Head and neck cancer chemotherapy recurrence animal model and construction method and application thereof
A head and neck cancer recurrence animal model was constructed by subcutaneously implanting fluorescently labeled head and neck cancer cells in the groin of mice and subjecting them to multiple cycles of chemotherapy. This model solves the problems of uncontrollable models and large individual differences in existing technologies, and achieves efficient simulation and stable monitoring of the recurrence process after chemotherapy. It is suitable for research on recurrence mechanisms and drug screening.
Patent Information
- Application Number
- CN202511711040.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-20
- Publication Date
- 2026-03-06
Smart Images
Figure CN121606408A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal model construction technology, and more specifically to an animal model of head and neck cancer recurrence after chemotherapy, its construction method, and its application. Background Technology
[0002] Head and neck cancer is one of the most common types of malignant tumors worldwide, ranking sixth in incidence among all cancers. Head and neck cancer includes malignant tumors originating from the oral cavity, pharynx, larynx, nasal cavity, and sinuses, with over 90% of cases being squamous cell carcinoma. Currently, the standard clinical treatment for head and neck cancer includes a combination of surgical resection, radiotherapy, and chemotherapy.
[0003] Despite advancements in treatment methods, head and neck cancer patients still face a high risk of recurrence after radical therapies (such as chemotherapy). Tumor recurrence is a leading cause of treatment failure and patient death in head and neck cancer. Therefore, in-depth research into the mechanisms of recurrence after chemotherapy in head and neck cancer and the development of effective strategies to prevent and control recurrence have become major challenges in clinical practice.
[0004] However, research in this field severely lacks animal models that can effectively simulate the recurrence process after clinical chemotherapy. Existing animal models primarily focus on the construction of the primary tumor or induce tumor shrinkage and regrowth through a single method (such as high-dose chemotherapy), failing to systematically simulate the dynamic evolution of the "chemotherapy-residual-recurrence" process in clinical treatment. These models suffer from drawbacks such as long treatment cycles, significant individual variability, uncontrollable recurrence processes, and poor correlation with clinicopathological features, making it difficult to meet the reliability and stability requirements of in-depth research on recurrence mechanisms and drug screening.
[0005] Therefore, developing an animal model that can highly simulate the entire process of recurrence after chemotherapy in clinical head and neck cancer, and that is easy to operate and highly stable, is of urgent need and great significance for promoting basic and translational research on head and neck cancer recurrence. Summary of the Invention
[0006] In view of this, the present invention provides an animal model of head and neck cancer recurrence after chemotherapy, its construction method and application.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing an animal model of head and neck cancer recurrence after chemotherapy, comprising the following steps: Under S1 in vitro conditions, fluorescently labeled head and neck cancer tumor cells were cultured. S2. The fluorescent cells of the head and neck cancer tumor are subcutaneously implanted in the groin area of the model animal; Five days after S3 implantation, subcutaneous tumor formation in the model animals was observed using imaging, and chemotherapy was administered. S4 underwent periodic chemotherapy, and the regression of subcutaneous xenografts in the model animals was observed using an in vivo imaging system until the tumor fluorescence signal basically disappeared. S5 was given a 5-6 week feeding period after chemotherapy was stopped, and the tumor recurrence was observed in the model animals to obtain the head and neck cancer chemotherapy recurrence animal model.
[0008] Furthermore, the head and neck cancer cells are selected from one or more human or mouse head and neck cancer cells.
[0009] Furthermore, the head and neck cancer tumor cells are FaDu cells.
[0010] Furthermore, the fluorescent label is a Luciferase fluorescent label and a GFP fluorescent label.
[0011] Furthermore, the model animal is an immunodeficient mouse.
[0012] Furthermore, the chemotherapy uses cisplatin and is administered via tail vein injection; The dosage of cisplatin is 5 mg / kg, administered once every 3 days. Typically, 3-5 consecutive administrations are sufficient to achieve the endpoint of tumor regression.
[0013] Furthermore, after stopping chemotherapy, the animals were fed for another 5-6 weeks, and the tumor recurrence was observed weekly using an in vivo imaging system.
[0014] A method for constructing an animal model of head and neck cancer recurrence after chemotherapy.
[0015] The application of a method for constructing an animal model of head and neck cancer recurrence after chemotherapy in the study of the mechanism of head and neck cancer recurrence after chemotherapy.
[0016] The application of a method for constructing an animal model of head and neck cancer recurrence after chemotherapy in screening drugs for head and neck cancer treatment.
[0017] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides an animal model of head and neck cancer recurrence after chemotherapy, its construction method, and its applications. By subcutaneously inoculating fluorescently labeled head and neck cancer cells and combining this with a multi-cycle chemotherapy regimen, this invention can highly simulate the recurrence process after clinical head and neck cancer chemotherapy, facilitating the study of recurrence mechanisms and the development of new treatment methods. This method is simple to operate, has a short experimental cycle, and low costs. Furthermore, it allows for non-invasive dynamic monitoring using in vivo imaging technology, resulting in low variability and a high success rate, providing an ideal platform for head and neck cancer recurrence research and drug screening. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0019] Figure 1 A schematic diagram illustrating the process of constructing an animal model of head and neck cancer recurrence after chemotherapy, as provided by this invention; Figure 2 This is a schematic diagram of tumor growth curves in the head and neck cancer chemotherapy recurrence model provided by the present invention. Figure 3 This is an in vivo imaging monitoring image of a small animal model of head and neck cancer recurrence after chemotherapy, provided by the present invention. Figure 4 This is a schematic diagram of HE staining results of tumor tissue in the head and neck cancer chemotherapy recurrence model provided by the present invention. Figure 5 This is a schematic diagram of immunofluorescence staining of tumor tissue in a head and neck cancer chemotherapy recurrence model provided by the present invention. Detailed Implementation
[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0021] This invention provides a method for constructing an animal model of head and neck cancer recurrence after chemotherapy, comprising the following steps: S1. Head and neck cancer cells with fluorescent reporter genes cultured in vitro; S2. The fluorescently labeled cell suspension was inoculated into the subcutaneous groin area of nude mice to establish a head and neck cancer xenograft model; S3. After the tumor has grown to a suitable size, the tumor-bearing mice are given multiple cycles of chemotherapy. S4. Monitor tumor regression using in vivo imaging systems and visual observation until the tumor fluorescence signal disappears and no obvious tumor tissue can be observed with the naked eye; S5. Continue feeding the animals after chemotherapy is stopped, and regularly observe and record tumor recurrence to obtain an animal model of head and neck cancer recurrence after chemotherapy.
[0022] In some embodiments, the head and neck cancer cells are selected from human or mouse head and neck squamous cell carcinoma cell lines, including but not limited to FaDu, CAL-27, SCC-25, or SCCVII cell lines.
[0023] In some preferred embodiments, the head and neck cancer cells are human FaDU cell lines.
[0024] In some embodiments, the fluorescent reporter gene is a luciferase gene or a green fluorescent protein gene, or a fusion gene of both.
[0025] In one specific embodiment, the head and neck cancer cells carrying the fluorescent reporter gene are commercially available FaDu-Luc-GFP cell lines.
[0026] In some embodiments, the model animal is an immunodeficient mouse, preferably a 4-6 week old male BALB / c nude mouse.
[0027] In some implementations, the cell seeding density is 1×10⁻⁶. 6 Cells / 100μL PBS, inoculated subcutaneously in the groin.
[0028] In some implementations, five days after implantation, the subcutaneous tumor formation is assessed using a live imaging system and visual observation, and chemotherapy intervention begins once the tumor has formed.
[0029] In some specific implementations, the chemotherapy regimen is as follows: cisplatin is diluted with normal saline to the required concentration and administered via tail vein injection at a dose of 5 mg / kg every 3 days. Changes in tumor fluorescence signal intensity are monitored using a live imaging system, combined with visual observation, and tumor regression is used as the criterion for discontinuing chemotherapy. Typically, this criterion is achieved after 3-5 consecutive administrations.
[0030] In some implementations, after chemotherapy, changes in tumor fluorescence signal intensity are monitored using an in vivo imaging system, and tumor volume is measured using calipers to assess tumor regression.
[0031] In some implementations, after chemotherapy is stopped, the animals are fed routinely for 5-6 weeks, and the recurrence of tumors at the original inoculation site is monitored at least once a week by in vivo imaging system and visual observation. When a continuously enhanced fluorescence signal is detected and tumor tissue regeneration is visible to the naked eye, it indicates that the animal model of chemotherapy recurrence of head and neck cancer has been successfully established.
[0032] The animal model of head and neck cancer recurrence after chemotherapy constructed by the above method can be used to study the recurrence mechanism of head and neck cancer after chemotherapy, screen anti-recurrence drugs, and evaluate individualized treatment plans.
[0033] Figure 1This is a schematic diagram illustrating the construction process of the animal model for head and neck cancer recurrence after chemotherapy provided by this invention. As shown in the figure, this invention establishes the model through the following steps: First, head and neck cancer cells are cultured and fluorescently labeled in vitro; then, the labeled cells are seeded into the subcutaneous groin area of experimental animals to establish a xenograft tumor model; after the tumor grows to an appropriate size, it is treated with a multi-cycle chemotherapy regimen; finally, the recurrence of the tumor is observed through regular monitoring.
[0034] The animal model established in this invention can highly simulate the recurrence process of head and neck cancer patients after chemotherapy. By establishing the primary tumor through subcutaneous inoculation and then inducing tumor regression through multiple cycles of chemotherapy, the natural recurrence process is realistically reproduced, showcasing the dynamic evolution of clinical tumor treatment. This model is not only simple to operate and has a controllable experimental cycle, but also allows for non-invasive and quantitative monitoring through in vivo imaging technology, providing a reliable experimental platform for studying the recurrence mechanism of head and neck cancer after chemotherapy.
[0035] This invention also provides the application of the above-described method in the study of the recurrence mechanism of head and neck cancer after chemotherapy, enabling the exploration of recurrence-related molecular mechanisms and signaling pathways. Furthermore, this model can also be used for the screening and evaluation of drugs against head and neck cancer recurrence, providing an important preclinical research tool for developing new treatment strategies.
[0036] The present invention will be further illustrated below through specific embodiments. It should be noted that the cell culture, molecular biology, and animal experiment techniques involved in this invention all employ conventional operating methods and standard experimental protocols in the art, and specific details can be found in relevant professional literature. Unless otherwise specified, all experimental operations are performed in accordance with conventional standards in the art.
[0037] The Examples section details the specific implementation methods for culturing, fluorescently labeling, animal inoculation, chemotherapy regimens, and monitoring of FaDu human head and neck cancer cells. These examples are for illustrative purposes only and do not constitute a limitation on the scope of protection of this invention.
[0038] Example 1: Culture and preparation of fluorescently labeled head and neck cancer cells In this embodiment, the head and neck cancer cells selected were FaDu cells, labeled with Luciferase and GFP fluorescent markers, and designated as FaDu-Luc-GFP cells. This FaDu-Luc-GFP cell line was purchased from [Wuhan Sewell Biotechnology Co., Ltd.], product code [STCC00109P]. The FaDu-Luc-GFP cell line was cultured and passaged in high-glucose DMEM medium (containing 10% FBS + 1% penicillin / streptomycin + 5 μg / mL puromycin) at 37°C in a 5% CO2 incubator. When the cell confluence reached 80%, the medium was discarded, the cells were washed with PBS, and then digested with 0.25% trypsin digestion solution. Digestion was stopped with twice the volume of complete medium. After centrifugation, the supernatant was discarded, the cells were resuspended in PBS, washed, counted, and diluted to 1×10⁻⁶. 7 The concentration of cells / mL was used for subsequent experiments.
[0039] Example 2: Establishment of an animal model of primary head and neck cancer and chemotherapy Male BALB / c nude mice aged 4-6 weeks were selected as the model animals. Mice were anesthetized with 1-2% isoflurane inhalation. FaDu-Luc-GFP cell suspension (resuspended in PBS, density 1×10⁻⁶) was aspirated using a microsyringe. 6 (cells / 100μL). After disinfecting the skin with alcohol, the cell suspension was slowly injected subcutaneously into the groin area of mice. Routine feeding was followed post-operatively.
[0040] Tumor growth was monitored every 3 days after inoculation. On day 5, tumor growth was monitored and recorded using an in vivo imaging system before treatment. Mice were anesthetized 2 minutes after intraperitoneal injection of fluorescein potassium (150 mg / kg) for imaging, with exposure time typically 1-5 minutes. Chemotherapy intervention was initiated when the tumor fluorescence signal became stable and strong (approximately day 5 post-inoculation).
[0041] Tumor-bearing mice were randomly divided into groups. Mice in the treatment group received a tail vein injection of cisplatin (5 mg / kg body weight) every three days, and tumor fluorescence signals were monitored using in vivo imaging. In this embodiment, all mice reached the regression standard of near-complete disappearance of tumor fluorescence signals after five consecutive administrations. The control group received an equal volume of physiological saline.
[0042] Example 3: Construction and Validation of a Relapse Animal Model 1. Construction of the relapse model Tumor-bearing mice were administered cyclical cisplatin chemotherapy (5 mg / kg, administered via tail vein every 3 days), and tumor fluorescence signal intensity was monitored weekly using an in vivo imaging system, combined with gross observation of tumor volume changes. Chemotherapy was discontinued when the tumor fluorescence signal significantly decreased to near background levels and the tumor volume noticeably shrank or disappeared upon gross observation. In this embodiment, all mice reached this discontinuation criterion after 5 consecutive administrations. Chemotherapy was then discontinued, and the mice were fed according to standard feeding practices.
[0043] 2. Monitoring of the relapse process After tumor regression was achieved and chemotherapy was discontinued, changes in fluorescence signals at the original inoculation site were monitored weekly using a live in vivo imaging system, and the presence of regrowth was observed visually. During this process, the tumor growth curve clearly demonstrated the typical dynamic process of "chemotherapy-regression-recurrence" (see [link to relevant documentation]). Figure 2 Typically, a weak fluorescence signal can be detected at the original site 5-6 weeks after chemotherapy is stopped. The fluorescence signal significantly increases after 7-8 weeks, and regrowth of tumor tissue is visible to the naked eye, indicating successful establishment of the tumor recurrence model (see [link to relevant documentation]). Figure 3 ).
[0044] 3. Model Validation When the fluorescence signal significantly increased and the recurrent tumor volume reached 200-300 mm³, some mice were sacrificed, and the recurrent tumor tissue was completely dissected. The tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E staining and immunofluorescence analysis. H&E staining revealed that the recurrent tumor cells had large, deeply stained nuclei with varied morphologies and disordered arrangement, consistent with the characteristics of malignant tumors (see [link to relevant documentation]). Figure 4 ).like Figure 5 As shown, Ki-67 staining revealed a large number of positive cell nuclei, confirming the vigorous proliferative capacity of recurrent tumor cells. Simultaneously, the broad-spectrum epithelial cell marker CKpan (red) showed strong positivity, clearly confirming that these highly proliferating cells originated from the initially inoculated epithelial-derived FaDu cells, thus representing true tumor recurrence (see [link to original text]). Figure 5 These results collectively validate the successful establishment of an animal model for recurrent head and neck cancer after chemotherapy.
[0045] The above results collectively demonstrate that the present invention has successfully constructed an animal model that can simulate the recurrence process of human head and neck cancer after chemotherapy. This model has the characteristics of high tumor formation rate, stable recurrence process, and easy monitoring, and is suitable for the study of the recurrence mechanism of head and neck cancer and the screening of anti-recurrence drugs.
[0046] In summary, this invention successfully established an animal model that effectively simulates the recurrence process of head and neck cancer after chemotherapy. This model has a reasonable construction cycle, a high success rate, and is easy to monitor, providing a powerful tool for in-depth research on recurrence mechanisms and the development of new treatment strategies.
[0047] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for constructing a head and neck cancer chemotherapy relapse animal model, characterized in that, The method comprises the following steps: S1, culturing head and neck cancer tumor cells with fluorescent markers under in vitro conditions; S2, subcutaneously implanting the head and neck cancer tumor fluorescent cells in the groin of a model animal; S3, after 5 days of implantation, observing the subcutaneous tumor formation of the model animal by imaging and performing chemotherapy; S4, performing periodic chemotherapy and observing the regression of the subcutaneously implanted tumor of the model animal by using an in vivo imaging system until the tumor fluorescent signal is basically disappeared; S5, stopping the chemotherapy and continuing to feed the model animal for 5-6 weeks to observe the tumor recurrence of the model animal, and obtaining the head and neck cancer chemotherapy recurrence animal model.
2. The method of constructing a head and neck cancer chemotherapy-relapsed animal model according to claim 1, characterized in that, The head and neck cancer tumor cells are selected from one or more of human or murine head and neck cancer tumor cells.
3. The method of claim 2, wherein the head and neck cancer chemotherapy relapse animal model is constructed by, The head and neck cancer tumor cells are FaDu cells.
4. The method of claim 1, wherein the head and neck cancer chemotherapy relapse animal model is constructed by, The head and neck cancer tumor cells are labeled with Luciferase and GFP fluorescent markers.
5. The method of constructing a head and neck cancer chemotherapy-relapsed animal model according to claim 1, wherein, The model animal is an immunodeficient mouse.
6. The method of claim 1, wherein the head and neck cancer is selected from the group consisting of squamous cell carcinoma, adenocarcinoma, and lymphoma. The chemotherapy adopts cisplatin, and the administration mode is tail vein injection. The administration dose of cisplatin is 5 mg / kg, and the administration is performed once every 3 days. The tumor regression is monitored by using an in vivo imaging system, and the basic disappearance of the tumor fluorescent signal is the endpoint of chemotherapy.
7. The method of constructing a head and neck cancer chemotherapy-relapsed animal model according to claim 1, wherein, After stopping the chemotherapy, the model animal is continued to be fed for 5-6 weeks, and the tumor recurrence is observed by using an in vivo imaging system every week.
8. A head and neck cancer chemotherapy recurrence animal model obtained by the method for constructing a head and neck cancer chemotherapy recurrence animal model according to any one of claims 1-7.
9. The method for constructing a head and neck cancer chemotherapy recurrence animal model according to any one of claims 1-7 is applied to the research of the mechanism of head and neck cancer chemotherapy recurrence.
10. The method for constructing a head and neck cancer chemotherapy recurrence animal model according to any one of claims 1-7 is applied to the screening of head and neck cancer treatment drugs.