CN114075539A

CN114075539A - Method for constructing orthotopic primary bladder cancer animal model

Info

Publication number: CN114075539A
Application number: CN202010834645.0A
Authority: CN
Inventors: 陈崇; 王漫丽; 刘玉
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-02-22
Anticipated expiration: 2040-08-18
Also published as: CN114075539B

Abstract

The invention discloses a preparation method of an in-situ primary bladder cancer tumor model, and belongs to the field of tumor animal models. The invention cultures the normal bladder cells of the mouse into organoid by a specific culture medium, then carries out gene editing on the organoid, and injects the organoid into the bladder of the mouse to lead the organoid to develop 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 in-vivo microenvironment with tumor occurrence and development and the occurrence and development process from normal to tumor are more close to the most real state of the occurrence and development of bladder cancer.

Description

Method for constructing in-situ primary bladder cancer animal model

Technical Field

The invention belongs to the field of tumor animal models.

Background

Bladder Cancer (BC) is a common malignancy of the urinary system. According to the latest global statistical data, about 55 ten thousand cases (3.0%) of new bladder cancer are globally discovered in 2018, accounting for the twelfth position of the tumor morbidity, about 20 ten thousand cases (2.1%) of new bladder cancer are killed, and accounting for the fourteenth position of the tumor mortality. Bladder cancer is at about 3-4 times the risk of developing in men: the incidence of bladder cancer accounts for the sixth (4.5%) in men and the ninth (2.8%) in women. In China, bladder cancer is the most common malignant tumor of the urinary system, and the incidence rate is the first and is in a year-by-year increasing trend in the malignant tumor of the male genitourinary system in China; the bladder cancer in men has risen to the sixth position of the incidence rate of tumors, and in the eleventh position of women, the bladder cancer will inevitably cause huge burden on the medical and health system in China.

At present, few animal models can be used for bladder cancer research, and primary in-situ bladder cancer animal models are lacked, so that the research on molecular mechanisms in the occurrence and development of bladder cancer is greatly restricted. Currently available bladder cancer models include: chemical induction bladder cancer model, transplantation tumor model, PDX model and gene mouse model. The carcinogen induction model mostly uses N-butyl-N- (4-hydroxybutyl) nitrosamine to induce bladder cancer orally, and has the advantages that the carcinogenic process is spontaneous and complete immunogenicity is achieved; however, the gene background of the tumor formation is complex, the tumor types are various, the metastasis is rarely generated, and the phenotype difference with clinical patients is large. The bladder cancer transplantation tumor model has short tumor formation time, and the genetic background of the tumor formation is clear, but the defect is that the tumor spontaneous formation process and immunogenicity are lacked. The tumor cells transplanted by the human PDX model are derived from tumor specimens of patients, can specifically reflect the tumor characteristics of the patients, but the tumorigenic process of the tumor cells lacks immunogenicity and is a non-primary tumor model. The gene mouse model is a spontaneous model with clear gene background, but the technical difficulty is high and the time consumption is long; recently, the Michael m.shen team has constructed a mouse animal model of bladder cancer organoids derived from clinical patients, and the model has the advantages that the tumor organoids can retain the heterogeneity of the patient's tumor to a greater extent, the tumor formation time is short, and the model can be used for researches such as tumor development and drug screening, but cannot be used for researching the tumor generation process. In view of the existing models, an animal model which can better simulate the generation and development of tumors of clinical patients is still lacked.

Disclosure of Invention

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

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

a method for culturing a bladder organoid, comprising the steps of:

mixing normal bladder cells with Matrigel, and adding an organoid culture medium for culturing after the Matrigel is solidified;

the term "normal bladder cell" is used in relation to a bladder cancer cell and refers to a healthy bladder cell that is not cancerous.

The organoid culture medium takes DMEM/F12 as a basic culture medium and contains the following additives: 50-100ng/ml Wnt3a and 300-750ng/ml R-spondin 1.

The culture method is as described above, and the concentration of the Wnt3a is 75 ng/ml.

The culture method as described above, wherein the concentration of R-spondin 1 is 500 ng/ml.

As with the previous culture method, the organoid culture medium is supplemented with the following amounts of additives:

B27	1％(v/v)	N-acetylcysteine	0.125mM
				EGF	50ng/ml	Noggin	50ng/ml
R-spondin 1	500ng/ml	A83-01	200nM
				FGF10	100ng/ml	Nicotinamide	1mM
Y-27632	10uM	Wnt3a	75ng/ml
				Glutamax	100 x dilution	N2		1％(v/v)
Penicillin	10000units/ml	Streptomycin	10000ug/ml

。

A method for constructing an in situ primary bladder cancer animal model comprises the following steps:

1) performing primary culture on normal bladder cells of a human or an animal;

2) culturing primary cells into organoid, and performing expanded culture;

3) dispersing the obtained organoids into single cells, performing gene editing, and then culturing into organoids;

4) injecting the organoids successfully edited by the genes into animal bladder tissues;

the organoid culture method in step 2) and step 3) is as shown above;

the gene editing in the step 3) refers to knocking out the cancer suppressor gene and/or increasing the copy number of the protooncogene.

As the method for constructing the animal model of the primary carcinoma of bladder in situ, the gene editing in the step 3) is specifically as follows:

knocking out Trp53, Pten and Rb1 genes and overexpressing cMyc and Kras^G12DA gene;

or, knocking out Trp53 gene and overexpressing cMyc gene.

Note: kras^G12DIt means that the Kras gene has G12D mutation, i.e. the 12 th amino acid is mutated from G (glycine) to D (aspartic acid).

The method for constructing the animal model of orthotopic primary bladder cancer as described above, wherein the gene editing of step 3) further comprises transferring a fluorescent marker gene and/or a luciferase gene into the organoid.

The method for constructing an animal model of primary carcinoma in situ as described above, wherein the animals in steps 1) and 4) are mice.

The animal model prepared by the method is applied to non-disease treatment purpose drug screening, drug toxicity tests or immunotherapy tests.

The technical scheme of the invention is shown in figure 1.

Compared with a gene engineering animal model, the tumor model construction period is greatly shortened, the death of the animal before the tumor formation can hardly be caused, and the overall efficiency is high.

The in-situ primary mouse bladder tumor model constructed by the invention 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 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 bladder cancer model which is closer to the characteristics of the bladder cancer and meets the requirements of clinical research; the model can provide a favorable tool in the research fields of exploring the occurrence and development mechanism of bladder cancer, searching and optimizing possible treatment modes of new bladder 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. 1 is a technical roadmap for primary in situ bladder cancer model construction.

FIG. 2 Normal bladder organoid culture in mice.

FIG. 3 is white light and fluorescence images after organoid gene editing.

Figure 4 is an image of a living body.

FIG. 5 genotype verification after mouse organoid gene editing (top: restriction enzyme digestion verification of Trp53, Rb1, Pten mutation; middle: Kras;)^G12DOver-expression in tumor cells; the following: cMyc is overexpressed in tumor cells).

FIG. 6 survival curves after mouse transplantation.

FIG. 7 bladder tumors after tumor formation in mice; BF, white light; GFP, green fluorescent protein.

FIG. 8 is a graph showing HE staining of bladder tumor.

FIG. 9 shows immunohistochemical staining patterns of bladder tumors.

FIG. 10 is a white light and fluorescence image of normal mouse after organ editing.

FIG. 11 mutation detection map of gene Trp53 (left) and live imaging luciferase detection map of mice one week after orthotopic transplantation (right).

FIG. 12 white light and fluorescence images of mouse bladders 47 days after orthotopic transplantation.

FIG. 13 bladder pathology H & E staining.

FIG. 14 is a schematic diagram of in vivo chemotherapy treatment in a primary orthotopic mouse model.

FIG. 15. luciferase signal detection map of mouse bladder after chemotherapy treatment; left: detecting a direct result; and (3) right: a signal strength histogram.

FIG. 16 shows the effect of medium screening.

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 corning incorporated.

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

Gastrin, purchased from Sigma.

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

EXAMPLE 1 organoid culture and method of expansion culture of the invention

Comprises obtaining fresh bladder tissue cells, and digesting pancreatin into single cells; bladder tissue organoids were cultured in vitro 3D culture conditions.

The method comprises the following steps:

(1) 1-2 fresh mouse bladder tissues are cut into pieces on ice;

(2)8mL of digest (1.5mg/mL collagenase I and 1mg/mL collagenase IV +10uM Y27632) resuspended in the minced tissue mass (Y27632 inhibits cell death and maintains the activity of the cells during digestion);

(3) the resuspended tissue was then digested for 50min by shaking the digestion solution at 37 ℃ on a shaker at a speed of 220rpm, and blown up every 10 min. Fully dispersing the tissue cells;

(4) filtering the digested tissue fluid by using a 100-micron cell screen;

(5) after filtration, the supernatant is removed by centrifugation at room temperature at 1500rpm for 5 min;

(6) adding 3ml DMEM/F12 for resuspension, centrifuging at room temperature and 1500rpm for 5min, and removing supernatant;

(7) after counting cells, about 35 μ L Matrigel per 10000 cells were mixed and dropped in the middle of a 48-well plate;

(8) transferring to a 37 deg.C incubator containing 5% CO2, and coagulating Matrigel for 20-30 min;

(9) adding 180. mu.L of organoid culture medium (the components of the culture medium are shown in Table 1) into each well, and culturing in a cell culture box;

(10) replacing the culture medium every 2-3 days to culture the normal mouse bladder organoid.

(11) Taking organoids cultured for about 7 days, re-suspending and digesting the organoids by using TrypLE, transferring the organoids into a 15mL centrifugal tube, blowing and beating for 10-20 times according to the calculation of adding 2mL TrypLE in one hole of a 48-hole plate until matrigel is completely disintegrated, and digesting for 5min in water bath at 37 ℃;

(12) taking out from the water bath, blowing and beating for 20-30 times again, digesting for 5min at 37 ℃, and then blowing and beating for the third time (20-30 times). When organoids were viewed under a microscope, they were digested into single cells. If the cells are not single cells, the water bath and the air blowing can be repeated for one time until the cells become single cells.

(13) Centrifuging at 1500rpm at room temperature for 5min, and removing supernatant;

(14) after counting cells, 30. mu.L of Matrigel was added to 5000 cells for resuspension and dropped into a well of a 48-well plate;

(15) transferring to an incubator, and solidifying the Matrigel for 20-30 min;

(16) adding 180 μ L organoid culture medium into each well, and culturing at 37 deg.C in 5% CO2 cell culture box;

(17) replacing the culture medium every 2-3 days to culture enough mouse bladder organoids.

TABLE 1 cell culture media

Example 2 Gene editing

Gene editing in this example includes gene knock-out and gene overexpression.

The gene knockout is as follows: the mouse bladder organoid is subjected to gene knockout by using CRISPR/cas9 technology. Namely, the sgRNA vector and the Cas9 expression vector are packaged into lentivirus, and the lentivirus is transfected into organoids, cultured, and the gene knockout effect is verified by using T7E1 enzyme. In CRISPR, the sgRNA vector used in CRISPR/cas9 technology is pLenti-sgRNA-EFs-mCherry (with mCherry red fluorescent reporter gene).

The gene overexpression is: overexpression of genes in organoids is carried out using an overexpression vector plasmid, which also contains sequences for expressing luciferase, and when cells are transferred into the vector plasmid, the cells express luciferase, and when the enzyme binds to its substrate (luciferin), bioluminescence is detected. The overexpression of the gene was analyzed by RNA sequencing results.

The following combinations of gene editing were involved:

combination A: trp53 Rb1 Pten cMyc Kras, namely, Trp53, Rb1 and Pten genes are knocked out by using CRISPR/cas9 technology, and then the cMyc gene and the Kras mutant (G12D) gene are overexpressed.

Combination B: trp53 cMyc, namely, the CRISPR/cas9 technology is used for knocking out the Trp53 gene and then overexpressing the cMyc gene.

Injecting the edited mouse bladder organoid into the mouse bladder, and feeding for 100 days to obtain the bladder cancer animal model.

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

Experimental example 1 construction of muscle-layer invasive bladder cancer model

1. Method of producing a composite material

Organoid culture, passage, gene editing (combination a) were performed in sequence using the methods of examples 1, 2, and the resulting gene-edited organoids were transplanted into mouse bladders, the size and location of tumors were monitored by in vivo imaging techniques, 30 days after transplantation, mice were sacrificed, and bladder tissues were observed for direct visualization, HE staining, and Immunohistochemical (IHC) staining.

2. Results

2.1 organoid culture

As shown in fig. 2, over time, individual bladder cells gradually grow into organoids.

2.2 Gene editing and identification

The results of fluorescence detection after organoid gene editing are shown in FIG. 3, which shows that organoids have been successfully transfected with gene editing viruses.

As shown in FIG. 5, it can be seen that, in the organoid DNA after gene editing, the Trp53, Rb1 and Pten genes can be cut into multiple fragments by the T7E1 endonuclease, which indicates that the gene editing generates DNA mutation and the gene editing is successful.

Kras^G12DGene and cMyc gene overexpression also detected by RNA-Seq sequencing that the expression level of cMyc gene after cell editing was increased relative to that of untreated group, and Kras^G12DThe expression level is higher than that of the control group, which indicates that the Kras is successfully over-expressed^G12DGenes and the cMyc gene (fig. 5).

2.3 in vivo imaging

One week after transplantation, fluorescein in vivo imaging was performed and significant luciferase signal was observed in the mouse bladder site, indicating that the transplanted organoids survived and proliferated in the bladder (fig. 4).

2.4 survival Observation

As the time to transplant organoids passed, mice all died within 30 days after transplantation, it could be assumed that all transplanted mice developed tumors and died from the tumors (fig. 6); the success rate of the invention reaches 100%.

2.5 tumor Observation

Fluorescence microscopy showed a positive signal throughout the bladder (fig. 7), indicating that the implanted cells, tumor cells, proliferated into the positive bladder cavity and invaded the muscle layer.

2.6 histological section Observation

HE staining is shown in FIG. 8, the bladder is occupied by a large number of tumor cells, and the tumor cells are low-differentiation cancer and invade the nearly whole muscle layer, and are malignant muscle-invasive urothelial carcinoma.

The immunohistochemical staining result is shown in fig. 9, the tumor highly expresses Ki67 and CK5 markers, and the tumor is high in proliferation capacity and derived from epithelial cells.

Experimental example 2 construction of bladder squamous carcinoma model

1. Method of producing a composite material

The only difference from experimental example 1 was that the combination B of example 2, i.e. knockout Trp53, was selected for the genomic organization to overexpress cMyc.

2. Results

2.1 Gene editing and identification

The inventor also adopts a CRISPR/Cas9 and an overexpression method to carry out gene editing on mouse organoid organs, the sgRNA is connected to pLenti-sgRNA-EFs-mCherry plasmid (with mChery red fluorescent reporter gene), and the result of organoid culture fluorescence detection by using the urinary bladder of a mouse expressing Cas9 protein is shown in FIG. 10, which shows that the organoids have been successfully transfected with viruses for gene editing.

As shown in the left panel of FIG. 11, it can be seen that multiple fragments (middle lanes) of Trp53 can be cut out from the DNA of the organoid after gene editing, indicating that the gene editing has been successful due to DNA mutation.

2.2 in vivo imaging

One week after transplantation, fluorescein in vivo imaging was performed and significant luciferase signal was observed in the mouse bladder site, indicating that the transplanted organoids survived and proliferated in the bladder (right panel of fig. 11).

2.3 mouse bladder Observation

Fluorescence microscopy showed a positive signal throughout the bladder (figure 12), indicating that the implanted cells invaded the entire bladder.

The HE staining results are shown in fig. 13, where the tumor cell nuclei were deeply stained and the tumor cells invaded the bladder full-thickness and adventitia, so the pathological condition showed that urothelial cancer was accompanied by squamous differentiation, and the tumor type was bladder squamous carcinoma.

The results of experimental examples 1 and 2 show that the method can effectively construct the in-situ primary bladder cancer animal model.

Experimental example 3 in vivo drug Effect test

1. Method of producing a composite material

The bladder cancer model obtained in Experimental example 1 was treated with the first-line chemotherapy regimen for bladder cancer (gemcitabine + cisplatin), and the specific administration time is shown in FIG. 14, in which Gem + DDP indicates gemcitabine + cisplatin administration at a dose of 5mg/kg, once per week, 100mg/kg, once per week, and by intraperitoneal injection.

The organoids after gene editing are injected into the urinary bladder of the mouse in situ, the tumor size of the mouse is evaluated according to the signal value of luciferase expression evaluation, the drug administration is carried out in groups according to the tumor size, and the tumor size range is monitored by in vivo imaging every week during the treatment period.

2. Results

As shown in fig. 15, the results: after the mouse in-situ model is successfully constructed, the mice are treated by the current first-line chemotherapy scheme, the treatment group mice better reflect the chemotherapy sensitivity and tolerance recurrence process in the drug administration process, the tumor size of the treatment group mice is firstly reduced and then increased, and the tumor size of the control group mice is continuously increased along with the time. The mouse model of bladder cancer better simulates the treatment response of clinical patients to first-line chemotherapy drugs, and can be used for the research of more chemotherapy problems.

Experimental example 4 screening experiment of organoid culture Medium composition

The organoid comprises a self-renewing stem cell population which can differentiate into a plurality of organ-organ specific cell types, so that the organoid can be subcultured in vitro for a long time; wnt pathway activation helps bladder epithelial basal cells maintain the dryness, so we add two factors Wnt3a and Rspondin (i.e. R-spondin 1) which activate Wnt pathway into the culture medium to maintain the dryness of the cells, thereby subculturing for a long time.

As shown in FIG. 16, we initially ensured that the organoids were consistent in number, and that the numbers of the two groups of cells differed greatly when passaged to P2. Organoids added with Wnt3a and rsponin (additive as in table 1 of example 1) grew very well to P2, whereas control (no Wnt3a and rsponin added based on table 1 of example 1) showed apoptosis and significantly reduced organoid formation.

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

Claims

1. a bladder organoid culture method, is characterized in that, comprises the steps:

Mix normal bladder cells with Matrigel, and after Matrigel solidifies, add organoid culture medium for culture.

The organoid culture medium was based on DMEM/F12 and contained the following additives: 50-100 ng/ml Wnt3a and 300-750 ng/ml R-spondin 1.

2. culture method as claimed in claim 1 is characterized in that:

The Wnt3a concentration was 75ng/ml.

3. culture method as claimed in claim 1 is characterized in that:

The R-spondin 1 concentration was 500ng/ml.

4. The culture method according to any one of claims 1 to 3, wherein:

The organoid culture medium is added with the following additives:

B27 1%(v/v) N-acetylcysteine 0.125mM EGF 50ng/ml Noggin 50ng/ml R-spondin 1 500ng/ml A83-01 200nM FGF10 100ng/ml Nicotinamide 1mM Y-27632 10uM Wnt3a 75ng/ml Glutamax 100× dilution N2 1%(v/v) penicillin 10000units/ml Streptomycin 10000ug/ml

.

5. a method for constructing an in situ primary bladder cancer animal model, is characterized in that, comprises the steps:

1) Primary culture of human or animal normal bladder cells;

2) Use primary cells to cultivate into organoids and expand the culture;

3) Disperse the resulting organoids into single cells, perform gene editing, and then culture them into organoids;

4) Inject the successfully gene-edited organoids into the animal bladder tissue;

The method for culturing the organoids in step 2) and step 3) is as shown in any one of claims 1 to 4;

Step 3) The gene editing refers to knocking out tumor suppressor genes, and/or increasing the copy number of proto-oncogenes.

6. The method of claim 5, wherein:

The gene editing in step 3) is specifically:

Knock out Trp53, Pten and Rb1 genes, overexpress cMyc and Kras ^G12D genes;

Or, knock out the Trp53 gene and overexpress the cMyc gene.

7. The method of claim 5 or 6, wherein:

The gene editing in step 3) also includes transferring a fluorescent marker gene and/or a luciferase gene into the organoid.

8. The method of claim 5 or 6, wherein:

The animals described in steps 1) and 4) are mice.

9. The animal model prepared by the method of any one of claims 5 to 8 is used in drug screening, drug toxicity test or immunotherapy test for non-disease treatment purposes.