CN114369573A - Method for constructing in-situ 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 mouse nasopharyngeal cell is cultured into organoid in specific culture medium, and the organoid is gene edited and injected back to mouse nasopharynx to develop 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 model has an in-vivo microenvironment for tumorigenesis and development, and is closer to the most real state of nasopharyngeal carcinoma.

Description

Method for constructing in-situ primary nasopharyngeal carcinoma animal model

Technical Field

The invention belongs to the field of tumor animal models.

Background

Nasopharyngeal carcinoma refers to a malignant tumor that occurs in the top and side walls of the nasopharyngeal cavity. Epidemic disease areas are mainly in east Asia and south-east Asia, are one of high-incidence malignant tumors in China, and the incidence rate is the first of the malignant tumors of ear, nose and throat.

In the process of researching the occurrence and development mechanism of nasopharyngeal carcinoma and developing a medicine for treating nasopharyngeal carcinoma, an animal model of nasopharyngeal carcinoma can not be opened.

The nasopharyngeal carcinoma 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 Xenograft tumor models (PDX).

The genetic engineering animal model has good tumor microenvironment and good repeatability, and an immune system is free of defects, but the genetic engineering animal model needs to prepare a transgenic animal, so that the cost is high, and the preparation period is long. The tumor cell line transplanted tumor model only needs to implant the human tumor cell line into a model animal, is easy to prepare and has high repeatability, but an immunodeficiency mouse is needed, and the obtained tumor ex-situ primary tumor is greatly different from the actual development condition and the pathophysiological condition of the tumor. The PDX model is prepared by inoculating tumor tissues in a patient body into a model animal body, is easy to prepare, has a genotype close to that of an actual tumor, but can not provide an in-situ microenvironment of nasopharyngeal tissues due to non-in-situ tumors, further possibly causes the loss of relevant biological characteristics of human tumors in an experimental process, can not simulate the conditions in the human body, is very precious in clinical tumor specimens at present, has less tissue cell amount for experimental research of some special clinical specimens such as puncture specimens and the like, has lower construction success rate of the PDX model of the nasopharyngeal carcinoma, and can not meet the construction requirements of the model.

In conclusion, in order to explore the occurrence and development mechanism of nasopharyngeal carcinoma and develop novel therapeutic drugs for nasopharyngeal carcinoma, an animal model with nasopharyngeal carcinoma, which is close to the biological characteristics of nasopharyngeal carcinoma, short operation time, high repeatability and high throughput, is urgently needed.

Disclosure of Invention

The invention aims to provide an in-situ primary mouse nasopharyngeal carcinoma model which is closer to the biological characteristics of nasopharyngeal carcinoma, has short preparation period and is determined by genotype.

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

a culture medium for culturing human or animal nasopharyngeal tissue cell organoid has the following formula:

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			Dilution of 100 + -10 times concentration
	N2	Dilution of 100 + -10 times concentration
Gastrin
		1±0.1nM

。

Further, the formula of the culture medium is as follows:

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	Dilution by 100 times concentration
		N2	Dilution by 100 times concentration
Gastrin	1nM

。

A method for constructing an in-situ primary nasopharyngeal carcinoma animal model comprises the following steps:

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

2) fixing cells in Matrigel, adding a culture medium to culture the cells into organoids;

3) resuspending the organoid into single cells, performing genetic transformation, and culturing into organoid;

4) injecting the organoid obtained in step 3) into the nasopharynx of the animal;

the genetic modification in the step 3) refers to knockout of an anti-cancer gene and/or over-expression of a proto-oncogene.

As in the previous method, the culture medium used in step 2) for culturing the organoids is the culture medium described above.

As in the previous method, step 1) includes:

a. using pancreatin with a final concentration of 0.25% to digest nasopharyngeal tissues;

b. filtering with 80-130 μm sieve, and collecting cells in the filtrate;

c. neutralizing the filtrate with cell culture medium to stop digestion;

d.400G, centrifuging for 5min to remove the supernatant, adding a cell culture medium to resuspend the cells, and centrifuging to remove the supernatant;

preferably, a 100 μm sieve is used in step b.

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

As in the previous method, step 2) includes:

i. mixing the cells with 30ul of Matrigel, and adding a culture medium to culture after the Matrigel is solidified to obtain organoids;

resuspending organoids with 1X TrypLE for 15 min;

adding cell culture medium to wash the cells and terminating digestion;

centrifuging to remove the supernatant, adding a cell culture medium to resuspend the cells, dispersing the cells, and centrifuging;

v. adding 30ul of Matrigel to resuspend the cells, and adding a culture medium to culture after the Matrigel is solidified to obtain the organoid.

The method as described above, wherein step 3) further comprises transferring the fluorescence-labeled gene into the organoid.

As the method, the gene editing in the step 4) is specifically as follows: overexpresses cMYC gene, Kras G12D mutant gene, knocks out Cdkn2a gene;

the nasopharyngeal carcinoma is hypo-differentiated squamous carcinoma.

As in the previous method, it further comprises:

step 5): in vivo imaging observations were performed every 2-3 weeks to detect tumor formation.

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

The method of the invention has the following beneficial effects:

1) compared with a tumor cell line transplanted tumor model and a human source xenotransplantation tumor model, the model constructed by the method does not need immunodeficient animals, is an in-situ tumor, can simulate the process of transforming normal cells into tumor cells in a human body due to genetic change, can dynamically represent the process of tumor development and development, and is closer to the real condition of tumor development and development in the aspects of gene level, tumor microenvironment, tumor development, pathophysiology and the like.

2) Compared with a genetic engineering animal model, the tumor model construction method does not need to be constructed from fertilized eggs or embryos, the period can be obviously shortened, and the problem of early death of animals caused by whole-body gene mutation can be avoided.

3) The success rate of the tumor model construction method is high and can reach 75%.

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. 1: the process schematic diagram of constructing the mouse in situ primary nasopharyngeal tumor.

FIG. 2: the nasopharyngeal tissue of the fresh mouse is trypsinized into single cells, and then the growth condition of the mouse is cultured in Martrilgel on the 1 st to 11 th days.

FIG. 3: mouse nasopharyngeal organoids are cultured in vitro.

FIG. 4: mouse nasopharyngeal organoid HF staining.

FIG. 5: mouse nasopharyngeal organoid gene editing. A. The nasopharyngeal carcinoma model is constructed schematically; B. schematic diagram of mouse nasopharyngeal organoid gene editing original, editing Cdkn2a gene original: u6 promoter and EFS are promoters, Cdkn2a is an sgRNA sequence of a targeted Cdkn2a gene, and mCherry is a fluorescent protein nucleic acid sequence; overexpression of the original cMYC gene: pMSCV and iRES are promoters, and cMYC/Kras (G12D) is the gene sequence; C. detecting the infection efficiency of the Cdkn2a original in the nasopharynx organoid by mCherry (red fluorescence) after lentivirus transfection; t7 detecting Cdkn2a gene mutation; E. the detection of the cMYC/Kras (G12D) expression original element in organoid infection efficiency, and the infection efficiency is considered to be higher when the Luciferase value is more than 10 ten thousand.

FIG. 6: gene editing mice nasopharyngeal organoid in situ transplantation after 15 days, 30 days live imaging and mice after death collected tumor samples. A, in vivo imaging; b, performing fluorescence living body imaging from the skull; and C, carrying out fluorescence imaging on the tumor tissue.

FIG. 7: in situ primary mouse nasopharyngeal tumor pathology HF staining and Ki67, CK5 and P63 immunohistochemical staining.

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, isolated from tumors of EHS mice rich in extracellular matrix proteins, consisting of laminin, type iv collagen, entactin, heparin sulfate glycoprotein, and the like, as well as growth factors and matrix metalloproteinases, and the like. Purchased from BD corporation.

B27, a B27 supplement, a commercially available product, can be used to formulate the media. The B27 supplement 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, commercially available from R & D.

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

R-spondin 1, human cell growth-encoding protein, commercially available product, purchased from Peprotech corporation.

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

FGF10, fibroblast growth factor, purchased from Peprotech.

Nicotinamide, niacinamide, purchased from Sigma.

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

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

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

The N2, N2 supplement is provided as a 100-fold liquid concentrate comprising 500 μ g/ml human transferrin, 500 μ g/ml

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

Gastrin, purchased from Sigma.

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

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

EXAMPLE 1 Molding method of the present invention

1. Primary culture of cells

Dissecting mouse nasopharyngeal tissue with a split microscope, and shearing the mouse nasopharyngeal tissue with small scissors, preferably on ice or in the same low-temperature environment, or mechanically crushing in the same low-temperature environment; taking the cut tissue blocks, digesting with 5mL of pancreatin, incubating for 1h at 37 ℃ in a shaking table, blowing and beating for more than ten minutes at intervals of about 15-20min by using a liquid transfer gun to prevent tissue agglomeration, and fully digesting out single cells.

Filtration the cells are filtered through a 80-130 μm cell screen, preferably a 100 μm cell screen; after filtration, 15-20mL of DMEM/F12 was added to flush the membrane and stop digestion, and the supernatant was removed by centrifugation; centrifuging to remove supernatant; preferably, the centrifugation temperature is 2-8 ℃, and the centrifugation is carried out for 4-6min at the temperature of 400-450 g; the supernatant is resuspended by adding DMEM/F12, preferably 10mL DMEM/F12, and then centrifuged.

2. Organoid culture

2.1 preculture

Counting cells, mixing Matrigel, 20000 cells per 30 μ L, and dropping in a well of 48-well plate; transferring to incubator, at 36-38 deg.C, under 3-8% CO2 environment for 10-20min, and solidifying Matrigel; adding 150 μ L cell culture medium into each well, preferably the cell culture medium is conditioned medium, culturing at 36-38 deg.C in 3-8% CO2 cell culture box; replacing the culture medium every 4-6 days to culture mouse nasopharyngeal organs. The growth conditions in Martrilgel on days 1-11 of culture after pancreatin of the nasopharyngeal tissues of fresh mice into single cells are shown in FIG. 2.

The formula of the conditioned medium is as follows:

note: 50X means 50 times concentration, 100X means 100 times concentration, and so on.

2.2 expansion culture

Collecting organoids cultured in a culture dish for about 8-12 days, re-suspending and digesting the organoids with 1X TrypLE, and digesting at 36-38 deg.C for 5-15 min; preferably 1ml TrypLE resuspends the organoids and digests at 37 ℃ for 15 min. DMEM/F12 was added to stop digestion; digestion is preferably stopped by addition of 5ml DMEM/F12.

Through the heavy suspension treatment, the dispersibility of the cells in the organoid is restored, and original cells are provided for the amplification culture.

Centrifuging to remove the supernatant, preferably centrifuging at 2-8 ℃ for 5min at 400-; preferably 400g at 4 ℃ for 5 min. Add Matrigel for resuspension and drop into a well of a 48-well plate. Preferably, a suitable amount of Matrigel reagent is added for resuspension. Preferably, the Matrigel is first melted on ice and then added to the previously treated cell sap. Transferring to a culture dish, and solidifying the Matrigel at 36-39 ℃ under the environment of 3-8% CO 2. The Matrigel is preferably solidified by placing the petri dish in an environment at 37 deg.C (5% CO2) for 10 min. The Matrigel was allowed to complete a phase transition at body temperature to form a gel-like transition. 150 μ L of conditioned medium was added to each well and cultured in a 3-8% CO2 cell culture chamber at 36-39 deg.C. The composition of the conditioned medium is the same as that of the conditioned medium applied in the process of preparing the nasopharyngeal tissue. The culture medium is changed every 4-6 days to culture nasopharyngeal tissue organoids, as shown in FIG. 3.

And H & E staining of the organoids, as shown in figure 4, the nasopharyngeal tissue organoids are composed of multiple cells, are hollow or solid, and have good activity characteristics.

3. Genetic modification of nasopharyngeal cells

The Cdkn2a gene of nasopharyngeal cells was knocked out by using CRISPR/Cas9 gene editing technology, and the cMYC gene and Kras G12D mutant gene (abbreviated as "Kras (G12D)) of nasopharyngeal cells were overexpressed by using gene overexpression technology (fig. 5A and B).

3.1 the editing operation method is as follows:

(1) packaging lentivirus (or retrovirus) carrying genetic modification elements (a vector for CRISPR/Cas9 gene editing, a vector carrying sgRNA and Cas9 protein coding genes and an m-Cherry red fluorescence reporter gene), culturing 293T cells in a 6-well plate by using a DMEM (DMEM) culture medium, wherein 2mL of culture solution is used in each well, packaging the virus by using a calcium phosphate precipitation method after the cells grow to be full of the 6-well plate, replacing the culture solution every 12 hours, collecting 36 th and 48 th hour virus solutions, filtering the virus solution by using a 022um filter membrane, storing the virus solution at 4 ℃ for later use, and using the virus solution within one week is suitable.

(2) Digesting the cultured mouse nasopharyngeal organoid to form single cells, mixing the single cells with mouse nasopharyngeal cells according to the volume of 500-: virus liquid volume ratio 1: adding ploybrene 1000, transferring the cell virus suspension to a 24-well plate, centrifuging at 2000rpm for 1h, and then placing in an incubator at 37 ℃ for 1.5-2 h; blowing virus liquid to resuspend cells, centrifuging to remove supernatant, and centrifuging for 5min at 400-500 g.

(3) Add Matrigel for resuspension and drop into a well of a 48-well plate. Preferably, a suitable amount of Matrigel reagent is added for resuspension. Preferably, the Matrigel is first melted on ice and then added to the previously treated cell sap. Transfer to petri dish, place petri dish to 37 ℃ (5% CO2) environment for 10min, solidify Matrigel. mu.L of conditioned medium was added to each well and cultured in a cell incubator at 37 deg.C (5% CO 2). The composition of the conditioned medium is the same as that of the conditioned medium applied in the process of preparing the nasopharyngeal tissue. Replacing the culture medium every 4-6 days to culture nasopharyngeal tissue organoid or cell group.

The efficiency of viral infection and gene editing was identified by fluorescence signal, fluorescence value and cleavage with T7 (FIGS. 5C and D).

3.2 Gene overexpression protocol was as follows:

the cMYC gene and the Kras (G12D) gene were inserted into a multiple cloning site in a commercially available overexpression vector to obtain a recombinant overexpression vector carrying the Luciferase gene (FIG. 5B). And packaging the recombinant overexpression vector into the lentivirus to obtain the recombinant lentivirus.

The recombinant lentivirus is then transferred into nasopharyngeal cells in the same manner as in sections 3.1 (2) and (3).

The expression condition of the gene in the overexpression vector can be monitored by detecting the Luciferase signal value, and the infection efficiency is considered to be higher when the Luciferase value is more than 10 thousands (FIG. 5E).

4. Mouse nasopharyngeal organ in-situ transplantation

Collecting nasopharyngeal cells after gene editing, culturing for 5-7 days to obtain nasopharyngeal organoid, resuspending organoid according to 1ml TrypLE per well, and digesting at 37 deg.C for 5 min; the digestion is stopped by adding DMEM/F12, preferably 5ml DMEM/F12; centrifuging at 400g for 5min, and removing supernatant; cells were resuspended using 10-15ul Matrigel and placed on ice.

Anaesthetizing a Nude mouse by using isoflurane in an anaesthesia machine, lying the anaesthetized mouse in a look-up manner, and fixing the four limbs of the mouse by using an adhesive tape; sucking the Matrigel cell suspension by using an insulin needle, opening the oral cavity of the mouse by using a surgical forceps, and transplanting the cell suspension to the nasopharynx part of the mouse; after operation, the mice were returned to the SPF animal house for feeding, the sterilized water and feed were periodically replaced, and the condition of the mice was examined, and live imaging was performed once every 2 weeks. When the state of the mice begins to deteriorate, the mice are killed by breaking the neck, and tumor samples are collected.

FIG. 6A shows the images of the live images at 15 th and 30 th days after transplantation, and it can be seen that the transplanted gene-editing cells occupy a significantly larger area. FIGS. 6B and 6C show mCherry and GFP fluorescence signal detection maps of the cranium and nasopharynx positions of post-mortem mice, respectively, mCherry and GFP fluorescence signals can be detected, infected DNA master carries mCherry, Trp53-/-Cas9 gene mice endogenously express GFP protein, and mChery and GFP positive signal tissues are from exogenously transplanted cells.

Pathological HE staining and immunohistochemical staining are carried out on nasopharyngeal tumors, as shown in figure 7, the result shows that the nasopharyngeal part of the mouse transplanted with the gene editing organoid forms bump space occupation, the cell nucleus is deeply stained, the heterogeneity is large, the nuclear-cytoplasmic ratio is large, and part of the cell nucleus is naked; the tumor tissue Ki67 is positive, the tumor tissue Ki is positive, the tumor tissue classic squamous Marker CK5 and the tumor tissue P63 are positive, and the tumor tissue Ki67 is low-differentiation squamous carcinoma and is consistent with the nasopharyngeal carcinoma mainly comprising low-differentiation squamous carcinoma clinically.

In the present example, a total of 4 mice were modeled, and 3 of them had solid tumors detected, with a high success rate of 75% for modeling.

In conclusion, the method can efficiently prepare the nasopharyngeal carcinoma model which is more close to the nasopharyngeal carcinoma characteristics and meets the clinical research requirements; the model can provide a beneficial tool in research fields such as research on occurrence and development mechanisms of nasopharyngeal carcinoma, search and optimization of possible treatment modes of nasopharyngeal carcinoma and the like.

Claims (10)

1. A culture medium for culturing human or animal nasopharyngeal tissue cell organoid is characterized in that 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 Dilution of 100 + -10 times concentration N2 Dilution of 100 + -10 times concentration Gastrin 1±0.1nM

。

2. The culture medium of claim 1, wherein the formulation 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 Dilution by 100 times concentration N2 Dilution by 100 times concentration Gastrin 1nM

。

3. A method for constructing an in-situ primary nasopharyngeal carcinoma animal model is characterized by comprising the following steps:

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

2) fixing cells in Matrigel, adding a culture medium to culture the cells into organoids;

3) resuspending the organoid into single cells, performing genetic transformation, and culturing into organoid;

4) injecting the organoid obtained in step 3) into the nasopharynx of the animal;

the genetic modification in the step 3) refers to knockout of an anti-cancer gene and/or over-expression of a proto-oncogene.

4. The method of claim 3, wherein:

step 2) the medium for culturing organoids is the medium according to claim 1 or 2.

5. The method of claim 3 or 4, wherein:

the step 1) comprises the following steps:

a. using pancreatin with a final concentration of 0.25% to digest nasopharyngeal tissues;

b. filtering with 80-130 μm sieve, and collecting cells in the filtrate;

c. neutralizing the filtrate with cell culture medium to stop digestion;

d.400G, centrifuging for 5min to remove the supernatant, adding a cell culture medium to resuspend the cells, and centrifuging to remove the supernatant;

preferably, a 100 μm sieve is used in step b.

6. The method of claim 3 or 4, wherein:

the step 2) comprises the following steps:

i. mixing the cells with 30ul of Matrigel, and adding a culture medium to culture after the Matrigel is solidified to obtain organoids;

resuspending organoids with 1X TrypLE for 15 min;

adding cell culture medium to wash the cells and terminating digestion;

centrifuging to remove the supernatant, adding a cell culture medium to resuspend the cells, dispersing the cells, and centrifuging;

v. adding 30ul of Matrigel to resuspend the cells, and adding a culture medium to culture after the Matrigel is solidified to obtain the organoid.

7. The method of claim 3 or 4, wherein:

the step 3) also comprises transferring a fluorescence labeling gene into the organoid.

8. The method of claim 3 or 4, wherein:

the gene editing in the step 4) is specifically as follows: overexpresses a cMYC gene and a KrasG12D mutant gene, and knocks out a Cdkn2a gene;

the nasopharyngeal carcinoma is hypo-differentiated squamous carcinoma.

9. The method of claim 3 or 4, wherein:

it still includes:

step 5): in vivo imaging observations were performed every 2-3 weeks to detect tumor formation.

10. The method of claim 3 or 4, wherein:

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

Images