Human hypopharyngeal carcinoma tissue block PDX-HPC1 and application thereof

Abstract

The invention relates to the field of cell engineering, in particular to a human hypopharyngeal carcinoma tissue block PDX-HPC1 and application thereof. The invention provides a human hypopharyngeal carcinoma tissue block PDX-HPC1 or a cancer cell derived from the tissue block. The preservation number of the human hypopharyngeal carcinoma tissue block PDX-HPC1 is CCTCC (China Center For Type Culture Collection) No: C2025106. The tumor tissue of the patient is directly inoculated to the mouse, sample pretreatment and cell digestion processes are not involved, operation is easy, the tumor formation rate is high, the constructed hypopharyngeal carcinoma PDX model can make up the huge vacancy of domestic and overseas transformation research of the hypopharyngeal carcinoma PDX model, and the development of the hypopharyngeal carcinoma PDX model is promoted. And research on the anti-tumor effect of palbociclib on the basis of the constructed hypopharyngeal carcinoma PDX model is helpful for determining the anti-tumor mechanism of palbociclib in hypopharyngeal carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of cell engineering, and in particular to a human hypopharyngeal cancer tissue block PDX-HPC1 and uses thereof. Background Art

[0002] Hypopharyngeal squamous cell carcinoma (HSCC) accounts for approximately 3% of head and neck squamous cell carcinomas (HNSCCs), yet it has the worst prognosis of all HNSCCs. Due to its low incidence, large-scale prospective clinical studies are not feasible. To date, clinical guidelines for HNSCC have mostly been derived from subgroup analyses of clinical trials of laryngeal cancer, oropharyngeal cancer, or HNSCC. However, these results fall far short of fully reflecting the unique biological characteristics and treatment outcomes of this cancer. Due to the lack of effective preclinical research models, research into the pathogenesis, drug screening, and treatment of HNSCC has been slow, and new effective treatments are urgently needed.

[0003] The preclinical evaluation of tumor-targeted drugs requires a suitable animal model as a drug evaluation system. Due to the problem of tumor heterogeneity, cell lines and cell line-derived xenograft models (CDX) cannot represent the heterogeneity and diversity of tumors, resulting in significant results in preclinical trials but poor results in actual clinical trials. Patient-derived xenograft models (PDX) are different from traditional cell lines and CDX. PDX is a method of directly inoculating patient tumor tissue into immunodeficient mice. Because it is directly derived from the patient's lesion tissue, its genetic information is less changed than the patient's tumor from which it is derived. It is currently considered to be a drug screening model that is closer to clinical patients. However, there is no PDX model for patients with hypopharyngeal cancer in the existing technology.

[0004] Palbociclib is the first CDK4 / 6 inhibitor approved by the US FDA for first-line treatment of postmenopausal ER+ / HER2- metastatic breast cancer, but there are no reports of its use in hypopharyngeal cancer. Given the limitations of existing technologies, a method for studying the efficacy of palbociclib in hypopharyngeal cancer PDX models is urgently needed. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a human hypopharyngeal carcinoma tissue block PDX-HPC1 and uses thereof, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from the tissue. The human hypopharyngeal cancer tissue block PDX-HPC1 is deposited in the China Center for Type Culture Collection (CCTCC), with a deposit date of 2025-3-25, a deposit number of CCTCC No: C2025106, and a deposit address of Wuhan University, Wuhan, China.

[0007] Preferably, the human hypopharyngeal cancer tissue block PDX-HPC1 is an HPV-negative tumor tissue block.

[0008] The present invention also provides the use of the aforementioned human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from the tissue block PDX-HPC1 in preparing a hypopharyngeal cancer PDX animal model construction product.

[0009] The present invention also provides a method for constructing a hypopharyngeal cancer PDX animal model, the method comprising the following steps:

[0010] 1) Human hypopharyngeal carcinoma tissue blocks (PDX-HPC1) obtained from the primary tumor lesion of a patient with primary hypopharyngeal carcinoma were processed into tissue fragments after removal of connective tissue and necrotic tissue, and then placed in pre-chilled serum-free culture medium;

[0011] 2) Transplanting the tissue fragments obtained after treatment in step 1) into mice to obtain a P0 generation hypopharyngeal cancer PDX animal model.

[0012] The present invention also provides the use of the aforementioned human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from the human hypopharyngeal cancer tissue block PDX-HPC1 in preparing the following products:

[0013] 1) Development of products targeting tumor treatment targets;

[0014] 2) Tumor diagnosis / treatment drug screening products;

[0015] 3) Products for screening biomarkers for hypopharyngeal cancer or research on the mechanisms of hypopharyngeal cancer development;

[0016] 4) Use of drugs in products for studying the mechanism of action of tumor treatment; preferably, the drug is selected from one or more of palbociclib, entrectinib, larotrectinib, belizatinib, AZ23, Cpd5n, PHA-E429, milcilib, GNF-5837, cabozantinib, sitravatinib, or altiratinib; more preferably, the drug is palbociclib.

[0017] As described above, the human hypopharyngeal cancer tissue block PDX-HPC1 of the present invention and its use have the following beneficial effects:

[0018] 1. This method directly inoculates patient tumor tissue into mice, without involving sample pretreatment and cell digestion, resulting in simple operation and high tumor formation rate;

[0019] 2. The constructed hypopharyngeal cancer PDX model can fill the huge gap in translational research of hypopharyngeal cancer PDX models at home and abroad;

[0020] 3. The method of studying the anti-tumor effect of palbociclib based on the hypopharyngeal cancer PDX model facilitates confirmation of whether palbociclib is effective for patients with hypopharyngeal cancer, thereby avoiding ineffective drug use;

[0021] 4. The method of studying the anti-tumor effects of palbociclib based on hypopharyngeal cancer PDX models will help expand the indications of palbociclib, identify new treatments for hypopharyngeal cancer, and facilitate personalized analysis of patients;

[0022] 5. Studying the anti-tumor effect of palbociclib based on the hypopharyngeal cancer PDX model will help determine its anti-tumor mechanism in hypopharyngeal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure shows the P0 generation PDX model constructed in Example 1 of the present invention.

[0024] Figure 2 The figure shows the P3 generation PDX model constructed in Example 1 of the present invention.

[0025] Figure 3 Shown are the HE staining results of the hypopharyngeal cancer PDX model in Example 2 of the present invention and the corresponding clinical patient tumor pathological tissue sections.

[0026] Figure 4 Shown is the tumor growth curve of the hypopharyngeal cancer PDX model after administration in Example 3 of the present invention;

[0027] Figure 5 The results show that after the administration of Example 3 of the present invention, mice were dissected to remove the tumor tissue and the tumor tissue was photographed.

[0028] Figure 6 The results show that after the administration of Example 3 of the present invention, mice were dissected and tumor tissues were removed, and the tumor tissues were weighed.

[0029] Figure 7 Shown are the results of E2F1 immunohistochemical staining of two groups of tumor tissues in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] The present invention provides a human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from the tissue. The human hypopharyngeal cancer tissue block PDX-HPC1 is deposited in the China Center for Type Culture Collection (CCTCC), with a deposit date of 2025-3-25, a deposit number of CCTCC No: C2025106, and a deposit address of Wuhan University, Wuhan, China.

[0031] The human hypopharyngeal cancer tissue block PDX-HPC1 is derived from a human primary tumor.

[0032] The human hypopharyngeal cancer tissue block PDX-HPC1 is an HPV-negative tumor tissue block.

[0033] In some embodiments, the human hypopharyngeal carcinoma tissue block PDX-HPC1 has one or more of the following morphological characteristics:

[0034] 1) PDX-HPC1 tissue block has pathological mitotic figures;

[0035] 2) PDX-HPC1 tissue blocks showed angular beading;

[0036] 3) Irregular intercellular bridges were found in the tissue block PDX-HPC1.

[0037] The present invention also provides the use of the aforementioned human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from the tissue block PDX-HPC1 in preparing a hypopharyngeal cancer PDX animal model construction product.

[0038] In some embodiments, the hypopharyngeal cancer PDX animal model is a mouse model. Specifically, the mouse model is an immunodeficient mouse. Preferably, the immunodeficient mouse is selected from NPSG mice or BALB / c nude mice.

[0039] The present invention also provides a method for constructing a hypopharyngeal cancer PDX animal model, the method comprising the following steps:

[0040] 1) Human hypopharyngeal carcinoma tissue blocks (PDX-HPC1) obtained from the primary tumor lesion of a patient with primary hypopharyngeal carcinoma were processed into tissue fragments after removal of connective tissue and necrotic tissue, and then placed in pre-chilled serum-free culture medium;

[0041] 2) Transplanting the tissue fragments obtained after treatment in step 1) into mice to obtain a P0 generation hypopharyngeal cancer PDX animal model.

[0042] In some specific embodiments, the human hypopharyngeal cancer tissue block PDX-HPC1 is an HPV-negative tumor tissue block.

[0043] In some embodiments, the size of the tissue fragments is 1.6-2.2 mm*1.6-2.2 mm*1.6-2.2 mm. Preferably, the size of the tissue fragments is 1.8-2 mm*1.8-2 mm*1.8-2 mm.

[0044] In some embodiments, the pre-cooling temperature is 0-8° C. Specifically, the temperature is 0-2° C., 2-4° C., 4-6° C., or 6-8° C. Preferably, the temperature is 4-6° C.

[0045] In some specific embodiments, the serum-free culture medium is RPMI 1640 culture medium.

[0046] In some embodiments, the time for transplantation in step 2) is less than 6 hours. Specifically, the time is less than 4-6 hours, 2-4 hours or 2 hours. Preferably, the time is less than 2 hours.

[0047] In some specific embodiments, the mice in step 2) are NPSG mice.

[0048] In some embodiments, the transplantation site in step 2) is the axilla.

[0049] In some embodiments, the construction method further comprises the following steps:

[0050] 3) Raising the P0 generation hypopharyngeal cancer PDX animal model in step 2), processing the tumor tissue into tissue fragments after the breeding is completed, and transplanting them into the armpit of the mouse to obtain the P1 generation hypopharyngeal cancer PDX animal model.

[0051] In some embodiments, the volume of the tumor tissue in the hypopharyngeal cancer PDX animal model raised to P0 in step 3) is 800-1200 mm 3 Specifically, the volume is 800-900, 900-1000, 1000-1100 or 1100-1200 mm 3 Preferably, the volume is 900-1100mm 3 .

[0052] In some specific embodiments, the mice in step 3) are BALB / c nude mice.

[0053] The present invention also provides the use of the aforementioned human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from the human hypopharyngeal cancer tissue block PDX-HPC1 in preparing the following products:

[0054] 1) Development of products targeting tumor treatment targets;

[0055] 2) Tumor diagnosis / treatment drug screening products;

[0056] 3) Products for screening biomarkers for hypopharyngeal cancer or research on the mechanisms of hypopharyngeal cancer development;

[0057] 4) Use of drugs in products for studying the mechanism of action of tumor treatment; preferably, the drug is selected from one or more of palbociclib, entrectinib, larotrectinib, belizatinib, AZ23, Cpd5n, PHA-E429, milcilib, GNF-5837, cabozantinib, sitravatinib, or altiratinib; more preferably, the drug is palbociclib.

[0058] Preferably, the aforementioned tumor is hypopharyngeal cancer.

[0059] The aforementioned tumor treatment product is selected from a pharmaceutical drug. For example, the aforementioned use involves using human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from it to study the metabolism, efficacy, or safety of drugs used to treat hypopharyngeal cancer. Another example is using human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from it to screen for drugs to treat hypopharyngeal cancer.

[0060] In some embodiments, the drug is selected from one or more of palbociclib, entrectinib, larotrectinib, belizatinib, AZ23, Cpd5n, PHA-E429, milcilib, GNF-5837, cabozantinib, sitravatinib, or altiratinib.

[0061] The present invention also provides a method for establishing cancer cells derived from the aforementioned human hypopharyngeal cancer tissue block PDX-HPC1, the method comprising the following steps:

[0062] 1) Tissue obtained from the primary tumor lesion of a primary human hypopharyngeal carcinoma (PDX-HPC1) patient was processed into tissue fragments after removal of connective tissue and necrotic tissue;

[0063] 2) digesting the above tissue fragments;

[0064] 3) Cultivate and obtain hypopharyngeal cancer cells.

[0065] In some specific embodiments, in step 1), the tissue obtained from the primary tumor lesion is washed with 5% penicillin-streptomycin-containing PBS, and then the connective tissue and necrotic tissue are removed.

[0066] In some embodiments, the size of the tissue fragments in step 1) is 0.8-1.2 mm * 0.8-1.2mm *0.8-1.2mm.

[0067] In some embodiments, the digestion in step 2) is performed by incubating the tissue fragments in a serum-free DMEM high-glucose medium containing type II collagenase and DNase I. More specifically, based on the serum-free DMEM high-glucose medium, the concentration of type II collagenase is 1-3 mg / ml, and the concentration of DNase I is 0.05-2 mg / ml.

[0068] In some embodiments, the culturing method in step 3) comprises one or more of the following steps:

[0069] 1a) The suspension obtained after digestion in step 2) is filtered sequentially at 100 μm, 70 μm, and 40 μm, washed, and collected;

[0070] 1b) Add 1-3 ml of culture medium containing human hypopharyngeal carcinoma tissue piece PDX-HPC1 to the culture flask in step 1a);

[0071] 1c) Continue culturing until the cells adhere to the wall.

[0072] In some embodiments, the culture medium for human hypopharyngeal carcinoma tissue piece PDX-HPC1 comprises one or more of DMEM / F12 medium, serum, or antibiotics. Preferably, the serum is fetal bovine serum; more preferably, the fetal bovine serum accounts for 15-25% of the culture medium volume.

[0073] In the present invention, the term "pathological mitosis" refers to abnormal nuclear division, that is, the two poles of the cell nucleus are different in size and shape, resulting in asymmetric nuclear division.

[0074] As used herein, the term "keratin beads" refers to clusters of keratinized material formed during the keratinization process of epithelial cells. These structures are often formed by the aggregation of keratin within the cytoplasm of cancer cells and are typically round or oval in shape. Microscopic observation reveals that the center of a keratin bead is primarily composed of homogeneous keratin, surrounded by concentric circles of cancer cells.

[0075] As used herein, the term "intercellular bridge" refers to a structure formed by localized fusion and adhesion of cell membranes between adjacent cells, connecting the cells. Due to the heterogeneity and growth characteristics of cancer cells, intercellular bridges between cancer cells may change, becoming irregular, sparse, or even absent.

[0076] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0077] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0078] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0079] Example 1 Construction of hypopharyngeal cancer PDX model

[0080] Tumor tissue: Fresh hypopharyngeal cancer tumor tissue removed surgically. After tumor tissue resection, a detailed pathological evaluation will be performed by a head and neck specialist pathologist. The pathological characteristics of hypopharyngeal squamous cell carcinoma include significant cellular atypia (such as enlarged and darkly stained nuclei, increased mitotic figures), keratinization, and an invasive growth pattern. Immunohistochemistry results show positive expression of indicators such as CK5 / 6, p63, p40, and CKpan; while negative results for CD45 and CD20 help rule out the possibility of lymphoma, and negative results for CK7, CK20, and TTF-1 can rule out the diagnosis of adenocarcinoma; in addition, the p16 test result is negative, indicating that the tumor is not related to human papillomavirus (HPV) infection.

[0081] RPMI 1640: cell culture medium

[0082] Experimental mice: NPSG and BALB / c nude mice. All mice were housed in an SPF animal facility.

[0083] 1) Screening patients: Patients with a confirmed pathological diagnosis of hypopharyngeal cancer and access to in situ tissue specimens after surgery are considered for screening. For hypopharyngeal cancer tumors, patients with advanced tumor stages should be selected to significantly increase the tumorigenicity rate of PDX models.

[0084] 2) Sampling: Fresh tumor tissue was excised under a sterile environment, and connective tissue, blood clots, and necrotic tissue were removed. Immediately after sample collection, the sample was placed in serum-free RPMI 1640 culture medium precooled to 4°C.

[0085] As a further improvement of the present invention, the patient screening method described above includes cases with HPV-negative hypopharyngeal cancer. Transplantation of HPV-positive malignant tumors significantly reduces the tumorigenicity rate of the model. Because HPV-positive tumor tissue is rich in lymphocytes, xenografts derived from these tissues may promote co-transfer of stroma rich in lymphoid tissue, leading to the development of lymphoproliferative disorders and ultimately significantly reducing the tumorigenicity rate of the model.

[0086] As a further improvement of the present invention, in the above method, tumor tissue is transplanted into mice within two hours of sampling. This minimizes tumor tissue destruction, improves the tumorigenicity rate of PDX models, and ensures the accuracy of PDX models. Beyond two hours, factors such as tissue autolysis and antigen denaturation will significantly affect sample preservation and tissue survival.

[0087] 3) Preparation of transplant blocks: Trim the specimen into tumor blocks of 1.8-2 mm.

[0088] 4) Subcutaneous inoculation to construct P0 generation immunodeficient mice ( Figure 1 NPSG mice were selected and anesthetized with isoflurane inhalation. The axillary skin of the mice was disinfected with 70% alcohol. A small incision was made subcutaneously 2 cm below the axilla. The tumor was inserted between the skin and muscle toward the forelimb using ophthalmic forceps and pushed to the axilla. The wound was sutured and marked.

[0089] As a further improvement of the present invention, in the above method, the mouse strain used is NPSG. If BALB / c nude mice are directly used for transplantation, the tumor formation rate is extremely low. The transplantation success rate of head and neck tumor tissue samples in PDX ranges from 11% to 80%. This significant difference is mainly attributed to the different mouse strains used in the experiments. Specifically, in NPSG or NOD-SCID mice, the transplantation engraftment rate can be as high as about 80%, while in BALB / c nude mice, the success rate is only 15%-17%.

[0090] 5) Observe tumor formation: Observe tumor size and volume every other day, and calculate tumor volume according to the formula = (long diameter × short diameter) 2 ) / 2.

[0091] 6) Subculture and inoculation to construct P1, P2, and P3 generation PDX models: wait until the tumor grows to 1000 mm 3 When the mice were sacrificed, the tumors were carefully dissected out, cut into 2mm×2mm×2mm tissue blocks and transplanted into BALB / c nude mice to obtain the P1 generation PDX model. After three passages of inoculation, the P3 generation PDX model ( Figure 2 ) for subsequent experiments.

[0092] Example 2 Identification of consistency between hypopharyngeal cancer PDX model and patient tumor tissue

[0093] HE staining

[0094] Tumor tissue, typically no thicker than 0.5 cm, is placed in a pre-prepared fixative (10% formaldehyde, Bouin's fixative). Fixation is then performed using a dehydrating agent, alcohol, and xylene. After successful fixation, the tissue is trimmed, placed in an embedding cassette, and rinsed in running water for 30 minutes. Dehydration with alcohol is performed using a dehydrating agent that progresses from low to high concentrations, gradually removing water from the tissue. The tissue block is placed in xylene to make it transparent, and the alcohol in the tissue block is replaced with xylene; the transparent tissue block is placed in melted paraffin, embedded, and cooled to solidify into a block; the embedded wax block is sliced ​​and placed in a 45-degree constant temperature box for drying; the paraffin sections are placed in xylene to dissolve the wax, and an appropriate dehydration solution is used to dewax; the dewaxed sections are placed in hematoxylin staining solution for staining; the sections are placed in acidic alcohol for acid washing to remove excess hematoxylin dye; the sections are placed in a blue agent to enhance the hematoxylin staining effect, and the sections are rinsed again with distilled water to remove excess dye; the sections are stained with eosin solution; dehydrated through an ethanol concentration gradient, the sections are air-dried, and finally sealed.

[0095] The morphology of the tumor tissue was examined under a microscope to compare the pathological type, degree of differentiation, degree of keratinization and nuclear atypia of the tumor tissue. Figure 3 Figures (A) show patient tumor tissue, B shows P0 generation PDX model mouse tumor tissue, and C shows P3 generation PDX model mouse tumor tissue. HE staining reveals distinct hypopharyngeal carcinoma features, with pathological mitotic figures, cornified beads, and intercellular bridges visible in all three tumor tissues. The pathological findings between the patient and PDX tumor tissues are remarkably similar.

[0096] The results showed that the established PDX model retained the pathological characteristics of the patient's tumor tissue and was highly consistent with the patient's histology.

[0097] Example 3 Drug sensitivity test of palbociclib and cetuximab in hypopharyngeal cancer PDX model

[0098] Experimental drugs: Palbociclib (Selleck Chemicals), cetuximab (Merck, Germany).

[0099] Experimental grouping: 18 P3 generation PDX model mice were selected for drug testing experiments. When the tumor volume grew to 70 mm 3 At about 6 s, the mice were randomly divided into 3 groups, 6 in each group: negative control group, palbociclib group and cetuximab group.

[0100] Medication interventions:

[0101] 1) Negative control group: 100 μl of normal saline per day, gavage, qd*4w (d1-d28);

[0102] 2) Palbociclib monotherapy group: 100 mg / kg / d, oral gavage, every 4 weeks, d1-d28.

[0103] 3) Cetuximab group: 1 mg / d, intraperitoneal injection, qw*4w, specific days d1, d8, d15, d21.

[0104] Observational Indicators: Tumor volume and mouse body weight were measured twice weekly. Tumor volume was calculated using the formula = (longest diameter × shortest diameter squared) / 2, and growth curves were plotted. The mice's mental state, hair growth, and gait were observed twice weekly.

[0105] Experimental endpoint: After 28 days of drug intervention, mice were killed by carbon dioxide. The tumor growth curve of mice from the start of drug administration to the end of the experiment is as follows Figure 4 As shown, the horizontal axis represents the time after the start of the experiment, and the vertical axis represents the tumor volume (*p<0.05, **p<0.01, ***p<0.001).

[0106] After killing the mice, the tumor tissues were dissected and photographed. Figure 5 As shown, from top to bottom are the control group, palbociclib group and cetuximab group.

[0107] After killing the mice, the tumor tissues were dissected and weighed. Figure 6 shown.

[0108] The above results show that palbociclib significantly inhibited tumor growth, and its tumor inhibitory effect was comparable to that of the EGFR monoclonal antibody cetuximab, a targeted drug commonly used in the treatment of head and neck malignant tumors. No statistically significant difference was observed between the two.

[0109] Example 4 Immunohistochemical Analysis of the Mechanism of Palbociclib Inhibiting Hypopharyngeal Cancer Growth

[0110] Hypopharyngeal carcinoma paraffin sections were baked: the sections were placed in an oven at 58°C for 2 hours; dewaxing: two cylinders of xylene were prepared, each for 15 minutes; hydration: gradient alcohol: 100% X2 cylinders (each for 10 minutes), 95%, 90%, 80%, 70%, 60%, ddH2OX2 cylinders, each for 5 minutes; endogenous peroxidase removal: soak in 3% H2O2 at room temperature for 10-20 minutes; EDTA repair; PBS rinse; circle drawing; blocking, A solution, 3 Incubate at 7°C for 15 minutes, discard solution A; add primary antibody: prepare primary antibody according to the required antibody concentration using primary antibody diluent, and incubate at 4°C overnight; wash with PBS for 5 minutes*3 times; add secondary antibody, incubate at 37°C for 15-30 minutes; wash with PBS for 5 minutes*3 times; develop with DAB; stop with water and rinse for 20 minutes; counterstain with hematoxylin for 1 minute; rinse with water; differentiate with alcohol and hydrochloric acid; rinse with running water for >2 hours; dry in a 37°C incubator; and seal with neutral gum.

[0111] E2F1 plays a very important role in controlling the cell cycle and can be used to assess whether the cell cycle is operating normally or blocked. Figure 7 shown.

[0112] The results showed that E2F1 was expressed in both the control group and the drug intervention group. Compared with the control group, E2F1 expression was significantly decreased in the palbociclib group. These data suggest that palbociclib exerts its anti-cancer effects by arresting the cell cycle and promoting cell transformation.

[0113] Statistical methods: Statistical analyses and graphs were performed using GraphPad Prism software (version 9.0, San Diego, CA, United States). Multiple group analyses were performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. A p < 0.05 was considered statistically significant.

[0114] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A human hypopharyngeal carcinoma tissue block PDX-HPC1 or cancer cells derived from said tissue, characterized in that: The deposit number of the human hypopharyngeal cancer tissue block PDX-HPC1 is CCTCC No: C2025106.

2. The human hypopharyngeal carcinoma tissue block PDX-HPC1 according to claim 1, characterized in that The human hypopharyngeal cancer tissue block PDX-HPC1 is an HPV-negative tumor tissue block.

3. The human hypopharyngeal cancer tissue block PDX-HPC1 according to claim 1, characterized in that The human hypopharyngeal carcinoma tissue block PDX-HPC1 has one or more of the following morphological characteristics: 1) PDX-HPC1 tissue block has pathological mitotic figures; 2) PDX-HPC1 tissue blocks showed angular beading; 3) Irregular intercellular bridges were found in the tissue block PDX-HPC1.

4. Use of the human hypopharyngeal cancer tissue block PDX-HPC1 or cancer cells derived from the tissue block PDX-HPC1 according to any one of claims 1 to 3 in preparing a hypopharyngeal cancer PDX animal model product.

5. The use according to claim 4, characterized in that The hypopharyngeal cancer PDX animal model is a mouse model. Preferably, the mouse model is an immunodeficient mouse; more preferably, the immunodeficient mouse is selected from NPSG mice or BALB / c nude mice.

6. A method for constructing a hypopharyngeal cancer PDX animal model, characterized in that: The construction method comprises the following steps: 1) processing the human hypopharyngeal carcinoma tissue block PDX-HPC1 according to any one of claims 1 to 3 into tissue fragments, and placing them in pre-cooled serum-free culture medium; 2) Transplanting the tissue fragments obtained after treatment in step 1) into mice to obtain a P0 generation hypopharyngeal cancer PDX animal model.

7. The construction method according to claim 6, characterized in that: The construction method may further include one or more of the following features: a) the human hypopharyngeal cancer tissue block PDX-HPC1 is obtained from the primary tumor lesion of a primary hypopharyngeal cancer patient; b) removing connective tissue and necrotic tissue from the human hypopharyngeal carcinoma tissue block PDX-HPC1 and then processing it into tissue fragments; Preferably, the size of the tissue fragments is 1.6-2.2 mm*1.6-2.2 mm*1.6-2.2 mm; c) the pre-cooling temperature is 0-8°C; d) the serum-free culture medium is RPMI 1640 culture medium; e) the transplantation time in step 2) is less than 6 hours; f) the mice in step 2) are NPSG mice; g) The transplant site in step 2) is the axilla.

8. The construction method according to claim 6, characterized in that: The construction method further comprises the following steps: 3) Raising the P0 generation hypopharyngeal cancer PDX animal model in step 2), processing the tumor tissue into tissue fragments after the raising is completed, and transplanting them into the armpit of the mouse to obtain the P1 generation hypopharyngeal cancer PDX animal model.

9. The construction method according to claim 8, characterized in that: The construction method may further include one or more of the following features: h) The volume of the tumor tissue in the hypopharyngeal cancer PDX animal model raised to P0 is 800-1200 mm 3 When the feeding ends; i) The mice in step 3) are BALB / c nude mice.

10. Use of the human hypopharyngeal carcinoma tissue block PDX-HPC1 or cancer cells derived from the human hypopharyngeal carcinoma tissue block PDX-HPC1 according to any one of claims 1 to 3 in the preparation of the following products: 1) Development of products targeting tumor treatment targets; 2) Tumor diagnosis / treatment drug screening products; 3) Products for screening biomarkers for hypopharyngeal cancer or research on the mechanisms of hypopharyngeal cancer development; 4) Use of drugs in products for studying the mechanism of action of tumor treatment; preferably, the drug is selected from one or more of palbociclib, entrectinib, larotrectinib, belizatinib, AZ23, Cpd5n, PHA-E429, milcilib, GNF-5837, cabozantinib, sitravatinib, or altiratinib; more preferably, the drug is palbociclib.

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