CN114369573B - Methods for constructing orthotopic primary nasopharyngeal carcinoma animal model - Google Patents

Abstract

The invention discloses a preparation method of an in-situ primary nasopharyngeal carcinoma tumor model, and belongs to the field of tumor animal models. The invention cultures the mouse nasopharyngeal cells into organoids by a specific culture medium, then carries out gene editing on the organoids, and injects the organoids back into the mouse nasopharynx to enable the organoids to develop into tumors. Compared with a genetic engineering tumor animal model, the method provided by the invention has the advantages of short time consumption and high tumor formation rate; compared with an animal model of transplanted tumor, the method has the in-vivo microenvironment for tumor generation and development, and is more similar to the truest state of nasopharyngeal carcinoma.

Description

Methods for constructing orthotopic primary nasopharyngeal carcinoma animal model

Technical field

The invention belongs to the field of tumor animal models.

Background technique

Nasopharyngeal cancer refers to malignant tumors that occur on the top and side walls of the nasopharyngeal cavity. The epidemic areas are mainly in East Asia and Southeast Asia. It is one of the most common malignant tumors in my country, and its incidence rate ranks first among malignant tumors in the ear, nose and throat.

In the process of studying the occurrence and development mechanism of nasopharyngeal cancer and developing drugs to treat nasopharyngeal cancer, animal models of nasopharyngeal cancer are inseparable.

Currently, nasopharyngeal cancer animal models commonly used in scientific research are mainly divided into three categories, including genetically engineered animal models, tumor cell line transplanted tumor models, and human-derived xenograft tumor models (PDX, patient derived Xenograft model).

Genetically engineered animal models have a good tumor microenvironment, good reproducibility, and no defects in the immune system. However, they require the preparation of transgenic animals, which is costly and requires a long preparation period. The tumor cell line transplanted tumor model only needs to implant human tumor cell lines into model animals, which is easy to prepare and has high reproducibility. However, it requires the use of immunodeficient mice, and the obtained tumors are not primary tumors in situ, which is consistent with the actual tumor. The development situation is quite different from the pathophysiological situation. The PDX model is a patient's tumor tissue inoculated into a model animal. It is easy to prepare, and its genotype is close to the actual tumor. However, it is not an orthotopic tumor and cannot provide an in situ microenvironment of the nasopharyngeal tissue, which may lead to human tumors during the experiment. Relevant biological characteristics are lost and cannot simulate the situation in the human body. Moreover, clinical tumor specimens are very rare at present. Some special clinical specimens such as puncture specimens and other small specimens have less tissue cells for experimental research. The PDX model of nasopharyngeal carcinoma was successfully constructed. The rate is also relatively low and cannot meet the needs of model construction.

In summary, in order to explore the occurrence and development mechanism of nasopharyngeal carcinoma and develop new therapeutic drugs for nasopharyngeal carcinoma, there is an urgent need for an animal model of nasopharyngeal carcinoma that is close to the biological characteristics of nasopharyngeal carcinoma, has short operation time, is highly reproducible, and has high throughput. .

Contents of the invention

The purpose of the present invention is to provide an orthotopic primary mouse nasopharyngeal carcinoma model that is closer to the biological characteristics of nasopharyngeal carcinoma, has a short preparation period, and has a determined genotype.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

A culture medium for culturing human or animal nasopharyngeal tissue cell organoids, the formula is as follows:

B27 50±5 times concentration dilution N-acetylcysteine 1±0.1mM EGF 50±5ng/mL Noggin 100±10ng/mL R-spondin 1 250±25ng/mL A83-01 200±20nM FGF10 500±50ng/mL Nicotinamide 10±1mM Y-27632 10±1uM WNT3a 25±2.5ng/mL Glutamax 100±10 times concentration dilution N2 100±10 times concentration dilution Gastrin 1±0.1nM

Further, the formula of the culture medium is:

B27 50 times dilution N-acetylcysteine 1mM EGF 50ng/mL Noggin 100ng/mL R-spondin 1 250ng/mL A83-01 200nM FGF10 500ng/mL Nicotinamide 10mM Y-27632 10uM WNT3a 25ng/mL Glutamax 100 times dilution N2 100 times dilution Gastrin 1nM

A method for constructing an orthotopic primary nasopharyngeal carcinoma animal model, including the following steps:

1) Primary culture of human or animal nasopharyngeal tissue cells;

2) Fix the cells in Matrigel, add culture medium and culture them into organoids;

3) Resuspend the organoids into single cells, perform genetic modification, and then culture them into organoids;

4) Inject the organoids obtained in step 3) into the nasopharynx of the animal;

Step 3) The genetic modification refers to knocking out tumor suppressor genes and/or overexpressing proto-oncogenes.

As in the aforementioned method, the medium used for culturing organoids in step 2) is the aforementioned medium.

As mentioned above, step 1) includes:

a. Use trypsin at a final concentration of 0.25% to digest nasopharyngeal tissue;

b. Filter through a 80-130 μm mesh and collect the cells in the filtrate;

c. Use cell culture medium to neutralize the filtrate and terminate digestion;

d. 400G, centrifuge for 5 minutes to remove the supernatant, then add cell culture medium to resuspend the cells, and centrifuge again to remove the supernatant;

Preferably, a 100 μm filter is used in step b.

As in the aforementioned method, the cell culture medium in steps c and/or d is DMEM/F12 culture medium.

As mentioned above, step 2) includes:

i. Mix the cells with 30ul of Matrigel. After the Matrigel solidifies, add culture medium for culture to obtain organoids;

ii. Resuspend the organoids in 1X TrypLE for 15 minutes;

iii. Add cell culture medium to wash the cells and terminate digestion;

iv. Centrifuge to remove the supernatant, add cell culture medium to resuspend the cells, disperse the cells, and centrifuge;

v. Add 30ul of Matrigel to resuspend the cells. After the Matrigel solidifies, add culture medium and culture to obtain organoids.

As in the aforementioned method, step 3) also includes transferring a fluorescent marker gene into the organoid.

As mentioned above, the gene editing in step 4) is specifically: overexpressing the cMYC gene, the Kras G12D mutant gene, and knocking out the Cdkn2a gene;

The nasopharyngeal carcinoma is poorly differentiated squamous cell carcinoma.

As mentioned above, it also includes:

Step 5): Perform in vivo imaging observations every 2-3 weeks to detect tumor formation.

As in the aforementioned method, the animals described in steps 1) and 4) are mice.

The method of the present invention has the following beneficial effects:

1) Compared with the tumor cell line transplanted tumor model and the human xenografted tumor model, the model constructed by the method of the present invention does not require the use of immunodeficient animals, and is an orthotopic tumor, which can simulate the normal cells caused by genetic changes in the human body. The process of transformation into tumor cells can dynamically represent the process of tumor occurrence and development, and is closer to the actual situation of tumor occurrence and development in terms of genes, tumor microenvironment, tumor development and pathophysiology.

2) Compared with genetically engineered animal models, the tumor model construction method of the present invention does not need to start from fertilized eggs or embryos, its cycle can be significantly shortened, and there will be no problem of early death of animals caused by systemic gene mutations.

3) The tumor model construction method of the present invention has a high success rate, which can reach 75%.

Obviously, according to the above content of the present invention, according to the common technical knowledge and common means in the field, without departing from the above basic technical idea of the present invention, various other forms of modifications, replacements or changes can also be made.

The above contents of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

Description of the drawings

Figure 1: Schematic diagram of the process for constructing orthotopic primary nasopharyngeal tumors in mice.

Figure 2: Growth status of fresh mouse nasopharyngeal tissue from days 1 to 11 after trypsin digestion into single cells and culture in Martrilgel.

Figure 3: In vitro culture of mouse nasopharyngeal organoids.

Figure 4: HF staining of mouse nasopharyngeal organoids.

Figure 5: Gene editing of mouse nasopharyngeal organoids. A. Schematic diagram of the construction of the nasopharyngeal cancer model of the present invention; B. Schematic diagram of the original gene editing of mouse nasopharyngeal organoids, editing the original Cdkn2a gene: U6 Promotor and EFS are promoters, Cdkn2a is the sgRNA sequence targeting the Cdkn2a gene, and mCherry is the fluorescence Protein nucleic acid sequence; overexpression of cMYC gene original: pMSCV and iRES are promoters, cMYC/Kras (G12D) is the gene sequence; C. After lentivirus transfection, nasopharyngeal organoids, mCherry (red fluorescence) detects the Cdkn2a original in the organoids Organ infection efficiency; D.T7 detects Cdkn2a gene mutation; E. Detects expression of cMYC/Kras (G12D) original in organoid infection efficiency. Luciferase value above 100,000 is considered to have higher infection efficiency.

Figure 6: In vivo imaging on days 15 and 30 after orthotopic transplantation of nasopharyngeal organoids in gene-edited mice and tumor samples collected after the mice were sacrificed. A, In vivo imaging; B, Fluorescence in vivo imaging of the skull; C, Fluorescence imaging of tumor tissue.

Figure 7: Pathological HF staining and immunohistochemical staining of Ki67, CK5 and P63 in primary mouse nasopharyngeal tumors in situ.

Detailed ways

Some English abbreviations in the present invention are explained as follows:

DMEM: It is a very widely used culture medium that can be used for many mammalian cell cultures. It is purchased from GIBCO.

DMEM/F12: The combination of F12 medium and DMEM medium at a ratio of 1:1 is called DMEM/F12 medium. It combines the advantages of F12 containing richer ingredients and DMEM containing higher concentrations of nutrients. Purchased from GIBCO Company.

Matrigel is isolated from EHS mouse tumors rich in extracellular matrix proteins. Its main components are composed of laminin, type IV collagen, nestin, heparin sulfate glycoprotein, etc. It also contains growth factors and matrix metalloproteinases. Purchased from BD.

B27, B27 supplement, is a commercially available product that can be used to prepare culture media. The B27 supplement is supplied as a 50x liquid concentrate and contains, among other ingredients, biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, and selenite. Sodium, triiodothyronine (T3), DL-alpha-tocopherol (vitamin E), albumin, insulin, and transferrin. Purchased from Life Technologies. N-acetylcysteine: N-acetylcysteine, purchased from Sigma.

EGF, epidermal growth factor, a commercial product purchased from R&D Company.

Noggin, a cell growth protein component, is a commercial product purchased from Peprotech.

R-spondin 1, a human cell growth-encoding protein, is a commercial product purchased from Peprotech.

A83-01, TGF-β inhibitor, was purchased from Tocris Bioscience.

FGF10, fibroblast growth factor, was purchased from Peprotech.

Nicotinamide was purchased from Sigma.

Y-27632*, ROCK-specific pathway blocker. Purchased from Abmole Bioscience.

WNT3a, WNT agonist, a factor that activates TCF/LEF-mediated transcription in cells, was purchased from PeproTech.

Glutamax, a commercially available cell culture additive, was purchased from GIBCO.

N2, N2 supplement is supplied as a 100x liquid concentrate containing 500μg/ml human transferrin, 500μg/ml

Bovine insulin, 0.63 μg/ml progesterone, 1611 μg/ml putrescine and 0.52 μg/ml sodium selenite. Purchased from Life Technologies.

Gastrin, purchased from Sigma.

TrypLE, a recombinant digestive enzyme used to dissociate adherent mammalian cells, was purchased from GIBCO.

Kras (G12D), Kras G12D mutant gene, that is, the mutant gene obtained by mutating the 12th amino acid of the Kras gene from G to D.

Embodiment 1 Modeling method of the present invention

1. Primary cell culture

Use a stereomicroscope to dissect the mouse nasopharyngeal tissue, use small scissors to cut the mouse nasopharyngeal tissue, preferably on ice or in an equivalent low-temperature environment, or mechanically crush in an equivalent low-temperature environment; take the chopped tissue pieces, Digest with 5 mL trypsin, incubate on a shaking table at 37°C for 1 hour, and pipette a dozen times with a pipette every 15-20 minutes to prevent tissue clumping and fully digest single cells.

Filtration: Use 80-130μm cell mesh to filter cells, preferably 100μm cell mesh; after filtration, add 15-20mL DMEM/F12 to rinse the filter membrane and terminate digestion, centrifuge to remove the supernatant; centrifuge to remove the supernatant; the centrifugation temperature is preferred Centrifuge at 400-450g for 4-6 minutes at 2-8°C; add DMEM/F12 to resuspend, preferably add 10 mL DMEM/F12 to resuspend, and then centrifuge to remove the supernatant.

2. Organoid culture

2.1 Pre-culture

Count the cells, mix Matrigel, 20,000 cells per 30 μL, drop into a 48-well plate well; transfer to incubator, 36-38°C, 3-8% CO2 environment for 10-20 minutes, solidify Matrigel; add 150 μL cell culture medium to each well , the preferred cell culture medium is a conditioned medium, cultured in a 36-38°C, 3-8% CO2 cell incubator; the culture medium is replaced every 4-6 days to culture mouse nasopharyngeal organoids. The growth status of fresh mouse nasopharyngeal tissue in Martrilgel from days 1 to 11 after trypsin digestion into single cells is shown in Figure 2.

The formula of the conditioned medium is as follows:

Note: 50X refers to 50 times the concentration, 100X refers to 100 times the concentration, and so on.

2.2 Expand training

Collect the organoids cultured in the culture dish for about 8-12 days, resuspend and digest the organoids in 1X TrypLE, and digest at 36-38°C for 5-15 minutes; preferably resuspend the organoids in 1ml TrypLE and digest at 37°C for 15 minutes. Digestion is terminated by adding DMEM/F12; preferably, 5 ml DMEM/F12 is added to terminate digestion.

After resuspension treatment, the dispersion of cells in the organoids is restored and original cells are provided for expansion and culture.

Centrifuge to remove the supernatant. The preferred centrifugation process is 2-8°C, 400-500g for 5 minutes; preferably 400g, 4°C for 5 minutes. Add Matrigel to resuspend and drop into the wells of the 48-well plate. It is preferred to add an appropriate amount of Matrigel reagent for resuspension. Preferably, Matrigel is first melted on ice and then added to the previously treated cell solution. Transfer to a petri dish and solidify Matrigel at 36-39°C and 3-8% CO2. It is best to place the Petri dish in a 37°C (5% CO2) environment for 10 minutes to solidify Matrigel. Matrigel completes phase transition at body temperature to form a gel-like transition. Add 150 μL of conditioned medium to each well and culture in a 36-39°C, 3-8% CO2 cell culture incubator. The composition of the conditioned medium was identical to that used in the preparation of nasopharyngeal tissue. The culture medium was changed every 4-6 days, and nasopharyngeal tissue organoids were cultured, as shown in Figure 3.

H&E staining of organoids, as shown in Figure 4, nasopharyngeal tissue organoids are composed of multi-cells, hollow or solid, and have good activity characteristics.

3. Genetic modification of nasopharyngeal cells

CRISPR/Cas9 gene editing technology was used to knock out the Cdkn2a gene in nasopharyngeal cells, and gene overexpression technology was used to overexpress the cMYC gene and Kras G12D mutant gene (abbreviated as "Kras (G12D)") in nasopharyngeal cells (Figure 5A and B).

3.1 The editing operation method is as follows:

(1) Pack the lentivirus (or retrovirus) carrying the genetic modification original component (vector for CRISPR/Cas9 gene editing, carrying sgRNA and Cas9 protein coding genes, and m-Cherry red fluorescent reporter gene), and use DMEM culture medium to Culture 293T cells in a 6-well plate with 2 mL of culture medium per well. After the cells have grown to fill the 6-well plate, use calcium phosphate precipitation to package the virus. Change the culture medium every 12 hours. Collect the virus liquid at 36 and 48 hours and use Filter the virus liquid with a 022um filter membrane and store it at 4°C for later use. The virus liquid should be used within a week.

(2) Digest the cultured mouse nasopharyngeal organoids into single cells, mix 500-800ul of each virus liquid with mouse nasopharyngeal cells evenly, add ploybrene at a ploybrene: virus liquid volume ratio of 1:1000, and mix the cell viruses Transfer the suspension to a 24-well plate, centrifuge at 2000 rpm for 1 hour, and then place it in a 37°C incubator for 1.5-2 hours; pipette the virus liquid to resuspend the cells, centrifuge to remove the supernatant, and centrifuge at 400-500g for 5 minutes.

(3) Add Matrigel to resuspend and drop into the wells of the 48-well plate. It is preferred to add an appropriate amount of Matrigel reagent for resuspension. Preferably, Matrigel is first melted on ice and then added to the previously treated cell solution. Transfer to a Petri dish and place the Petri dish in a 37°C (5% CO2) environment for 10 minutes to solidify Matrigel. Add 150 μL of conditioned medium to each well and culture in a 37°C (5% CO2) cell culture incubator. The composition of the conditioned medium is identical to that used in the preparation of nasopharyngeal tissue. The culture medium is changed every 4-6 days to culture nasopharyngeal tissue organoids or cell groups.

Viral infection efficiency and gene editing status can be identified by fluorescence signal, fluorescence value and T7 enzyme digestion (Figure 5C and D).

3.2 Gene overexpression operation methods are as follows:

The cMYC gene and the Kras (G12D) gene were inserted into the multiple cloning sites of the commercially available overexpression vector to obtain a recombinant overexpression vector, which carries the Luciferase gene (Figure 5B). The recombinant overexpression vector is then packaged into a lentivirus to obtain a recombinant lentivirus.

Then transfer the recombinant lentivirus into nasopharyngeal cells. The steps are the same as steps (2) and (3) in Section 3.1.

By detecting the Luciferase signal value, the expression of genes in the overexpression vector can be monitored. Luciferase values above 100,000 are considered to have high infection efficiency (Figure 5E).

4. Orthotopic transplantation of mouse nasopharyngeal organoids

Collect the gene-edited nasopharyngeal cells and culture them for 5-7 days to obtain nasopharyngeal organoids. Resuspend the organoids in 1ml TrypLE per well and digest at 37°C for 5 minutes. Add DMEM/F12 to terminate the digestion. It is best to add 5ml DMEM/F12 to terminate the digestion. Digest; centrifuge at 400g for 5 minutes, remove the supernatant; resuspend the cells in 10-15ul Matrigel and place on ice.

Use isoflurane to anesthetize Nude mice in an anesthesia machine. After anesthesia, the mice lie flat and look up, and use tape to fix the limbs of the mice. Use an insulin needle to absorb the Matrigel cell suspension, use surgical tweezers to open the mouth of the mouse, and suspend the cells. The liquid was transplanted into the nasopharynx of the mice; after the operation, the mice were returned to the SPF animal room for rearing. Sterile water and feed were replaced regularly. The condition of the mice was detected, and in vivo imaging was performed every 2 weeks. When the mice's condition began to deteriorate, the mice were killed by cervical dissection and tumor samples were collected.

Figure 6A shows the live imaging on days 15 and 30 after transplantation. It can be seen that the area occupied by the transplanted gene-edited cells has obviously become larger. Figure 6B and Figure 6C respectively show the detection of mCherry and GFP fluorescence signals in the skull and nasopharynx of mice after sacrifice. mCherry and GFP fluorescence signals can be detected. The infected DNA original carries mCherry, and the Trp53-/-Cas9 gene is endogenous in mice. Sexually expressing GFP protein, mCherry and GFP positive signal tissue are derived from exogenous transplanted cells.

Pathological HE staining and immunohistochemical staining were performed on nasopharyngeal tumors, as shown in Figure 7. The results showed that mice transplanted with gene-edited organoids formed masses in the nasopharynx, with deeply stained nuclei and large heterogeneity. The nuclear-to-cytoplasmic ratio is large, and the nuclei of some cells are exposed; the tumor tissue is Ki67-positive, and the classic squamous markers CK5 and P63 are positive. It is a poorly differentiated squamous cell carcinoma, which is consistent with clinical nasopharyngeal carcinoma, which is mainly poorly differentiated squamous cell carcinoma.

In this example, a total of 4 mice were used for modeling, and solid tumors were detected in 3 of them. The modeling success rate was 75%, which is a high success rate.

In summary, the method of the present invention can efficiently prepare a nasopharyngeal carcinoma model that is closer to the characteristics of nasopharyngeal carcinoma and meets the needs of clinical research; this model can be used to explore the occurrence and development mechanism of nasopharyngeal carcinoma, and to find and optimize new nasopharyngeal carcinoma. Areas of research such as possible treatments provide helpful tools.

Claims (8)

1. A method for constructing an orthotopic primary nasopharyngeal carcinoma animal model, characterized by comprising the following steps: 1) Primary culture of mouse nasopharyngeal tissue cells; 2) Fix the cells in Matrigel, add culture medium and culture them into organoids; 3) Resuspend the organoids into single cells, perform genetic modification, and then culture them into organoids; 4) Inject the organoid obtained in step 3) into the nasopharynx of the mouse; Step 3) The genetic modification refers to knocking out tumor suppressor genes, and/or overexpressing proto-oncogenes, specifically: overexpressing cMYC gene, Kras G12D mutant gene, knocking out Cdkn2a gene; the nasopharyngeal carcinoma is poorly differentiated. Squamous cell carcinoma. 2. The method of claim 1, characterized in that: Step 2) The medium formula used to culture organoids is as follows: B27 50±5 times concentration dilution N-acetylcysteine 1±0.1mM EGF 50±5ng/mL Noggin 100±10ng/mL R-spondin 1 250±25ng/mL A83-01 200±20nM FGF10 500±50ng/mL Nicotinamide 10±1mM Y-27632 10±1uM WNT3a 25±2.5ng/mL Glutamax 100±10 times concentration dilution N2 100±10 times concentration dilution Gastrin 1±0.1nM . 3. The method according to claim 2, characterized in that: the formula of the culture medium is: 4. The method according to any one of claims 1-3, characterized in that: Step 1) includes: a. Use trypsin at a final concentration of 0.25% to digest nasopharyngeal tissue; b. Filter through a 80-130 μm mesh and collect the cells in the filtrate; c. Use cell culture medium to neutralize the filtrate and terminate digestion; d. Centrifuge at 400G for 5 minutes to remove the supernatant, then add cell culture medium to resuspend the cells, and centrifuge again to remove the supernatant. 5. The method of claim 4, characterized in that step b uses a 100 μm filter. 6. The method according to any one of claims 1-3, characterized in that: Step 2) includes: i. Mix the cells with 30ul of Matrigel. After the Matrigel solidifies, add culture medium for culture to obtain organoids; ii. Resuspend the organoids in 1X TrypLE for 15 minutes; iii. Add cell culture medium to wash the cells and terminate digestion; iv. Centrifuge to remove the supernatant, add cell culture medium to resuspend the cells, disperse the cells, and centrifuge; v. Add 30ul of Matrigel to resuspend the cells. After the Matrigel solidifies, add culture medium and culture to obtain organoids. 7. The method according to any one of claims 1-3, characterized in that: Step 3) also includes transferring a fluorescent marker gene into the organoid. 8. The method according to any one of claims 1-3, characterized in that: It also includes: Step 5): Perform in vivo imaging observations every 2-3 weeks to detect tumor formation.