Bionic hydrogel for culturing tongue cancer organoid and application

Abstract

The invention is applicable to the technical field of biology, and provides bionic hydrogel for culturing tongue cancer organoid and application. The bionic hydrogel is prepared by taking type I collagen, alginate and periostin polypeptide as cores according to the microenvironment characteristics of tongue cancer, and is adapted to the mechanical characteristics of tongue squamous cell carcinoma tissues and the three-dimensional growth space requirements of organoids in the physical aspect; in the biochemical level, the tongue cancer microenvironment is accurately simulated by virtue of the effects of promoting cell adhesion of the I-type collagen and maintaining the dryness of the cancer cells by virtue of the periostin polypeptide, so that a complete culture method is formed. The bionic hydrogel is stable in property and capable of supporting the culture requirement for a long time and enhancing the interaction between cells and a matrix so as to improve the cell adhesion capacity, the cultured tongue cancer organoid can well maintain the heterogeneity and tumor malignant phenotype of tongue cancer cells, and the bionic effect of the organoid is optimized. The result provides a high-precision in-vitro model for tongue cancer drug screening, chemotherapy sensitivity prediction and individualized treatment, and has practical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a biomimetic hydrogel for tongue cancer organoid culture and application. BACKGROUND

[0002] Tongue squamous cell carcinoma is one of the common malignant tumors of the head and neck, which has strong invasiveness and metastatic potential, and the five-year survival rate of patients is low. In the face of this serious clinical situation, through basic research and clinical research, in-depth exploration of tongue cancer treatment strategies and improvement of patient prognosis has become an important task that needs to be promoted at present. The basic research of tumor depends on a reliable tumor model, and the scientificity and applicability of the model directly affect the accuracy and translation value of the research results.

[0003] Traditional two-dimensional cell culture models cannot simulate the complex three-dimensional structure of tumors in vivo, and there is a large difference between their molecular characteristics and patient tissues. Although patient-derived xenograft tumor models can reflect the phenotypic characteristics of patients to some extent, they have problems such as long experimental period, high cost, and difficulty in accurately simulating the specific microenvironment of the patient's primary tumor. In recent years, tumor organoid technology has become a new direction for tumor model research due to its ability to better preserve the genetic and phenotypic characteristics of primary tumors. However, current organoid culture generally relies on commercialized Matrigel, which is derived from mouse sarcoma and has limitations such as large batch differences, high cost, and difficulty in accurately simulating the specific microenvironment of tumor tissue. This leads to a "distortion" phenomenon between the molecular characteristics of the organoid and the patient's primary tissue, thereby affecting its application in basic research and drug development. These limitations indicate that the extracellular matrix is crucial for maintaining the genetic and phenotypic characteristics of tumors. The biochemical composition of the tumor extracellular matrix provides important biological signals for tumor cell proliferation and survival, and the mechanical force stimulation exerted by the matrix also has an important impact on the phenotype of the tumor. Therefore, it is crucial to design a cell culture environment that can accurately simulate the biochemical signals and physical characteristics of the tongue cancer microenvironment. Artificially synthesized hydrogels have become an important research direction for optimizing organoid culture due to their clear composition, strong controllability, and good reproducibility. Based on this, the present application proposes a biomimetic hydrogel for tongue cancer organoid culture and application. SUMMARY

[0004] The purpose of the present application is to provide a biomimetic hydrogel for tongue cancer organoid culture and application, which aims to solve the problems raised in the background art.

[0005] The purpose of the present application is achieved by the following technical solutions: A kind of biomimetic hydrogel for tongue cancer organoid culture, the component thereof includes type I collagen, alginate and periostin polypeptide;The type I collagen includes two alpha 1 chains and an alpha 2 chain, the amino acid sequence of alpha 1 chain is as shown in SEQ ID NO.1, and the amino acid sequence of alpha 2 chain is as shown in SEQ ID NO.2;The amino acid sequence of the periostin polypeptide is as shown in SEQ ID NO.3.

[0006] Further, the alginate is sodium alginate;The final concentration of each component is: 2% (w / v) sodium alginate, 15 μM periostin polypeptide, 1.8 mg / mL type I collagen.

[0007] A preparation method of the above-mentioned biomimetic hydrogel, comprising the following steps: Preparation of sodium alginate solution and periostin polypeptide solution: dissolve sodium alginate powder and periostin polypeptide powder in double distilled water respectively to form a uniform solution; Neutralization of type I collagen solution: place the type I collagen solution on ice, and adjust the pH to 7.0 with a mixed solution; Preparation of biomimetic hydrogel: mix the neutralized type I collagen solution, sodium alginate solution and periostin polypeptide solution to obtain a hydrogel precursor solution;After thoroughly mixing the hydrogel precursor solution at 4℃, immerse it in a calcium chloride solution for cross-linking and solidification to obtain the biomimetic hydrogel.

[0008] Further, the mixed solution comprises 10x Hanks' balanced salt solution and 1M HEPES.

[0009] Further, the temperature of cross-linking and solidification is 37℃, and the time is 15 minutes.

[0010] The biomimetic hydrogel according to the above-mentioned application as a culture matrix in culturing tongue cancer organoids.

[0011] A culture method of tongue cancer organoids, using the above-mentioned biomimetic hydrogel as a culture matrix, comprising the following steps: Resuspend the tumor cells obtained by dissociating tongue squamous cell carcinoma tissue in the biomimetic hydrogel precursor solution; After cross-linking and gelation, add organoid culture medium for culture to obtain tongue cancer organoids that retain the heterogeneity of tongue cancer cells and the malignant phenotype of tumors.

[0012] Compared with the prior art, the beneficial effects of the present application are: The present application is directed to the physical and biochemical characteristics of the tongue cancer microenvironment, and a biomimetic hydrogel is prepared with easily available and low-cost type I collagen, alginate and periostin polypeptide as the core, wherein the physical level adapts to the mechanical characteristics of tongue squamous cell carcinoma tissue and the three-dimensional growth space demand of organoids, the biochemical level relies on the cell adhesion function of type I collagen and the role of periostin polypeptide in maintaining the stemness of cancer cells to accurately simulate the tongue cancer microenvironment, and then a complete method for culturing tongue cancer organoids based on the biomimetic hydrogel is provided. The present application successfully builds a highly biomimetic tongue cancer organoid culture system and a new three-dimensional culture platform, the biomimetic hydrogel used has stable properties and can support the culture demand for a long time, and can also enhance the interaction between cells and the matrix to improve the cell adhesion capacity, and the tongue cancer organoids cultured by the biomimetic hydrogel can well retain the heterogeneity and malignant phenotype of tongue cancer cells, greatly optimizing the biomimetic effect of the organoid model. This achievement has practical significance in tongue cancer drug screening and basic research, which not only provides a high-precision in vitro model for predicting chemotherapy sensitivity, but also opens up a new path for drug screening and individualized treatment of tongue squamous cell carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Fig. 1 is a macro photograph of the biomimetic hydrogel in liquid and gel states; wherein (a) is a macro photograph of the biomimetic hydrogel in liquid state (i.e. the hydrogel precursor solution), and (b) is a macro photograph of the biomimetic hydrogel in gel state.

[0014] Figure 2 Fig. 4 is the elastic modulus of the biomimetic hydrogel and tongue cancer tissue.

[0015] Figure 3 Fig. 5 is the degradation curve of the biomimetic hydrogel.

[0016] Figure 4 Fig. 6 is a scanning electron microscope of the biomimetic hydrogel.

[0017] Figure 5 Fig. 7 is a photograph of tongue cancer organoids cultured by the biomimetic hydrogel and Matrigel.

[0018] Figure 6 Fig. 8 is the HE staining experimental results of tongue cancer organoids cultured by the biomimetic hydrogel and Matrigel.

[0019] Figure 7 Fig. 9 is a whole staining diagram of tongue cancer organoids cultured by the biomimetic hydrogel; wherein (a) is a three-dimensional diagram reconstructed by imaris software, and (b) is a real staining diagram.

[0020] Figure 8 Fig. 10 is the circularity analysis results of tongue cancer organoids cultured by the biomimetic hydrogel and Matrigel.

[0021] Figure 9 Fig. 11 is the gene expression quantity of tongue cancer organoids cultured by the biomimetic hydrogel.

[0022] Figure 10 The expression of "tongue cancer-related oncogenes" in tongue cancer organoids cultured in bionic hydrogel and matrix gel.

[0023] Figure 11 The expression of malignancy-related genes in tongue cancer organoids cultured on biomimetic hydrogel and matrix gel.

[0024] Figure 12 The results of KEGG pathway enrichment analysis of differentially expressed genes in tongue cancer organoids cultured in biomimetic hydrogel and matrix gel.

[0025] Figure 13 The expression of integrin-related genes in tongue cancer organoids cultured on biomimetic hydrogel and matrigel.

[0026] Figure 14 Immunofluorescence staining of ITGB1 in tongue cancer organoids cultured on biomimetic hydrogels and matrigel. DETAILED DESCRIPTION

[0027] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0028] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0029] Example 1: Design and preparation of biomimetic hydrogel; 1. Design basis; The mechanical and biochemical properties of the tumor microenvironment play an important role in tumor cell growth and phenotype. The average elastic modulus of primary tissue from tongue cancer patients (tongue squamous cell carcinoma tissue, hereinafter referred to as "tongue cancer tissue") is 10.7 kPa; type I collagen is a protein widely expressed in tongue cancer and has the function of promoting cell adhesion; periostin is a protein specifically expressed in head and neck squamous cell carcinoma and has the function of maintaining the stemness of cancer cells. Therefore, this embodiment designs a biomimetic hydrogel with type I collagen and periostin polypeptides as the main components and incorporated with sodium alginate. The goal is to simultaneously simulate the physical and biochemical properties of the tongue cancer microenvironment and provide an adaptive matrix for the in vitro culture of tongue cancer organoids.

[0030] Type I collagen is composed of three chains (two α1 chains and one α2 chain). The amino acid sequence of the α1 chain is shown in SEQ ID NO. 1, and the amino acid sequence of the α2 chain is shown in SEQ ID NO. 2. The three chains form a stable triple helical structure through a variety of interactions. These include secondary bonds between peptide chains, such as ionic bonds, hydrogen bonds, and van der Waals interactions, as well as aldol condensation crosslinks, aldehyde-amine condensation crosslinks, and aldol-histidine crosslinks between and within collagen molecules.

[0031] The amino acid sequence of the periostin polypeptide is shown in SEQ ID NO. 3.

[0032] 2. Preparation method (take the preparation of 3 mL hydrogel precursor solution as an example); Preparation of sodium alginate solution: Dissolve 60 mg of sodium alginate powder in 0.8 ml of double distilled water to form a uniform solution.

[0033] Preparation of periostin polypeptide solution: Dissolve 159.2 μg of periostin polypeptide powder in 70 μL of double distilled water to form a uniform solution.

[0034] Neutralization of type I collagen solution (operation on ice): Place 1.8 mL of type I collagen solution (Cellmatrix type I collagen solution (637-00653, Nitta Gelatin)) on ice (0~4°C), and use the mixed solution to adjust the pH value of the type I collagen solution to 7.0. The mixed solution contains: 300 μL of 10×Hanks' balanced salt solution and 30 μL of 1M HEPES (final concentration 10 mM).

[0035] Preparation of biomimetic hydrogel: Mix the neutralized type I collagen solution, sodium alginate solution and periostin polypeptide solution to obtain a hydrogel precursor solution (final concentration: 2% (w / v) sodium alginate, 15 μM periostin polypeptide, 1.8 mg / mL type I collagen); after the hydrogel precursor solution is thoroughly mixed at 4°C, immerse it in a calcium chloride solution (150 mM, 4 mL) and cross-link and solidify at 37°C for 15 minutes to obtain a biomimetic hydrogel. Figure 1 Figures (a) and (b) are macroscopic photographs of the biomimetic hydrogel in liquid state (i.e. hydrogel precursor solution) and gel state, respectively.

[0036] Example 2: Characterization of key properties of biomimetic hydrogel; This example is directed to the biomimetic hydrogel prepared in Example 1, and verifies its elastic modulus, degradability and microstructure. The specific method and results are as follows: 1. Elastic modulus test; An electronic universal testing machine (INSTRON 5944, USA) was used to test the elastic modulus of tongue cancer tissue and biomimetic hydrogel. The tongue cancer tissue and biomimetic hydrogel were prepared into cylindrical samples (diameter 5 mm x height 1.5 mm), and compression was performed at a rate of 1 mm / min. The elastic modulus was calculated based on the linear part of the stress-strain curve. The results are shown in Figure 2As shown, the elastic modulus of the biomimetic hydrogel is 10 kPa, which has no statistical difference from the elastic modulus of tongue cancer tissue, indicating that the biomimetic hydrogel can better simulate the physical properties of the tongue cancer microenvironment.

[0037] 2. Degradability test; The initial mass (W0) of the biomimetic hydrogel was weighed, immersed in culture medium, and placed in a 37°C, 5% CO2 incubator for 14 days. The biomimetic hydrogel was removed from the culture medium at predetermined time points (days 1-14), and the residual liquid on the surface was gently absorbed with filter paper. The remaining mass (Wt) at each time point was recorded using an analytical balance. The mass retention percentage was calculated according to the formula (mass retention rate (%) = Wt / W0 × 100%) to characterize the degradation. The degradation curve of the biomimetic hydrogel is shown in Figure 2. Figure 3 As shown in the figure, the biomimetic hydrogel showed no obvious degradation within 14 days, meeting the matrix stability requirements for long-term culture of tongue cancer organoids.

[0038] 3. Scanning electron microscopy observation; The biomimetic hydrogel was fixed with 2.5% glutaraldehyde, frozen in liquid nitrogen, and then freeze-dried; gold nanoparticles were sputtered on the surface of the dried sample, and its microstructure morphology was observed using a scanning electron microscope. Figure 4 This is a scanning electron microscope image of the bionic hydrogel. The image shows that the pores of the bionic hydrogel are 50-100μm, which is highly compatible with the growth space requirements of tongue cancer organoids and can provide sufficient physical support for the three-dimensional growth of organoids.

[0039] Example 3: Tongue cancer organoids cultured on biomimetic hydrogels are highly heterogeneous; Surgically resected tongue cancer tissue was dissociated using tumor tissue digestion solution. The isolated tumor cells were resuspended in commercial Matrigel or biomimetic hydrogel precursor solution. After cross-linking to form a gel, organoid culture medium (composition: 1% double-streptomycin (penicillin-streptomycin), 1× B27, 1.25mM N-acetyl-L-cysteine, 10mM nicotinamide, 50ng / mL EGF, 500nM A83-01, 10ng / mL FGF10, 5ng / mL FGF2, 1µM prostaglandin E2, 3µM CHIR 99021, 1µM forskolin, 125ng / mL R-spondin 1, 125ng / mL Noggin) was added and cultured in a 37°C, 5% CO2 incubator for 7-10 days. The morphology of tongue cancer organoids cultured in different hydrogels was observed microscopically. Cryosections of tongue cancer organoids cultured in different hydrogels were then processed for hematoxylin and eosin staining. Figure 5 、 Figure 6Pictures of different patient-derived tongue cancer organoids cultured in two hydrogels respectively and HE staining pictures. It can be seen that tongue cancer organoids cultured in Matrigel are round, while tongue cancer organoids cultured in biomimetic hydrogel show irregular morphology, which are oval or surface budding, indicating that tongue cancer organoids cultured in biomimetic hydrogel have strong heterogeneity inside. Whole staining of tongue cancer organoids cultured in biomimetic hydrogel was performed using phalloidin, and then confocal microscope was used to take pictures, and then imaris software was used to reconstruct three-dimensional structure, and the results are shown in Figure 7 Figures (a) and (b). From the appearance, tongue cancer organoids cultured in biomimetic hydrogel show three-dimensional structure, and cystic and solid regions appear inside tongue cancer organoids, which is consistent with the results of HE staining, further confirming the heterogeneity of tongue cancer organoids cultured in biomimetic hydrogel. As shown in Figure 8 Figure, the roundness of tongue cancer organoids cultured in two hydrogels was evaluated, and compared with tongue cancer organoids cultured in Matrigel, the roundness of tongue cancer organoids cultured in biomimetic hydrogel is lower, indicating that the heterogeneity is stronger.

[0040] The above results show that tongue cancer organoids cultured in biomimetic hydrogel are a kind of organoid model with high heterogeneity, which can better reproduce the structural heterogeneity of tongue cancer tumor.

[0041] Example 4: Tongue cancer organoids cultured in biomimetic hydrogel can retain the malignant phenotype of tongue cancer; To evaluate the biomimicry of the hydrogel system, high-throughput RNA sequencing was performed on tongue cancer tissues, Matrigel-cultured organoids and biomimetic hydrogel-cultured organoids. As shown in Figure 9 Figure, transcriptome analysis showed that more than 70% of genes were co-expressed in biomimetic hydrogel and tongue cancer tissues, indicating that tongue cancer organoids cultured in biomimetic hydrogel can effectively retain the transcriptional characteristics of the original tumor. Further define the "tongue cancer-related oncogene" set composed of 1199 genes, whose gene characteristics are high expression in tongue cancer tissues and high expression in epithelial cells in head and neck squamous cell carcinoma (HNSCC) data set. As shown in Figure 10 Compared with tongue cancer organoids cultured in Matrigel, tongue cancer organoids cultured in biomimetic hydrogel have higher expression levels of "tongue cancer-related oncogenes", and the expression levels are similar to those in tongue cancer tissues, indicating that they maintain the malignant phenotype of tongue cancer. Further analysis (see Figure 11 ) shows that tongue cancer organoids cultured in biomimetic hydrogel retain key malignant characteristics, such as high expression levels of malignant epithelial cell differentiation (EPCAM, KRT10, etc.), cell cycle (CDK1, CCNB1, etc.), reflection of cell proliferation characteristics), stem cell stemness (CD133, ALDH1A1, etc.) and hypoxia (HIF1A, etc.).

[0042] The above results show that the biomimetic hydrogel can regulate gene expression by simulating the tumor microenvironment, so that the cultured tongue cancer organoids retain the malignant phenotype at the transcriptome level, and their biological behavior is closer to in vivo tongue cancer. The biomimetic hydrogel significantly optimizes the biomimetic performance of the organoids, providing more reliable model support for tongue cancer basic research and precise diagnosis and treatment.

[0043] Example 5: Biomimetic hydrogel promotes the interaction of tongue cancer organoids with the matrix environment The biological functional significance of the differential genes was analyzed by KEGG pathway analysis. Gene set enrichment analysis (see Figure 12 ) showed that the differential genes up-regulated in tongue cancer organoids cultured in the biomimetic hydrogel were enriched in the cell adhesion molecule (CAMs) related pathway. Further analysis of the expression levels of the integrin family, which is a receptor for cell sensing the environment, showed that, as shown in Figure 13 , the integrin level was higher in tongue cancer organoids cultured in the biomimetic hydrogel than in the Matrigel, and the expression level of ITGB1 was significantly higher than that of other integrin molecules. Immunofluorescence staining also confirmed the high expression level of ITGB1 in tongue cancer organoids cultured in the biomimetic hydrogel (see Figure 14 ). This indicates that the biomimetic hydrogel is conducive to tongue cancer cell adhesion, and the interaction between tongue cancer organoids cultured in the biomimetic hydrogel and the hydrogel is stronger.

[0044] The above results show that the biomimetic hydrogel significantly improves the adhesion ability of tongue cancer organoids by enhancing integrin (especially ITGB1) mediated cell-matrix interaction, and further strengthens the interaction between tongue cancer organoids and the hydrogel interface.

[0045] The above is only a preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the patent.

Claims

1. A biomimetic hydrogel for culturing tongue cancer organoids, characterized in that: Its components include type I collagen, alginate and periostin polypeptide; the type I collagen contains two α1 chains and one α2 chain, the amino acid sequence of the α1 chain is shown in SEQ ID NO.1, and the amino acid sequence of the α2 chain is shown in SEQ ID NO.2; the amino acid sequence of the periostin polypeptide is shown in SEQ ID NO.

3.

2. The biomimetic hydrogel according to claim 1, characterized in that The alginate is sodium alginate; the final concentrations of the components are: 2% (w / v) sodium alginate, 15 μM periostin polypeptide, and 1.8 mg / mL type I collagen.

3. A method for preparing the biomimetic hydrogel according to claim 1 or 2, characterized in that: The following steps are involved: Prepare sodium alginate solution and periostin polypeptide solution: dissolve sodium alginate powder and periostin polypeptide powder in double-distilled water to form a homogeneous solution; Neutralize the type I collagen solution: Place the type I collagen solution on ice and adjust the pH to 7.0 with the mixture; Preparation of biomimetic hydrogel: mixing neutralized type I collagen solution, sodium alginate solution and periostin polypeptide solution to obtain a hydrogel precursor solution; The hydrogel precursor solution was fully mixed at 4° C., and then immersed in a calcium chloride solution for cross-linking and curing to obtain the biomimetic hydrogel.

4. The preparation method according to claim 3, characterized in that The mixture contained 10× Hanks' balanced salt solution and 1 M HEPES.

5. The preparation method according to claim 3, characterized in that The cross-linking and curing temperature is 37° C. and the time is 15 minutes.

6. Use of the biomimetic hydrogel according to claim 1 or 2 as a culture matrix in culturing tongue cancer organoids.

7. A method for culturing tongue cancer organoids, characterized in that: Using the biomimetic hydrogel according to claim 1 or 2 as a culture matrix comprises the following steps: Tumor cells obtained by dissociation of tongue squamous cell carcinoma tissue were resuspended in a biomimetic hydrogel precursor solution; After cross-linking to form a gel, organoid culture medium was added for culture to obtain tongue cancer organoids that retained the heterogeneity of tongue cancer cells and the malignant phenotype of the tumor.

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