CN114369573B - 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 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

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 malignancy that occurs in the top and side walls of the nasopharyngeal cavity. The epidemic disease area is mainly in east Asia and southeast Asia, is one of the high malignant tumors in China, and the incidence rate is the first malignant tumor of ear, nose and throat.

In the process of researching the occurrence and development mechanism of nasopharyngeal carcinoma and developing the nasopharyngeal carcinoma therapeutic drug, the nasopharyngeal carcinoma animal model is not separated.

The nasopharyngeal carcinoma animal models currently used in scientific research are mainly classified into three types, including genetically engineered animal models, tumor cell line transplantation tumor models, and human xenograft tumor models (PDX, patient derived Xenograft model).

The genetically engineered animal model has good tumor microenvironment and good repeatability, and the immune system is free of defects, but the genetically engineered animal model needs to be used for preparing transgenic animals, and has high cost and long preparation period. The tumor cell line transplantation tumor model only needs to implant a human tumor cell line into a model animal, is easy to prepare and high in repeatability, but an immunodeficiency mouse is needed, and the obtained tumor is not in-situ primary tumor and is greatly different from the actual development condition and pathophysiological condition of the tumor. The PDX model is prepared by inoculating tumor tissue in a patient into a model animal body, is easy to prepare, has genotype close to that of an actual tumor, but is not in-situ tumor, and cannot provide in-situ microenvironment of nasopharyngeal tissue, so that related biological characteristics of human tumor in the experimental process are lost, the condition in a human body cannot be simulated, at present, clinical tumor specimens are very precious, a few special clinical specimens such as puncture specimens and other small specimens can be used for experimental research, the tissue cell quantity is less, the success rate of constructing the nasopharyngeal carcinoma PDX model is low, and the model construction requirement cannot be met.

In summary, in order to explore the mechanism of development of nasopharyngeal carcinoma and develop a novel nasopharyngeal carcinoma therapeutic drug, there is an urgent need for a nasopharyngeal carcinoma animal model that is close to the biological characteristics of nasopharyngeal carcinoma, has short operation time, high repeatability and high flux.

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, short in preparation period and definite in genotype.

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

a culture medium for culturing human or animal nasopharyngeal tissue cell organoids comprises the following formula:

B27	dilution at 50+ -5-fold concentration
		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-fold dilution
		N2	100+ -10-fold dilution
Gastrin	1±0.1nM

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

B27	dilution by 50 times
		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
		N2	Dilution by 100 times
Gastrin	1nM

A method of constructing an in situ primary nasopharyngeal carcinoma animal model comprising the steps of:

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

2) Fixing cells in Matrigel, and adding culture medium to culture into organoids;

3) Resuspending the organoids into single cells, performing genetic engineering, and then culturing the organoids;

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

step 3) the genetic engineering refers to knocking out the oncogene and/or over-expressing the proto-oncogene.

The method as described above, step 2) the medium used to culture the organoids is the medium described above.

The method as described above, step 1) comprising:

a. nasopharyngeal tissue was digested with 0.25% final concentration pancreatin;

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

c. neutralizing the filtrate with a cell culture medium, and terminating digestion;

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

preferably, step b uses a 100 μm sieve.

The cell culture medium in step c and/or d is DMEM/F12 medium as in the previous method.

The method as described above, step 2) comprising:

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

re-suspending organoids with 1X TrypLE for 15min;

washing the cells with cell culture medium to terminate digestion;

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

adding 30ul Matrigel to re-suspend cells, and adding a culture medium to culture after Matrigel is solidified to obtain organoids.

The method as described above, step 3) further comprising transferring the organoid with a fluorescent marker gene.

The method as described above, the gene editing of step 4) is specifically: overexpressing cMYC genes and Kras G12D mutant genes, and knocking out Cdkn2a genes;

the nasopharyngeal carcinoma is hypodifferentiation squamous carcinoma.

The method as described above, further comprising:

step 5): living imaging observation was performed every 2 to 3 weeks to detect tumor formation.

The method as described above, wherein the animals in steps 1) and 4) are mice.

The method of the invention has the following beneficial effects:

1) Compared with a tumor cell line transplanted tumor model and a human xenograft tumor model, the model constructed by the method does not need to use an immunodeficiency animal, is an in-situ tumor, can simulate the process of converting normal cells into tumor cells in a human body due to genetic change, can dynamically characterize the process of generating and developing tumors, and is closer to the real situation of generating and developing tumors 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 construct from fertilized eggs or embryos, the period can be obviously shortened, and the problem of early death of animals caused by systemic gene mutation can be avoided.

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

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

Fig. 1: a schematic flow chart of the in-situ primary nasopharyngeal tumor of the mice is constructed.

Fig. 2: fresh mouse nasopharyngeal tissue was digested with pancreatin into single cells and cultured in Martrilgel for day 1-11 growth conditions.

Fig. 3: the nasopharyngeal organoids of the mice were cultured in vitro.

Fig. 4: HF staining of mouse nasopharyngeal organoids.

Fig. 5: mouse nasopharyngeal organoid gene editing. A. The nasopharyngeal carcinoma model of the invention constructs the schematic diagram; B. schematic diagram of mouse nasopharyngeal organoid gene editing original, edit Cdkn2a gene original: u6 promoter and EFS are promoters, cdkn2a is sgRNA sequence targeting Cdkn2a gene, mCherry is fluorescent 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) detected the efficiency of infection of the Cdkn2a original at organoids; t7 detecting the Cdkn2a gene mutation; E. the cMYC/Kras (G12D) expression original was tested for its efficiency in organoid infection, and higher infection efficiency was considered to be found with a Luciferase number of over 10 ten thousand.

Fig. 6: tumor samples collected after gene editing mice nasopharyngeal organoids were imaged in vivo on day 15, day 30 after in situ transplantation and mice were sacrificed. A, living body imaging; b, performing fluorescence living imaging from the skull; and C, performing fluorescence imaging on tumor tissues.

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

Detailed Description

Some of the english abbreviations in the present invention are explained as follows:

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

DMEM/F12: f12 medium and DMEM medium were used according to 1:1, called DMEM/F12 medium. Combines the advantages of the F12 containing rich components and the DMEM containing high-concentration nutrients. Purchased from GIBCO.

Matrigel is separated from EHS mouse tumor rich in extracellular matrix protein, and its main components are laminin, IV type collagen, nidogen, heparan sulfate glycoprotein, etc., and also contains growth factor, matrix metalloproteinase, etc. Purchased from BD company.

B27, a B27 supplement, a commercially available product, can be used to formulate the culture medium. B27 supplement was provided as a 50-fold liquid concentrate comprising, 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 company. N-acetylcysteine: n-acetylcysteine, purchased from Sigma company.

EGF, epidermal growth factor, commercially available from R & D company.

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

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

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

FGF10, fibroblast growth factor, purchased from Peprotech company.

Nicotinamide, nicotinamide, purchased from Sigma.

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

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

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

N2, N2 supplement was provided as a 100-fold liquid concentrate comprising 500 μg/ml human transferrin, 500 μg/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 company.

Gastin, gastrin, purchased from Sigma company.

TrypLE, a recombinant digestive enzyme for dissociating adherent mammalian cells, purchased from GIBCO corporation.

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

Example 1A method of Forming a mold according to the invention

1. Primary cell culture

Cutting out the nasopharynx tissue of the mouse by using a microscope, and shearing the nasopharynx tissue of the mouse by using a small pair of scissors, wherein the shearing is preferably performed on ice or in the same low-temperature environment; taking sheared tissue blocks, using 5mL pancreatin digestion treatment, incubating for 1h at 37 ℃ by a shaking table, and blowing with a pipetting gun at intervals of about 15-20min for more than ten times to prevent tissue from agglomerating and fully digesting single cells.

Filtering the cells with a 80-130 μm cell screen, preferably a 100 μm cell screen; after filtration, the filter membrane is rinsed by adding 15-20mL of DMEM/F12 and digestion is stopped, and the supernatant is removed by centrifugation; centrifuging to remove supernatant; preferably centrifuging at 2-8deg.C for 4-6min at 400-450 g; the supernatant is removed by adding DMEM/F12 for resuspension, preferably 10mL DMEM/F12 for resuspension, and then centrifuging.

2. Organoid culture

2.1 Pre-culture

Cell count, mix Matrigel,20000 cells per 30 μl, drop in 48 Kong Bankong; transferring to an incubator at 36-38deg.C under 3-8% CO2 for 10-20min, and solidifying Matrigel; 150 μl of cell culture medium, preferably conditioned medium, is added to each well and cultured in a 3-8% CO2 cell incubator at 36-38deg.C; the culture medium is replaced every 4-6 days to culture the nasopharyngeal organoids of the mice. Growth in Martrilgel on days 1-11 after pancreatin digestion of fresh mouse nasopharyngeal tissue into single cells is shown in FIG. 2.

The formula of the conditioned medium is as follows:

note that: 50X means a 50-fold concentration, 100X means a 100-fold concentration, and so on.

2.2 expanded culture

Collecting organoids cultured in a culture dish for about 8-12 days, re-suspending the digested organoids with 1X TrypLE, and digesting for 5-15min at 36-38deg.C; preferably 1ml of TrypLE, the organoids are resuspended and digested at 37℃for 15min. Adding DMEM/F12 to terminate digestion; digestion is preferably terminated by adding 5ml of DMEM/F12.

After the resuspension treatment, the dispersibility of cells in the organoids is recovered, and primitive cells are provided for expansion cultivation.

Centrifuging to remove supernatant, preferably centrifuging at 2-8deg.C for 5min at 400-500 g; preferably 400g, are centrifuged at 4℃for 5min. Matrigel was added to resuspend and dropped into 48 well wells. Preferably, a proper amount of Matrigel reagent is added for the resuspension treatment. Preferably, matrigel is thawed on ice and then added to the previously treated cell fluid. Transfer to a petri dish and solidify Matrigel at 36-39deg.C in the presence of 3-8% CO 2. Preferably, the dish is left in an environment of 37℃C (5% CO 2) for 10min, and the Matrigel is coagulated. Allowing the Matrigel to complete phase inversion at body temperature, forming a gel-like inversion. 150. Mu.L of conditioned medium was added to each well and incubated at 36-39℃in a 3-8% CO2 cell incubator. The composition of the conditioned medium is identical to the composition of the conditioned medium used in the preparation of the nasopharyngeal tissue. The culture medium was changed every 4-6 days to culture nasopharyngeal tissue organoids as shown in FIG. 3.

Organoid H & E staining, as shown in FIG. 4, nasopharyngeal tissue organoids are multicellular in composition, hollow or solid, with good activity profile.

3. Nasopharyngeal cell genetic engineering

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

3.1 editing operation method is as follows:

(1) Packaging a slow virus (or retrovirus) carrying a genetic modification original (a carrier for CRISPR/Cas9 gene editing, carrying sgRNA and Cas9 protein coding genes and m-Cherry red fluorescent reporter genes), culturing 293T cells in a 6-well plate by using a DMEM culture medium, packaging the virus by using a calcium phosphate precipitation method after the cells grow into the 6-well plate to be low, changing the culture solution every 12 hours, collecting 36 th and 48 th hours virus solutions, filtering the virus solution by using a 022um filter membrane, and preserving the virus solution at 4 ℃ for later use, wherein the virus solution is preferably used within one week.

(2) The cultured mouse nasopharyngeal organoids are digested into single cells, and the single cells are uniformly mixed with the mouse nasopharyngeal cells according to the volume of 500-800ul of each virus liquid, and the single cells are mixed according to the following steps: the volume ratio of the virus liquid is 1:1000 adding ploybrene, transferring the cell virus suspension to a 24-well plate, centrifuging at 2000rpm for 1h, and then placing in a 37 ℃ incubator for 1.5-2h; the cells are resuspended by blowing in a virus liquid, the supernatant is removed by centrifugation, and 400-500g is centrifuged for 5min.

(3) Matrigel was added to resuspend and dropped into 48 well wells. Preferably, a proper amount of Matrigel reagent is added for the resuspension treatment. Preferably, matrigel is thawed on ice and then added to the previously treated cell fluid. Transfer to a Petri dish, place the dish in 37℃ (5% CO 2) environment for 10min, solidify Matrigel. 150. Mu.L of conditioned medium was added to each well and incubated in a 37℃C (5% CO 2) cell incubator. The composition of the conditioned medium is identical to the composition of the conditioned medium used in the preparation of the nasopharyngeal tissue. The culture medium is changed every 4-6 days to culture nasopharyngeal tissue organoids or cell populations.

Viral infection efficiency and gene editing can be identified by fluorescent signal, fluorescent value and T7 cleavage (fig. 5C and D).

3.2 Gene overexpression procedure as follows:

the cMYC gene and the Kras (G12D) gene were inserted into a commercially available overexpression vector at multiple cloning sites to obtain a recombinant overexpression vector carrying the Luciferase gene (fig. 5B). And packaging the recombinant over-expression vector into the lentivirus to obtain the recombinant lentivirus.

And transferring the recombinant lentivirus into nasopharyngeal cells, wherein the steps are the same as those in the steps (2) and (3) in section 3.1.

By detecting the signal value of luciferases, the expression condition of genes in the over-expression vector can be monitored, and the luciferases have a value of more than 10 ten thousand and are considered to have higher infection efficiency (figure 5E).

4. Mouse nasopharyngeal organoids in situ transplantation

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

Isoflurane is used for anesthetizing the Nude mice in an anesthesia machine, the anesthetized mice lie on the back, and the four limbs of the mice are fixed by using an adhesive tape; sucking Matrigel cell suspension with insulin needle, opening the mouth of the mouse by using surgical forceps, and transplanting the cell suspension to the nasopharynx of the mouse; the mice were returned to the SPF animal house for feeding, periodically replaced with sterile water and feed, and the condition of the mice was examined, and imaged in vivo every 2 weeks. When the state of the mice starts to worsen, the mice are sacrificed by cervical fracture, and tumor samples are collected.

FIG. 6A shows in vivo imaging at day 15 and day 30 post-implantation, showing that the area occupied by transplanted gene-editing cells is significantly larger. FIGS. 6B and 6C show the detection of mCherry and GFP fluorescence signals at the cranium and nasopharynx sites, respectively, of mice after sacrifice, where mCherry and GFP fluorescence signals were detectable, infected DNA elements carrying mCherry, trp53-/-Cas9 gene mice endogenously expressing GFP protein, and mCherry and GFP positive signal tissues from exogenously transplanted cells.

Pathological HE staining and immunohistochemical staining are carried out on nasopharyngeal tumors, as shown in fig. 7, and the result shows that the nasopharyngeal of the mouse transplanted with the gene editing organoids forms a bump space, the cell nucleus is deeply stained, the heterogeneity is large, the nuclear mass ratio is large, and part of cell nuclei are exposed; the Ki67 positive tumor tissue and the classical squamous Marker CK5 and P63 positive tumor tissues are hypodifferentiation squamous cancers, and are consistent with the clinical nasopharyngeal carcinoma which is mainly hypodifferentiation squamous cancers.

In the embodiment, the modeling is performed on 4 mice in total, wherein 3 mice detect solid tumors, the modeling success rate is 75%, and the success rate is high.

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

Claims (8)

1. A method for constructing an in situ primary nasopharyngeal carcinoma animal model, comprising the steps of:

1) Primary culture of mouse nasopharyngeal tissue cells;

2) Fixing cells in Matrigel, and adding culture medium to culture into organoids;

3) Resuspending the organoids into single cells, performing genetic engineering, and then culturing the organoids;

4) Injecting the organoid obtained in the step 3) into the nasopharynx of the mouse;

step 3) refers to knocking out the oncogene and/or over-expressing the proto-oncogene, specifically: overexpressing cMYC genes and Kras G12D mutant genes, and knocking out Cdkn2a genes; the nasopharyngeal carcinoma is hypodifferentiation squamous carcinoma.

2. The method of claim 1, wherein:

step 2) the formulation of the medium used to culture organoids is as follows:

B27 dilution at 50+ -5-fold concentration 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-fold dilution N2 100+ -10-fold dilution Gastrin 1±0.1nM

。

3. The method of claim 2, wherein: the formula of the culture medium is as follows:

4. a method according to any one of claims 1-3, wherein:

step 1) comprises:

a. nasopharyngeal tissue was digested with 0.25% final concentration pancreatin;

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

c. neutralizing the filtrate with a cell culture medium, and terminating digestion;

d 400G, centrifuging for 5min to remove the supernatant, adding cell culture medium to re-suspend the cells, and centrifuging to remove the supernatant.

5. The method of claim 4, wherein: step b uses a 100 μm sieve.

6. A method according to any one of claims 1-3, wherein:

step 2) comprises:

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

re-suspending organoids with 1X TrypLE for 15min;

washing the cells with cell culture medium to terminate digestion;

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

adding 30ul Matrigel to re-suspend cells, and adding a culture medium to culture after Matrigel is solidified to obtain organoids.

7. A method according to any one of claims 1-3, wherein:

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

8. A method according to any one of claims 1-3, wherein:

it also includes:

step 5): living imaging observation was performed every 2 to 3 weeks to detect tumor formation.