Biomimetic hydrogel for tongue cancer organoid culture and application thereof

Abstract

The application belongs to the technical field of biotechnology, and provides a biomimetic hydrogel for tongue cancer organoid culture and application.According to the characteristics of the tongue cancer microenvironment, the biomimetic hydrogel is prepared by taking collagen type I, alginate and periostin polypeptide as the core, the mechanical characteristics of tongue squamous cell carcinoma tissue and the three-dimensional growth space requirements of organoids are adapted on the physical level, the tongue cancer microenvironment is precisely simulated on the biochemical level by relying on the cell adhesion promotion of collagen type I and the role of periostin polypeptide in maintaining the stemness of cancer cells, and a complete culture method is formed.The biomimetic hydrogel is stable in nature, can support the culture requirements for a long time, can 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 of tongue cancer cells and the malignant phenotype of tumors, and the biomimetic effect of the organoids is optimized.The achievement 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:

[0006] 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.

[0007] 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.

[0008] A preparation method of the above-mentioned biomimetic hydrogel, comprising the following steps:

[0009] Prepare 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;

[0010] Neutralize the type I collagen solution: place the type I collagen solution on ice, and adjust the pH to 7.0 using a mixed solution;

[0011] Prepare the 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.

[0012] Further, the mixed solution comprises 10x Hanks' balanced salt solution and 1M HEPES.

[0013] Further, the temperature of cross-linking and solidification is 37℃, and the time is 15 minutes.

[0014] The biomimetic hydrogel as described above is used as a culture matrix in the culture of tongue cancer organoids.

[0015] A culture method of tongue cancer organoids uses the above-mentioned biomimetic hydrogel as a culture matrix, comprising the following steps:

[0016] Resuspend the tumor cells obtained by dissociating tongue squamous cell carcinoma tissue in the biomimetic hydrogel precursor solution;

[0017] 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.

[0018] Compared with the prior art, the beneficial effects of the present application are:

[0019] 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 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

[0020] Figure 1 Fig. 1 is a macroscopic photograph of the biomimetic hydrogel in liquid and gel states; wherein (a) is a macroscopic photograph of the biomimetic hydrogel in liquid state (i.e. the hydrogel precursor solution), and (b) is a macroscopic photograph of the biomimetic hydrogel in gel state.

[0021] Figure 2 Fig. 4 is the elastic modulus of the biomimetic hydrogel and tongue cancer tissue.

[0022] Figure 3 Fig. 5 is the degradation curve of the biomimetic hydrogel.

[0023] Figure 4 Fig. 6 is a scanning electron microscope of the biomimetic hydrogel.

[0024] Figure 5 Fig. 7 is a photograph of tongue cancer organoids cultured by the biomimetic hydrogel and Matrigel.

[0025] Figure 6 Fig. 8 is the HE staining experimental results of tongue cancer organoids cultured by the biomimetic hydrogel and Matrigel.

[0026] 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.

[0027] Figure 8 Fig. 10 is the roundness analysis results of tongue cancer organoids cultured by the biomimetic hydrogel and Matrigel.

[0028] Figure 9 Fig. 11 is the gene expression quantity of tongue cancer organoids cultured by the biomimetic hydrogel.

[0029] Figure 10 Expression of tongue cancer related oncogenes in tongue cancer organoids cultured by biomimetic hydrogel and matrigel.

[0030] Figure 11 Expression of tumor related genes in tongue cancer organoids cultured by biomimetic hydrogel and matrigel.

[0031] Figure 12 KEGG pathway enrichment analysis results of differentially expressed genes in tongue cancer organoids cultured by biomimetic hydrogel and matrigel.

[0032] Figure 13 Expression of integrin related genes in tongue cancer organoids cultured by biomimetic hydrogel and matrigel.

[0033] Figure 14 ITGB1 immunofluorescence staining of tongue cancer organoids cultured by biomimetic hydrogel and matrigel. DETAILED DESCRIPTION

[0034] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the present application.

[0035] The specific implementation of the present application will be described in detail below in combination with specific examples.

[0036] Example 1: Design and preparation of biomimetic hydrogel

[0037] 1. Design basis

[0038] The mechanical properties and biochemical characteristics of the tumor microenvironment play an important role in tumor cell growth and phenotype. The average elastic modulus of the primary tissue of tongue cancer patients (tongue squamous cell carcinoma tissue, hereinafter referred to as "tongue cancer tissue") is 10.7 kPa; collagen type I is a protein widely expressed in tongue cancer, which has the function of promoting cell adhesion; periostin is a protein specifically expressed in head and neck squamous cell carcinoma, which has the function of maintaining cancer stem cell stemness. Therefore, this embodiment designs a biomimetic hydrogel with collagen type I and periostin polypeptide as the main components and sodium alginate incorporated, aiming to simultaneously simulate the physical properties and biochemical characteristics of the tongue cancer microenvironment and provide an adaptive matrix for the in vitro culture of tongue cancer organoids.

[0039] Collagen type I is composed of three chains (two α1 chains and one α2 chain), the amino acid sequence of α1 chain is shown as SEQ ID NO. 1, and the amino acid sequence of α2 chain is shown as SEQ ID NO. 2. The three chains form a stable triple helix structure through various forces. Including inter-peptide secondary bonds, such as ionic bonds, hydrogen bonds, van der Waals forces, and alcohol aldehyde condensation cross-linking, aldehyde amine condensation cross-linking and alcohol aldehyde histidine cross-linking between and within collagen molecules.

[0040] The amino acid sequence of periostin polypeptide is shown as SEQ ID NO. 3.

[0041] 2. Preparation method (take the preparation of 3 mL hydrogel precursor solution as an example);

[0042] 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.

[0043] 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.

[0044] Neutralization of collagen type I solution (operation on ice): place 1.8 mL of collagen type I solution (Cellmatrix type I collagen solution (637-00653, Nitta Gelatin)) on ice (0~4°C), and use the mixed solution to neutralize the collagen type I solution to adjust the pH value to 7.0. The mixed solution contains: 10×Hanks' balanced salt solution 300 μL and 1M HEPES 30 μL (final concentration 10 mM).

[0045] Preparation of biomimetic hydrogel: mix the neutralized collagen type I 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 collagen type I); after the hydrogel precursor solution is thoroughly mixed at 4°C, it is immersed in a calcium chloride solution (150 mM, 4 mL) and cross-linked and solidified 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.

[0046] Example 2: Characterization of key properties of biomimetic hydrogel;

[0047] This example is directed to the biomimetic hydrogel prepared in Example 1, to verify its elastic modulus, degradability and microstructure, and the specific method and results are as follows:

[0048] 1. Elastic modulus test;

[0049] The elastic modulus of tongue cancer tissue and biomimetic hydrogel was tested by electronic universal testing machine (INSTRON 5944, USA). 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 2 It can be seen that the elastic modulus of the biomimetic hydrogel is 10 kPa, which has no statistical difference with the elastic modulus of the tongue cancer tissue, indicating that the biomimetic hydrogel can better simulate the physical properties of the tongue cancer microenvironment.

[0050] 2. Degradation test;

[0051] The initial mass (W0) of the biomimetic hydrogel was weighed, and it was immersed in the culture medium and placed in a 37°C, 5% CO2 incubator for 14 days. At the preset time points (1-14 days), the biomimetic hydrogel was taken out from the culture medium, the surface residual liquid was lightly absorbed with filter paper, and 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 x 100%) to characterize the degradation. The degradation curve of the biomimetic hydrogel is shown in Figure 3 It can be seen that the biomimetic hydrogel does not show obvious degradation within 14 days, which meets the stability requirement of the matrix for long-term culture of tongue cancer organoids.

[0052] 3. Scanning electron microscope observation;

[0053] The biomimetic hydrogel was fixed with 2.5% glutaraldehyde, and then freeze-dried after liquid nitrogen freezing. The surface of the dried sample was sputtered with gold nanoparticles, and the microstructure morphology was observed using a scanning electron microscope. Figure 4 The scanning electron microscope image of the biomimetic hydrogel is shown in the figure, which shows that the pores of the biomimetic hydrogel are 50-100 μm, which is highly compatible with the growth space requirement of tongue cancer organoids, and can provide sufficient physical support for the three-dimensional growth of organoids.

[0054] Example 3: Tongue cancer organoids cultured by biomimetic hydrogel have high heterogeneity;

[0055] Surgically removed tongue cancer tissue was dissected using tumor tissue digestion fluid. The isolated tumor cells were resuspended in commercially available matrix gel or biomimetic hydrogel precursor solutions, cross-linked into gels, and then added to organoid culture medium (components: 1% penicillin-streptomycin, 1×B27, 1.25mM N-acetyl-L-cysteine, 10mM nicotinamide, 50ng / mL EGF, 500nMA83-01, 10ng / mL FGF10, 5ng / mL FGF2, 1µM prostaglandin E2, 3µM CHIR 99021, 1µM laryngin, 125ng / mL R-Spondin 1, 125ng / mL Noggin). The culture was incubated at 37℃ in a 5% CO2 incubator for 7–10 days. The morphology of tongue cancer organoids cultured in different hydrogels was observed under a microscope, and frozen sections and HE staining were then performed on the organoids cultured in different hydrogels. Figure 5 , Figure 6 Images of tongue cancer organoids from different patients cultured using two different hydrogels, along with HE-stained images, are shown. It is evident that tongue cancer organoids cultured in Mastolte exhibit a nearly round shape, while those cultured in biomimetic hydrogel show irregular morphologies, appearing oval or with budding surfaces, indicating stronger internal heterogeneity in the biomimetic hydrogel-cultured organoids. The biomimetic hydrogel-cultured tongue cancer organoids were then stained using phalloidin, photographed using a confocal microscope, and their three-dimensional structures reconstructed using Imaris software. The results are shown below. Figure 7 As shown in (a) and (b), the tongue cancer organoids cultured in the biomimetic hydrogel exhibit a three-dimensional structure. Both cystic and solid regions are present within the organoids, consistent with HE staining results, further confirming the internal heterogeneity of the tongue cancer organoids cultured in the biomimetic hydrogel. Figure 8 As shown, the morphological differences of tongue cancer organoids cultured by the two hydrogels were assessed using roundness. Compared with tongue cancer organoids cultured by matrix gel, organoids cultured by biomimetic hydrogel had lower roundness, indicating stronger heterogeneity.

[0056] The above results indicate that biomimetic hydrogel-cultured tongue cancer organoids are highly heterogeneous organoid models that can better reproduce the structural heterogeneity of tongue cancer tumors.

[0057] Example 4: Tongue cancer organoids cultured in biomimetic hydrogels can retain the malignant phenotype of tongue cancer;

[0058] To evaluate the biomimetic properties of the hydrogel system, high-throughput RNA sequencing was performed on tongue cancer tissue, matrix gel-cultured organoids, and biomimetic hydrogel-cultured organoids in this embodiment. The results are as follows: Figure 9 As shown, transcriptomic analysis revealed that the biomimetic hydrogel co-expressed more than 70% of the contents of tongue cancer tissue. ), indicating that the tongue cancer organoids cultured in the biomimetic hydrogel effectively retained the transcriptional characteristics of the original tumor. Further, a "tongue cancer-related oncogene" set consisting of 1199 genes was defined, which was characterized by genes that were simultaneously highly expressed in tongue cancer tissues and in epithelial cells in the head and neck squamous cell carcinoma (HNSCC) dataset. As shown in Figure 10 , tongue cancer organoids cultured in the biomimetic hydrogel had higher expression levels of "tongue cancer-related oncogenes" than tongue cancer organoids cultured in Matrigel, and the expression levels were similar to those in tongue cancer tissues, indicating that they maintained the malignant phenotype of tongue cancer. Further analysis showed (see Figure 11 ) that tongue cancer organoids cultured in the biomimetic hydrogel retained high expression levels of key malignant characteristics, such as malignant epithelial cell differentiation (EPCAM, KRT10, etc.), cell cycle (CDK1, CCNB1, etc.), reflecting the retention of cell proliferation characteristics), stem cell stemness (CD133, ALDH1A1, etc.), and hypoxia (HIF1A, etc.) related genes.

[0059] The above results show that the biomimetic hydrogel can regulate gene expression by simulating the tumor microenvironment, allowing tongue cancer organoids cultured in the biomimetic hydrogel to retain the malignant phenotype at the transcriptome level, and their biological behavior is closer to that of tongue cancer in vivo. The biomimetic hydrogel significantly optimizes the biomimetic performance of the organoids, providing more reliable model support for basic research and precise diagnosis and treatment of tongue cancer.

[0060] Example 5: Biomimetic hydrogel promotes the interaction of tongue cancer organoids with the matrix environment;

[0061] The biological functional significance of the differential genes was analyzed by KEGG pathway analysis. Gene set enrichment analysis showed (see Figure 12 ) that the up-regulated differential genes of tongue cancer organoids cultured in the biomimetic hydrogel were enriched in the cell adhesion molecule (CAMs) pathway compared to Matrigel. Further analysis of the expression levels of the integrin family, which are receptors for cell sensing the environment, showed (see Figure 13 ) that the integrin levels of tongue cancer organoids cultured in the biomimetic hydrogel were higher than those of tongue cancer organoids cultured in Matrigel, with ITGB1 expression levels significantly higher than those of other integrin molecules. Immunofluorescence staining also confirmed the high expression levels 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 and the hydrogel is stronger.

[0062] 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, thereby strengthening the interaction between tongue cancer organoids and the hydrogel interface.

[0063] The above are only preferred embodiments of the present application, it should be pointed out that, for those skilled in the art, without departing from the concept of the present application, can also make several variations and improvements, these should also be considered as the protection scope of the present application, these will not affect the effect and the practicality of the patent of the present application.

Claims

1. A biomimetic hydrogel for culturing tongue cancer organoids, characterized in that, Its components include type I collagen, alginate, and periosteal protein polypeptide; the type I collagen comprises 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 periosteal protein polypeptide is shown in SEQ ID NO.

3. The alginate is sodium alginate; the final concentrations of each component are: 2% w / v sodium alginate, 15 μM periosteal protein polypeptide, and 1.8 mg / mL type I collagen.

2. A method for preparing the biomimetic hydrogel according to claim 1, characterized in that, Includes the following steps: Preparation of sodium alginate solution and periosteum protein polypeptide solution: Sodium alginate powder and periosteum protein polypeptide powder were dissolved in double-distilled water to form homogeneous solutions; Neutralize type I collagen solution: Place type I collagen solution on ice and adjust pH to 7.0 using a mixture; Preparation of biomimetic hydrogel: Neutralized type I collagen solution, sodium alginate solution and periosteal protein polypeptide solution were mixed to obtain hydrogel precursor solution; The hydrogel precursor solution was thoroughly mixed at 4°C and then immersed in calcium chloride solution for cross-linking and curing to obtain the biomimetic hydrogel.

3. The preparation method according to claim 2, characterized in that, The mixture contains 10×Hanks' balanced salt solution and 1M HEPES.

4. The preparation method according to claim 2, characterized in that, The cross-linking curing temperature is 37°C and the time is 15 minutes.

5. The application of the biomimetic hydrogel according to claim 1 as a culture substrate in the preparation of tongue cancer organoids.

6. A method for culturing tongue cancer organoids, characterized in that, Using the biomimetic hydrogel of claim 1 as a culture substrate includes the following steps: Tumor cells obtained from the dissociation of tongue squamous cell carcinoma tissue were resuspended in a biomimetic hydrogel precursor solution; After cross-linking into a gel, the gel was added to organoid culture medium for culturing, resulting in tongue cancer organoids that retained the heterogeneity of tongue cancer cells and the malignant phenotype of the tumor.

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