Construction method of vascularized tumor microenvironment based on genetic code recoding technology

Abstract

The invention discloses a method for constructing a vascularized tumor microenvironment based on a genetic code recoding technology, which comprises the following steps of: constructing biological orthogonal translation systems PiggyBac-(7sk-tRNAPyl) x4-EF1a-MmPylRS and PiggyBac-ETV2122 *, transfecting the biological orthogonal translation systems and transposase plasmids into a liver cancer cell line, and screening a Huh-7 cell line which stably expresses the biological orthogonal translation systems; and carrying out balling culture on the obtained Huh-7 cell line, and adding NAEK in the culture process to induce partial Huh-7 cells to be transdifferentiated into endothelial cells, so as to obtain vascularized liver cancer cell balls and realize the construction of a tumor microenvironment. The genetic code recoding technology is used for regulating and controlling formation of the functional vascular tumor microenvironment, and the method has the advantages of being simple and easy to operate and high in repeatability and universality. The technology is a near-physiological protein translation regulation and control means, the regulation and control speed is high, protein translation regulation and control can be rapidly started or stopped, a traditional two-dimensional culture mode can be replaced, multi-cell interaction is achieved, and the biomimetic property is better.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic code re-encoding technology, cell transdifferentiation and tissue engineering, and particularly relates to a method for constructing a vascularized tumor microenvironment based on genetic code re-encoding technology. BACKGROUND

[0002] In recent years, more and more researches focus on the influence of tumor microenvironment (TME) on tumor treatment. TME is a complex system, including cellular components: cancer cells, vascular endothelial cells, fibroblasts, immune cells, etc. and non-cellular components: extracellular matrix, soluble factors, hypoxia, etc. TME not only plays a key role in tumor occurrence, progression and metastasis, but also significantly affects the treatment effect. In recent years, in vitro tumor models have developed rapidly. Traditional cancer anti-tumor drug development mostly relies on 2D monolayer cell culture models, which are simple to operate and easy to carry out high-throughput drug screening, but the 2D tumor cell culture environment is quite different from the TME in vivo, which may lead to unreliable results. Compared with 2D tumor models, 3D tumor models are more similar to solid tumors in cell proliferation, gene expression and anti-cancer drug response, and can more truly simulate the behavior of cells in the natural growth environment. In addition, 3D tumor models can also reveal the complexity of tumor tissues, including oxygen and nutrient gradients and other non-cellular component factors. Therefore, in vitro 3D tumor models are increasingly widely used in the fields of drug screening and pathological research, which has become an inevitable trend in the development of tumor drug screening technology. Therefore, it is crucial to construct an in vitro 3D model that can dynamically interact with the tumor and the surrounding environment. 3D tumor spheres are cancer cell aggregates formed by in vitro 3D cell culture. 3D spheres replicate the interaction between cells, growth kinetics, and ECM deposition of human solid tumors. Due to its tumor-like properties, tumor spheres have been used as a conventional drug screening model to reduce drug development costs and unnecessary animal experiments. However, tumor spheres with a diameter greater than 400 μm are affected by the limitation of nutrient and oxygen transport, resulting in necrosis in the core of the tumor sphere, which affects drug development.

[0003] Therefore, it is a challenge to construct a biomimetic microtissue with a tumor microenvironment in vitro, mainly because the construction of a vascularized tumor microtissue is a difficult problem. There are currently a variety of methods to achieve vascularization of tumors in vitro, including 3D printing technology, co-culture with endothelial cells, and the use of microfluidic chips. However, none of them can well simulate the development regulation of blood vessels. Therefore, it is necessary to couple multiple technologies to create a more physiologically representative 3D tumor model. SUMMARY

[0004] The purpose of the present application is to provide a new strategy for constructing a vascularized tumor microenvironment based on genetic code re-encoding technology.

[0005] The technical scheme adopted by the present application is:

[0006] A biological orthogonal translation system, comprising a transposon plasmid PiggyBac-(7sk-tRNA Pyl )x4-EF1a-MmPylRS and PiggyBac-ETV2 122* . ETV2 122* That is, by introducing a premature stop codon by point mutation at the 122nd amino acid encoded by the ETV2 gene, the MmPylRS / tRNA system will accurately insert NAEK into the 122nd amino acid site, realizing the readthrough of the full-length protein of ETV2.

[0007] Preferably, used in conjunction with the transposase plasmid of the PiggyBac vector system, so as to integrate the MmPylRS / 4xtRNA Pyl system and ETV2 122* gene into the genomic chromosome.

[0008] The aforementioned biological orthogonal translation system is used in the construction of a vascularized tumor microenvironment, wherein the construction of the vascularized tumor microenvironment is for non-therapeutic use.

[0009] The application also discloses a method for constructing a vascularized tumor microenvironment based on genetic code recoding technology, and the steps of the method comprise:

[0010] (1) Constructing the aforementioned biological orthogonal translation system, and transiently transfecting the system into Huh-7 cells together with a transposase plasmid to screen a cell line Huh-7 MmPylRS / *ETV2 stably expressing the aforementioned biological orthogonal translation system;

[0011] (2) Culturing tumor spheres in the cell line Huh-7 MmPylRS / *ETV2 , and adding NAEK during the culturing process to induce endothelial cell transdifferentiation, so as to obtain vascularized tumor spheres.

[0012] Preferably, step (2) specifically comprises adding a culture medium DMEM+10% FBS medium containing 1500 Huh-7 MmPylRS / *ETV2 cells in each well of a cell culture multi-well plate, centrifuging at 100g for 3 minutes, and aggregating the Huh-7 MmPylRS / *ETV2 cells into tumor spheres; on the sixth day, adding a small molecule switch 1 mM NAEK to regulate the expression of the full-length protein of ETV2 122* , replacing the fresh culture medium every day and continuing to add NAEK, and during the development process, NAEK will regulate part of the cells to transdifferentiate into endothelial cells, so as to realize the regulation of the vascularized tumor spheres.

[0013] The Huh-7 cell line is a commercially available cell line.

[0014] The application provides a method for constructing a vascularized tumor sphere based on genetic code recoding technology, and non-natural amino acid NAEK is inserted into position 122 of ETV2 through a biological orthogonal translation system, ETV2 overexpression controlled by NAEK in space and time is realized, cell transdifferentiation into endothelial cells is regulated, and a dynamic regulation vascular endothelial cell transdifferentiation system based on genetic code recoding technology is constructed, so that the construction of a functional vascularized tumor sphere is realized, and non-passaged continuous long-term culture and function maintenance of the vascularized tumor sphere are carried out. The application uses genetic code recoding technology to control the formation of a vascular tumor sphere, has the characteristics of simple operation, strong repeatability and universality, is a near-physiological protein translation regulation means, the protein translation regulation speed is fast and the protein translation regulation can be quickly started or terminated, the vascularization is uniformly regulated, the traditional two-dimensional culture mode can be replaced, the interaction of multiple cells is realized, the vascular tumor sphere is more bionic, and the application provides a high-bionic vascular tumor sphere in vitro model for the fields of new drug research and development and regenerative medicine.

[0015] The application has the advantages that: 1) the translation system of eukaryotes is expanded, and near-physiological protein translation regulation is realized; 2) non-natural amino acids can be introduced into target proteins in a site-specific manner, and the disturbance to the structure of the proteins is small; 3) protein expression is directly regulated at the translation level, and the regulation speed is fast; 4) compared with drug (such as DOX) regulation, the genetic code recoding technology can realize fast start / stop of protein expression; and 5) the vascularization regulated based on the genetic code recoding technology is more uniformly distributed in the tumor sphere. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A flowchart of a method for constructing a vascularized tumor microenvironment based on genetic code recoding technology is provided in the application.

[0017] Figure 2 A plasmid map of the orthogonal translation system constructed in the application, wherein A is a PiggyBac-(7sk-tRNA Pyl )x4-EF1a-MmPylRS plasmid map, B is a PiggyBac-*ETV2 plasmid map, and C is a transposase plasmid map;

[0018] Figure 3 Huh-7 cells stably expressing the orthogonal translation system in the application MmPylRS / *ETV2 Cell and drug screening

[0019] Figure 4 RT-qPCR is used to identify whether the orthogonal translation system is successfully constructed;

[0020] Figure 5 NAEK regulation is verified at the two-dimensional cell level MmPylRS / *ETV2Differentiation of cells into endothelial cells;

[0021] Figure 6 Schematic diagram and light microscope photos of regulating vascularized tumor spheres;

[0022] Figure 7 Immunofluorescence detection of endothelial cell differentiation in 14-day vascularized tumor spheres;

[0023] Figure 8 Transendothelial electrical resistance (TEER) detection of barrier resistance value of vascularized tumor spheres. DETAILED DESCRIPTION

[0024] The application will be further described below in conjunction with the accompanying drawings and examples. The specific examples described herein are merely intended to explain the application, but not to limit the application. In addition, it should be noted that, for the convenience of description, only the parts related to the application are shown in the drawings, but not all the results.

[0025] Example 1

[0026] Step 1, Huh-7 stably expressing orthogonal translation system MmPylRS / *ETV2 Cell construction.

[0027] The biological orthogonal translation system was constructed into the PiggyBac transposon plasmid, i.e. PiggyBac-(7sk-tRNA Pyl )x4-EF1a-MmPylRS and PiggyBac-*ETV2 transposon plasmids (for specific construction, see Figure 2 The *ETV2 here is the ETV2 described above 122* , i.e. introducing a premature stop codon by point mutation at the 122th amino acid encoded by the ETV2 gene; the unmutated ETV2 is in NCBI ACCESSION#: NM_001300974, https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001300974.2), and the two plasmids were transiently transfected into Huh-7 cells together with the transposase plasmid; selection was performed by adding 200ug / mL G418+2ug / mL Puromycin antibiotics, and Huh-7 cells stably expressing orthogonal translation system MmPylRS / tRNA and *ETV2 were obtained MmPylRS / *ETV2 , and were expanded and cultured (for specific details, see Figure 3 . Figure 3 Flowchart in A showing construction of Huh-7 stably expressing translation system MmPylRS / *ETV2 ; Figure 3 Light microscope photos in B showing Huh-7 MmPylRS / *ETV2Cell expansion culture). Stable expression of orthogonal translation system was demonstrated by qRT-PCR (see Figure 4 ) for details).

[0028] Step 2, differentiation of vascularized tumor spheroids

[0029] Resuspend the cells with DMEM + 10% FBS medium, and add 150ul medium (containing 1500 Huh-7 MmPylRS / *ETV2 cells per well) into each well of ultra-low attachment 96-well U-shaped plate. After 100g centrifugation for 3min, tumor spheroids were formed after 24h culture. On the 6th day of tumor spheroid culture, small molecule switch NAEK was added to regulate ETV2 full-length protein expression, and part of the cells were transdifferentiated into endothelial cells, realizing the regulation of vascularized tumor spheroids (see Figure 1 for details).

[0030] Figure 5 The experimental results confirmed that in the two-dimensional culture of Huh-7 MmPylRS / *ETV2 cells, after adding 1mM NAEK directly and continuing to culture for 4 days, the fluorescence photos Figure 5 (A) proved that NAEK could directly regulate the differentiation of Huh-7 MmPylRS / *ETV2 cells into endothelial cells without adding any induction medium. The RT-qPCR results Figure 5 (B) proved that the endothelial cell markers CD31 and VE-Cadherin also presented overexpression state.

[0031] Figure 6 A shows the regulation process of vascularized tumor spheroids. We observed the morphology and size changes of tumor spheroids at different stages under a microscope, and collected the photos of the morphology and size of tumor spheroids after 2 days, 9 days and 14 days of culture, as shown in Figure 6 B, the spheroid diameter of NAEK+ group was significantly smaller than that of the control group on the 9th day, but recovered to similar size on the 14th day, and the core area of the spheroid was bright and transparent.

[0032] Figure 7 We performed cryosection on the tumor spheroids cultured for 30 days, and found that the vascularized tumor spheroids highly expressed ALB+ hepatocarcinoma cells and CD31+ endothelial cells by immunofluorescence detection. The 3D staining of spheroids (~40 microns) showed high expression of endothelial markers and hepatocarcinoma cell markers Figure 7 (A); the 3D cross-section of spheroids showed vascular lumen-like structure Figure 7 (B).

[0033] Figure 8 The transendothelial electrical resistance experiment showed that the TEER value of the vascularized tumor spheroids cultured for 30 days was significantly higher than that of the non-vascularized tumor spheroids, indicating that the vascularized tumor spheroids formed tighter intercellular connections. Figure 8A shows how to detect the barrier inside the sphere; Figure 8 B shows the comparison of the detected TEER values.

[0034] It should be noted that the above only describes the preferred embodiments of the present application and the principles of the applied technology. Those skilled in the art will understand that the present application is not limited to the specific embodiments described herein, and that various obvious changes, re-adjustments and substitutions can be made by those skilled in the art without departing from the scope of the present application. Therefore, although the present application has been described in more detail through the above embodiments, the present application is not limited to the above embodiments, and can include more other equivalent embodiments without departing from the concept of the present application, and the scope of the present application is determined by the scope of the appended claims.

Claims

1. A bio-orthogonal translation system, characterized in that Transposon plasmid PiggyBac-(7sk-tRNA Pyl )x4-EF1a-MmPylRS and PiggyBac-ETV2 122* .

2. The bio-orthogonal translation system of claim 1, wherein The MmPylRS / 4xtRNA system is used in conjunction with the transposase plasmid of the PiggyBac vector system to integrate the MmPylRS / 4xtRNA system into the genome chromosome. Pyl system and ETV2 122* gene into the genome chromosome.

3. The bio-orthogonal translation system of claim 1, wherein the ETV2 122* means an ETV2 gene in which the codon corresponding to the 122nd amino acid of the amino acid sequence is mutated to a stop codon.

4. Use of the bio-orthogonal translation system of any one of claims 1-3 in the construction of a vascularized tumor microenvironment, wherein the construction of a vascularized tumor microenvironment is a non-therapeutic use.

5. A method for constructing a vascularized tumor microenvironment based on genetic code re-encoding technology, characterized in that The steps include: (1) Constructing the biological orthogonal translation system of claim 1, transiently transfecting it into hepatoma cells Huh-7 together with the transposase plasmid, screening the cell line Huh-7 stably expressing the biological orthogonal translation system of claim 1 MmPylRS / *ETV2 ; (2) To the cell line Huh-7 MmPylRS / *ETV2 Spheroids were cultured and NAEK was added at specific stages of the culture to induce partial transdifferentiation of Huh-7 MmPylRS / *ETV2 cells to endothelial cells and obtain vascularized spheroids.

6. The method of claim 5, wherein the tumor spheroid is a tumor spheroid of a solid tumor. Step (2) is specifically adding 1500 Huh-7 cells per well in a cell culture multi-well plate MmPylRS / *ETV2 DMEM + 10% FBS medium, 100g centrifugation for 3 minutes, Huh-7 cells MmPylRS / *ETV2 Cell aggregation into tumor spheres; on day 6, add small molecule switch 1 mM NAEK to regulate ETV2 122* Full-length protein expression, replace fresh medium every day and continue to add NAEK, NAEK will regulate part of the cell transdifferentiation into endothelial cells during development, realize the regulation of vascularized tumor spheres.

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