A head and neck squamous cell carcinoma-associated fibroblast and organoid co-culture system and its construction method and application

Abstract

The present invention discloses a head and neck squamous cell carcinoma-related fibroblast and organoid co-culture system and its construction method and application, which belongs to the field of tumor biology. The construction method of the co-culture system is to enzymatically digest the head and neck squamous cell carcinoma tissue, culture it into a fibroblast cell line and organoid tissue respectively, and then use organoid culture medium for co-culture. The organoid cells obtained by co-culture of the present invention have a 3-5-fold increase in stemness and a 5-7-fold increase in drug resistance. Moreover, the method is simple to operate, takes little time, and can complete cell separation and organoid culture within 1 hour. The present invention provides a basis for establishing an organoid library, conducting drug screening, and promoting clinical transformation.

Description

Technical Field

[0001] The present invention belongs to the field of tumor biology, and specifically relates to a head and neck squamous cell carcinoma-associated fibroblast and organoid co-culture system, and its construction method and application. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) originates in the oropharynx, oral cavity, larynx, and hypopharynx, accounting for nearly 90% of hand and neck cancer cases and is the sixth most common malignant tumor in the world. [1,2] For the treatment of head and neck squamous cell carcinoma patients, chemotherapy such as fluorouracil (5'FU) or cisplatin (DDP) is routinely given to patients before and after surgery. However, the development of drug resistance has jeopardized the prognosis of head and neck squamous cell carcinoma patients. [3-5] .

[0003] The tumor microenvironment is a highly complex ecosystem composed of different types of stromal cells including fibroblasts, blood vessels and immune cells as well as the tumor cells themselves, which together form a communication network to facilitate tumor cell growth. [6-8] Cancer-associated fibroblasts (CAFs), in particular, have been reported to influence cancer progression in different cancers. [9] Studies have shown that direct crosstalk between tumor cells and fibroblasts determines cancer growth and progression [6,9,10] Fibroblasts are known to be abundant in head and neck squamous cell carcinoma, suggesting they play a crucial role. However, when organoids are used as drug screening models, the absence of fibroblasts during their growth can compromise the accuracy of experimental results.

[0004] As a revolutionary disease model, organoids have broad application prospects in many fields such as stem cell and disease research and drug development. The most classic cytokine culture program in organoid culture is WNER (standing for: Wnt-3a, Noggin, EGF and R-Spondins). Noggin is an important regulator of Wnt signaling pathway and BMP signaling, playing a role in many aspects.

[11] . The R-spondin family of secreted proteins (Rspo1-4) are microenvironmental factors that maintain adult stem cells in multiple organs. Among them, Rspo1 has been proven to be a key factor in the in vitro expansion culture of various adult stem cells. R-sponding1 and Noggin are important components of the most classic growth factor culture scheme in organoid culture. Currently, the culture of organoids mainly relies on expensive commercial cytokines (R-sponding1, Noggin, etc.), which makes the entire culture cost too high, seriously hindering the research and promotion of organoids.

[0005] References:

[0006] 1. Johnson, D. E., et al., Head and neck squamous cell carcinoma. Nat Rev Dis Primers, 2020. 6(1): p. 92.

[0007] 2. Klein, J. D. and J. R. Grandis, The molecular pathogenesis of head and neck cancer. Cancer Biol Ther, 2010. 9(1): p. 1 - 7.

[0008] 3. Joshi, P., et al., Role of neoadjuvant chemotherapy in advanced carcinoma of the hypopharynx and larynx. South Asian J Cancer, 2017. 6(1): p. 15 - 19.

[0009] 4. Li, R., et al., Induction chemotherapy of modified docetaxel, cisplatin, 5 - fluorouracil for laryngeal preservation in locally advanced hypopharyngeal squamous cell carcinoma. Head Neck, 2022. 44(9): p. 2018 - 2029.

[0010] 5. Won, H. S., et al., Clinical outcome of induction chemotherapy in locally advanced head and neck squamous cell carcinoma. Anticancer Res, 2014. 34(10): p. 5709 - 14.

[0011] 6. Kalluri, R., The biology and function of fibroblasts in cancer. Nat Rev Cancer, 2016. 16(9): p. 582 - 98.

[0012] 7. Wong, PP, et al., Cancer Burden Is Controlled by Mural Cell-beta3-Integrin Regulated Crosstalk with Tumor Cells. Cell, 2020.181(6):p.1346-1363e21.

[0013] 8. Wong, PP, N. Bodrug, and KMHodivala-Dilke, Exploring Novel Methods for Modulating Tumor Blood Vessels in Cancer Treatment. Curr Biol, 2016.26(21):p.R1161-R1166.

[0014] 9. Chen, Y., KMMcAndrews, and R. Kalluri, Clinical and therapeuticrelevance of cancer-associated fibroblasts. Nat Rev Clin Oncol, 2021.18(12):p.792-804.

[0015] 10.Ma, J., et al., Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11Targeting JAK / STAT3 / Bcl2Pathway. Cancer Res Treat, 2019.51(1):p.194-210.

[0016] 11.Fatehullah, A., SHTan, and N.Barker, Organoids as an in vitro model of human development and disease. Nat Cell Biol, 2016.18(3):p.246-54. Summary of the Invention

[0017] In view of the shortcomings and deficiencies of the existing technology, the purpose of the present invention is to provide a method for constructing a co-culture system of head and neck squamous cell carcinoma-associated fibroblasts and organoids.

[0018] Another object of the present invention is to provide a head and neck squamous cell carcinoma-associated fibroblast and organoid co-culture system obtained by the above-mentioned construction method.

[0019] Another object of the present invention is to provide an application of the above-mentioned head and neck squamous cell carcinoma-associated fibroblast and organoid co-culture system.

[0020] In order to achieve the above object, the present invention adopts the following technical solutions:

[0021] A method for constructing a co-culture system of head and neck squamous cell carcinoma-associated fibroblasts and organoids, comprising the following steps:

[0022] (1) Fresh head and neck squamous cell carcinoma tissue was obtained and cut into two parts;

[0023] (2) One portion was taken, digested with tissue digestion solution, filtered with a 100 μm filter, the filtrate was centrifuged to obtain the precipitate, and cultured with DMEM complete medium to obtain head and neck squamous cell carcinoma-associated fibroblasts;

[0024] (3) Taking another portion, adding organoid digestion solution for digestion, filtering with a 70 μm filter, centrifuging the filtrate to obtain a precipitate, adding red blood cell lysis solution, mixing, reacting, adding cleaning solution for washing, centrifuging to obtain a precipitate, and the precipitate is the head and neck squamous cell carcinoma laryngeal cancer single cell; mixing the obtained head and neck squamous cell carcinoma laryngeal cancer single cell with matrix gel, adding improved organoid culture medium for culture, and obtaining head and neck squamous cell carcinoma tissue organoids;

[0025] (4) digesting the head and neck squamous cell carcinoma-associated fibroblasts obtained in step (2) and the head and neck squamous cell carcinoma tissue organoids obtained in step (3) separately; placing the Transwell chamber in a culture plate, the chamber inside the chamber is called the upper chamber, and the culture plate inside the chamber is called the lower chamber; inoculating the head and neck squamous cell carcinoma-associated fibroblasts obtained by digestion into the upper chamber, inoculating the head and neck squamous cell carcinoma tissue organoids obtained by digestion into the lower chamber, adding the improved organoid culture medium for co-culturing, thereby obtaining the head and neck squamous cell carcinoma-associated fibroblast and organoid co-culture system;

[0026] Furthermore, the composition of the improved organoid culture medium described in the above method is as follows: Advanced DMEM / F-12 medium containing 20 ng / ml Wnt-3a, 5 ng / ml R-spondin 1, 1× B27 Supplement, 10 mmol / L Nicotinamide, 1.25 mmol / L N-acetylcsteine, 100 μg / ml Primocin, 50 ng / ml mNoggin, 50 ng / ml hEGF, 100 ng / ml hFGF, 10 nmol / L Gastrin I, 500 nmol / L A83-01, 10.5 μmol / L Y-27632, 10 mmol / L HEPES, and 1× GlutaMAX Supplement.

[0027] Furthermore, the head and neck squamous cell carcinoma described in step (1) includes neck tumors, otolaryngology tumors, and oral and maxillofacial tumors, such as thyroid tumors, laryngeal cancer, paranasal sinus cancer, tongue cancer, gum cancer, cheek cancer, etc.

[0028] Furthermore, the shearing in step (1) refers to cutting into 0.5-1 cm 3 Small piece of.

[0029] Furthermore, the composition of each tissue digestion solution in step (2) is as follows: DMEM culture medium containing 25 μg / ml Collagenase III and 25 μg / ml DNAse I.

[0030] Furthermore, the digestion condition of adding tissue digestion solution in step (2) is digestion at 37±2°C for 30±5 minutes.

[0031] Furthermore, the composition of the DMEM complete medium described in step (2) is as follows: DMEM medium with 1% (v / v) double antibody and 10% (v / v) serum.

[0032] Furthermore, the composition of each portion of the organoid digestion solution described in step (3) is as follows: advanced DMEM / F12 culture medium containing 10 μg / ml Collagenase II, 10 μg / ml DNAse I and 10.5 μmol / L Y-27632.

[0033] Furthermore, the red blood cell lysis solution in step (3) is ACK red blood cell lysis solution.

[0034] Furthermore, the cleaning solution in step (3) is 1 mg / ml BSA (ie, 0.1% BSA).

[0035] Furthermore, the centrifugation conditions in step (3) are: 4±2°C, 300±50g, 3±1min.

[0036] Furthermore, the matrix glue described in step (3) is Matrigel matrix glue.

[0037] Furthermore, the ratio of the head and neck squamous cell carcinoma or laryngeal cancer single cell to the matrix gel in step (3) is 1-1.2:1-1.2 by mass.

[0038] Furthermore, the culture conditions in step (3) are 37±2° C. and 95±2% relative humidity.

[0039] Furthermore, the Transwell chamber described in step (4) is a Transwell chamber with a pore size of 0.4 μm.

[0040] Furthermore, the specific operation of the digestion described in step (4) is as follows: take the head and neck squamous cell carcinoma-associated fibroblasts obtained in step (2), add 0.25% trypsin and digest at 37±2°C for 2±1 minutes; take the head and neck squamous cell carcinoma-associated fibroblasts obtained in step (3), blow 10±2 times with a pipette tip to perform physical digestion.

[0041] Furthermore, the inoculation amount of the head and neck squamous cell carcinoma tissue organoid cells in step (4) is (1-1.2)×10 per well of a 24-well plate. 4 A plan.

[0042] Furthermore, the ratio of the number of head and neck squamous cell carcinoma-associated fibroblasts to the number of head and neck squamous cell carcinoma tissue organoid cells in step (4) is 1-1.2:1-1.2.

[0043] Furthermore, the co-cultivation conditions in step (4) are 37±2° C. and 95±2% relative humidity.

[0044] A head and neck squamous cell carcinoma-associated fibroblast and organoid co-culture system is obtained by the above-mentioned construction method.

[0045] A method for constructing head and neck squamous cell carcinoma tissue organoids with high drug resistance and high stemness, comprising taking the above-mentioned head and neck squamous cell carcinoma-related fibroblast and organoid co-culture system, removing the transwell chamber, and taking away the fibroblasts in the upper layer to obtain the head and neck squamous cell carcinoma tissue organoids with high drug resistance and high stemness.

[0046] A head and neck squamous cell carcinoma tissue organoid with high drug resistance and high stemness is obtained through the above-mentioned construction method.

[0047] The application of the above-mentioned head and neck squamous cell carcinoma tissue organoids with high drug resistance and high stemness in the preparation of drug evaluation or screening models.

[0048] The method of the present invention reduces the concentration of R-spondin1 and Noggin in the culture medium, saving culture costs. Compared with single culture, the co-culture of the present invention can effectively promote the growth of organoids, as well as improve the drug resistance and cell stemness of organoids. As a drug screening model, organoids will affect the accuracy of experimental results if there is no participation of fibroblasts during the growth process. The co-culture of the present invention better simulates the drug-resistant tumor microenvironment and ensures the accuracy of experimental results. The co-culture of the present invention has played a good effect in the co-culture of organoids of other solid tumors. The present invention provides a basis for establishing an organoid library, conducting drug screening and promoting clinical transformation.

[0049] The present invention has the following advantages and effects compared to the prior art:

[0050] 1. Fibroblasts are highly abundant in head and neck squamous cell carcinoma, suggesting their crucial role. Without the involvement of fibroblasts during the growth process, organoids, as drug screening models, can compromise the accuracy of experimental results. The co-culture method of the present invention better simulates the microenvironment of drug-resistant tumors, ensuring the accuracy of experimental results.

[0051] 2. Current organoid culture relies heavily on the expensive commercial cytokine R-spondin 1, which results in high overall culture costs and severely hinders organoid research and expansion. This invention reduces the concentration of R-spondin 1 in the culture medium, saving culture costs.

[0052] 3. Noggin, as an important component of the most classic growth factor culture protocol for organoid culture, can enable stem cells to proliferate in an undifferentiated state and maintain their stemness. In the co-culture of the present invention, hNoggin (human Noggin) is replaced with mNoggin (mouse Noggin), and the concentration is reduced by half. The addition of mNoggin is beneficial to the survival and growth of head and neck squamous cell carcinoma organoids. The use of this culture medium can highly effectively culture head and neck squamous cell carcinoma organoids, with an extremely high success rate of establishment, and its culture success rate can reach 100%.

[0053] 4. Compared with single culture, the co-cultured organoids of the present invention have faster growth ability and can be passaged in 3 to 5 days.

[0054] 5. The stemness of the organoid cells co-cultured in the present invention is enhanced by 3 to 5 times.

[0055] 6. The drug resistance of the co-cultured organoid cells of the present invention is enhanced by 5 to 7 times under the stimulation of the chemotherapy drug DDP / 5'FU.

[0056] 7. The present invention adopts an on-ice operation method to mince tumor tissue, which can effectively improve the cell yield and cell activity.

[0057] 8. The method of the present invention is simple to operate, takes little time, and can complete cell separation and organoid culture within 1 hour.

[0058] 9. The method of the present invention requires a small amount of tissue specimen, and at least 0.1 g of tissue can be used to separate enough cells to establish fibroblast cell lines and organoids, respectively.

[0059] 10. Compared with other methods for isolating and establishing organoids from head and neck squamous cell carcinoma organoid tissue, the cells isolated by the method of the present invention have high activity and can form obvious tumor organoid tissue 3 to 4 days after cell isolation. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a flow chart of drug addition testing after laryngeal cancer patient-derived fibroblasts and organoids are isolated and co-cultured in vitro.

[0061] Figure 2 The figure shows the morphology of organoids isolated from laryngeal cancer tissue after 4 days of growth. A shows the growth morphology of organoids cultured in conventional culture after 4 days, and B shows the growth morphology of organoids co-cultured with the invention after 4 days (scale bar: 200 μm).

[0062] Figure 3 Figures 2 and 3 are growth morphologies of cells after culture with the addition of chemotherapy drugs (scale bar: 200 μm); A shows the growth after the addition of cisplatin DDP; B shows the growth after the addition of fluorouracil 5'FU.

[0063] Figure 4 After culture, the organoids were used to conduct a mouse subcutaneous tumor experiment to detect the cell stemness; among them, A is the stemness morphology of mouse subcutaneous tumor cells; B is the relative tumor volume statistics. DETAILED DESCRIPTION

[0064] The present invention will be described in further detail below with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

[0065] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional in the art. Experimental methods in the following examples, where specific experimental conditions are not specified, are generally performed under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0066] The following reagents were used in the preparation of the examples:

[0067] 1. Composition of tissue digestion solution: DMEM medium containing 25 μg / ml Collagenase III and 25 μg / ml DNAse I.

[0068] 2. Composition of organoid digestion medium: advanced DMEM / F12 medium containing 10 μg / ml Collagenase II, 10 μg / ml DNAse I, and 10.5 μmol / L Y-27632.

[0069] 3. The composition of common organoid culture medium is shown in Table 1 below:

[0070] Table 1 Composition of common organoid culture medium

[0071] Reagents Specification volume Storage concentration Final concentration Advanced DMEM / F-12 500mL 100mL B27Supplement 10mL 2mL 50× 1× Primocin 50mg / mL 200 μL 50mg / mL 100 μg / mL Wnt3a 10 μg 10 μL 200 μg / mL 20 ng / mL Nicotinamide 100g (take 1g) 1mL 1mol / l 10mmol / l N-acetylcysteine 30mg 250 μL 500mmol / L 1.25mmol / L R-spondin1 100 μg 50 μL 1mg / mL 500ng / mL hNoggin 20 μg 100 μL 100 μg / mL 100ng / mL hEGF 100 μg 10 μL 500 μg / mL 50 ng / mL hFGF 25 μg 10 μL 1000 μg / mL 100ng / mL hGastrin I 250 μg 10 μL 100 μmol / L 10nmol / L A 83-01 10mg 1 μL 50mmol / L 500nmol / L Y-27632 1mg 100 μL 10.5mmol / L 10.5 μmol / L HEPES pH 7.2-7.5 20mL (1mol / L) 1mL 1mol / L 10mmol / L GlutaMAX Supplement 100mL(100×) 1mL 100× 1× Primocin 1mL (50mg / mL) 0.2mL 50mg / mL 100 μg / mL

[0072] 4. The composition of the improved organoid culture medium is as follows: Advanced DMEM / F-12 medium containing 20 ng / ml Wnt-3a, 5 ng / ml R-spondin 1, 1× B27 Supplement, 10 mmol / L Nicotinamide, 1.25 mM N-acetylcsteine, 100 μg / ml Primocin, 50 ng / ml mNoggin, 50 ng / ml hEGF, 100 ng / ml hFGF, 10 nmol / L Gastrin I, 500 nM A83-01, 10.5 μmol / L Y-27632, 10 mmol / L HEPES, and 1× GlutaMAX Supplement.

[0073] Example 1

[0074] 1. Immediately after surgery, fresh laryngeal cancer tumor tissue was obtained for cell isolation. The tissue was rinsed with 0.9% NaCl saline in a clean bench to remove blood, placed in a 10-cm culture dish, photographed, and weighed. The patient's tissue number was recorded (this experiment was approved by the ethics committee of our hospital).

[0075] 2. Place the tissue on ice and quickly chop it into pieces of 0.5-1 cm 3 If the tissue piece is too large, it will affect the yield of single cells and can be divided into two parts;

[0076] 3. Place one of the chopped tissues in tissue digestion solution and digest at 37°C for 30 minutes. Filter the digested tissue using a 100 μm filter in a clean bench, collect the filtrate, and discard impurities on the filter membrane.

[0077] 4. The filtrate was centrifuged at 300 g for 3 min at 4°C, the supernatant was discarded, and the pellet was washed once with physiological saline. The resulting pellet was resuspended in DMEM medium (with 1% double antibody and 10% serum) and cultured on a six-well plate to obtain laryngeal cancer-associated fibroblasts.

[0078] 5. Add another portion of minced tissue to the organoid digestion solution and digest at 37°C for 30 minutes. Filter through a 70μm filter and rinse the filter three to four times with saline to remove any cells adhering to the filter to prevent loss. Centrifuge the filtrate at 300g for 3 minutes at 4°C. Carefully aspirate the supernatant, retaining only the pellet. Add 2ml of ACK red blood cell lysis buffer and rapidly invert for 1 minute. Add 0.1% BSA wash buffer and centrifuge at 300g for 3 minutes at 4°C. Carefully aspirate the supernatant, retaining only the pellet. This pellet is the desired laryngeal cancer single cell. Mix the laryngeal cancer single cells with Matrigel at a 1:1 mass ratio. Pipet 50μL into the center of a well of a 24-well plate. Add modified organoid culture medium and culture at 37°C and 95% relative humidity to generate laryngeal cancer tissue organoids.

[0079] 6. After 3-5 days, the laryngeal cancer-associated fibroblasts and laryngeal cancer tissue organoids obtained above were digested separately. The fibroblasts were digested with 1 ml of 0.25% trypsin at 37°C for 2 minutes. The organoids were directly pipetted 10 times with a 1 ml pipette tip to break the organoids into single cells and counted. Co-culture was performed: a 0.4 μm Transwell chamber was placed in a 24-well plate, with the chamber inside the chamber called the upper chamber and the culture plate inside the chamber called the lower chamber. 1×10 4 Laryngeal cancer-associated fibroblasts were seeded into the upper chamber, 1×10 4 Single cells of laryngeal cancer tissue organoids were inoculated into the lower chamber, and improved organoid culture medium was added to both the upper and lower chambers, and the cells were co-cultured in an incubator at 37°C and 95% relative humidity.

[0080] 7. Observe the growth morphology of the organoids in co-culture under a microscope. After 7 days of co-culture, remove the transwell chamber, remove the fibroblasts in the upper layer, and perform drug addition and cell stemness tests only on the organoids in the lower layer.

[0081] Comparative Example 1

[0082] Organoid culture was performed using standard organoid culture medium according to traditional methods. The specific steps are as follows:

[0083] 1. Immediately after surgery, fresh laryngeal cancer tumor tissue was obtained for cell isolation. The tissue was rinsed with 0.9% NaCl saline in a clean bench to remove blood, placed in a 10-cm culture dish, photographed, and weighed. The patient's tissue number was recorded (this experiment was approved by the ethics committee of our hospital).

[0084] 2. Place the tissue on ice and quickly chop it into pieces of 0.5-1 cm 3 Add the organoid digestion solution to the small pieces and digest at 37°C for 30 minutes. Filter through a 70μm filter and rinse the filter three to four times with saline to remove any cells adhering to the filter to prevent loss. Centrifuge the filtrate at 300g for 3 minutes at 4°C. Carefully aspirate the supernatant, retaining only the pellet. Add 2ml of ACK red blood cell lysis buffer and quickly invert to mix for 1 minute. Add 0.1% BSA wash solution and centrifuge at 300g for 3 minutes at 4°C. Carefully aspirate the supernatant, retaining only the pellet. This pellet is the desired laryngeal cancer single cell. Mix the laryngeal cancer single cells with Matrigel at a 1:1 mass ratio. Pipet 50μL into the center of a well of a 24-well plate. Add standard organoid culture medium and culture at 37°C and 95% relative humidity to obtain laryngeal cancer tissue organoids.

[0085] The growth morphology of the organoids in monoculture was observed under a microscope. After 7 days of monoculture, the obtained organoids were subjected to drug addition testing and cell stemness testing according to the method of Example 1.

[0086] result:

[0087] The morphology of cells isolated from laryngeal cancer tissue after 4 days of growth is as follows Figure 2 As shown, the organoids co-cultured in Example 1 can form obvious tumor organoid tissue within 4 days, while the cells of Control Example 1 (control) have no obvious changes. The drug resistance of the organoid cells co-cultured in Example 1 is enhanced by 5 to 7 times compared with Control Example 1 under the stimulation of chemotherapy drugs DDP / 5'FU ( Figure 3 The stemness of the organoid cells co-cultured in Example 1 was enhanced by 3 to 5 times compared with that in Comparative Example 1 ( Figure 4 ).

[0088] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for constructing head and neck squamous cell carcinoma tissue organoids with high drug resistance and high stemness, characterized by: The steps include: (1) Take fresh head and neck squamous cell carcinoma tissue, cut it and divide it into two parts; (2) Take one of the samples, add tissue digestion solution to digest, filter with a 100 μm filter, centrifuge the filtrate to obtain the precipitate, and culture with DMEM complete medium to obtain head and neck squamous cell carcinoma-associated fibroblasts; (3) Take another portion, add organoid digestion solution to digest, filter with a 70 μm filter, centrifuge the filtrate to obtain the precipitate, add red blood cell lysis solution, mix, react, add washing solution to wash, centrifuge to obtain the precipitate, and the precipitate is the head and neck squamous cell carcinoma laryngeal cancer single cell; after mixing the obtained head and neck squamous cell carcinoma laryngeal cancer single cell with matrix gel, add improved organoid culture medium and culture to obtain head and neck squamous cell carcinoma tissue organoids; (4) digesting the head and neck squamous cell carcinoma-associated fibroblasts obtained in step (2) and the head and neck squamous cell carcinoma tissue organoids obtained in step (3) separately; placing the Transwell chamber in a culture plate, with the inner chamber being called the upper chamber and the inner chamber being called the lower chamber; inoculating the head and neck squamous cell carcinoma-associated fibroblasts obtained by digestion into the upper chamber, and inoculating the head and neck squamous cell carcinoma tissue organoids obtained by digestion into the lower chamber, adding the improved organoid culture medium for co-culture, thereby obtaining the head and neck squamous cell carcinoma-associated fibroblast and organoid co-culture system; (5) Taking the head and neck squamous cell carcinoma-associated fibroblast and organoid co-culture system obtained in step (4), removing the transwell chamber, and taking away the fibroblasts on the upper layer, thereby obtaining the head and neck squamous cell carcinoma tissue organoid with high drug resistance and high stemness; The composition of the improved organoid culture medium is as follows: Advanced DMEM / F-12 medium containing 20 ng / ml Wnt-3a, 5 ng / ml R-spondin1, 1× B27 Supplement, 10 mmol / L Nicotinamide, 1.25 mmol / L N-acetylcsteine, 100 μg / ml Primocin, 50 ng / ml mNoggin, 50 ng / ml hEGF, 100 ng / ml hFGF, 10 nmol / L Gastrin I, 500 nmol / L A83-01, 10.5 μmol / L Y-27632, 10 mmol / L HEPES, and 1× GlutaMAX Supplement; The composition of each organoid digestion solution is as follows: advanced DMEM / F12 medium containing 10 μg / ml Collagenase II, 10 μg / ml DNAse I and 10.5 μmol / L Y-27632.

2. The method according to claim 1, wherein: The composition of each tissue digestion solution described in step (2) is as follows: DMEM medium containing 25 μg / ml Collagenase III and 25 μg / ml DNAse I; The digestion conditions for adding tissue digestion solution in step (2) are digestion at 37±2°C for 30±5 minutes; The composition of the DMEM complete medium described in step (2) is as follows: DMEM medium with 1% v / v double antibody and 10% v / v serum.

3. The method according to claim 1, wherein: The red blood cell lysis solution in step (3) is ACK red blood cell lysis solution; The washing solution described in step (3) is 1 mg / ml BSA; The centrifugation conditions in step (3) are: 4 ± 2°C, 300 ± 50 g, 3 ± 1 min; The matrix glue described in step (3) is Matrigel matrix glue; The ratio of the head and neck squamous cell carcinoma and laryngeal cancer single cells to the matrix gel in step (3) is 1-1.2:1-1.2 by mass; The culture conditions described in step (3) are 37±2°C and 95±2% relative humidity.

4. The method according to claim 1, wherein: The Transwell chamber described in step (4) is a Transwell chamber with a pore size of 0.4 μm; The specific digestion operation described in step (4) is as follows: take the head and neck squamous cell carcinoma-associated fibroblasts obtained in step (2), add 0.25% trypsin and digest at 37±2°C for 2±1 minutes; take the head and neck squamous cell carcinoma-associated fibroblasts obtained in step (3), blow 10±2 times with a pipette tip to perform physical digestion; The seeding amount of head and neck squamous cell carcinoma tissue organoid cells described in step (4) is 1-1.2×10 per well of a 24-well plate. 4 a plan; The ratio of the number of head and neck squamous cell carcinoma-associated fibroblasts to the number of head and neck squamous cell carcinoma tissue organoid cells in step (4) is 1-1.2:1-1.2; The co-cultivation conditions described in step (4) are 37±2°C and 95±2% relative humidity.

5. The method according to any one of claims 1 to 4, characterized in that: The head and neck squamous cell carcinoma described in step (1) is thyroid tumor, laryngeal cancer, paranasal sinus cancer, tongue cancer, gum cancer or cheek cancer.

6. A head and neck squamous cell carcinoma tissue organoid with high drug resistance and high stemness, characterized by: Obtained by the construction method described in any one of claims 1-5.

7. Use of the head and neck squamous cell carcinoma tissue organoids with high drug resistance and high stemness as described in claim 6 in preparing a drug evaluation or screening model.

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