CN113943755A

CN113943755A - Method for constructing in-situ primary esophageal cancer animal model

Info

Publication number: CN113943755A
Application number: CN202010684524.2A
Authority: CN
Inventors: 陈崇; 王健; 刘玉
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-07-15
Filing date: 2020-07-15
Publication date: 2022-01-18
Anticipated expiration: 2040-07-15
Also published as: CN113943755B

Abstract

The invention discloses a preparation method of an in-situ primary esophageal cancer tumor model, and belongs to the field of tumor animal models. The mouse esophagus cells are cultured into organoid by specific culture medium, then the organoid is subjected to gene editing and injected back to mouse esophagus, so that the organoid can be developed into tumor. Compared with a gene engineering tumor animal model, the method has the advantages of short time consumption and high tumor formation rate; compared with a transplanted tumor animal model, the tumor model has the characteristics of clear tumor generation driving factors, truer organ microenvironment and the like.

Description

Method for constructing in-situ primary esophageal cancer animal model

Technical Field

The invention belongs to the field of tumor animal models.

Background

The incidence rate of the esophageal cancer in China is as high as 13.9/10 ten thousand people, which is far higher than the average level in the world. Meanwhile, the five-year survival rate of the esophageal cancer at the middle and late stages is only 10% -20%, so that the esophageal cancer becomes a malignant tumor which seriously affects the health of the national people. The mouse esophagus organ culture and the establishment of a tumor model provide a possible technical platform for researching the generation and development of esophagus cancer and exploring various treatment means.

Traditional cell culture techniques, or cell line culture, are often of a single cell type, and during the process of cell immortalization, genetic information is shifted, lost or certain specific chromosome segments are added. Normal tissues or tumors have strong heterogeneity, and cell lines of a single species have great difference from the in vivo state, and the influence of genes on the resistance of some drugs cannot reflect the true condition of patients. Organoids (organoids) are cultured using tissue cells under in vitro 3D culture conditions and can exhibit cell composition and physiological functions similar to those of cells in vivo in vitro. Scientists have succeeded in culturing tissues and organoids such as liver, small intestine, etc. using mature human tissue cells under in vitro 3D culture conditions. The technology can be used in the related fields of tissue development, tumorigenesis, drug testing and the like.

At present, no primary in-situ model exists for esophageal tumor, and the existing model can not well reflect the influence of genes in the process of tumorigenesis and development.

TABLE 1 animal models of esophageal carcinoma

A subcutaneous transplantation model needs to transplant a large number of tumor cell lines under the skin of an immunodeficient mouse, the cell lines are difficult to characterize the tumor characteristics of a patient, and subcutaneous tumors cannot reflect the ecology of esophageal tissues. Besides the defect that the PDX model derived from a patient is transplanted under the skin, the success rate of the model depends on the specimen due to the difference of the patient and the difference of the specimen material, and the breeding cost of the severe immunodeficiency mouse is too high and the difficulty is very high. The mouse esophagus is slender, the operation difficulty is very high, and special equipment is needed for monitoring tumor formation in the orthotopic transplantation model, but the model can simulate the ecological environment of esophageal tumor. The background of the gene mouse model is very clear, the position of the tumor is also possible to be in situ in the esophagus, but the model has very high cost, and the breeding period of the gene mouse is too long. The carcinogen-induced model is the oldest one of the esophageal cancer models, and depends largely on the genetic background of the mouse, which is difficult to simulate in practice the tumorigenesis of patients.

Disclosure of Invention

The invention aims to provide an in-situ primary esophageal cancer model which is closer to the biological characteristics of esophageal cancer and has short preparation period.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method for constructing an in situ primary esophageal cancer animal model comprises the following steps:

1) primary culture of human or animal esophageal cells;

2) culturing the primary cells into organoids;

3) performing gene editing on the organoids obtained in the step 2);

4) injecting the organoid successfully edited by the gene into the esophageal tissue of the animal;

the gene editing in the step 3) refers to mutation of the cancer suppressor gene and/or high-expression protooncogene.

As the method, the isolation step of the mouse esophageal primary cells is also included before the step 1):

a. digesting the esophagus by using Trypsin with the final concentration of 0.25% and DNase I with the final concentration of 0.1 mg/mL;

b. neutralizing Trypsin in a DMEM medium containing 10% FBS, filtering by using a screen to obtain single cells, washing the medium, and centrifuging to stop the enzymatic digestion reaction;

preferably, the medium in step b is DMEM/F12 medium.

As in the previous method, step 2) includes the following steps:

mixing the primary esophageal cells of the mouse with Matrigel with the protein concentration of 8-12mg/ml, and adding an organoid culture medium to culture after the Matrigel is solidified;

the culture medium is DMEM/F12, and is obtained by adding 12-13 of the following cytokines:

cytokine	Alternative concentration ranges	Cytokine	Alternative concentration ranges
				B27
	50 times dilution	EGF	50～100ng/ml
				R-spondin 1	250～1000ng/ml	FGF-10	500～1000ng/ml
Y-27632	10～50uM	Glutamax	100 times dilution
				Gastrin	1～5nM	N-acetylcysteine	1～2mM
Noggin
		100～1000ng/ml	A83-01	200～500nM
Nicotinamide					10～50mM	Wnt-3a	50～100ng/ml
	N2	Diluting by 100-400 times

Wherein Gastrin, N2 and Y-27632 are indispensable;

the Glutamax is a GIBCO product;

the N2 is a GIBCO product: n-2 Supplement;

the B27 is a GIBCO product: b27Supplement serum free.

As in the previous method, the organoid culture medium additives are:

cytokine	Alternative concentration ranges	Cytokine	Alternative concentration ranges
				B27
	50 times dilution	EGF	50ng/ml
				R-spondin 1	250ng/ml	FGF-10	500ng/ml
Y-27632	10uM	Glutamax	100 times dilution
				Gastrin	1nM	N-acetylcysteine	1mM
Noggin	100ng/ml	A83-01	200nM
				Nicotinamide	10mM	Wnt-3a	50ng/ml
N2
		100 times dilution

The method is as described above, wherein the temperature of the enzymolysis is 37 ℃;

and/or the enzymolysis time is 5-10 min.

The method as described above, wherein the esophageal cancer is esophageal squamous carcinoma;

the gene editing in the step 3) is specifically as follows:

mutant Trp53, Pten, and Smad4 genes; high expression of cMyc and Kras^G12D(mutation of the 12 th amino acid of Kras protein from G to D) gene.

The method as described above, wherein the esophageal cancer is esophageal adenocarcinoma;

the gene editing in the step 3) is specifically as follows:

mutation of Trp53 and Kmt2d genes to highly express cMyc and Kras^G12DA gene.

As in the previous method, the gene editing of step 3) further comprises transferring a fluorescent marker gene into the organoid.

The method as described above, wherein the animal is a mouse in steps 1) and 4).

The animal model prepared by the method is applied to drug screening, drug toxicity tests or immunotherapy tests.

The invention has the beneficial effects that:

compared with a gene engineering animal model, the tumor model construction period of the invention is greatly shortened, and the death of the animal before the tumor formation can not be caused, and the molding success rate reaches 100 percent.

The mouse esophageal tumor model in situ can simulate the process of transformation from normal cells to tumor cells caused by genetic change in a human body, can dynamically represent the process of tumor development and development, and is closer to the real situation of tumor development and development in the aspects of gene level, tumor microenvironment, tumor development, pathophysiology and the like.

In a word, the method can efficiently prepare the esophageal cancer model which is closer to the esophageal cancer characteristics and meets the requirements of clinical research; the model can provide a favorable tool in research fields of exploring the esophageal cancer occurrence and development mechanism, searching and optimizing a new possible treatment mode of esophageal cancer and the like.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 13D schematic of culturing of esophageal organoids.

FIG. 2 shows an esophageal organoid photomontage.

FIG. 3 esophageal organoid passage.

FIG. 4 fluorescence detection of esophageal organoid gene editing.

FIG. 5 mouse organoid gene editing T7E1 assay.

FIG. 6 mice were observed for neoplasia over time after esophageal surgery.

FIG. 7 mouse esophageal adenocarcinoma formed by organoid transplantation.

FIG. 8 esophageal squamous carcinoma following mouse organoid transplantation.

FIG. 9 statistics of clonogenic of mouse organoids after deletion of a cytokine; some factors in the figures are abbreviated: NAC means N-acetVlcystein; nico refers to Nicotinamide; y27632 refers to Y-27632; r-spondin refers to R-spondin 1; wnt3a refers to Wnt-3 a.

FIG. 10 diameter statistics of mouse organoids after deletion of certain cytokines; some factors in the figures are abbreviated: NAC means N-acetylcysteine; nico refers to Nicotinamide; y27632 refers to Y-27632; r-spondin refers to R-spondin 1; wnt3a refers to Wnt-3 a.

Detailed Description

The partial english abbreviations in the present invention are explained as follows:

DMEM: is a very widely used culture medium, can be used for culturing a plurality of mammalian cells and is purchased from GIBCO company.

DMEM/F12: is F12 medium and DMEM medium according to 1: 1 in combination, designated DMEM/F12 medium. Combines the advantages of the F12 containing richer components and the DMEM containing higher concentrations of nutrients. Purchased from GIBCO corporation.

Matrigel: is separated from EHS mouse tumor rich in extracellular matrix protein, and mainly comprises laminin, type IV collagen, nidogen, heparin sulfate glycoprotein, growth factor, matrix metalloproteinase and the like. Purchased from BD corporation.

B27, a B27supplement, a commercially available product, can be used to formulate the media. The B27supplement is provided as a 50-fold liquid concentrate that contains, among other ingredients, biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol acetate, sodium selenite, triiodothyronine (T3), DL-alpha-tocopherol (vitamin E), albumin, insulin, and transferrin. Purchased from Life Technologies, Inc. N-acetyl cysteine: n-acetylcysteine, purchased from Sigma.

EGF: epidermal growth factor, a commercially available product, purchased from R & D.

Noggin, a cell growth protein component, a commercially available product, purchased from Peprotech corporation.

R-spondin 1: human cell growth encoded protein, commercially available from Peprotech corporation.

A83-01: TGF-. beta.inhibitors, purchased from Tocris Bioscience, Inc.

FGF 10: fibroblast growth factor, purchased from Peprotech corporation.

Nicotinamide, niacinamide, purchased from Sigma.

Y-27632: a ROCK-specific pathway blocker. Purchased from Abmole Bioscience, Inc.

Wnt3 a: wnt agonists, a factor in cells that activates TCF/LEF-mediated transcription, were purchased from PeproTech, Inc.

Glutamax: commercially available cell culture additives, purchased from: gibco Corp.

N2: the N2 supplement is provided as a 100-fold liquid concentrate containing 500 μ g/ml human transferrin.

Gastrin: gastrin, purchased from Sigma.

TrypLE: recombinant digestive enzymes for dissociation of adherent mammalian cells, purchased from GIBCO.

EXAMPLE 1 organoid culture method of the present invention

Comprises the steps of obtaining fresh esophageal tissue cells, and digesting the esophageal tissue cells into single cells by pancreatin; in vitro 3D culture conditions for esophageal tissue organoids are shown in FIG. 1, and the organoid effects are shown in FIG. 2.

Required reagents:

1. mouse esophagus organ culture medium (ingredients in Table 2)

2.TryplE(GIBCO)

3. 0.25％Trypsin-EDTA(GIBCO)

4.DNaseI(5mg/ml)

5.Matrigel(corDing)

6.75% ethanol

7.DPBS basic(gobo)

8.1X ACK

9.DMEM(GIBCO)+10％FBS

Table 2 mouse esophageal organoid medium composition (in DMEM/F12):

the required instruments are as follows:

1. scissor tweezers

2. Six-hole plate

3.50 ml BD tube

4.48 orifice plate

5. Ice box

6. Water-absorbing paper

7.70 um cell sieve

The method comprises the following steps:

1. mice were euthanized and the carcasses were sprayed repeatedly with 75% ethanol.

2. The mice were placed on absorbent paper, the abdominal cavity and thoracic cavity were opened, and the trachea and esophagus were cut at the neck. The stomach was pulled with forceps and the esophagus was removed.

3. Esophageal tissue was repeatedly rinsed in pre-chilled PBS and forceps were used to remove any significant blood stain from the esophageal tissue.

4. The esophageal tissue was minced with scissors.

5. The esophageal tissue was resuspended in 15ml Trypsin in 50ml BD tube, 10ul DNase I was added and digested in a 37 deg.C water bath for 1 hour, during which time the tube was inverted every ten minutes.

6. Trypsin was neutralized with an equal volume of DMEM + 10% FBS.

7. The tissue suspension from the sixth step is passed through a 70um sieve.

8.1500 rpm 10 mins.

9. The supernatant was removed, the cells were resuspended in 5ml of ACK on ice and allowed to stand for 3 mins.

10.1500rpm 5 mins.

11. The supernatant was removed and 120ul matrigel was added to resuspend the cells on ice, seeded in 48 well plates at 30 ul/well.

12. And (4) solidifying for 15mins in a 37-degree incubator.

13. 150ul of medium was added to each well and PBS was added around to prevent evaporation to dryness.

14. Observed daily, the medium turned yellow and was replaced with new medium or passaged.

Note that:

1. when pulling the stomach with forceps, care should be taken to remove the esophagus sufficiently free, otherwise the esophagus will break at the cardia.

2. In the step 5, the digestion time is not too long, and pancreatin has great damage to cells.

3. Step 9 may be omitted if no visible red blood cells are visible.

4. The supernatant in the 11 th step cannot be removed completely, and the gel strength is insufficient due to insufficient setting time in the 12 th step.

5. The Matrigel of step 11 was melted in a refrigerator at 4 degrees 20min in advance and the whole operation was performed on ice.

Example 2 organoid passaging

Required reagents:

1. mouse esophagus organ culture medium (same table 2)

2.TryplE(GIBCO)

3.Matrigel(corning)

4.DPBS basic(GIBCO)

The required instruments are as follows:

1.50 ml BD tube

2.15 ml BD tube

3.48 orifice plate

4. Ice box

5.70 um cell sieve

The method comprises the following steps:

1. the medium was aspirated off the organoids and 1ml TryplE was added to each well.

2. Triturate matrigel with 1ml of the tip of the gun, aspirate all into BD tubes and replenish TryplE to 15 ml.

The mixture was incubated in a water bath at 3.37 ℃ for 15 mins.

4. The organoids were repeatedly blown with a 10ml pipette and filtered through a 70um cell sieve into a new BD tube.

5.1500 rpm 10 mins.

6. Remove supernatant, add 120ul matrigel in ice heavy suspension cells, seed in 48 well plates, 30 ul/well.

7. And (4) solidifying for 15mins in a 37-degree incubator.

8. 150ul of medium was added to each well and PBS was added around to prevent evaporation to dryness.

9. Observed daily, the medium turned yellow and was replaced with new medium or passaged.

Note that:

1. the generation is distinguished according to different purposes, if the organoid is used for amplifying, the organoid is as dense as possible and is beneficial to growth of the organoid, and if the organoid is used for photographing and dyeing, the organoid is as sparse as possible and is beneficial to differentiation of the organoid.

The selection of 2.15 ml BD tubes and 50ml BD tubes should depend on the number of organoids, and if the number is larger, 50ml BD tubes should be used, and the number is smaller, 15ml BD tubes should be used.

3. And a 70um filter screen is used for filtering during passage to remove larger organoids, and the organoids occupy a large space in the new matrigel and influence the growth of the new organoids.

The effect of passaging is shown in FIG. 3.

Example 3 Gene editing

Required reagents:

1. mouse esophagus organ culture medium (same table 2)

2.TryplE(GIBCO)

3.Matrigel(corning)

pLenti-Virus (vector carrying Cas9 gene, and vector expressing sgRNA targeting Trp53, Kmt2d, Smad4 and Pten gene, frame shift mutation of Trp53 gene, frame shift mutation of Kmt2d gene, frame shift mutation of Smad4 gene, and frame shift mutation of Pten gene.)

Retro-Virus (carrying cMyc, Kras)^G12DProtooncogene, and D-luciferase fluorescein

Enzyme reporter genes, enabling cMyc and Kras^G12DHigh expression of protooncogene

6.Polybrane(4mg/ml)

7.DPBS basic(GIBCO)

The required instruments are as follows:

1.50 ml BD tube

2.15 ml BD tube

3.48 orifice plate

4.24 orifice plate

5. Ice box

6.70um cell screen

The method comprises the following steps:

The mixture was incubated in a water bath at 3.37 ℃ for 15 mins.

5.1500 rpm 10 mins.

6. Cells were resuspended in organoid, polybrane 1: 1000 using virus and added to 24-well plates.

7.2000 rpm for 1 hour at 32 degrees.

Incubate at 8.37 ℃ for 4 hours.

9. The organoids were collected into 15ml small BD tubes.

10.1500rpm 10 mins.

11. Remove supernatant, add 120ul matrigel in ice heavy suspension cells, seed in 48 well plates, 30 ul/well.

12. And (4) solidifying for 15mins in a 37-degree incubator.

Fluorescence was observed for 14.48 hours.

Note that:

1. before infection, as many cells as possible should be ensured, since a considerable number of cells die due to infection.

2. The virus is preferably filtered by a 0.22um filter membrane during infection, so that pollution is avoided. Or centrifugation at 800rpm to remove 293T cells when they were enveloped.

3. The organoids should be planted as densely as possible after the infection is over, in order for the organoids to grow.

And (3) identification result:

the fluorescence identification of organoid gene editing is shown in FIG. 4, and the cleavage identification of T7E1 for gene editing is shown in FIG. 5.

The advantageous effects of the present invention are further illustrated in the form of experimental examples.

Experimental example 1 construction of esophageal adenocarcinoma model

Organoid culture, passage and gene editing were performed in sequence by using the methods of examples 1 to 3, and the obtained organoids with gene editing were transplanted into mouse esophagus, the size and position of tumor were monitored by in vivo imaging technique (see fig. 6), 45 days after transplantation, the mouse was sacrificed, and direct observation and HE staining observation were performed on esophageal tissue.

Mutations in Trp53, Smad4, Pten, cMyc, Kras^G12DIn the high expression experiment group, 5 mice all formed esophageal squamous cell carcinoma in about 45 days, and the success rate is 100%. FIG. 7 is a drawing showingThe appearance graph and the HE staining graph of the esophageal adenocarcinoma show that severe hyperplasia is formed at the junction of the esophagus and the stomach of the mouse, and the pathological result shows that a large amount of malignant hyperplasia of the karyotic gland epithelium is formed.

Mutations in Trp53, Kmt2d, cMyc, Kras^G12DIn the high expression experiment group, 5 mice all formed esophageal adenocarcinoma in about 44 days, and the success rate is 100%. FIG. 8 is an appearance view of esophageal squamous carcinoma and a staining graph by HE, showing that nodules are formed at the junction of mouse esophagus and stomach, and pathological results show that squamous epithelium is malignantly proliferated with a large amount of keratinization.

Experimental example 2 screening experiment of organoid culture Medium composition

The cell factors added into the culture medium during the mouse organoid culture are 12, wherein the cell factors with complex components such as N2, B27 and the like are not lacked, the cost of the cell factors is high, and the cost of the mouse esophageal organoid during the tumor formation test is greatly improved. The inventor removes one of the cytokines one by one, plants the same number of first generation esophagus organoid single cells (3000 cells in 2ul of matrigel) in the same volume of matrigel, evaluates the effect of each cytokine according to the number and the diameter of mouse esophagus organoid formation by taking complete culture medium as a control, and the experiment is totally provided with 3 technical repeats and 3 biological repeats.

As shown in FIG. 9, 3000 cells formed an average of 80 organoids in complete medium with 2ul of matrigel, and removal of any cytokine affected organoid formation efficiency, with removal of N2 resulting in organoids forming only 40 under the same conditions and Y-27632 also resulting in similar results. However, the efficiency of mouse esophageal organoid formation after the removal of A83-01 is not greatly different, and in conclusion, A83-01 is not a critical cytokine in terms of organoid formation efficiency, and the rest various cytokines have more or less negative effects, wherein N2 and Y-27632 are indispensable cytokines for culturing mouse esophageal organoids.

As shown in FIG. 10, 3000 esophageal organoid cells were counted in 2ul of matrigel, and organoid sizes were observed after various organoid cytokines had been deleted. As shown, in complete medium, the organoid size is approximately 55 μm, and after deletion of B27 or EGF, the esophageal organoid size decreases significantly, indicating a deterioration in differentiation ability of esophageal organoids, and after deletion of N2 and Nico, the esophageal organoids develop toward a larger size, but after deletion of N2, the resulting decrease in organoid formation is observed.

In summary, under different requirements (pursuing organoid formation efficiency or size of individual organoids), various cytokines can be reduced as appropriate, further reducing the culture cost.

Claims

1. A method for constructing an in-situ primary esophageal cancer animal model is characterized by comprising the following steps:

1) primary culture of human or animal esophageal cells;

2) culturing the primary cells into organoids;

3) performing gene editing on the organoids obtained in the step 2);

2. The method of claim 1, wherein:

the method also comprises the following steps of 1) separating the primary mouse esophageal cells before the step:

a. esophagus was digested with Trypsin and 0.1mg/mLDNase I at a final concentration of 0.25%;

preferably, the medium in step b is DMEM/F12 medium.

3. The method of claim 1, wherein step 2) comprises the steps of:

Among them, Gastrin, N2 and Y-27632 are indispensable.

4. The method of claim 3, wherein:

the organoid culture medium comprises the following additives:

5. the method of claim 1, wherein:

the temperature of the enzymolysis is 37 ℃;

and/or the enzymolysis time is 5-10 min.

6. The method of claim 1, wherein:

the esophageal cancer is esophageal squamous carcinoma;

the gene editing in the step 3) is specifically as follows:

mutant Trp53, Pten, and Smad4 genes; high expression of cMyc and Kras^G12DA gene.

7. The method of claim 1, wherein:

the esophageal cancer is esophageal adenocarcinoma;

the gene editing in the step 3) is specifically as follows:

mutation of Trp53 and Kmt2d genes to highly express cMyc and Kras^G12DA gene.

8. The method of claim 1, wherein:

the gene editing of step 3) further comprises transferring a fluorescence-labeled gene into the organoid.

9. The method of claim 1, wherein:

the animals of steps 1) and 4) are mice.

10. The use of an animal model prepared by the method of claims 1-9 in drug screening, drug toxicity testing, or immunotherapy testing.