A rhesus monkey skin squamous cell carcinoma cell line mcsc14397 and its application

Abstract

The application discloses a rhesus monkey skin squamous carcinoma cell line MCSCC14397 and application thereof. The rhesus monkey skin squamous carcinoma cell line is named as the rhesus monkey skin squamous carcinoma cell line MCSCC14397, and the preservation number is CCTCC NO: C2025156. The cell line of the application has similar protein expression as primary tumor tissue, has abnormal karyotype, in-vitro tumorigenic ability and stable proliferation characteristics, and simultaneously shows significant genomic instability (such as high-frequency C>T mutation, structural variation) and inflammation signal activation. MYO10 gene expression is up-regulated in the cell line and drives DNA damage response and inflammation factor expression, which provides unique advantages for the application of the cell line in skin squamous carcinoma mechanism research, drug screening and efficacy evaluation. The cell line of the application can be used as an effective cell model, can establish a xenotransplantation animal model, and can provide a research basis for in-depth research on the occurrence, development, metastasis mechanism and drug resistance mechanism of human skin squamous carcinoma and innovative drug development and screening.

Description

Technical Field

[0001] This invention relates to the field of tumor biology technology, specifically to a rhesus monkey skin squamous cell carcinoma cell line MCSCC14397 and its applications. Background Technology

[0002] Cutaneous squamous cell carcinoma (cSCC) is a malignant tumor originating from the malignant proliferation of keratinocytes in the epidermis or its appendages (such as pilosebaceous units and eccrine sweat glands). It accounts for 20-50% of all skin cancers, making it the second most common type of skin cancer, with an overall mortality rate of 1.5-3.4%. CSCCC has a strong tendency to metastasize and invade, and its poor prognosis contributes to the increasing number of deaths from skin cancer. Clinically, it presents as a single, firm papule or a erythematous nodule with distinct scaly patches, accompanied by a tendency to bleed, varying degrees of hyperkeratosis, and ulceration. A definitive diagnosis is made through lesion biopsy and histopathological examination. The pathogenesis of cSCC is currently unclear. Complete excision under histopathological guidance has a high recurrence rate. Topical medications and cryotherapy are associated with adverse reactions such as pain, local irritation, and irreversible pigmentation, leading to low patient compliance and a high treatment failure rate. Therefore, studying its pathogenesis, constructing its disease model, and developing clinical intervention strategies has become a task with both scientific significance and practical value.

[0003] In the field of disease research, isolating and establishing cell lines from patient tumor tissues is a crucial foundation for translational medicine research. Cell lines provide abundant experimental materials for studying disease pathogenesis, constructing disease models, screening drugs, and identifying therapeutic targets. Therefore, establishing skin squamous cell carcinoma cell lines that are similar in occurrence to human skin squamous cell carcinoma and constructing suitable animal models are important prerequisites for studying the disease mechanisms of skin squamous cell carcinoma and developing more effective intervention and treatment strategies.

[0004] Squamous cell carcinoma cell lines originate from various tissues, including the lung (SK-MES-1, NCI-H520), skin, and head and neck. Lung squamous cell carcinoma lines are often derived from surgically removed cancerous tissue or pleural effusion. Cell morphology is predominantly epithelial-like adherent growth, retaining typical squamous cell carcinoma characteristics such as keratin pearl formation and intercellular bridge structures. Long-term passage of some classic cell lines (such as SK-MES-1, established in 1970) may lead to alterations in genetic characteristics, weakening their relevance to the primary tumor. Tumor heterogeneity is not adequately reflected; single-cell sequencing reveals significant heterogeneity in squamous cell carcinoma, including cancer cells, immune cells, and stromal cell subsets, but traditional cell lines struggle to mimic this complex diversity. In vitro culture loses the immune cell interactions within the tumor microenvironment, failing to reflect in vivo immune escape mechanisms. Since many cell lines originate from advanced-stage patients, there is a lack of models of early carcinogenesis, making it difficult to study the mechanisms of squamous cell carcinoma development (such as the dynamic process of bronchial epithelial proliferation leading to carcinogenesis). Existing models are poor at predicting responses to immunotherapy; for example, the response rate to PD-1 inhibitors in clinical trials is only about 10%. While existing squamous cell carcinoma lines provide a basic platform for research, their genetic stability, lack of a suitable microenvironment, and insufficient reduction of heterogeneity remain core bottlenecks hindering clinical translation. Future efforts should focus on optimizing model reliability through technological innovation and multidisciplinary collaboration, combining single-cell transcriptomics and genomic analysis to construct molecular subtyping models that more closely approximate clinical outcomes.

[0005] Rhesus monkeys with spontaneous squamous cell carcinoma of the skin show a high degree of consistency with human patients in clinical manifestations, pathological features, and disease progression. They can effectively reproduce the typical pathological changes in human patients, demonstrating significant clinical translational potential. This helps researchers simulate human disease progression and provides a reliable platform for preclinical validation of novel treatment strategies, potentially accelerating the translation from basic research to clinical applications. MYO10 is an unstable protein that is upregulated in both human and mouse tumors. MYO10 may promote tumor progression by inducing genomic instability (such as chromosomal abnormalities and DNA damage repair disorders). Existing research, through analysis of 519 head and neck squamous cell carcinoma tissue samples and 44 adjacent normal tissue samples from the TCGA database, found that MYO10 is significantly highly expressed in head and neck squamous cell carcinoma, and its elevated expression is positively correlated with advanced tumor stage and lymph node metastasis status, suggesting that MYO10 may play an important role in the progression of human head and neck squamous cell carcinoma. Existing research also indicates that MYO10, as a non-traditional myosin, plays a crucial role in the occurrence and development of cutaneous squamous cell carcinoma by regulating cytoskeleton dynamics, cell adhesion and migration, and interactions with the tumor microenvironment. In particular, the core function of MYO10 is to drive the formation of filamentous pseudopodia and enhance cell motility. Therefore, cell lines with high MYO10 expression exhibit significantly stronger invasive, migratory, and metastatic potential than ordinary cutaneous squamous cell carcinoma cell lines, more closely resembling the clinically predisposed high-risk cutaneous squamous cell carcinoma (such as cSCC with lymph node metastasis). Therefore, establishing macaque cutaneous squamous cell carcinoma cell lines carrying abnormal MYO10 expression is of significant value for elucidating its molecular mechanisms, screening targeted drugs, and evaluating treatment strategies.

[0006] Existing studies mostly employ artificially induced or human cell line models, but these models have limitations in simulating natural pathogenesis, genomic instability, and the inflammatory microenvironment. Spontaneous squamous cell carcinoma of the skin in rhesus monkeys is highly similar to human models in clinical presentation and pathological features, making it an ideal model for translational medicine. However, there is currently a lack of rhesus monkey squamous cell carcinoma cell lines with clearly defined genomic characteristics and mechanistic research value. Therefore, establishing rhesus monkey squamous cell carcinoma cell lines with a natural mutational background and stable characteristics is of great significance for further elucidating the pathogenesis of cSCC and developing novel therapeutic strategies. Summary of the Invention

[0007] Therefore, this invention provides a rhesus monkey skin squamous cell carcinoma cell line MCSCC14397 and its applications. To achieve the above objectives, this invention adopts the following technical solution:

[0008] A rhesus monkey skin squamous cell carcinoma cell line, named rhesus monkey skin squamous cell carcinoma cell line MCSCC14397, was deposited at the China Center for Type Culture Collection (CCTCC) on June 25, 2025, with accession number CCTCC NO: C2025156.

[0009] This invention also provides a progeny cell line of the rhesus monkey skin squamous cell carcinoma cell line described above. This cell line not only possesses stable in vitro passage ability and tumorigenicity, but also exhibits unique MYO10-driven genomic instability and inflammatory signaling pathway activation characteristics, providing a novel experimental tool for skin squamous cell carcinoma research.

[0010] The present invention also protects the use of the described macaque skin squamous cell carcinoma cell line or progeny cell line as a cell model of skin squamous cell carcinoma cell line.

[0011] The present invention also protects the use of the macaque skin squamous cell carcinoma cell line or progeny cell line as a cell model for the occurrence, development and metastasis of skin squamous cell carcinoma.

[0012] This invention also protects the application of the macaque skin squamous cell carcinoma cell line or its progeny in establishing an animal model of skin squamous cell carcinoma.

[0013] This invention also protects the application of the described macaque skin squamous cell carcinoma cell line in screening targeted drugs that inhibit skin squamous cell carcinoma with MYO10 as the target, as well as in the study of the mechanism of MYO10 in the occurrence, development and metastasis of skin squamous cell carcinoma.

[0014] This invention provides the aforementioned rhesus monkey skin squamous cell carcinoma cell line or its progeny cell line, which is obtained by isolation, culture, and purification from spontaneous rhesus monkey skin squamous cell carcinoma tissue. This invention also provides a method for establishing the aforementioned rhesus monkey skin squamous cell carcinoma cell line or its progeny cell line: obtaining cancer tissue specimens from spontaneous tumors in rhesus monkeys, performing primary in vitro explant culture, and after 2-4 weeks of primary culture, performing digestion and passage, and repeatedly purifying and culturing to obtain tumor epithelial-like cells. This invention further provides the identification of the aforementioned rhesus monkey skin squamous cell carcinoma cell line or its progeny cell line: squamous cell carcinoma marker identification, karyotype analysis, mycoplasma detection, and cell growth curve determination, as well as verification of the tumorigenicity of squamous cell carcinoma cells.

[0015] The present invention provides the application of the macaque skin squamous cell carcinoma cell line or its progeny cell line as described above in the cell model of macaque skin squamous cell carcinoma cell line, and uses the macaque skin squamous cell carcinoma cell line to detect the sensitivity of antitumor drugs in vitro, and screens out the most sensitive paclitaxel.

[0016] The present invention provides the application of the macaque skin squamous cell carcinoma cell line or its progeny cell line as described above in animal models of macaque skin squamous cell carcinoma cell lines: the cell suspension of the macaque skin squamous cell carcinoma cell line is subcutaneously injected into Ba1b / cNude nude mice to obtain a transplanted skin squamous cell carcinoma nude mouse model, and the success rate of model establishment is 100%.

[0017] This invention provides the application of the macaque skin squamous cell carcinoma cell line or its progeny cell line as an animal model of macaque skin squamous cell carcinoma cell line in drug efficacy evaluation. Furthermore, the successfully constructed transplanted macaque skin squamous cell carcinoma nude mouse model was treated with paclitaxel to observe the intervention effect.

[0018] This invention also provides a study on the cellular characteristics of the rhesus monkey skin squamous cell carcinoma cell line or its progeny cell line as described above. This cell line has the following characteristics: abnormal chromosome number (47-98 chromosomes) and unstable karyotype; whole-genome sequencing shows high frequency C>T mutations, indicating UV-induced DNA damage characteristics; transcriptomic and proteomic analysis reveals significant upregulation of cell cycle and cytoskeleton regulatory pathways, and downregulation of related pathways such as lysosomal function; the MYO10 gene is significantly upregulated in tumor tissues, and its expression is positively correlated with genomic instability (such as micronucleus formation and γ-H2AX expression) and inflammatory factor (such as IL-6 and TNF-α) expression; the cells grow rapidly, have strong tumorigenicity, and the tumor formation rate in nude mice after transplantation reaches 100%.

[0019] The cell lines of this invention can be used as cell models to study the genomic instability and inflammatory transduction mechanisms of squamous cell carcinoma of the skin; to establish xenotransplantation animal models for efficacy evaluation and drug screening; to develop innovative therapies targeting MYO10 and other targets; and to study the association between natural ultraviolet radiation-induced DNA damage and cancer cell characteristics.

[0020] Compared with the prior art, the advantages of the present invention are as follows:

[0021] 1) The rhesus monkey skin squamous cell carcinoma cell line of the present invention is a novel single cell line, free from contamination by other cells; the cells are stable and can be passaged multiple times; they have strong proliferative activity, can be expanded in large quantities and passaged in vitro for a long time; and can serve as an effective cell model. The rhesus monkey skin squamous cell carcinoma cell line of the present invention enriches the skin squamous cell carcinoma cell line library.

[0022] 2) The rhesus monkey skin squamous cell carcinoma cell line of the present invention showed high expression of p63, cytokeratin 5 / 6 (CK5 / 6) and Ki67, all of which were protein positive.

[0023] 3) The rhesus squamous cell carcinoma cell line provided by this invention has the following characteristics: abnormal chromosome number (47-98 chromosomes) and unstable karyotype; whole-genome sequencing shows high-frequency C>T mutations, indicating UV-induced DNA damage; transcriptomic and proteomic analyses reveal significant upregulation of cell cycle and cytoskeleton regulatory pathways, and downregulation of related pathways such as lysosomal function; protein levels of MYO10 gene, γ-H2AX, and phosphorylated-NFκB-p65 (S468) are significantly upregulated in tumor tissues, and MYO10 expression is positively correlated with genomic instability (such as micronucleus formation and γ-H2AX expression) and inflammatory factor (such as IL-6 and TNF-α) expression; rapid cell growth, strong tumorigenicity, and a 100% tumorigenesis rate in nude mice after transplantation, enabling the establishment of in vitro transplantation animal models. This indicates that the rhesus squamous cell carcinoma cell line provided by this invention provides crucial support for developing precision treatment strategies targeting "MYO10-related malignant phenotypes".

[0024] 4) This invention is the first to obtain the macaque skin squamous cell carcinoma cell line MCSCC14397 with a natural mutant background and stable characteristics. The cell line combines genomic instability, inflammatory activation, and rapid growth characteristics, which are superior to artificially induced models. It provides a unique platform for studying the complex association between DNA damage, inflammation, and cancer cell characteristics, and has high translational potential in drug screening and mechanism research. From clinical discovery to model development and treatment validation, this invention has strong innovation and application value. Attached Figure Description

[0025] Figure 1 The image shows the results of identifying spontaneous tumors in macaques. Figure 1 A represents the appearance of a tumor-bearing monkey; Figure 1 B and Figure 1 C represents 4x and 20x HE staining images of normal rhesus monkey skin and subcutaneous tissue; Figure 1 D and Figure 1 E represents HE staining images of macaque mandibular tumors at 4x and 20x, respectively. Figure 1 F, Figure 1 G, Figure 1 H represents the immunohistochemical (IHC) detection results of p63, CK5 / 6, and Ki67 in the mandibular tumors of rhesus monkeys;

[0026] Figure 2 This image shows the results of tumor cell isolation, culture, and identification. Figure 2 A is a schematic diagram of the tumor tissue explant culture method; Figure 2 B is a schematic diagram of the tissue block culture method; Figure 2 C represents fibroblasts and epithelial-like cells; Figure 2 D represents purified tumor epithelial-like cells; Figure 2 E, Figure 2 F, Figure 2 G, Figure 2H represents the immunofluorescence detection results of tumor epithelioid cells CK, Vimentin, Ki67, and SCCA1.

[0027] Figure 3 The image shows the karyotype analysis and mycoplasma detection results of squamous cell carcinoma cells in the skin of rhesus monkeys. Figure 3 A and Figure 3 B represents 50 chromosomes and 57 chromosomes, respectively. Figure 3 C represents the chromosome distribution in the cell; Figure 3 D and Figure 3 E represents the mycoplasma detection results of F5 and F30 generation cells, respectively; Figure 3 F is the negative control; Figure 3 G positive control;

[0028] Figure 4 Growth curve determination for squamous cell carcinoma cells in the skin of rhesus monkeys;

[0029] Figure 5 The image shows the results of tumorigenicity verification of squamous cell carcinoma cells in macaque skin. Figure 5 A represents a nude mouse model; Figure 5 B and Figure 5 C represents 4x and 20x HE staining images of normal nude mouse dorsal skin tissue; Figure 5 D and Figure 5 E represents 4x and 20x HE staining of skin tumors on the back of nude mice; Figure 5 F, Figure 5 G, Figure 5 H represents the immunohistochemical (IHC) detection results of p63, CK5 / 6, and Ki67 in skin tumors on the back of nude mice;

[0030] Figure 6 This image shows the application results of rhesus monkey skin squamous cell carcinoma cells in a cell model. Figure 6 A and Figure 6 Figure B shows the in vitro drug sensitivity test results of squamous cell carcinoma cells of macaque skin and normal macaque epithelial cells;

[0031] Figure 7 The image shows the application results of rhesus monkey skin squamous cell carcinoma cells in an animal model. Figure 7 A shows representative nude mouse photos of tumor-forming mice (days 6, 33, 72, and 99 post-inoculation). Figure 7 B is a tumor growth curve of Balb / c Nude nude mice with subcutaneous xenografted rhesus squamous cell carcinoma cell line from macaques.

[0032] Figure 8 This is a diagram illustrating the genomic and transcriptomic characteristics of MCSCC14397 cells. Figure 8 A is a Circos diagram of genomic structural variations in MCSCC14397 cells, showing large-scale variations such as translocations and inversions; Figure 8 B is a radar diagram of point mutations in the MCSCC14397 cell genome, showing that C>T mutations are dominant;

[0033] Figure 9 The image shows a Bulk RNA-seq analysis of MCSCC14397 cells. Figure 9 A is a Bulk RNA-seq volcano diagram. Figure 9 B is a Bubble graph of Bulk RNA-seq, showing differentially expressed genes and pathway enrichment.

[0034] Figure 10 This is a diagram of proteomics analysis of MCSCC14397 cells, in which... Figure 10 A is a cell thermogram. Figure 10 B is the result graph of the Western blot;

[0035] Figure 11 This is a functional experimental diagram of MYO10, in which... Figure 11 a) Western blot analysis of MYO10 knockdown efficiency and γ-H2AX expression level in cSCC cells; Figure 11 b represents the quantitative analysis of γ-H2AX expression levels; Figure 11 c represents a representative image of micronuclei in the control group and MYO10 knockdown cSCC cells; Figure 11 d represents the quantitative analysis of micronuclei in MYO10 knockdown cSCC cells; Figure 11 e Figure 11 f、 Figure 11 g and 11h are figures showing the mRNA expression of ATM, ATR, IL6 and TNF-α in MYO10 knockdown cSCC cells detected by qPCR. Figure 11 i represents the Western blot results of proteins overexpressing MYO10 and γ-H2AX in normal epithelial cells; Figure 11 j represents the quantitative analysis of γ-H2AX overexpression; Figure 11 k represents a representative image of micronuclei in normal epithelial cells overexpressing MYO10; Figure 11 l represents quantitative analysis of micronuclei in normal epithelial cells overexpressing MYO10; Figure 11 m、 Figure 11 n、 Figure 11 o、 Figure 11 p represents the mRNA expression of ATM, ATR, IL6 and TNF-α in normal epithelial cells overexpressing MYO10, as detected by qPCR.

[0036] Figure 12 The figure shows the application results of the rhesus monkey skin squamous cell carcinoma cell line animal model in drug efficacy evaluation. Figure 12Figure A shows the results of paclitaxel intervention in nude mice treated with subcutaneous xenograft transplantation of the rhesus monkey skin squamous cell carcinoma cell line Balb / c Nude nude mice; Figure 12 B is a comparison graph of weight changes in diseased nude mice during paclitaxel intervention treatment;

[0037] Figure 13 The expression level of MYO10 in xenografted nude mice is shown in the figure. Figure 13 Figure A shows the results of the Western blot (WB) experiment. Figure 13 B is Figure 13 Quantification of A. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The specific materials used and their sources listed in the following embodiments are merely exemplary and are not intended to limit the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0039] Sample Sources: The rhesus squamous cell carcinoma cell line MCSCC14397 in this study was obtained from a male rhesus monkey (14397) with spontaneous squamous cell carcinoma at the Experimental Animal Center of Kunming Institute of Zoology, Chinese Academy of Sciences. Tumor cells were isolated and cultured from the mandibular tumor tissue. Normal epithelial cells were obtained from the mandibular skin tissue of a healthy rhesus monkey. Tissue samples for the multi-omics study were obtained from head and neck tumor tissues and adjacent normal tissues from four of the five rhesus monkeys with spontaneous squamous cell carcinoma (including 14397).

[0040] Example 1: Identification of spontaneous tumors in rhesus monkeys

[0041] HE staining of tissue samples: Tissue samples fixed with 10% neutral formalin were dehydrated using a gradient of ethanol (70%, 80%, 90%, 95%, 100%), treated with xylene, and then embedded in paraffin. Paraffin sections of 2 μm thickness were prepared using a microtome, attached to glass slides, and baked in an oven at 60–65°C for 2–4 hours. The prepared sections were dewaxed sequentially in xylene I, xylene II, and xylene III, and then hydrated using a gradient of ethanol (100% ethanol I, 100% ethanol II, 95% ethanol, and 75% ethanol). The sections were stained with hematoxylin for 5–10 minutes, rinsed with running water for 2–3 seconds to remove excess stain, differentiated in hydrochloric acid alcohol for 2–5 seconds, and then blued again in running water for 10–15 minutes. Immerse in eosin stain for 1-3 minutes, then dehydrate in 75% ethanol, 95% ethanol I, 95% ethanol II, 100% ethanol I, and 100% ethanol II. Add an appropriate amount of neutral mounting adhesive to the slide, cover with a coverslip, and observe and photograph under a microscope.

[0042] Immunohistochemistry: Tissue samples fixed in 10% neutral formalin and embedded in paraffin were prepared into 2μm thick paraffin sections. After baking at 60-65℃ for 2-4 hours, the sections were dewaxed with xylene and then hydrated in a gradient of ethanol (100%, 95%, 70%). Antigen retrieval was achieved by boiling the slides in citrate buffer (pH 6.0), followed by treatment with 3% H2O2 for 10 minutes to block endogenous peroxidase. The slides were washed three times with PBS. The slides were permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature, blocked with 10% goat serum for 1 hour, and then incubated overnight at 4℃ in a humidified chamber. The next day, diluted secondary antibody was added, and after incubation for 1 hour, the slides were developed with DAB for 3 minutes, washed with water, and counterstained with hematoxylin. The slides were then dehydrated in a gradient of ethanol (70%, 95%, 100%) and xylene, mounted with mounting medium, and observed and photographed under a microscope.

[0043] Tumor tissue and adjacent tissue were collected from monkeys with tumors. After formalin fixation, the tissues were dehydrated, embedded, and prepared into paraffin blocks for pathological diagnosis. HE staining of the tumor tissue revealed large cancer cell nuclei with clear nucleoli, significant atypia, frequent mitotic figures, and nested or sheet-like infiltrative growth. Keratin beads were visible within the cancer nests. Figure 1 China D and Figure 1 (Central E). Preliminary diagnosis: well-differentiated squamous cell carcinoma. Further immunohistochemical (IHC) identification revealed high expression of p63, cytokeratin 5 / 6 (CK5 / 6), and Ki67, all of which were protein positive. Figure 1 China F, Figure 1 China G, Figure 1 The presence of H indicates that the tumor is an epithelial-derived squamous cell carcinoma. P63 is a marker of squamous epithelial differentiation, while CK5 / 6 is a specific marker for squamous epithelium or basal cells. High Ki67 expression indicates the degree of tumor malignancy.

[0044] Example 2: Isolation, Culture, and Identification of Tumor Cells

[0045] Tumor cell isolation and culture: The obtained tissue samples were transferred to 6 cm cell culture dishes and washed repeatedly with DPBS containing 2% penicillin-dextrose antibody and 2.5 μg / mL amphotericin B until the tissue turned white. After removing blood vessels, fat, connective tissue, and hair from the tissue block, it was cut into pieces of approximately 1 mm. 3Small tissue fragments were evenly spread on the wall of a T25 cell culture flask, and a small amount of complete culture medium was added. After the tissue fragments adhered firmly, the culture medium was added to a final volume of 5 mL, and the flask was placed in a CO2 incubator. The next day, the culture flask was observed under an inverted microscope to check for bacterial contamination. Appropriate amounts of culture medium were added based on the color change of the medium until cells were observed swimming out from around the tissue fragments. Once the cells had filled the culture flask, the tissue fragments were rinsed off and aspirated.

[0046] Tumor cell purification: Remove the old culture medium from the cell culture flask. Wash the cells with 2-3 mL L PBS buffer, then digest the cells with 0.25% trypsin. Immediately add an appropriate amount of complete culture medium to stop the digestion. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge, discard the supernatant, and collect the cell pellet. Resuspend the cell pellet in fresh complete culture medium and transfer it to a culture flask labeled "①". Incubate for 1 hour. When some cells have adhered, aspirate the culture medium and suspended cells and transfer them to a culture flask labeled "②". Add an appropriate amount of culture medium to flask ①. Incubate both flasks ① and ② for 1 hour. When some cells have adhered to flask ②, gently aspirate the culture medium and suspended cells and transfer them to a culture flask labeled "③". Add an appropriate amount of culture medium to flask ②. Incubate both flasks ② and ③ for 1 hour. After culturing for a period of time, the above steps can be repeated for the cells in bottles ② and ③ until highly purified epithelial-like cells are obtained.

[0047] Tumor cell culture: Cell growth was observed under a microscope daily, and the medium was changed every 1-2 days. The culture medium was Pneu:DEME / F12 (3:2). Pneu medium additives included 1‰ hydrocortisone, 1% penicillin antibiotics, 1% L-glutamine, 2% growth factor additives, and 5% FBS. DMEM / F12 medium additives included 1% penicillin antibiotics and 5% FBS.

[0048] Tumor cell passage: When cells reach approximately 80-90% confluence, passage can be performed. Aspirate the old culture medium, wash cells 1-2 times with 2 mL L PBS buffer, add 0.25% trypsin to digest the cells (adjust digestion time according to digestion effect), add an appropriate amount of complete culture medium to stop digestion, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge, discard the supernatant, and collect the cell pellet. Resuspend the cell pellet in fresh complete culture medium, seed cells into new culture flasks at a passage ratio of 1:2, add an appropriate amount of fresh complete culture medium, and continue culturing in a CO2 cell culture incubator.

[0049] Cryopreservation of tumor cells: After the cells have grown to the bottom of the cell culture flask, digest and centrifuge to discard the supernatant, add 1 mL of freshly prepared cell cryopreservation solution (basal culture medium + 20% FBS + 8% DMSO), resuspend the cell pellet with a pipette and transfer it to a cell cryopreservation tube, then place the cryopreservation tube in a gradient cooling box and put it in a -80℃ freezer. The next day, take out the cell sample and transfer it to a liquid nitrogen tank.

[0050] Tumor cell identification: Pretreated cell slides were placed in 48-well cell culture plates. Epithelial-like cells to be identified in culture flasks were digested with 0.25% trypsin, and an appropriate amount of complete culture medium was added to stop the digestion. The cell suspension was then transferred to a 15mL centrifuge tube, centrifuged, and the supernatant was discarded. The cell pellet was collected. The cells were resuspended in fresh complete culture medium, and 500μL of the cell suspension was seeded into each well of a 48-well plate. The cells were cultured in an incubator until a monolayer was formed. The culture medium was aspirated from the wells, the cells were washed with DPBS, and an appropriate amount of 4% paraformaldehyde was added to fix the cell sample for 15 minutes, followed by washing with DPBS. 0.5% Triton X-100 was added, and the cells were permeated at room temperature for 20 minutes, followed by washing with DPBS. Excess DPBS on the cell slides was blotted dry with absorbent paper, and 500μL of 1% BAS blocking solution was added to each well. The cells were blocked at room temperature for 30 minutes. Aspirate the blocking solution, add 200 μL of diluted cytokeratin (CK), vimentin, Ki67, and SCCA1 primary antibodies to each well, and incubate overnight at 4°C. The next day, remove cell samples, wash with DPBS, blot dry with absorbent paper, add an appropriate amount of diluted fluorescent secondary antibody, and incubate at room temperature in the dark for 2 hours, followed by washing with DPBS. Add an appropriate amount of DAPI staining solution, incubate at room temperature in the dark for 2-3 minutes, and wash with DPBS. Remove the slide, blot dry with absorbent paper, and add an appropriate amount of mounting medium containing anti-fluorescence quencher to a glass slide. Flip the slide over and place it on the glass slide. After the mounting medium dries naturally, observe the acquired images under a fluorescence microscope.

[0051] By culturing tumor tissue explants, fibroblasts and epithelioid cells were freed from the tissue blocks and grew rapidly. After repeated adhesion purification, the epithelioid cells were then subjected to immunofluorescence experiments. High expression of cytokeratin (CK), Ki67, and squamous cell carcinoma antigen 1 (SCCA1) was detected, while Vimentin was not expressed. Figure 2 Chinese E, Figure 2 China F, Figure 2 China G, Figure 2 (H) proves that the isolated, purified, and cultured tumor epithelial-like cells are squamous cell carcinoma cells.

[0052] In this invention, primary cells derived from head and neck tumor tissue of spontaneous squamous cell carcinoma in macaques were passaged to obtain a stable passageable macaque skin squamous cell carcinoma cell line and its progeny cell lines, named macaque skin squamous cell carcinoma cell line MCSCC14397, which was deposited at the China Center for Type Culture Collection (CCTCC) on June 25, 2025, with accession number CCTCCNO: C2025156. This macaque skin squamous cell carcinoma cell line MCSCC14397 and its progeny cell lines are not the same cell line as the "primary macaque skin squamous tumor cells" derived from the left upper limb tumor tissue of spontaneous squamous cell carcinoma in macaques mentioned in Yang Chengmei's graduation thesis "Characteristic Analysis of Macaque Skin Squamous Cell Carcinoma and Establishment of Methods for Isolation and Culture of Cancer Cells," and the culture conditions were optimized during the isolation and culture of these cells. It is worth noting that since 2022, the inventors' team has discovered six cases of spontaneous squamous cell carcinoma in macaques. Five of these cases involved the head and neck, while one case involved the limbs (the cell source mentioned in Yang Chengmei's thesis). Two spontaneous squamous cell carcinoma cell lines were obtained from tumor tissue samples and cultured. Only the MCSCC14397 cell line provided in this invention could be primary cultured and stably passaged for more than 30 generations. The "primary squamous cell carcinoma cells of macaque skin" mentioned in Yang Chengmei's thesis could not be passaged further after the 8th generation.

[0053] Example 3: Karyotype analysis and mycoplasma detection of squamous cell carcinoma cells in rhesus monkey skin.

[0054] Karyotype analysis of rhesus monkey skin squamous cell carcinoma cells: Colchicine (final concentration 0.2 μg / mL) was added to the test cells in the logarithmic growth phase, shaken well, and incubated in an incubator for 1-2 hours; after incubation, the cells were digested with trypsin, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded to collect the cell pellet; the cells were resuspended in 8 mL of 0.075 M KCl preheated at 37 °C, and after mixing, the cells were placed in a 37 °C water bath for 40 minutes, and then 2 mL of fixative (methanol: glacial acetic acid = 3:1) was added, shaken well, and fixed at room temperature for 10 minutes, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded (500 μL was retained to mix the bottom cells), and then 8 mL of fixative was added, centrifuged at 1200 rpm for 10 minutes, and repeated three times. The cells were then dropped onto slides, aged in an 80 °C oven for 3 hours, and digested with 0.25% trypsin for 1 minute. Then add an appropriate amount of Giemsa stain to the slide, stain for 5-10 minutes at room temperature, gently rinse off the stain with running water, and observe the results under a microscope.

[0055] Mycoplasma detection in rhesus monkey skin squamous cell carcinoma cells: Monolayer cells were fixed with acetic acid-ethanol or Carnoy's fixative and washed with DPBS for 5 minutes. Hoechst 33258 staining solution (0.5 μg / mL) was added to the cell samples, and staining was performed at room temperature for 15 minutes. The staining solution was aspirated, and the cells were washed three times with DPBS for 5 minutes each time. The cells were mounted with water-soluble mounting medium, and the staining results were observed under a fluorescence microscope.

[0056] Based on chromosome counting results from 108 metaphase cells, the chromosome number of this cell was determined to be 47-98; the normal chromosome number of a rhesus monkey cell is 42, indicating that this cell exhibits a karyotype abnormality with a variable chromosome number, classifying it as a malignant tumor cell. Fluorescent staining for mycoplasma showed negative results for mycoplasma in both F5 and F30 generations. Figure 3 China D and Figure 3 (E).

[0057] Example 4: Determination of growth curves of squamous cell carcinoma cells in macaque skin

[0058] Growth curve determination of squamous cell carcinoma cells in macaque skin: Cells were digested and divided into 10 cells per well. 3 Cells were seeded into two 24-well plates. Once most cells had adhered and grown, cells from three wells in each plate were randomly collected, and cell concentration was measured using a cell counter. The average cell count was calculated, and this average was used as the cell count for day one. This measurement was repeated for 16 consecutive days. A cell growth curve was plotted with time on the x-axis and the daily cell count on the y-axis. The population doubling time (PDT) was calculated using the formula: PDT = ... Calculations are performed (where t is the entire period for measuring the growth curve, N0 is the number of cells in the seed plate on the first day, and Nt is the number of cells after culture time t).

[0059] Growth curves of three passages (F7, F9, and F27) of the rhesus monkey skin squamous cell carcinoma line MCSCC14397 were measured, and the results are as follows: Figure 4 As shown, the horizontal axis represents culture time per day, and the vertical axis represents the number of cells per day × 10. 5 The cell / mL growth rate indicates that the growth curve of the squamous cell carcinoma cells in the macaque skin roughly conforms to the standard growth curve of general cells, which is approximately "S"-shaped.

[0060] Example 5: Validation of tumorigenicity of rhesus monkey skin squamous cell carcinoma cells

[0061] Nude mouse modeling: MCSCC14397 cells in the logarithmic growth phase were collected and resuspended in Matrigel (diluted with serum-free medium). Cells were then incubated at 1×10⁻⁶ cells / mL.6 Subcutaneously inoculate 4-week-old SPF-grade BALB / c Nude female nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., experimental animal license number SCXK (Beijing) 2021-0006) at a density of cells / 100 μL, and observe the tumor formation situation.

[0062] Subcutaneously inject the rhesus macaque squamous carcinoma cells into the back of the nude mice. Tumors can grow on the back. Take tumor tissues and adjacent tissues from the successfully modeled nude mice. After soaking and fixing with formalin, dehydrate and embed them to prepare paraffin blocks for pathological diagnosis. The results of HE staining and immunohistochemical detection of pathological sections of tumor tissues show that: obvious atypia of cancer cells and clear nucleoli are seen in the subcutaneous muscular layer by HE staining, showing infiltrative growth in nest-like clusters, and keratin pearls can be seen in the cancer nests. The preliminary diagnosis is: well-differentiated squamous cell carcinoma. Further immunohistochemical (IHC) identification can detect high expression of p63, cytokeratin 5 / 6 (CK5 / 6), and Ki67, all of which are protein positive ( Figure 5 medium F, Figure 5 medium G, Figure 5 medium H), proving that this tumor is a squamous cell carcinoma of epithelial origin.

[0063] Example 6 Application of rhesus macaque skin squamous carcinoma cells in cell models

[0064] In vitro drug sensitivity test of cells: Inoculate MCSCC14397 cells ( cells / well) into 96-well culture plates, and add 100 μL of complete medium to each well. After 24 hours, replace the medium with 200 μL of medium containing a specified dose (0.001 - 100 μM) of the compound, and incubate at 37 °C for three days. According to the manufacturer's instructions, use the CellTiter-Glo Luminescent Cell Viability Assay Kit (G1111, Promega, Madison, WI, USA) to detect cell viability. Use GraphPad v9.0.2 to plot the dose-response curve and calculate the half-maximal inhibitory concentration (IC 3 ). 50 ).

[0065] Perform in vitro drug sensitivity tests with rhesus macaque skin squamous carcinoma cells (MCSCC14397 cells) and normal rhesus macaque epithelial cells (10350 cells). The results show that rhesus macaque skin squamous carcinoma cells are particularly sensitive to paclitaxel ( Figure 6 ).

[0066] Example 7 Application of rhesus macaque skin squamous carcinoma cells in animal models

[0067] Nude mouse model establishment and tumor observation: Collect MCSCC14397 cells in the logarithmic growth phase and resuspend the cells with Matrigel (diluted with serum-free medium). Subcutaneously inoculate 4-week-old SPF-grade Balb / c nude female mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., experimental animal license number SCXK (Beijing) 2021-0006) at a density of 1×10 6 cells / 100 μL. After inoculation, observe and record the tumor growth every 2 days, measure the long diameter (L) and short diameter (W) with a vernier caliper, and calculate the volume according to the formula: V = 1 / 2×L×W 2 . Use GraphPad Prism 10 to plot the tumor growth curve. Each data point represents the tumor volume (mean ± SD) of each individual (N = 11 animals), and the error bars represent ±SEM. The P value comes from a one-sample t-test, showing that the change in tumor volume is statistically significant (t(11) = 10.16, p < 0.01).

[0068] Subcutaneously inject the cell suspension of the rhesus macaque skin squamous cell carcinoma cell line into Balb / c Nude nude mice to obtain a transplanted rhesus macaque skin squamous cell carcinoma nude mouse model, and the success rate of model establishment is 100% ( Figure 7 ).

[0069] Example 8 Analysis of the genomic and transcriptomic characteristics of MCSCC14397 cells

[0070] Whole-genome sequencing analysis: Perform whole-genome sequencing on the tumor and adjacent tissues of rhesus macaque squamous cell carcinoma. After quality control with fastp and adapter removal, use BWA-MEM to align the clean reads to the reference genome. After deduplication with Samtools and Picard, use GATK for base quality recalibration. Somatic mutations are systematically detected by Strelka2 (SNV / indel), CNV kit (CNV), and Manta (SV).

[0071] Single-cell RNA-seq analysis: Use Cell Ranger to process the raw data to obtain an expression matrix. After preprocessing with Seurat: filter low-quality cells (gene number 200 - 6000, mitochondrial gene proportion < 5%), and remove doublets through Doublet Finder. Subsequently, perform data normalization, screening of highly variable genes, and PCA dimensionality reduction. After correcting the batch effect with Harmony, perform SNN clustering (resolution 0.5) and visualization.

[0072] Cell identification and in-depth analysis: Cell types (malignant epithelial cells / fibroblasts / immune cells / endothelial cells / stromal cells) were defined by differentially expressed genes (log2FC>0.25, adj.p<0.05) and typical marker genes. Copy number variations were inferred using CNV inferr as a reference for immune / stromal cells, and differential expression analysis of tumor-normal cell subsets was conducted.

[0073] The results are as follows Figure 8 As shown, Figure 8 Image A shows a Circos diagram of genomic structural variations in MCSCC14397 cells, revealing large-scale structural variations observed in the genome of rhesus squamous cell carcinoma of the skin, including translocations (exchange of chromosome segments) and inversions (reverse rejoining of chromosome segments), which correspond to karyotype analysis. Figure 8 Image B shows a radar map of point mutations in the MCSCC14397 cell genome, revealing the relative abundance of six major mutation types, with C>T mutations being the most prevalent. This suggests that genomic mutations may originate from UV-induced damage. Overall, these findings indicate that rhesus squamous cell carcinoma of the skin possesses extensive genomic instability, involving high somatic mutational burden and recurrent structural variations.

[0074] Example 9 Bulk RNA-seq analysis of MCSCC14397 cells

[0075] RNA Sequencing and Analysis: RNA was extracted from six rhesus monkey cell samples using TRIzol reagents (15596018CN, ThermoFisher Scientific, MA, USA), each sample containing three replicates from cancer-associated epithelial cells and squamous cell carcinomas, according to the manufacturer's protocol. Poly(A)-rich RNA-seq libraries were prepared by Biolinker Technology (Kunming) Co., Ltd. and sequenced on the Illumina Nova Seq X Plus platform. Trimmed reads processed with trim_galore were aligned to the rhesus monkey mulatta genome using STARv2.7.11a, followed by file conversion using SAmtoolsv1.18. Gene expression was quantified using feature Countsv2.0.6, and differentially expressed genes (DEGs) were identified using DESeq2 (adjusted p<0.05). Functional enrichment analysis of the KEGG pathway was performed using Shiny GO v0.82, and bubble plots and volcano plots were generated using ggplot2 in R.

[0076] like Figure 9As shown, the Bulk RNA-seq volcano plot of MCSCC14397 cells reveals differentially expressed genes between squamous cell carcinoma of the rhesus monkey skin and adjacent normal tissue. Figure 9 (A). Bubble plot of MCSCC14397 cells using Bulk RNA-seq. The bubble plot illustrates the enrichment pathway of upregulated genes. Figure 9 (B) Analysis Results: Differential expression analysis revealed widespread transcriptional alterations in tumors (volcano plot). Pathway enrichment analysis showed that genes related to cell cycle regulation, cytoskeleton organization, and canonical oncogenic signaling were significantly upregulated, while pathways related to lysosomal function, cell adhesion, apoptosis, and peroxisome activity were downregulated (bubble plot).

[0077] Example 10: Proteomics Analysis of MCSCC14397 Cells

[0078] Peptide samples were separated on a μPAC Neo high-throughput column using a Vanquish Neo UHPLC system, with gradient elution from 4% to 99% of solvent B (an aqueous solution containing 0.1% formic acid and 80% acetonitrile) for 10 minutes. DIA analysis was performed on an Orbitrap Astral mass spectrometer in positive ion mode at an MS1 ​​resolution of 240,000. Data were processed using DIA-NN software for protein identification and quantification, searching the uniprotkb-Macamulata database. For data analysis, proteins with non-missing values ​​>50% were included, and missing values ​​were estimated. Differentially expressed proteins were identified based on fold changes >1.5 and p-values ​​<0.05.

[0079] The results are as follows Figure 10 As shown in the heatmaps of MCSCC14397 cells, and the heatmaps of squamous cell carcinoma and adjacent normal tissue in macaque skin, it is evident that the upregulated proteins play a role in DNA damage repair, inflammation, and cytoskeleton regulation in macaque skin squamous cell carcinoma cells. Figure 10 (A). Western blot results showed that, compared with adjacent tissues, squamous cell carcinoma of macaque skin in macaques exhibited upregulated protein levels of MYO10, γ-H2AX, and phosphorylated-NFκB-p65 (S468). Figure 10 (B) Further multi-omics studies revealed significant genomic instability and activation of inflammatory signaling pathways in rhesus squamous cell carcinoma of the skin. Abnormally high expression of the MYO10 gene was identified as a key driver, which was significantly upregulated in tumor tissue and closely related to the expression of DNA damage marker γ-H2AX and inflammatory factors IL-6 / TNF-α.

[0080] Example 11: The role of the MYO10 gene in regulating genome stability and inflammatory factors

[0081] Real-time quantitative PCR (qPCR) was used to detect gene expression levels in specific cells. Total RNA was extracted from cells or tissues using TRIzol reagent (15596018, Thermo Fisher Scientific, USA) according to the manufacturer's instructions, and RNA purity and concentration were determined spectrophotometrically. cDNA was synthesized using a reverse transcription kit (K1622, Thermo Fisher Scientific, USA). qPCR analysis was performed using gene-specific primers and SYBR Green premix (2× universal qPCR premix TSE201, Beijing Qingke Biotechnology Co., Ltd.). Through 2... -ΔΔCt The method calculates the relative expression level of gene mRNA.

[0082] The results are as follows Figure 11 As shown, Figure 11 Figure a shows the Western blot analysis of MYO10 knockdown efficiency and γ-H2AX expression level in cSCC cells, indicating that MYO10 expression decreased after siRNA-mediated knockdown. Figure 11 The results showed that micronucleus formation was significantly reduced after MYO10 deficiency. Data showed that micronucleus formation was significantly reduced compared to the control group. Figure 11 (d). qPCR analysis of the mRNA expression of ATM, ATR, IL6, and TNF-α in MYO10 knockdown cSCC cells showed that the expression of ATM, ATR, IL6, and TNF-α was significantly downregulated in MYO10-deficient cells. Figure 11 e, Figure 11 f, Figure 11 g, Figure 11 (h). Under overexpression conditions, the level of γ-H2AX protein was significantly increased (h). Figure 11 i. Figure 11 (j). MYO10 overexpression increased the frequency of micronucleus formation ( Figure 11 (k); compared to the control group, micronucleus formation was significantly increased (k). Figure 11 In MYO10-overexpressing cells, the expression of ATM, ATR, IL6, and TNF-α was significantly upregulated. Figure 11 m, Figure 11 n, Figure 11 middle o, Figure 11 (p).

[0083] MYO10 expression is associated with genomic instability and inflammation in macaque cSCC. Integrated analysis of whole-exome sequencing, transcriptomics, and proteomics in macaque cSCC tissues identified MYO10 (myosin X) as a key candidate gene. In head and neck tumor tissue samples from four spontaneously generated squamous cell carcinoma macaques (including 14397), somatic mutations in MYO10 were detected in three cases, meaning that 75% of the tumor samples showed somatic mutations in MYO10, suggesting potential functional impact. Transcriptomic and proteomic data showed that MYO10 was significantly upregulated in tumor tissues and isolated tumor epithelial cells compared to adjacent normal tissues and normal controls.

[0084] The sustained upregulation of MYO10 in tumor tissues and its correlation with disease progression suggest that this gene may regulate genomic stability and inflammatory processes. To verify this hypothesis, the inventors conducted MYO10 knockdown and overexpression functional experiments in rhesus monkey cSCC cells and normal epithelial cells, respectively.

[0085] MYO10 knockdown can inhibit DNA damage and inflammatory signaling. Silencing MYO10 in cSCC cells using siRNA ( Figure 11 After the efficiency of knockdown was verified in the middle α region, the expression of the DNA double-strand break marker γ-H2AX decreased. Figure 11 a, Figure 11 (b) The number of micronuclei, a marker of genomic instability, was significantly reduced. Figure 11 c in the middle Figure 11 qPCR showed that the expression of DNA damage response genes ATM and ATR, as well as inflammatory factors IL6 and TNF-α, was significantly reduced. Figure 11 e in the middle Figure 11 f, Figure 11 g, Figure 11 (h), consistent with inflammatory transcriptional features observed in tumor tissue.

[0086] MYO10 overexpression induces somatic DNA damage and inflammatory response. MYO10 overexpression also occurs in normal epithelial cells. Figure 11 After verifying efficiency in the middle, the level of γ-H2AX increased significantly ( Figure 11 (j), micronucleus formation increases ( Figure 11 k, Figure 11 (l). qPCR detected upregulated mRNA expression of ATM, ATR, IL6, and TNF-α. Figure 11 m, Figure 11 n Figure 11 o, Figure 11(p). This indicates that MYO10 overexpression alone is sufficient to induce a pro-damage and pro-inflammatory state in non-transformed epithelial cells, reinforcing its potential role as a driver of tumor-associated cell phenotypes. Functional experiments also confirmed that MYO10 not only promotes genomic instability and inflammatory responses but also exhibits a similar expression pattern in human head and neck squamous cell carcinoma, suggesting its conserved role in cross-species squamous cell carcinoma development. These findings demonstrate that MYO10 drives genomic instability and inflammatory responses in MCSCC14397 cells, providing unique value for its application in squamous cell carcinoma mechanism research and drug development. The MCSCC14397 cell line not only possesses stable in vitro and in vivo characteristics but also inherent genomic instability and inflammatory activation features, making it suitable for multifaceted research on cutaneous squamous cell carcinoma. This invention provides crucial support for developing precision treatment strategies targeting "MYO10-related malignant phenotypes" using the rhesus monkey cutaneous squamous cell carcinoma cell line.

[0087] Example 12 Application of the rhesus monkey skin squamous cell carcinoma cell line MCSCC14397

[0088] MCSCC14397 cells were subcutaneously seeded into nude mice to construct a xenograft model. The tumor volume in the subcutaneously tumor-forming nude mice reached 100 mm². 3 At that time, paclitaxel intervention was started on nude mice. Sixteen nude mice were divided into a control group (n=8) injected with physiological saline and an experimental group (n=8, 10mg / kg) injected with paclitaxel (drug concentration 2.4mg / mL). The tail vein injection intervention was performed every two days, and the changes in tumors and the weight of nude mice were observed and recorded.

[0089] Further treatment with paclitaxel was used to establish a successfully constructed nude mouse model of transplanted rhesus squamous cell carcinoma of the skin, and the effects of the intervention were observed. The results showed that paclitaxel had a very good therapeutic effect on tumor-bearing nude mice, and the weight of the tumor-bearing nude mice did not change significantly during the paclitaxel intervention treatment. Figure 12 ).

[0090] via Western Blot ( Figure 13 China A, Figure 13 (B) Detecting intratumoral MYO10 protein levels to verify the in vivo inhibitory effect of the candidate drug paclitaxel (PTX) on the target. Figure 13As shown, Skin represents skin tissue (adjacent tissue) from three nude mice, Tumor represents tumor tissue from three tumor-bearing nude mice, and Tumor+PTX represents tumor tissue from another three tumor-bearing nude mice treated with paclitaxel. The results indicate that, compared to adjacent tissue, MYO10 expression was upregulated in the tumor tissue of tumor-bearing nude mice, and MYO10 expression returned to normal levels after paclitaxel treatment. In other words, MYO10 was also highly expressed in the xenograft model established using the macaque skin squamous cell carcinoma cell line MCSCC14397, and reached normal levels after drug treatment. Therefore, this invention demonstrates that the macaque skin squamous cell carcinoma cell line MCSCC14399 provided by this invention can be used for research on targeted drugs and mechanisms for inhibiting skin squamous cell carcinoma targeting MYO10.

[0091] In summary, this cell line exhibits the following characteristics: abnormal chromosome number (47-98 chromosomes) and unstable karyotype; whole-genome sequencing revealed high-frequency C>T mutations, indicating UV-induced DNA damage; transcriptomic and proteomic analyses revealed significant upregulation of cell cycle and cytoskeleton regulatory pathways, and downregulation of related pathways such as lysosomal function; the MYO10 gene was significantly upregulated in tumor tissues, and its expression was positively correlated with genomic instability (such as micronucleus formation and γ-H2AX expression) and inflammatory factor (such as IL-6 and TNF-α) expression; the cells grew rapidly, exhibited strong tumorigenicity, and achieved a 100% tumorigenesis rate when transplanted into nude mice.

Claims

1. A rhesus monkey skin squamous cell carcinoma cell line, characterized in that, The macaque skin squamous cell carcinoma cell line described above is named macaque skin squamous cell carcinoma cell line MCSCC14397, and was deposited at the China Center for Type Culture Collection on June 25, 2025, with accession number CCTCC NO: C2025156.

2. The progeny cell line of the macaque skin squamous cell carcinoma cell line as described in claim 1.

3. The application of the macaque skin squamous cell carcinoma cell line as described in claim 1 or the progeny cell line as described in claim 2 as a cell model for skin squamous cell carcinoma cell lines.

4. The application of the macaque skin squamous cell carcinoma cell line as described in claim 1 or the progeny cell line as described in claim 2 as a cell model for the occurrence, development, and metastasis of skin squamous cell carcinoma.

5. The application of the rhesus monkey skin squamous cell carcinoma cell line as described in claim 1 or the progeny cell line as described in claim 2 in establishing an animal model of skin squamous cell carcinoma.

6. The use of the rhesus monkey skin squamous cell carcinoma cell line as described in claim 1 or the progeny cell line as described in claim 2 in screening targeted drugs that inhibit skin squamous cell carcinoma with MYO10 as the target.

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