invasion, high metastasis, and treatment resistance) to fill the current gap in cell models in this field is an urgent need to address the bottleneck in undifferentiated thyroid cancer research and promote the development of novel diagnostic and therapeutic technologies. Summary of the Invention

[0007] The purpose of this invention is to overcome the aforementioned shortcomings of the prior art, addressing the scarcity of existing murine undifferentiated thyroid cancer cell lines, especially the rarity of new murine undifferentiated thyroid cancer cell lines in recent years, which fails to meet the needs of new research. This invention provides a murine undifferentiated thyroid cancer cell line and its applications. The murine undifferentiated thyroid cancer cell line provided by this invention exhibits stable passage, good tumorigenicity, and can be extensively expanded and passaged in vitro for long periods, making it suitable for establishing animal models.

[0008] The inventors have independently induced, isolated, extracted, purified in vitro, and continuously passaged mouse thyroid undifferentiated cancer cells (MATC Mus musculus). These cells exhibit clear passage background, stable passage, and consistently express characteristic markers of thyroid undifferentiated carcinoma. They also possess typical malignant phenotypes of high proliferation, high invasion, high metastasis, and resistance to traditional therapeutic drugs. This makes them an ideal in vitro research tool model for thyroid undifferentiated carcinoma, with broad applications in the study of its pathogenesis, screening of novel targeted drugs, exploration of drug resistance mechanisms, and construction of in vivo xenograft models. They have significant scientific research value and application prospects, and can effectively promote technological advancements in the diagnosis and treatment of thyroid undifferentiated carcinoma.

[0009] The first objective of this invention is to provide a murine undifferentiated thyroid cancer cell line.

[0010] A second objective of this invention is to provide a progeny cell line of the aforementioned murine undifferentiated thyroid cancer cell line.

[0011] A third objective of this invention is to provide the application of the aforementioned murine undifferentiated thyroid cancer cell line in the preparation of thyroid cancer models.

[0012] The fourth objective of this invention is to provide the application of the aforementioned murine undifferentiated thyroid cancer cell line in studying the pathogenesis, drug resistance mechanism, or screening drugs for the treatment of undifferentiated thyroid cancer.

[0013] The fifth objective of this invention is to provide a product for preparing a thyroid cancer model.

[0014] The sixth objective of this invention is to provide a method for preparing an in vitro cell model of undifferentiated thyroid carcinoma.

[0015] The seventh objective of this invention is to provide a method for preparing a mouse model of undifferentiated thyroid carcinoma.

[0016] The eighth object of the present invention is to provide a method for screening drugs for the treatment of undifferentiated thyroid carcinoma.

[0017] To achieve the above objectives, the present invention is implemented through the following solution: A murine undifferentiated thyroid cancer cell line, wherein the murine undifferentiated thyroid cancer cell line is mouse thyroid undifferentiated cancer cell MATC Mus musculus, was deposited at the China Center for Type Culture Collection on October 10, 2024, with accession number: CCTCC NO: C2024159.

[0018] The progeny cell line of the murine undifferentiated thyroid cancer cell line.

[0019] Application of the mouse-derived undifferentiated thyroid cancer cell line and / or its progeny cell lines in the preparation of thyroid cancer models.

[0020] Furthermore, the thyroid cancer model includes an in vitro cell model of undifferentiated thyroid carcinoma and / or a mouse model of undifferentiated thyroid carcinoma.

[0021] The application of the murine undifferentiated thyroid cancer cell line and / or its progeny cell lines in studying the pathogenesis, drug resistance mechanism, or screening drugs for the treatment of undifferentiated thyroid cancer.

[0022] A product for preparing a thyroid cancer model, the product containing the murine undifferentiated thyroid cancer cell line and / or its progeny cell lines as described in claim 1.

[0023] A method for preparing an in vitro cell model of undifferentiated thyroid carcinoma, comprising passage culture of the mouse-derived undifferentiated thyroid carcinoma cell line and / or its progeny cell lines.

[0024] Furthermore, the cells were passaged using a medium containing serum, penicillin, streptomycin, and hydrocortisone.

[0025] Furthermore, the culture was passaged in DMEM / F12 medium containing 8%–12% (v / v) fetal bovine serum, 90–110 U / mL penicillin, 90–110 μg / mL streptomycin, and 4–6 mg / L hydrocortisone.

[0026] A method for preparing a mouse model of undifferentiated thyroid carcinoma, comprising injecting the mouse-derived undifferentiated thyroid carcinoma cell line, its progeny cell line, or the product into mice.

[0027] Further, the mouse-derived undifferentiated thyroid cancer cell line, its progeny cell line, or the product is injected into the thyroid gland of mice.

[0028] A method for screening drugs for treating undifferentiated thyroid carcinoma includes using a murine undifferentiated thyroid carcinoma cell line and / or its progeny cell lines as a test group and a commonly used murine undifferentiated thyroid carcinoma cell line and / or its progeny cell lines as a control group. A test drug is added to both the test group and the control group. If the cell lines in the test group grow better than those in the control group, the test drug is a candidate drug for treating undifferentiated thyroid carcinoma. If the cell lines in the control group grow better than those in the test group, the murine undifferentiated thyroid carcinoma cell line and / or its progeny cell lines are resistant to the test drug, and a drug-resistant thyroid carcinoma model can be prepared using the murine undifferentiated thyroid carcinoma cell line and / or its progeny cell lines.

[0029] Furthermore, the commonly used undifferentiated thyroid cancer cell lines include 8505C and / or CAL-62 cells.

[0030] The mouse undifferentiated thyroid cancer cell line MATC Mus musculus provided by this invention fills a gap in the current field of mouse-derived undifferentiated thyroid cancer cell lines and can serve as an effective research cell model. Furthermore, it exhibits stable passage, good tumorigenicity, and can be massively expanded and passaged in vitro for extended periods, successfully preparing animal models of undifferentiated thyroid cancer. MATC also provides an effective model for research on chemotherapy resistance mechanisms. Therefore, the mouse undifferentiated thyroid cancer cell line MATC Mus musculus of this invention can provide researchers with a new experimental model for in-depth studies of the pathogenesis, metastasis, screening of characteristic biomarkers of pathogenesis and metastasis, drug resistance mechanisms, and new drug screening of undifferentiated thyroid cancer, making it an ideal cell line for basic research and preclinical applications of mouse-derived undifferentiated thyroid cancer.

[0031] Compared with the prior art, the present invention has the following beneficial effects: (1) The mouse thyroid undifferentiated cancer cells MATC Mus musculus of the present invention have good adherence and stable cell characteristics, and can be stably passaged multiple times.

[0032] (2) The mouse thyroid undifferentiated cancer cell line MATC Mus musculus of the present invention was identified as a new single cell line by STR.

[0033] (3) The immunohistochemical markers of the mouse undifferentiated thyroid cancer cells MATC Mus musculus of the present invention provide new experimental materials for the study of undifferentiated thyroid cancer that are close to the biological characteristics of clinical tumors.

[0034] (4) The mouse thyroid undifferentiated cancer cell line MATC Mus musculus of the present invention is a mouse undifferentiated cell line, and its differentiation grade and type are different from the commonly used human thyroid undifferentiated cancer cell lines, thus enriching the thyroid undifferentiated differentiated cell line library.

[0035] (5) The mouse thyroid undifferentiated cancer cell line MATC Mus musculus of the present invention is negative for P53. P53 is an important tumor suppressor gene. Therefore, the mouse thyroid undifferentiated cancer cell line of the present invention can be used as an effective target cell line for research.

[0036] (6) The mouse thyroid undifferentiated cancer cell MATC Mus musculus of the present invention has a very strong in vitro tumorigenic ability and can successfully prepare an animal model of thyroid undifferentiated cancer for use in basic research and drug screening. Attached Figure Description

[0037] Figure 1 This is an optical morphological image of MATC cells, P0 generation, magnified 10 times.

[0038] Figure 2 This is an optical morphological image of MATC P6 generation cells, magnified 10 times.

[0039] Figure 3 This is an optical morphological image of MATC cells P17, magnified 10 times.

[0040] Figure 4 This is an optical morphological image of MATC cells from generation P50, magnified 10 times.

[0041] Figure 5 This is a cell growth curve of MATC cells.

[0042] Figure 6HE-stained image of a murine primary undifferentiated thyroid carcinoma tissue specimen, magnified 40 times.

[0043] Figure 7 This is an HE-stained image of MATC cells, magnified 40 times.

[0044] Figure 8 Crystal violet staining results of MATC cells after in vitro treatment with sorafenib for 24 h.

[0045] Figure 9 Crystal violet staining results of MATC cells treated with sorafenib for 48 h in vitro.

[0046] Figure 10 The results are for sorafenib susceptibility testing. A represents the susceptibility test results of MATC cells treated with sorafenib for 24 h; B represents the susceptibility test results of 8505C cells treated with sorafenib for 24 h; C represents the susceptibility test results of MATC cells treated with sorafenib for 48 h; and D represents the susceptibility test results of 8505C cells treated with sorafenib for 48 h.

[0047] Figure 11 Fluorescence imaging images of MATC cells in C57BL / 6J mice at times 9, 11, 13, and 15, showing in situ tumor formation of the thyroid gland.

[0048] Figure 12 Images of MATC cell line and nude mouse tumor masses stained with hematoxylin and eosin (HE). In the images, A is an undifferentiated thyroid carcinoma tissue specimen; B is MATC cells; and C is a thyroid orthotopic tumor tissue specimen.

[0049] Figure 13 Images show the CK expression detection results of MATC cell line and nude mouse tumor masses. In the images, A is an undifferentiated thyroid carcinoma tissue specimen; B is MATC cells; and C is a thyroid orthotopic tumor tissue specimen.

[0050] Figure 14 Images show the Pax-8 expression detection results of MATC cell line and nude mouse tumor masses. In the images, A is an undifferentiated thyroid carcinoma tissue specimen; B is MATC cells; and C is a thyroid orthotopic tumor tissue specimen.

[0051] Figure 15 Images show the TTF1 expression detection results of MATC cell line and nude mouse tumor masses. In the images, A is an undifferentiated thyroid carcinoma tissue specimen; B is MATC cells; and C is a thyroid orthotopic tumor tissue specimen.

[0052] Figure 16 Images show the SMA expression detection results of MATC cell line and nude mouse tumor masses. In the images, A is an undifferentiated thyroid carcinoma tissue specimen; B is MATC cells; and C is a thyroid orthotopic tumor tissue specimen.

[0053] Figure 17 Images show the results of TG expression detection in MATC cell line and nude mouse tumor masses. In the images, A is an undifferentiated thyroid carcinoma tissue specimen; B is MATC cells; and C is a thyroid orthotopic tumor tissue specimen. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The specific materials and their sources listed in the following embodiments are merely exemplary and are not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or functions as the following tissues, cells, reagents, and instruments can be used to implement the present invention.

[0055] Experimental methods not specifically described in the following examples may be performed using conventional methods in the art, such as referring to Molecular Cloning: A Laboratory Manual (3rd Edition, New York: Cold Spring Harbor Laboratory Press, 1989) or following the conditions recommended by the supplier.

[0056] Example 1: Establishment of the MATC cell line 1. Induction of undifferentiated thyroid carcinoma from murine sources

[0057] TPOCreER / J mice, BRAFtm1Mmcm / J mice, and Trp53tm1Brn / J mice were all purchased from The Jackson Laboratory in the United States. The mice were raised and bred in accordance with SPF-grade animal breeding standards. The temperature, humidity, and light cycle of the breeding environment were kept constant to ensure the normal growth and reproduction of the mice.

[0058] First, healthy, age-appropriate, and breeding-suitable TPOCreER / J transgenic mice and BRAFtm1Mmcm / J mutant mice were selected and mated together in a male-to-female ratio of 1:2. After 21 days of age, the pups were separated into individual cages of 5 mice each to avoid overcrowding and its negative impact on growth and development. Each pup was uniquely numbered using a toe-stripping method, and toe tissue from each pup was collected and placed in 1.5 mL EPPENDORF (EP) tubes, stored at -80°C for subsequent mouse genotyping.

[0059] Genotyping was performed using a mouse tail direct PCR kit (catalog number: B40015, manufacturer: SELLECK, country of origin: USA). The kit was used strictly according to the instructions, with preserved mouse toe tissue as samples for PCR amplification, thus completing the genotyping of the pups. After identification, double-positive mice (TB mice) of both the TPOCreER / J transgene and the BRAFtm1Mmcm / WZ / J mutation were selected and used as parent mice for subsequent breeding. Mice that did not meet the genotyping requirements were culled.

[0060] Subsequently, the TB mice and Trp53tm1Brn / J mice obtained from the above screening were selected and, following the same mating, breeding, separate rearing, and genotype identification methods, continued to be mated together, reared, and screened for genotypes. Finally, the three-gene mice (abbreviated as TBP mice) that were positive for TPOCreER / J transgene, BRAFtm1Mmcm / WZ / J mutation, and Trp53tm1Brn / tm1Brn / J knockout were obtained, which are the target mice for inducing undifferentiated thyroid carcinoma.

[0061] Once TBP mice reached 8 weeks of age (the optimal age for development), thyroid undifferentiated carcinoma induction was initiated: TBP mice were intraperitoneally injected with tamoxifen (concentration: 120 mg / kg, solvent: corn oil) for 5 consecutive days. Tamoxifen induced the activation of relevant genes in the TBP mice, thereby promoting the spontaneous formation of thyroid undifferentiated carcinoma. After the induction process, the TBP mice were fed for another 6 months. At this time, the formation of thyroid undifferentiated carcinoma in the mice was stable, and the TBP mice at this stage could be used for subsequent experiments such as the isolation, extraction, and culture of murine thyroid undifferentiated carcinoma cell lines.

[0062] 2. Specimen processing Fresh undifferentiated thyroid carcinoma tissue specimens from mice induced and cultured in step 1 were surgically removed, rinsed three times with physiological saline, and hemorrhagic necrotic tissue and adipose tissue were removed. The specimens were then soaked in PBS (containing 500 IU / mL penicillin, 500 μg / mL streptomycin and 5 μg / mL amphotericin B) at 4°C for 10 min. The undifferentiated thyroid carcinoma tissue specimens were repeatedly rinsed with physiological saline a total of three times.

[0063] Under sterile conditions, undifferentiated thyroid carcinoma tissue specimens were cut into tissue blocks of approximately 1 mm × 1 mm. During the cutting process, 1-2 drops of DMEM / F12 culture medium (purchased from Gibco) were added to the tissue blocks to keep them moist, resulting in the cut tissue.

[0064] The cut tissue was placed in a 15 mL sterile centrifuge tube, and 5 mL of 0.1% (w / v) collagenase (purchased from Sigma, USA) and 0.125% (w / v) trypsin-EDTA (purchased from Gibco, USA) were added. The mixture was shaken at 37°C for 4 h. Then, 5 mL of DMEM / F12 culture medium containing 10% (v / v) fetal bovine serum (purchased from Gibco, catalog number: 12400-024) was added, mixed well, and allowed to stand for 15 min to obtain the digested tissue.

[0065] After digestion, the tissue was filtered through a 70 μm sterile filter membrane. The filtrate was collected, centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. The cells were washed once with PBS, centrifuged, and collected. The cells were then cultured at 37℃ in a 5% CO2 incubator for 4 weeks in a culture medium consisting of DMEM / F12 + 10% (v / v) FBS + 100 U / mL penicillin + 100 μg / mL streptomycin + 5 mg / L hydrocortisone.

[0066] 3. Monoclonal purification Selected cell regions with strong viability after step 1 culture were prepared into single-cell suspensions of 10 cells / mL. 100 μL of each suspension was placed in 96-well plates to create single-cell proliferation wells for monoclonal purification, yielding monoclonal cell clusters. The monoclonal cell clusters were digested with 0.25% (w / v) trypsin for 1 min and then expanded in a DMEM / F12 medium containing 10% (v / v) FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, and 5 mg / L hydrocortisone. The culture was carried out at 37°C in a 5% CO2 incubator. Cells were passaged weekly; they grew well, exhibited relatively uniform morphology, and could be passaged more than 50 times. Mouse-derived undifferentiated thyroid carcinoma cells were obtained.

[0067] The mouse-derived undifferentiated thyroid cancer cells passaged for 15 generations were named mouse undifferentiated thyroid cancer cells (MATC Mus musculus) (hereinafter referred to as MATC cells). The mouse-derived undifferentiated thyroid cancer cells (MATC) were deposited on October 10, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China), with accession number CCTCC NO: C2024159.

[0068] Example 2: Biological characteristics and applications of MATC cells

[0069] 1. Morphological observation of MATC cells

[0070] MATC cells were passaged and cultured, and their viable cell growth was observed under a light microscope (Zeiss inverted microscope) at 10×. The results are as follows: Figures 1-4As shown, MATC cells grow vigorously with a clear background and few impurities. Adherent cells are spindle-shaped, while cascaded cells are round, exhibiting cell clusters of varying sizes.

[0071] 2. STR analysis of genetic material in MATC cells Short tandem repeats (STRs), also known as microsatellite DNA, are DNA sequences on chromosomes formed by tandem repetitions of several base pairs as core units (2-6 base pairs). The number of repetitions per core unit varies among individuals, resulting in alleles of varying lengths. Therefore, the number of repetitions of a set of STR sequences is almost unique across individuals, serving as a genetic identifier and a primary method in cell biology for identifying cell identity and origin.

[0072] P15 generation MATC cells were collected by trypsin digestion. Genomic DNA was extracted from the cells using the Axygen Genomic DNA Extraction Kit (AxyPrep™ Multisource Genomic DNA Miniprep Kit from Axygen). The DNA was amplified using the 10-STR amplification protocol, and the STR loci and sex gene Amelogenin were detected on an ABI 3730XL genetic analyzer.

[0073] The STR analysis of the genetic material of the MATC cell line is shown in Table 1.

[0074] STR sequence searches were performed in the German Society for the Preservation of Microbial Cultures (DSMZ) database and the American Type Culture Collection (ATCC) database, but no identical STR results were found. This indicates that the cell line is a mouse cell line, and the lack of matching cell lines in the DNA typing search indicates that this is a new cell line. The absence of multiple isomorphic sites suggests that this cell line is a single cell line, free from cross-contamination and human contamination.

[0075] Table 1. Genotyping results of STR and Amelogenin loci in MATC cells.

[0076] 3. Study on the growth kinetics of MATC cells Prepare 1×10⁻⁶ cells from passaged logarithmic growth cells. 4Cells / mL cell suspension were seeded at 100 μL per well in 96-well plates. Cell proliferation was detected sequentially at days 0, 1, 2, 3, 4, 5, 6, 7, and 8 using the Cell Counting Kit-8 (CCK-8) method. The CCK-8 assay followed the instructions of the kit (Shanghai Taoshu Biotechnology Co., Ltd., catalog number C0005). A 1:9 (CCK-8 stock solution: culture medium) CCK-8 working solution was prepared, and 100 μL was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 2 h, and the OD value was obtained at 450 nm. The MATC cell growth curve is shown below. Figure 5 As shown.

[0077] Depend on Figure 5 It can be seen that MATC cells enter a doubling state on days 1, 2, 3, 4 and 5, and enter a plateau phase on day 6, exhibiting strong proliferative capacity.

[0078] 4. Pathological identification of MATC cells (1) The MATC cells from Example 1 and the surgical tissue specimens of undifferentiated thyroid carcinoma were paraffin-embedded, sectioned, and stained with H&E.

[0079] Preparation of MATC cell paraffin blocks: P15 generation MATC cells were digested with 0.25% (w / v) trypsin and centrifuged at 1000 r / min for 5 min using a low-speed automatic balancing centrifuge (LD-Z5-2, Beijing). The supernatant was discarded, and 1 mL of 10% (v / v) neutral buffered formalin was added for fixation for 5 h. The bottom precipitate was then wrapped with tea leaves. The cells were dehydrated sequentially with 75% (v / v), 80% (v / v), 95% (v / v), 95% (v / v), 100% (v / v), and 100% (v / v) ethanol for 2 h each time. The cells were cleared with xylene for 30 min, and this process was repeated twice. The cells were then immersed in paraffin at 60℃ for 2 h. The cells were embedded using a Leica JUNG embedding machine. The cells were sectioned using a Leica 2135 paraffin microtome to a thickness of 4 μm. The sections were then baked in a 60℃ oven for 1 hour. h; dyeing was performed using a German Leica ST5020 dyeing machine; sealing was performed using a German Leica CV5030 sealing machine.

[0080] Preparation of paraffin blocks for undifferentiated thyroid carcinoma tissue specimens: The tissue was cut into pieces approximately 1 cm × 1 cm × 0.3 cm in size and placed in a tissue embedding cassette. It was fixed with 10% (v / v) neutral buffered formalin. The tissue was then dehydrated sequentially with 75% (v / v), 80% (v / v), 95% (v / v), 95% (v / v), 100% (v / v), and 100% (v / v) ethanol for 2 h each time. The tissue was cleared with xylene for 30 min, repeated twice. It was then immersed in paraffin at 60℃ for 2 h. The tissue was embedded using a Leica JUNG embedding machine. Sections were prepared using a Leica 2135 paraffin sectioner to a thickness of 4 μm. The sections were baked in a 60℃ oven for 1 h. Staining was performed using a Leica ST5020 staining machine. Finally, the sections were mounted using a Leica CV5030 mounting machine.

[0081] (2) Microscopic photographs were taken under an optical microscope, and HE-stained images (40×) of mouse in situ induced undifferentiated thyroid carcinoma tissue specimens and MATC cells were compared. The results are as follows: Figure 6 and Figure 7 As shown. The pathological diagnoses, as determined by three pathologists, are as follows: MATC cells have the same morphology as undifferentiated thyroid carcinoma tissue specimens, with numerous mitotic figures, confirming that MATC cells are cancer-derived cells and classified as an undifferentiated cell line. This cell line is a murine undifferentiated thyroid cancer cell line; currently, there is no corresponding cell line of this type. It can be used for in vivo immune-related tests to differentiate it from the human thyroid cancer cell line 8505C. It is currently only used for tumorigenesis experiments in nude mice to enrich the undifferentiated thyroid cancer cell bank.

[0082] 5. Monoclonal crystal violet staining detection 5×10⁶ MATC cells were prepared from passaged logarithmic growth. 4 Cell suspension of 100 μL / well was seeded into 6-well plates at a concentration of 100 cells / mL. After adhesion, the cells were treated with 10 μM sorafenib or without for 24 h, and then the culture medium was discarded. Cells were then treated with fresh, drug-free culture medium for 24 h and 48 h, respectively, and then the culture medium was discarded. 2 mL of PBS buffer was added to each well to wash away residual serum, and the wash was repeated twice. Then, 1.5–2 mL of 4% (v / v) paraformaldehyde fixative was added to each well, and the cells were fixed at 37°C for 15–20 min, after which the fixative was discarded. 1 mL of 0.1% (v / v) crystal violet staining solution was added to each well, and the cells were stained at room temperature in the dark for 5–10 min with gentle shaking occasionally. After staining, the staining solution was discarded, and the cells were slowly rinsed 3–4 times with sterile distilled water until the background was colorless. The cells were then blotted dry on filter paper. Finally, the staining effect and clonal pattern were observed and recorded under an inverted microscope using paraformaldehyde fixation.

[0083] The results are as follows Figure 8 and Figure 9As shown in the results of the clonogenic assay, the murine undifferentiated thyroid cancer cells in the blank control group formed a large number of dense, poorly defined continuous clones after 24 h of culture, exhibiting vigorous cell proliferation and extremely strong clonogenic ability. After treatment with sorafenib, cell clonogenicity was significantly inhibited in a time-dependent manner: the cell clones in the 24 h treatment group were significantly smaller, appearing as scattered small clusters with significantly increased interclonal gaps; in the 48 h treatment group, cell clonogenicity was almost completely blocked, leaving only a few scattered single cells or microcolonies, with no large areas of continuous clone formation, and the cell density was significantly lower than that of the control group. These results indicate that sorafenib can significantly inhibit the clonogenic ability of murine undifferentiated thyroid cancer cells, and this effect increases with prolonged treatment time.

[0084] 6. MATC cell drug sensitivity testing MATC cells and human 8505C cells obtained from passaged logarithmic growth were used to prepare 5×10⁻⁶ cells each. 4 Cells / mL cell suspension: 100 μL of cell suspension was seeded into each well of a 96-well plate. When the cell density reached 80%, sorafenib (Sora), a clinical treatment for locally recurrent or metastatic progressive refractory thyroid cancer, was added to each well at concentrations of 0, 10, 20, 30, 40, and 50 μM. Cell viability was detected in each well 24 h and 48 h after drug addition using the CCK-8 assay. The CCK-8 assay method was performed according to the kit instructions. A 1:9 (CCK-8 stock solution: culture medium) CCK-8 working solution was prepared, and 100 μL was added to each well. The plate was incubated at 37°C in a 5% CO2 incubator for 2 h, and the OD value was detected at 450 nm.

[0085] Drug sensitivity test results as follows Figure 10 A~ Figure 10 As shown in Figure D, the IC50 of sorafenib half-inhibition concentration at 24 h in MATC cells was 18.46 μM, and at 48 h it was 11.36 μM, which were significantly higher than the IC50 of sorafenib half-inhibition concentration at 24 h (11.32 μM) and at 48 h (8.185 μM) in human thyroid cancer undifferentiated cell line 8505C cells, respectively. MATC cells can be used as a cell model for studying sorafenib tolerance.

[0086] 7. Procedure for Fluorescence Imaging of In Situ Thyroid Tumors Luciferase-labeled MATC cells were resuscitated, cultured, and expanded to ensure optimal cell condition. Cells in the logarithmic growth phase were collected, resuspended in serum-free medium, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 7Cell suspensions were obtained by measuring cells / mL. Six-week-old C57BL / 6J mice were selected and acclimatized for 1-2 weeks before the experiment to ensure stable animal condition. MATC cells were then injected in situ. The MATC cell injection procedure was as follows: the neck area of ​​the mouse was prepared and disinfected with povidone-iodine; the mouse was anesthetized by inhalation with isoflurane, and after the anesthesia took effect, the mouse was fixed supine on the operating table; a 5 mm incision was made in the midline of the neck, and the subcutaneous tissue was dissected to expose the thyroid lobe; 10 μL of MATC cell suspension was slowly injected into one thyroid lobe using a microsyringe, avoiding leakage of the cell suspension during the injection; after the injection, the incision was sutured with absorbable sutures, and antibiotic ointment was applied locally to prevent infection. Imaging detection was performed on days 7, 9, 11, 13, and 15 after modeling. Mice were anesthetized by isoflurane inhalation and injected intraperitoneally with 100 μL of D-fluorescein potassium salt solution (3 mg / mouse). After 10-15 minutes, the anesthetized mice were placed supine in the dark chamber of the IVIS Lumina imaging device for imaging.

[0087] The results are as follows Figure 11 As shown in the in vivo imaging results, the tumor growth of MATC cells in the thyroid gland exhibited a significant time-dependent characteristic after orthotopic inoculation. On day 7 post-inoculation, no obvious fluorescence signal was observed in the mouse thyroid region; a weak, specific fluorescence signal began to appear on day 9, indicating successful tumor colonization; as time progressed, the intensity and extent of the fluorescence signal progressively increased on days 11 and 13, indicating a sustained increase in tumor burden; by day 15 post-inoculation, a high-intensity fluorescence signal was visible in the thyroid situ, with significant signal enhancement in the central region, suggesting that a stable orthotopic tumor growth model had been established. These results demonstrate that MATC cells can form progressively growing tumors in the mouse thyroid gland in situ, successfully constructing an orthotopic thyroid tumorigenesis model, providing a reliable research platform for subsequent in vivo intervention experiments.

[0088] 8. Pathological identification of tumors (1) In step 7, the tumors formed in the thyroid glands of C57BL / 6J mice 15 days after inoculation with MATC cells were paraffin-embedded, sectioned, and H&E stained according to the following steps: the tumor was cut into pieces of approximately 1 cm × 1 cm × 0.3 cm and placed in a tissue embedding cassette, and fixed with 10% (v / v) neutral formalin; dehydrated with 75% (v / v), 80% (v / v), 95% (v / v), 95% (v / v), 100% (v / v), and 100% (v / v) ethanol for 2 h in sequence; cleared with xylene for 30 min, twice; immersed in paraffin at 60℃ for 2 h; embedded using a Leica JUNG embedding machine; sectioned using a Leica 2135 paraffin sectioner to a thickness of 4 μm; and baked in a 60℃ oven for 1 hour. h; staining was performed using a Leica ST5020 staining machine; and the slides were mounted using a Leica CV5030 mounting machine to obtain paraffin blocks of thyroid orthotopic tumor tissue specimens.

[0089] (2) Take microscopic photographs under an optical microscope to observe the HE staining morphology of the thyroid in situ tumor tissue specimen formed by MATC cells in nude mice.

[0090] Depend on Figure 12 C indicates that the cells in the thyroid in situ tumor tissue specimen are round, with a small nucleus-cytoplasm ratio, and are similar to... Figure 12 The clinical undifferentiated thyroid carcinoma tissue specimen shown in A Figure 12 The MATC cells shown in B have similar HE staining morphology, thus establishing a correspondence between the primary undifferentiated thyroid carcinoma tissue specimen, MATC cells, and the in situ thyroid tumor tissue specimen.

[0091] 9. Immunohistochemical analysis of MATC cell line and mouse thyroid orthotopic tumor tissue MATC cell paraffin blocks and primary undifferentiated thyroid carcinoma tissue specimens were prepared according to step 4. Thyroid orthotopic tumor tissue specimens were prepared according to step 8. Sections were cut to 4 μm, baked at 60℃ for 1 h, dewaxed with xylene, and incubated with 3% (w / v) H2O2 for 10 min. Immunohistochemistry was performed using a kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., PV-9000). Mouse-derived primary antibodies (CK, CD68, DES, Ki67, Pax-8, SMA, TG, TTF1) were added, and the slides were incubated overnight at 4℃. The slides were washed three times with PBS for 2 min each time. Goat anti-mouse IgG antibody-HRP polymer was added and incubated at room temperature for 20 min. The slides were washed three times with PBS for 2 min each time. DAB staining was performed for 1 min, followed by thorough rinsing with tap water. The slides were then mounted using a Leica CV5030 mounting machine.

[0092] Immunohistochemical analysis results of MATC cells and undifferentiated thyroid carcinoma tissue specimens for CK (spectral cytokeratin), CD68, DES (desin), Ki67 (Ki67 nuclear antigen), Pax-8 (box-8), SMA (actin), TG (thyroglobulin), and TTF1 (thyroid transcription factor 1) are as follows: Figures 13-17 As shown. Among them, Figure 13 A to Figure 13 C showed positive CK expression (+); CD68 expression in MATC cells and patient tissues was negative (-); DES expression in MATC cells and patient tissues was negative; Ki67 expression in MATC cells and patient tissues was positive (+, 60%). Figure 14 A to Figure 14 C's Pax-8 expression is negative (-); Figure 15 A to Figure 15 C's TTF1 expression is negative (-); Figure 16 A to Figure 16 C's SMA expression is negative (-); Figure 17 A to Figure 17 C showed a negative (-) TG expression.

[0093] The results were interpreted by three pathologists. CK expression was positive, consistent with the protein expression characteristics of undifferentiated thyroid cancer cells. CD68, which can distinguish between monocytes and macrophages, was negative, indicating no macrophage contamination. DES, an intermediate filament protein, is widely present in tumors derived from skeletal and smooth muscle; its negative expression excludes spindle muscle cells and sarcoma cells. Ki67, a cell proliferation index, is closely related to the differentiation, invasion, metastasis, and prognosis of many tumors; MATC tissues and cell lines showed high Ki67 expression and strong cell proliferation activity. Pax-8 expression was negative. SMA, actin, is mainly found in spindle cells, fibromyalgia cells, or sarcoma cells; its negative expression indicates no contamination by other cells. In summary, combined with HE staining and immunohistochemical results, MATC was confirmed as an undifferentiated thyroid cancer cell line, free from other cellular contamination.

[0094] Based on the above experimental observations and verifications, the mouse-derived undifferentiated thyroid cancer cell line MATC provided by this invention exhibits adherent cell growth in a network pattern, stable cell characteristics, and stable multiple passages. Immunohistochemical analysis and pathological identification show that its morphology is identical to that of the specimen tissue, confirming it as a thyroid undifferentiated cancer cell line without contamination from other cells (such as fibroblasts and smooth muscle cells). STR analysis of the genetic material identifies this cell line as a novel single cell line. This mouse-derived undifferentiated thyroid cancer cell line MATC can proliferate significantly and possesses strong proliferative activity. This mouse-derived undifferentiated thyroid cancer cell line MATC can serve as an effective research cell model, providing researchers with a new experimental model for in-depth research on the etiology, metastasis mechanisms, drug resistance mechanisms, and new drug screening of undifferentiated thyroid cancer.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A murine undifferentiated thyroid cancer cell line, characterized in that, The mouse-derived undifferentiated thyroid cancer cell line is mouse thyroid undifferentiated cancer cell MATC Mus musculus, which was deposited at the China Center for Type Culture Collection on October 10, 2024, with accession number CCTCC NO: C2024159.

2. The progeny cell line of the murine undifferentiated thyroid cancer cell line of claim 1.

3. The use of the murine undifferentiated thyroid cancer cell line and / or its progeny cell lines as described in claim 1 in the preparation of thyroid cancer models.

4. The application according to claim 3, characterized in that, The thyroid cancer models include in vitro cell models of undifferentiated thyroid carcinoma and / or mouse models of undifferentiated thyroid carcinoma.

5. The use of the murine undifferentiated thyroid cancer cell line and / or its progeny cell lines as described in claim 1 in studying the pathogenesis, drug resistance mechanism, or screening drugs for the treatment of undifferentiated thyroid cancer.

6. A product for preparing a thyroid cancer model, characterized in that, The product contains the murine undifferentiated thyroid cancer cell line and / or its progeny cell line as described in claim 1.

7. A method for preparing an in vitro cell model of undifferentiated thyroid carcinoma, characterized in that, The mouse-derived undifferentiated thyroid cancer cell line and / or its progeny cell lines as described in claim 1 were passaged.

8. The method according to claim 7, characterized in that, Subculture was performed using a medium containing serum, penicillin, streptomycin, and hydrocortisone.

9. A method for preparing a mouse model of undifferentiated thyroid carcinoma, characterized in that, The mouse-derived undifferentiated thyroid cancer cell line of claim 1, its progeny cell line, or the product of claim 6 is injected into mice.

10. A method for screening drugs for treating undifferentiated thyroid carcinoma, characterized in that, Using the murine undifferentiated thyroid cancer cell line and / or its progeny cell line as described in claim 1 as the test group, and a commonly used murine undifferentiated thyroid cancer cell line and / or its progeny cell line as the control group, the test drug is added to both the test group and the control group. If the cell line in the test group grows better than that in the control group, then the test drug is a candidate drug for treating undifferentiated thyroid cancer. If the cell line in the control group grows better than that in the test group, then the murine undifferentiated thyroid cancer cell line and / or its progeny cell line as described in claim 1 is resistant to the test drug, and a drug-resistant thyroid cancer model can be prepared using the murine undifferentiated thyroid cancer cell line and / or its progeny cell line as described in claim 1.

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