CN116004539A - A co-culture system of fibroblasts and organoids related to head and neck squamous cell carcinoma and its construction method and application - Google Patents

Abstract

The invention discloses a head and neck squamous cell carcinoma-related fibroblast-organoid co-cultivation system and its construction method and application, belonging to the field of tumor biology. The construction method of the co-cultivation system is as follows: after the head and neck squamous cell carcinoma tissue is digested with enzymes, the fibroblast cell line and the organoid tissue are cultured respectively, and then the organoid medium is used for co-cultivation. The stemness of the organoid cells obtained by the co-cultivation of the present invention is increased by 3-5 times, and the drug resistance is increased by 5-7 times, and the method is simple to operate and less time-consuming, and the cell separation and the culture of the organoids can be completed within 1 hour. The invention provides a basis for establishing an organoid bank, conducting drug screening and promoting clinical transformation.

Description

A co-culture system of fibroblasts and organoids related to head and neck squamous cell carcinoma and its construction method and application

technical field

The invention belongs to the field of tumor biology, and in particular relates to a co-cultivation system of fibroblasts and organoids related to head and neck squamous cell carcinoma and its construction method and application.

Background technique

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

The tumor microenvironment is a highly complex ecosystem consisting of different types of stromal cells including fibroblasts, vascular and immune cells as well as tumor cells themselves, while they form a communication network to flourish tumor cell growth ^[6-8] . In particular, cancer-associated fibroblasts (CAFs) have been reported to affect 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] , and fibroblasts are known to be abundant in squamous cell carcinoma of the head and neck, suggesting that it plays a role in important role. Organoids are used as drug screening models. If there is no participation of fibroblasts during the growth process, the accuracy of the experimental results will be affected.

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 protocol in organoid culture is WNER (respectively: Wnt-3a, Noggin, EGF and R-Spondins), and Noggin, as an important regulator of Wnt signaling pathway and BMP signaling, plays 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 proved to be a key factor in the in vitro expansion and culture of various adult stem cells. R-sponding1 and Noggin are important components of the most classic growth factor culture scheme in organoid culture. At present, the culture of organoids mainly relies on expensive commercial cytokines (R-sponding1, Noggin, etc.), which causes the entire culture The high cost has seriously hindered the research and promotion of organoids.

references:

1. Johnson, D.E., et al., Head and neck squamous cell carcinoma. Nat Rev Dis Primers, 2020.6(1): p.92.

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.

3. Joshi, P., et al., Role of neoadjuvant chemotherapy in advanced cancer of the hypopharynx and larynx. South Asian J Cancer, 2017.6(1): p.15-19.

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.

5.Won,H.S.,et al.,Clinical outcome of induction chemotherapy inlocally advanced head and neck squamous cell carcinoma. Anticancer Res,2014.34(10):p.5709-14.

6. Kalluri, R., The biology and function of fibroblasts in cancer. Nat Rev Cancer, 2016.16(9): p.582-98.

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

8.Wong,P.P.,N.Bodrug,and K.M.Hodivala-Dilke,Exploring Novel Methods for Modulating Tumor Blood Vessels in Cancer Treatment.Curr Biol,2016.26(21):p.R1161-R1166.

9. Chen, Y., K.M. McAndrews, and R. Kalluri, Clinical and therapeutic relevance of cancer-associated fibroblasts. Nat Rev Clin Oncol, 2021.18(12): p.792-804.

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.

11. Fatehullah, A., S.H. Tan, and N. Barker, Organoids as an in vitro model of human development and disease. Nat Cell Biol, 2016.18(3): p.246-54.

Contents of the invention

In view of the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a method for constructing a co-culture system of head and neck squamous cell carcinoma-related fibroblasts and organoids.

Another object of the present invention is to provide a co-culture system of head and neck squamous cell carcinoma-related fibroblasts and organoids obtained by the above construction method.

Another object of the present invention is to provide the application of the co-culture system of fibroblasts and organoids related to head and neck squamous cell carcinoma.

In order to achieve the above object, the present invention adopts following technical scheme:

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

(1) Take fresh head and neck squamous cell carcinoma tissue, cut it and divide it into two parts;

(2) Take one part, add tissue digestion solution for digestion, filter with a 100 μm filter, centrifuge the filtrate to collect the precipitate, and culture it with DMEM complete medium to obtain fibroblasts related to head and neck squamous cell carcinoma;

(3) Take another part, add organoid digestion solution for digestion, filter with a 70 μm filter, centrifuge the filtrate to collect the precipitate, add red blood cell lysate, mix well, react, add cleaning solution to wash, centrifuge to collect the precipitate, and the precipitate is ready It is a single cell of head and neck squamous cell carcinoma of laryngeal carcinoma; after mixing the obtained single cell of head and neck squamous cell carcinoma of laryngeal carcinoma with Matrigel, it is added to an improved organoid medium for culture to obtain tissue organoids of head and neck squamous cell carcinoma of the head and neck;

(4) The head and neck squamous cell carcinoma-related fibroblasts obtained in step (2) and the head and neck squamous cell carcinoma tissue organoids obtained in step (3) were respectively digested; the Transwell chamber was placed in the culture plate, and the chamber was called the upper chamber, The inside of the culture plate is called the lower chamber; the digested head and neck squamous cell carcinoma-related fibroblasts are inoculated into the upper chamber, the digested head and neck squamous cell carcinoma tissue organoids are inoculated into the lower chamber, and the improved organoid medium is added for co-cultivation , that is, to obtain the co-culture system of head and neck squamous cell carcinoma-related fibroblasts and organoids;

Further, the composition of the improved organoid medium described in the above method is as follows: containing 20ng/ml Wnt-3a, 5ng/ml R-spondin1, 1×B27 Supplement, 10mmol/L Nicotinamide, 1.25mmol/LN-acetylcsteine, 100μg/ml Primocin, 50ng/ml mNoggin, 50ng/ml hEGF, 100ng/ml hFGF, 10nmol/L Gastrin I, 500nmol/L A83-01, 10.5μmol/L Y-27632, 10mmol/L HEPES, 1×GlutaMAX Supplement Advanced DMEM/F-12 medium.

Further, the squamous cell carcinoma of the head and neck described in step (1) includes neck tumors, ENT tumors, and oral and maxillofacial tumors, such as thyroid tumors, laryngeal cancer, paranasal sinus cancer, tongue cancer, gingival cancer, buccal cancer, etc. cancer etc.

Further, the shearing mentioned in step (1) refers to cutting into small pieces of 0.5-1 cm ³ .

Further, the composition of each portion of the tissue digestion solution described in step (2) is as follows: DMEM medium containing 25 μg/ml Collagenase III and 25 μg/ml DNAse I.

Further, the condition of adding tissue digestion solution for digestion in step (2) is to digest at 37±2° C. for 30±5 min.

Further, 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.

Further, the composition of each part of the organoid digestion solution described in step (3) 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.

Further, the erythrocyte lysate described in step (3) is ACK erythrocyte lysate.

Further, the cleaning solution described in step (3) is 1 mg/ml BSA (ie 0.1% BSA).

Further, the centrifugation conditions described in step (3) are: 4±2°C, 300±50g, 3±1min.

Further, the matrigel described in step (3) is Matrigel matrigel.

Further, the ratio of the head and neck squamous cell carcinoma laryngeal carcinoma single cells to Matrigel in step (3) is a mass ratio of 1-1.2:1-1.2.

Further, the cultivation conditions described in step (3) are 37±2°C and 95±2% relative humidity.

Further, the Transwell chamber described in step (4) is a Transwell chamber with a pore size of 0.4 μm.

Further, the specific operation of the digestion described in step (4) is as follows: take the head and neck squamous cell carcinoma-related fibroblasts obtained in step (2), add 0.25% trypsin to digest at 37±2°C for 2±1 minutes; (3) The resulting head and neck squamous cell carcinoma-related fibroblasts were pipetted 10±2 times with a pipette tip, and then physically digested.

Further, the inoculation amount of head and neck squamous cell carcinoma tissue organoid cells described in step (4) is calculated as (1-1.2)×10 ⁴ per well of a 24-well plate.

Further, the number ratio of the head and neck squamous cell carcinoma-related fibroblasts and head and neck squamous cell carcinoma tissue organoid cells described in step (4) is 1-1.2:1-1.2.

Further, the conditions of the co-cultivation described in step (4) are 37±2°C and 95±2% relative humidity.

A co-culture system of fibroblasts and organoids related to head and neck squamous cell carcinoma is obtained through the above construction method.

A method for constructing head and neck squamous cell carcinoma tissue organoids with high drug resistance and high stemness, taking the above co-culture system of head and neck squamous cell carcinoma-related fibroblasts and organoids, taking out the transwell chamber, and removing the upper layer Fibroblasts, that is, to obtain the head and neck squamous cell carcinoma tissue organoids with high drug resistance and high stemness.

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

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.

The method of the invention reduces the concentration of R-spondin1 and Noggin in the culture medium, saving the culture cost. Compared with single culture, the co-culture of the present invention can effectively promote the growth of organoids, and improve the drug resistance and cell stemness of organoids. Organoids are used as drug screening models. During the growth process, if there is no participation of fibroblasts, the accuracy of the experimental results will be affected. The co-culture of the present invention can better simulate the drug-resistant tumor microenvironment and ensure the accuracy of the experimental results. . The co-culture of the present invention has achieved good results in the co-cultivation of organoids of other solid tumors. The invention provides a basis for establishing an organoid bank, conducting drug screening and promoting clinical transformation.

Compared with the prior art, the present invention has the following advantages and effects:

1. Fibroblasts are very abundant in squamous cell carcinoma of the head and neck, indicating that it plays an important role. Organoids are used as drug screening models. If there is no participation of fibroblasts during the growth process, the accuracy of the experimental results will be affected. The co-cultivation of the present invention better simulates the drug-resistant tumor microenvironment and ensures the accuracy of the experimental results.

2. The current culture of organoids mainly relies on the expensive commercial cytokine R-spondin1, which results in too high cost of the entire culture and seriously hinders the research and promotion of organoids. The invention reduces the concentration of R-spondin1 in the culture medium and saves the culture cost.

3. As an important component of the most classic growth factor culture program in organoid culture, Noggin can make stem cells proliferate in an undifferentiated state and maintain the stemness of stem cells. In the co-culture of the present invention, hNoggin (human noggin) is replaced by 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 medium can highly effectively culture head and neck squamous cell carcinoma organoids, and the success rate of establishment is extremely high, and the success rate of its culture can reach 100%.

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

5. The stemness of the co-cultured organoid cells of the present invention is enhanced by 3-5 times.

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

7. The method of operating on ice is used in the present invention to shred tumor tissue, which can effectively improve cell yield and cell activity.

8. The method of the present invention is easy to operate and less time-consuming, and can complete cell separation and organoid culture within 1 hour.

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

10. Compared with other methods for isolating and establishing head and neck squamous cell carcinoma organoid tissue-derived organoids, the cells isolated by the method of the present invention have high activity and can form obvious tumor organoid tissues 3 to 4 days after cell separation.

Description of drawings

Figure 1 is a flow chart of the drug-dosing test after laryngeal cancer patient-derived fibroblasts and organoids were isolated and co-cultured in vitro.

Figure 2 shows the morphology of organoids isolated from laryngeal cancer tissue after 4 days of growth, where A is the growth morphology of ordinary cultured organoids after 4 days, and B is the growth morphology of inventive co-cultured organoids after 4 days (the scale is 200 μm).

Fig. 3 is the growth appearance diagram of adding chemotherapeutic drugs after culture (the scale bar is 200 μm); wherein, A is the growth situation of DDP with cisplatin; B is the growth situation of 5'FU with fluorouracil.

Figure 4 shows the cell stemness of organoids in mouse subcutaneous tumor experiments after culture; where, A is the topography of mouse subcutaneous tumor cell stemness; B is the statistical graph of relative tumor volume.

Detailed ways

The present invention will be further described in detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. For the test methods that do not indicate the specific experimental conditions in the following examples, usually follow the routine experimental conditions or the experimental conditions suggested by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

Preparation of reagents used in the following examples:

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

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

3. The composition of common organoid medium is shown in Table 1 below:

Table 1 Composition of common organoid medium

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

4. The composition of the improved organoid medium: containing 20ng/ml Wnt-3a, 5ng/ml R-spondin1, 1×B27Supplement, 10mmol/L Nicotinamide, 1.25mM N-acetylcsteine, 100μg/ml Primocin, 50ng/ml mNoggin, 50ng/ml hEGF, 100ng/ml hFGF, 10nmol/L Gastrin I, 500nM A83-01, 10.5μmol/L Y-27632, 10mmol/L HEPES, Advanced DMEM/F-12 medium with 1×GlutaMAX Supplement.

Example 1

1. Immediately after the operation, fresh tumor tissue from patients with laryngeal cancer was taken to separate the cells, rinsed with 0.9% NaCl saline in an ultra-clean bench to remove blood, put into a 10cm petri dish to take pictures and weigh them, and record the patient’s tissue number information ( This experiment was approved by the Ethics Committee of our hospital).

2. Place the tissue on ice, cut it into pieces quickly, and cut it into small pieces of ^0.5-1cm3 . If the tissue piece is too large, it will affect the yield of single cells, and divide it into two parts;

3. Put one part of the shredded tissue into the tissue digestion solution, and digest at 37°C for 30 minutes; filter the digested tissue with a 100 μm filter in an ultra-clean bench, collect the filtrate, and discard the impurities on the filter membrane.

4. The filtrate was centrifuged at 300g at 4°C for 3min, the supernatant was discarded, washed once with normal saline, the resulting precipitate was resuspended in DMEM medium (plus 1% double antibody, 10% serum), and cultured on a six-well plate to obtain Laryngeal carcinoma-associated fibroblasts.

5. Add the organoid digestion solution to another cut-up tissue, digest at 37°C for 30 minutes, then filter with a 70 μm filter, and then rinse the filter with saline repeatedly for 3 to 4 times to wash away the cells sticking to the filter , so as not to cause losses. The filtrate was centrifuged at 300g at 4°C for 3 minutes, and the supernatant was carefully sucked off, leaving only the precipitate. Add 2ml of ACK erythrocyte lysate to the precipitate, quickly invert and mix for 1 minute, then add 0.1% BSA cleaning solution, and centrifuge at 300g at 4°C for 3 minutes. Carefully suck off the supernatant, leaving only the pellet, which is the laryngeal cancer single cell we need. Mix laryngeal cancer single cells and Matrigel evenly at a mass ratio of 1:1, suck 50 μL and drop them into the center of the well of a 24-well plate, then add the improved organoid medium, and store at 37°C and 95% relative humidity Cultured and cultured to obtain laryngeal cancer tissue organoids.

6. After 3-5 days, digest the laryngeal cancer-related fibroblasts and laryngeal cancer tissue organoids respectively. The fibroblasts were digested with 1ml 0.25% trypsin at 37°C for 2 minutes; the organoids were pipetted directly with a 1ml pipette tip for 10 First, the organoids were blown into single cells and counted; for co-cultivation: 0.4 μm Transwell chamber was placed in a 24-well plate, the chamber was called the upper chamber, and the inside of the culture plate was called the lower chamber, and 1×10 ⁴ laryngeal cancer-related fibroblasts were inoculated In the upper chamber, 1× ¹⁰⁴ laryngeal cancer tissue organoid single cells were inoculated into the lower chamber, and the upper and lower chambers were respectively added with improved organoid medium, and co-cultivated in an incubator at 37°C and a relative humidity of 95%. .

7. Observe the growth morphology of the organoids in the co-culture through a microscope. After the 7-day co-cultivation, the transwell chamber was taken out, the fibroblasts in the upper layer were removed, and only the organoids in the lower layer were tested for drug addition and cell stemness. in:

Comparative example 1

Ordinary organoid medium was used to culture organoids according to traditional methods, and the specific operations were as follows:

1. Immediately after the operation, fresh tumor tissue from patients with laryngeal cancer was taken to separate the cells, washed with 0.9% NaCl saline in an ultra-clean bench to remove blood, put into a 10cm petri dish to take pictures and weigh them, and record the patient’s tissue number information ( This experiment was approved by the Ethics Committee of our hospital).

2. Place the tissue on ice, quickly cut it into small pieces of 0.5-1cm ³ , add organoid digestion solution, digest at 37°C for 30 minutes, then filter through a 70μm filter, and then rinse the filter repeatedly with saline 3 ~4 times, rinse the cells sticking to the filter to avoid loss. The filtrate was centrifuged at 300g at 4°C for 3 minutes, and the supernatant was carefully sucked off, leaving only the precipitate. Add 2ml of ACK erythrocyte lysate to the precipitate, quickly invert and mix for 1 minute, then add 0.1% BSA cleaning solution, and centrifuge at 300g at 4°C for 3 minutes. Carefully suck off the supernatant, leaving only the pellet, which is the laryngeal cancer single cell we need. Mix laryngeal cancer single cells and Matrigel evenly at a mass ratio of 1:1, suck 50 μL and drop them into the center of the wells of a 24-well plate, then add common organoid medium, and culture them at 37°C and 95% relative humidity Cultured to obtain laryngeal cancer tissue organoids.

The growth morphology of organoids in monoculture was observed by microscopy. After the 7-day monoculture, referring to the method in Example 1, the obtained organoids were subjected to a drug addition test and a cell stemness test.

result:

The morphology of laryngeal cancer tissue isolation cells after 4 days of growth is shown in Figure 2. The organoids co-cultured in Example 1 can form obvious tumor organoids within 4 days, while the cells in Comparative Example 1 (control) have no obvious changes. Under the stimulation of the chemotherapeutic drug DDP/5'FU, the drug resistance of co-cultured organoid cells in Example 1 was 5-7 times stronger than that in Comparative Example 1 (Fig. 3). The stemness of the co-cultured organoid cells in Example 1 was 3-5 times stronger than that in Comparative Example 1 ( FIG. 4 ).

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the described embodiment, and any other changes, modifications, substitutions, Combination and simplification should all be equivalent replacement methods, and are all included in the protection scope of the present invention.

Claims (10)

1. A method for constructing a head and neck squamous cell carcinoma-related fibroblast and organoid co-culture system, characterized in that: comprising the steps of: (1) Take fresh head and neck squamous cell carcinoma tissue, cut it and divide it into two parts; (2) Take one part, add tissue digestion solution for digestion, filter with a 100 μm filter, centrifuge the filtrate to collect the precipitate, and culture it with DMEM complete medium to obtain fibroblasts related to head and neck squamous cell carcinoma; (3) Take another part, add organoid digestion solution for digestion, filter with a 70 μm filter, centrifuge the filtrate to collect the precipitate, add red blood cell lysate, mix well, react, add cleaning solution to wash, centrifuge to collect the precipitate, and the precipitate is ready It is head and neck squamous cell carcinoma laryngeal cancer single cell; after mixing the obtained head and neck squamous cell carcinoma laryngeal carcinoma single cell with Matrigel, adding an improved organoid medium for culture to obtain head and neck squamous cell carcinoma tissue organoid; (4) The head and neck squamous cell carcinoma-related fibroblasts obtained in step (2) and the head and neck squamous cell carcinoma tissue organoids obtained in step (3) were respectively digested; the Transwell chamber was placed in the culture plate, and the chamber was called the upper chamber, The culture plate is called the lower chamber; inoculate the digested head and neck squamous cell carcinoma-related fibroblasts into the upper chamber, inoculate the digested head and neck squamous cell carcinoma tissue organoids into the lower chamber, and add the improved organoid medium for co-culture , that is, to obtain the co-culture system of head and neck squamous cell carcinoma-related fibroblasts and organoids. 2. The construction method of the head and neck squamous cell carcinoma related fibroblast and organoid co-culture system according to claim 1, characterized in that: The composition of the improved organoid medium is as follows: containing 20ng/ml Wnt-3a, 5ng/ml R-spondin1, 1×B27Supplement, 10mmol/L Nicotinamide, 1.25mmol/L N-acetylcsteine, 100μg/ml Primocin, 50ng /ml mNoggin, 50ng/ml hEGF, 100ng/ml hFGF, 10nmol/L Gastrin I, 500nmol/L A83-01, 10.5μmol/L LY-27632, 10mmol/L HEPES, Advanced DMEM/F-12 of 1×GlutaMAX Supplement Medium. 3. The construction method of the head and neck squamous cell carcinoma related fibroblast and organoid co-culture system according to claim 1, characterized in that: The composition of each portion of the 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 condition for adding the tissue digestive solution to digestion in step (2) is to digest at 37±2°C for 30±5min; The composition of the complete DMEM medium described in step (2) is as follows: DMEM medium with 1% v/v double antibody and 10% v/v serum. 4. The construction method of the head and neck squamous cell carcinoma related fibroblast and organoid co-culture system according to claim 1, characterized in that: The composition of each part of the organoid digestion solution described in step (3) 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; The erythrocyte lysate described in step (3) is ACK erythrocyte lysate; The cleaning solution described in step (3) is 1mg/ml BSA; The centrifugation conditions described in step (3) are: 4±2°C, 300±50g, 3±1min; Matrigel described in step (3) is Matrigel Matrigel; The ratio of the head and neck squamous cell carcinoma laryngeal carcinoma single cell to Matrigel described in step (3) is a mass ratio of 1-1.2:1-1.2; The cultivation conditions described in step (3) are 37±2°C and 95±2% relative humidity. 5. The construction method of the head and neck squamous cell carcinoma related fibroblast and organoid co-culture system according to claim 1, characterized in that: The Transwell cell described in step (4) is a Transwell cell with an aperture of 0.4 μm; The specific operation of the digestion described in step (4) is as follows: take the head and neck squamous cell carcinoma-related fibroblasts obtained in step (2), add 0.25% trypsin to digest at 37±2°C for 2±1 minutes; take step (3) The resulting head and neck squamous cell carcinoma-related fibroblasts were pipetted 10±2 times with a pipette, and then physically digested; The inoculation amount of head and neck squamous cell carcinoma tissue organoid cells described in step (4) is calculated by inoculating 1 to 1.2× ¹⁰⁴ per well of a 24-well plate; The number ratio of head and neck squamous cell carcinoma related fibroblasts and head and neck squamous cell carcinoma tissue organoid cells described 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. 6. The construction method of the head and neck squamous cell carcinoma related fibroblast and organoid co-culture system according to any one of claims 1-5, 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 buccal cancer. 7. A co-culture system of head and neck squamous cell carcinoma-related fibroblasts and organoids, characterized in that it is obtained by the construction method described in any one of claims 1-6. 8. A method for constructing head and neck squamous cell carcinoma tissue organoids with high drug resistance and high stemness, characterized in that: the head and neck squamous cell carcinoma-related fibroblasts and organoids described in claim 7 are used In the co-culture system, the transwell chamber is taken out, and the fibroblasts in the upper layer are removed to obtain the head and neck squamous cell carcinoma tissue organoid with high drug resistance and high stemness. 9. A head and neck squamous cell carcinoma tissue organoid with high drug resistance and high stemness, characterized in that it is obtained by the construction method described in claim 8. 10. The application of the head and neck squamous cell carcinoma tissue organoid with high drug resistance and high stemness described in claim 9 in the preparation of drug evaluation or screening models.

Images