In-vitro tumor cell 3D (three-dimensional) budding invasion model as well as construction method and application thereof

Abstract

The invention relates to an in-vitro tumor cell 3D budding invasion model and a construction method and application thereof. A chip model for researching tumor invasion behaviors is manufactured through a microlithography technology. The model can simulate the interaction between a tumor cell nest and an induced cell in an in-vivo tumor microenvironment. The model has the advantage that the influence and promotion effect of the induced cells on the invasion behavior of the tumor cell nests can be intuitively monitored in real time. Compared with the prior art, the 3D invasion model has remarkable advantages in the aspects of universality and practicability. The device is not only suitable for research of different types of tumors, but also capable of displaying the dynamic invasion process of tumor cell nests in different forms under the action of induced cells. The model provides important experimental basis and theoretical support for research on tumor invasion behaviors, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an in vitro tumor cell 3D budding invasion model and a construction method and application thereof. Background Art

[0002] The interaction between tumor cells and the tumor microenvironment (TME) is crucial for the invasive growth of oral squamous cell carcinoma (OSCC). The TME is composed of multiple cell types, including tumor cells, fibroblasts, and immune cells, which influence tumor progression and metastasis through complex interactions. Stromal cells in the TME, especially fibroblasts, have a significant impact on tumor invasion. These cells promote tumor cell invasion and metastasis through cytokine secretion, physical interaction, and remodeling of the extracellular matrix.

[0003] The tumor invasion front is the interface between tumor and normal tissue. Its invasion patterns are primarily categorized into two types: advancing invasion and budding invasion. Advancing invasion manifests as tumor cells forming large nests that push outward, while budding invasion consists of small islands of cancer cells or single cells, forming an irregular invasive margin. Studying tumor invasion requires appropriate models that can mimic this complex process. Currently, in vitro 2D models are widely used due to their ease of use, but they fail to fully replicate the in vivo microenvironment. In vivo models use stromal cells as inducing cells, mixing tumor cells with stromal cells and injecting them into animals to observe their invasion. While reliable, these models are costly and time-consuming. In vitro 3D models combine the advantages of both 2D and in vivo models. For example, fibroblasts and tumor cells are mixed in a specific ratio, first spheroidized in a low-adhesion U-shaped well plate, then seeded in Matrigel to observe fibroblast-guided tumor invasion. However, these models still have limitations, namely, they lack the geometrical morphology of in vivo tumor cell nests and cannot simulate the diverse morphologies of tumor cell nests.

[0004] In summary, in-depth research on the mechanisms of action of interstitial cells in the tumor microenvironment is of great significance for understanding tumor invasion and metastasis. At the same time, the development of more precise tumor invasion models will help to reveal the complex relationship between tumors and the microenvironment and provide new strategies for cancer treatment. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of existing technologies by providing an in vitro 3D tumor cell budding invasion model, its construction method, and its application. The present invention utilizes microlithography to create a chip model for studying tumor invasion behavior. By simulating the morphology of various tumor cell nests in vivo, the role of induced cells in inducing invasion of different tumor cell nests can be observed. The present invention prepares Matrigel microwells containing induced cells by digesting the induced cells and mixing them with Matrigel, allowing for further study of the role of induced cells in tumor invasion.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for constructing an in vitro tumor cell 3D budding invasion model, comprising the following steps:

[0008] S1, preparing PDMS membrane;

[0009] S2. Preparation of PDMS membrane containing bovine serum albumin:

[0010] Adding bovine serum albumin dropwise onto the surface of the PDMS membrane obtained in step S1, and allowing to stand at low temperature to obtain a PDMS membrane containing bovine serum albumin;

[0011] S3. Preparation of Matrigel Microwells Containing Induced Cells:

[0012] Digesting the induced cells and mixing them with the matrigel, then adding them dropwise onto the PDMS membrane containing bovine serum albumin obtained in step S2, solidifying them, and peeling the PDMS membrane off the solidified matrigel to obtain matrigel microwells containing the induced cells;

[0013] S4. Prepare tumor cell suspension;

[0014] S5. Preparation of 3D invasion model of tumor cell nests with different morphologies induced by induced cells:

[0015] The tumor cell suspension obtained in step S4 is added dropwise to the surface of the matrix gel micropores containing the induced cells obtained in step S3, and cultured at a constant temperature to obtain a method for constructing an in vitro tumor cell 3D budding invasion model.

[0016] Furthermore, in step S1, the method for preparing the PDMS membrane is as follows:

[0017] S1-1, design silicon wafer;

[0018] S1-2, mixing a silicon-based polymer and a cross-linking agent, coating the mixture on the silicon wafer obtained in step S1-1, and curing the mixture at high temperature to obtain a silicon wafer covered with PDMS;

[0019] S1-3. Peel off the PDMS from the silicon wafer to obtain a PDMS membrane, which is then sterilized by UV and set aside.

[0020] Furthermore, in step S1-1, the thickness of the silicon wafer is 4 to 6 mm, preferably 5 mm.

[0021] Furthermore, in step S1-1, the silicon wafer is an SU8 silicon wafer template, and its preparation method is as follows:

[0022] S1-1-1. Model design:

[0023] Use AutoCAD software to draw the model and print the mask;

[0024] S1-1-2, Silicon wafer cleaning:

[0025] Immerse the silicon wafer in anhydrous ethanol and ultrasonically clean it for 10 to 30 minutes, then ultrasonically clean it with deionized water for 10 to 30 minutes, blow dry it, bake it on a hot plate at 120°C for 10 to 30 minutes, and cool it to room temperature;

[0026] S1-1-3. Spin coating of photoresist:

[0027] Treat the silicon wafer with plasma for 5-10 minutes, then drop the photoresist onto the treated silicon wafer, remove bubbles in the resin, and then spin-coat at a speed of 1200 rpm for 20 seconds.

[0028] S1-1-4, pre-baking:

[0029] Bake on a hot plate at 120°C for 5-10 minutes to improve the adhesion between the photoresist and the silicon wafer, then cool to room temperature;

[0030] S1-1-5, Exposure:

[0031] Place the silicon wafer on the UV exposure machine stage, attach the mask to the silicon wafer with the adhesive coating, and expose the wafer under a light intensity of 11mW / cm 2 Expose for 45 seconds under the conditions of , at which point the designed model is transferred to a silicon wafer with photoresist;

[0032] S1-1-6, post-baking:

[0033] Bake on a hot plate at 100°C for 5-10 minutes, then cool to room temperature;

[0034] S1-1-7, Development:

[0035] Use ethyl lactate to remove the uncured photoresist and develop for 15 minutes; then wash the ethyl lactate with isopropyl alcohol; after washing, use ddH2O to remove the residual isopropyl alcohol, blow dry and then examine under a microscope;

[0036] S1-1-8, Hard membrane:

[0037] Bake on a hot plate at 165°C for 20-40 minutes to ensure a firm bond between the photoresist and the silicon wafer, then cool to room temperature.

[0038] S1-1-9, Surface treatment:

[0039] The hydrophobic film is formed by using trimethylchlorosilane for hydrophobic treatment, which facilitates the peeling of the cured PDMS from the silicon wafer without damaging the structure on the silicon wafer.

[0040] Furthermore, in step S1-2, the silicone polymer and cross-linking agent are of the type Sylgard Silicone elastomer 184;

[0041] The weight ratio of the silicon polymer to the cross-linking agent is (9-11):1, preferably 10:1;

[0042] The curing temperature is 20-150° C., preferably 80° C., and the curing time is 20-40 minutes, preferably 30 minutes.

[0043] Furthermore, in step S1-3, the PDMS membrane has a length of 4 to 6 mm, a width of 4 to 6 mm, and a thickness of 4 to 6 mm;

[0044] The length is preferably 5 mm, the width is preferably 5 mm, and the thickness is preferably 5 mm.

[0045] Furthermore, in step S2, the concentration of bovine serum albumin is 0.8-1.2%, preferably 1%;

[0046] The standing time is 10 to 20 hours, and the standing temperature is 4°C.

[0047] Furthermore, in step S2, the obtained PDMS membrane containing bovine serum albumin is cleaned, and the specific steps are as follows:

[0048] Matrigel was added dropwise onto the PDMS membrane containing bovine serum albumin, and the washing was repeated twice.

[0049] Furthermore, the matrix gel comprises Collagen I and Matrigel,

[0050] The concentration of Collagen I is 2.5-3.5 mg / mL, preferably 3 mg / mL;

[0051] The volume percentage of Matrigel in Collagen I is 20-30%, preferably 25%.

[0052] Furthermore, in step S3, the induced cells include normal fibroblasts GNFs and cancer-associated fibroblasts CAFs.

[0053] Furthermore, the normal fibroblasts and cancer-associated fibroblasts are derived from gingiva.

[0054] Furthermore, in step S3, the induced cells are digested and mixed with the matrigel, and then added dropwise onto the PDMS membrane containing bovine serum albumin obtained in step S2, which is then turned over to place the bottom surface of the matrigel on the support and solidified.

[0055] Furthermore, in step S3, the induced cell density is 2×10 5 ~4×10 5 / mL, preferably 2.5×10 5 / mL;

[0056] The amount of the matrix gel containing the induced cells is 20 to 40 μL, preferably 30 μL;

[0057] The curing temperature is 30-50°C, preferably 37°C, and the curing time is 40-80 minutes, preferably 60 minutes;

[0058] The spatial depth of the matrix gel micropores containing the induced cells is 100 to 200 μm, preferably 150 μm.

[0059] Furthermore, in step S4, the method for preparing the tumor cell suspension is as follows:

[0060] The adherent cells were washed twice with PBS buffer, digested with 0.25% trypsin for 3 min, and then digested with serum-containing culture medium to terminate the digestion. The cells were centrifuged to remove the supernatant and resuspended in culture medium for counting.

[0061] Furthermore, the components and mass ratio of the culture medium are: 89% DMEM high glucose, 10% FBS and 1% double antibody (a mixture of penicillin and streptomycin).

[0062] Furthermore, in step S4, the concentration of the tumor cell suspension is 5×10 6 ~1×10 7 pieces / mL.

[0063] Furthermore, in step S5, the amount of the tumor cell suspension is 20-40 μL, preferably 30 μL.

[0064] Furthermore, in step S5, the method for preparing the 3D invasion model of tumor cells cultured alone is as follows:

[0065] S5-1. The tumor cell suspension obtained in step S4 is added dropwise to the surface of the Matrigel micropores containing the induced cells obtained in step S3. After standing for a period of time, the Matrigel micropores are tilted at a certain angle, and culture medium is taken to flush out the tumor cells outside the Matrigel micropores. The tumor cells are then cultured at a constant temperature to stabilize the tumor cells within the Matrigel micropores.

[0066] S5-2. Place the Matrigel cover on the surface of the Matrigel micropores containing tumor cells to prevent the tumor cells in the Matrigel micropores from overflowing, then place it in culture medium, and culture at a constant temperature to obtain an in vitro tumor cell 3D budding invasion model.

[0067] Furthermore, in step S5-1, the standing time is 2 to 4 minutes, preferably 3 minutes;

[0068] The inclination angle is 40° to 50°, preferably 45°;

[0069] The constant temperature incubation temperature is 37°C, and the time is 4 to 6 minutes, preferably 5 minutes;

[0070] The components and mass ratio of the culture medium are: 89% DMEM high glucose, 10% FBS and 1% double antibody (a mixture of penicillin and streptomycin).

[0071] Furthermore, in step S5-2, the method for preparing the Matrigel cover is as follows: dropping Matrigel on a cover glass, placing the cover glass in an incubator for 30 minutes, to obtain a Matrigel cover;

[0072] The constant temperature culture temperature is 37° C., and the constant temperature culture condition is a CO 2 concentration of 5%.

[0073] The present invention also provides an in vitro tumor cell 3D budding invasion model, which is constructed by the above-mentioned in vitro tumor cell 3D budding invasion model construction method.

[0074] The present invention also provides an application of an in vitro tumor cell 3D budding invasion model, and the application of the in vitro tumor cell 3D budding invasion model in preparing a drug for evaluating the effect of inducing cells on inducing tumor invasion.

[0075] In addition, the present invention also provides a 3D invasion model for individually culturing tumor cell nests of different morphologies, the construction method of which comprises the following steps:

[0076] S1, preparing PDMS membrane;

[0077] S2. Preparation of PDMS membrane containing bovine serum albumin:

[0078] Adding bovine serum albumin dropwise onto the surface of the PDMS membrane obtained in step S1, and allowing to stand at low temperature to obtain a PDMS membrane containing bovine serum albumin;

[0079] S3. Preparation of Matrigel Microwells:

[0080] Adding Matrigel dropwise onto the PDMS membrane containing bovine serum albumin obtained in step S2, solidifying it, and peeling the PDMS membrane off the solidified Matrigel to obtain Matrigel micropores;

[0081] S4. Prepare tumor cell suspension;

[0082] S5. Preparation of 3D invasion model of tumor cells cultured alone:

[0083] The tumor cell suspension obtained in step S4 is added dropwise to the surface of the matrix gel micropores obtained in step S3, and cultured at a constant temperature to obtain a 3D invasion model of tumor cells cultured alone.

[0084] Furthermore, in step S1, the method for preparing the PDMS membrane is as follows:

[0085] S1-1, preparing silicon wafer;

[0086] S1-2, mixing a silicon-based polymer and a cross-linking agent, coating the mixture on the silicon wafer obtained in step S1-1, and curing the mixture at high temperature to obtain a silicon wafer covered with PDMS;

[0087] S1-3. Peel off the PDMS from the silicon wafer to obtain a PDMS membrane, which is then sterilized by UV and set aside.

[0088] Furthermore, in step S1-1, the thickness of the silicon wafer is 4 to 6 mm, preferably 5 mm.

[0089] Furthermore, in step S1-1, the silicon wafer is an SU8 silicon wafer template, and its preparation method is as follows:

[0090] S1-1-1. Model design:

[0091] Use AutoCAD software to draw the model and print the mask;

[0092] S1-1-2, Silicon wafer cleaning:

[0093] Immerse the silicon wafer in anhydrous ethanol and ultrasonically clean it for 10 to 30 minutes, then ultrasonically clean it with deionized water for 10 to 30 minutes, blow dry it, bake it on a hot plate at 120°C for 10 to 30 minutes, and cool it to room temperature;

[0094] S1-1-3. Spin coating of photoresist:

[0095] Treat the silicon wafer with plasma for 5-10 minutes, then drop the photoresist onto the treated silicon wafer, remove bubbles in the resin, and then spin-coat at a speed of 1200 rpm for 20 seconds.

[0096] S1-1-4, pre-baking:

[0097] Bake on a hot plate at 120°C for 5-10 minutes to improve the adhesion between the photoresist and the silicon wafer, then cool to room temperature;

[0098] S1-1-5, Exposure:

[0099] Place the silicon wafer on the UV exposure machine stage, attach the mask to the silicon wafer with the adhesive coating, and expose the wafer under a light intensity of 11mW / cm 2 Expose for 45 seconds under the conditions of , at which point the designed model is transferred to a silicon wafer with photoresist;

[0100] S1-1-6, post-baking:

[0101] Bake on a hot plate at 100°C for 5-10 minutes, then cool to room temperature;

[0102] S1-1-7, Development:

[0103] Use ethyl lactate to remove the uncured photoresist and develop for 15 minutes; then wash the ethyl lactate with isopropyl alcohol; after washing, use ddH2O to remove the residual isopropyl alcohol, blow dry and then examine under a microscope;

[0104] S1-1-8, Hard membrane:

[0105] Bake on a hot plate at 165°C for 20-40 minutes to ensure a firm bond between the photoresist and the silicon wafer, then cool to room temperature.

[0106] S1-1-9, Surface treatment:

[0107] The hydrophobic film is formed by using trimethylchlorosilane for hydrophobic treatment, which facilitates the peeling of the cured PDMS from the silicon wafer without damaging the structure on the silicon wafer.

[0108] Furthermore, in step S1-2, the silicone polymer and cross-linking agent are of the type Sylgard Silicone elastomer 184;

[0109] The weight ratio of the silicon polymer to the cross-linking agent is (9-11):1, preferably 10:1;

[0110] The curing temperature is 20-150° C., preferably 80° C., and the curing time is 20-40 minutes, preferably 30 minutes.

[0111] Furthermore, in step S1-3, the PDMS membrane has a length of 4 to 6 mm, a width of 4 to 6 mm, and a thickness of 4 to 6 mm;

[0112] The length is preferably 5 mm, the width is preferably 5 mm, and the thickness is preferably 5 mm.

[0113] Furthermore, in step S2, the concentration of bovine serum albumin is 0.8-1.2%, preferably 1%;

[0114] The standing time is 10 to 20 hours, and the standing temperature is 4°C.

[0115] Furthermore, in step S2, the obtained PDMS membrane containing bovine serum albumin is cleaned, and the specific steps are as follows:

[0116] Matrigel was added dropwise onto the PDMS membrane containing bovine serum albumin, and the washing was repeated twice.

[0117] Furthermore, the matrix gel comprises Collagen I and Matrigel,

[0118] The concentration of Collagen I is 2.5-3.5 mg / mL, preferably 3 mg / mL;

[0119] The volume percentage of Matrigel in Collagen I is 20-30%, preferably 25%.

[0120] Furthermore, in step S3, matrigel is added dropwise onto the PDMS membrane containing bovine serum albumin obtained in step S2, and the membrane is turned over so that the bottom surface of the matrigel is placed on the support and solidified.

[0121] Furthermore, in step S3, the amount of the matrix gel is 20 to 40 μL, preferably 30 μL;

[0122] The curing temperature is 30-50°C, preferably 37°C, and the curing time is 40-80 minutes, preferably 60 minutes;

[0123] The spatial depth of the matrix gel micropores is 100 to 200 μm, preferably 150 μm.

[0124] Furthermore, in step S4, the method for preparing the tumor cell suspension is as follows:

[0125] The adherent cells were washed twice with PBS buffer, digested with 0.25% trypsin for 3 min, and then digested with serum-containing culture medium to terminate the digestion. The cells were centrifuged to remove the supernatant and resuspended in culture medium for counting.

[0126] Furthermore, the components and mass ratio of the culture medium are: 89% DMEM high glucose, 10% FBS and 1% double antibody (a mixture of penicillin and streptomycin).

[0127] Furthermore, in step S4, the concentration of the tumor cell suspension is 5×10 6 ~1×107 pieces / mL.

[0128] Furthermore, in step S5, the amount of the tumor cell suspension is 20-40 μL, preferably 30 μL.

[0129] Furthermore, in step S5, the method for preparing the 3D invasion model of tumor cells cultured alone is as follows:

[0130] S5-1. Add the tumor cell suspension obtained in step S4 dropwise to the surface of the Matrigel micropores obtained in step S3. After a period of stabilization, tilt the Matrigel micropores to a certain angle, flush the tumor cells outside the Matrigel micropores with culture medium, and incubate at a constant temperature to stabilize the tumor cells within the Matrigel micropores.

[0131] S5-2. Place the Matrigel cover on the surface of the Matrigel micropores containing tumor cells to prevent the tumor cells in the Matrigel micropores from overflowing, then place it in the culture medium. After constant temperature culture, a 3D invasion model of different tumor cell nests cultured separately is obtained.

[0132] Furthermore, in step S5-1, the standing time is 2 to 4 minutes, preferably 3 minutes;

[0133] The inclination angle is 40° to 50°, preferably 45°;

[0134] The constant temperature incubation temperature is 37°C, and the time is 4 to 6 minutes, preferably 5 minutes;

[0135] The components and mass ratio of the culture medium are: 89% DMEM high glucose, 10% FBS and 1% double antibody (a mixture of penicillin and streptomycin).

[0136] Furthermore, in step S5-2, the method for preparing the Matrigel cover is as follows: dropping Matrigel on a cover glass, placing the cover glass in an incubator for 30 minutes, to obtain a Matrigel cover;

[0137] The constant temperature culture temperature is 37° C., and the constant temperature culture condition is a CO 2 concentration of 5%.

[0138] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0139] The present invention manufactures a chip model based on a microlithography method, and the advantages of the model are:

[0140] 1. The chip model is micron-scale and only requires a small number of cells, which is a great advantage for relatively precious cells or primary cells;

[0141] 2. It can simulate the structural morphology of different tumor cell nests to study the differences in the invasive ability and invasion sites of tumor cell nests with different morphologies;

[0142] 3. The in vitro 3D tumor cell budding invasion model enables real-time observation of the dynamic interaction between inducer cells and tumor cells. It can also monitor the influence and promotion of inducer cells on the invasive behavior of tumor cell nests in real time and intuitively. It can also demonstrate the dynamic invasion process of tumor cell nests of different morphologies under the influence of inducer cells, providing an intuitive and specific research platform for studying the role of inducer cells in inducing tumor invasion.

[0143] 4. The in vitro 3D tumor cell budding invasion model has high throughput and good reproducibility. It can observe the invasion of tumors in multiple structures at a time, which is beneficial for later drug screening and other related research.

[0144] Therefore, the in vitro tumor cell 3D budding invasion model designed by the present invention has strong universality and practicality, can study the invasion of different tumors, provides an important experimental basis and theoretical support for studying tumor invasion behavior, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 This is a flow chart for constructing tumor cell nest models of different morphologies in Example 1 of the present invention;

[0146] Figure 2 Schematic diagram of Matrigel microwells;

[0147] Figure 3 Schematic diagram of cells seeded in microwells;

[0148] Figure 4 Schematic diagram of the effect of tumor cell nest morphology on tumor invasion and proliferation in Example 1, wherein A is a bright field image of CAL-27 inoculated into different microwells at 0 h and 5 days, as well as Ki67 (green) and F-actin (red) staining; B is a bright field image of UM-SCC-1 inoculated into different microwells at 0 h and 5 days, as well as Ki67 (green) and F-actin (red) staining; (Scale bars = 100 μm for bright field images, 50 μm for fluorescence images);

[0149] Figure 5 This is a quantitative analysis of the effect of tumor cell nest morphology on tumor invasion in Example 1. Figure A is a quantitative analysis of the advancing invasion area of ​​CAL-27 and UM-SCC-1 in different microwells, ***p<0.001, **p<0.01, *p<0.05; Figure B is a diagram of the advancing invasion pattern of OSCC in different microwells.

[0150] Figure 6 This is a flow chart for constructing a co-culture model of tumor cell nests of different morphologies and induced cells in Example 2 of the present invention;

[0151] Figure 7 Schematic diagram of the effect of CAFs density on tumor invasion in Example 2, wherein A is a bright field image of CAL-27 co-cultured with different densities of CAF-L1 for 5 days (Scale bars = 50 μm), B is a quantitative analysis of OSCC cell nest budding induced by different CAFs densities, and C is a schematic diagram of marginal Matrigel contraction caused by CAF-L1 (Scale bars = 50 μm);

[0152] Figure 8 Bright field images of OSCC cell nest budding invasion induced by GNF-4 and CAF-L1 at 5 days (Scale bars = 100 μm);

[0153] Figure 9 This is a confocal image of GNF-4 and CAF-L1-induced OSCC cell nest budding invasion induced by E-cadherin / Vimentin fluorescence staining (Scale bars = 50 μm);

[0154] Figure 10 Figure 1 shows quantitative analysis of OSCC cell nest budding and invasion induced by GNF-4 and CAF-L1. A shows quantitative analysis of the number of budding and invasion in CAL-27 cell nests co-cultured with GNF-4 and CAF-L1; B shows quantitative analysis of the number of budding and invasion in UM-SCC-1 cell nests co-cultured with GNF-4 and CAF-L1; C and D show quantitative analysis of the number of budding and invasion in different locations of CAL-27 and UM-SCC-1 co-cultured with GNF-4 and CAF-L1, respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

[0155] Figure 11 This is a diagram showing the pattern of OSCC cell nest budding invasion induced by GNFs / CAFs. DETAILED DESCRIPTION

[0156] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0157] In the following examples, the required product and reagent models are as follows:

[0158] The photoresist model is SU8-2050;

[0159] Polydimethylsiloxane (PDMS) (Sylgard Silicone elastomer 184; Dow Corning Corp) includes a silicon polymer and a crosslinking agent;

[0160] Matrigel product number is 354234, purchased from Corning.

[0161] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0162] The following describes some embodiments of the present invention in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0163] Example 1

[0164] This embodiment provides a 3D invasion model for individually culturing tumor cell nests of different morphologies, and the construction method thereof comprises the following steps:

[0165] (1) Designing a silicon wafer, wherein the silicon wafer is an SU8 silicon wafer template, and the preparation method thereof is as follows:

[0166] 1) Model design: Use AutoCAD software to draw the model and print the mask;

[0167] 2) Silicon wafer cleaning: Immerse the silicon wafer in anhydrous ethanol and ultrasonically clean it for 30 minutes, then ultrasonically clean it in deionized water for 30 minutes, blow dry, bake it on a hot plate at 120°C for 30 minutes, and cool it to room temperature;

[0168] 3) Spin coating of photoresist: Treat the silicon wafer with plasma for 10 min, then drop the photoresist onto the treated silicon wafer. After removing any bubbles in the photoresist, spin coat at 1200 rpm for 20 s.

[0169] 4) Pre-baking: Bake on a hot plate at 120°C for 10 minutes to improve the adhesion between the photoresist and the silicon wafer, and then cool to room temperature;

[0170] 5) Exposure: Place the silicon wafer with photoresist on the UV exposure machine stage, fit the mask to the silicon wafer with the photoresist coating, and expose the silicon wafer under a light intensity of 11mW / cm 2 Expose for 45 seconds under the conditions of , at which point the designed model is transferred to a silicon wafer with photoresist;

[0171] 6) Post-baking: bake on a hot plate at 100°C for 10 minutes, then cool to room temperature;

[0172] 7) Development: First, use ethyl lactate to remove the uncured photoresist and develop for 15 minutes; then use isopropyl alcohol to wash away the ethyl lactate; after washing, use ddH2O to remove the residual isopropyl alcohol, blow dry and examine under a microscope;

[0173] 8) Hardening: Bake on a hot plate at 165°C for 40 minutes to ensure a firm bond between the photoresist and the silicon wafer, then cool to room temperature.

[0174] 9) Surface treatment: Trimethylchlorosilane is used for hydrophobic treatment to form a hydrophobic film, which facilitates the peeling of the cured PDMS from the silicon wafer without damaging the structure on the silicon wafer.

[0175] (2) Preparation of PDMS membrane

[0176] 1) Polydimethylsiloxane (PDMS) (Sylgard Siliconelastomer 184; Dow Corning Corp). A silicone polymer and a crosslinker were thoroughly mixed at a ratio of 10:1. The mixture was then applied to a designed silicon wafer to a thickness of approximately 5 mm and placed in an 80°C oven for 30 minutes to solidify.

[0177] 2) Peel the PDMS off the silicon wafer and cut it into 5 mm long and wide cubes, called PDMS membranes, and sterilize them with UV light for later use;

[0178] (3) Preparation of PDMS membrane containing bovine serum albumin:

[0179] 1) Add 1% bovine serum albumin dropwise to the surface of the PDMS membrane and block overnight at 4°C (to prevent adhesion of the added Matrigel to the PDMS membrane);

[0180] 2) 30 μL of Matrigel (3 mg / mL Collagen I containing 25% Matrigel) was added dropwise to the PDMS membrane and washed twice to neutralize the residual bovine serum albumin on the surface and prevent adhesion between the PDMS membrane and the Matrigel, thus obtaining a cleaned PDMS membrane.

[0181] (4) Preparation of Matrigel Micropores:

[0182] 1) Add 30 μL of Matrigel to the cleaned PDMS membrane, flip it over, and place the bottom of the Matrigel on two 1 mm thick PDMS supports to prevent the PDMS membrane from sinking to the bottom of the culture dish; place it in an incubator for 1 hour to solidify;

[0183] 2) Use tweezers to gently peel off the PDMS membrane from the matrigel to obtain the matrigel micropores. The spatial depth of the matrigel micropores is 150 μm. Observe the integrity of the micropores under a microscope (e.g. Figure 2 shown);

[0184] (5) Preparation of digested tumor cell suspension:

[0185] The adherent cells were washed twice with PBS buffer, digested with 0.25% trypsin for 3 min, and then digested with serum-containing medium. The cells were centrifuged to remove the supernatant and resuspended in medium for counting.

[0186] (6) Preparation of a 3D invasion model of tumor cells cultured alone:

[0187] 1) Place the Matrigel microwells in a culture dish and drop 30 μL of tumor cell suspension onto the surface of the Matrigel microwells. Allow to stand for 3 minutes. After the tumor cells have settled in the Matrigel microwells, tilt the dish 45° and slowly flush out the tumor cells outside the Matrigel microwells with 400 μL of culture medium.

[0188] 2) Place the culture dish in an incubator for 5 minutes to stabilize the tumor cells in the matrix gel micropores;

[0189] 3) Add 30 μL of Matrigel to a 15 mm diameter circular coverslip and place in an incubator for 30 min to obtain a Matrigel cover.

[0190] 4) Gently place the prepared Matrigel cover on the surface of the microwell to prevent the cells in the microwell from overflowing;

[0191] 5) Add 3 mL of culture medium to the culture dish and place it in a 37°C / 5% CO2 incubator to obtain a 3D invasion model of tumor cells cultured alone (e.g. Figure 3 shown).

[0192] like Figure 4 As shown, CAL-27 and UM-SCC-1 cells invaded the surrounding area in a propulsive manner after being cultured in microwells for 5 days.

[0193] Confocal imaging of F-actin (red) and Ki67 (green) staining of CAL-27 and UM-SCC-1 cells cultured in microwells for 5 days. Figure 4 Ki67 staining showed that the proliferation ability of cells in different parts of the 3D invasion model of tumor cells cultured alone was different, that is, the proliferation ability of tumor cells in the peripheral area in contact with the extracellular matrix was stronger, while the proliferation ability of the tumor core was weaker, which was similar to the tumor cell nests in vivo, indicating that the 3D invasion model of tumor cells cultured alone can simulate the in vivo state.

[0194] like Figure 5As shown in the figure, the quantitative analysis of the invasion area and invasion mode pattern of CAL-27 and UM-SCC-1 in different microwells; among them, the rod-shaped invasion area is significantly higher than the triangle and circle invasion areas, indicating that the invasion ability of tumor cell nests with different morphologies is quite different.

[0195] Example 2

[0196] This example provides an in vitro tumor cell 3D budding invasion model, in which CAFs are used to induce 3D invasion of tumor cell nests of different morphologies. The construction method includes the following steps:

[0197] (1) Designing a silicon wafer, wherein the silicon wafer is an SU8 silicon wafer template, and the preparation method thereof is as follows:

[0198] 1) Model design: Use AutoCAD software to draw the model and print the mask;

[0199] 2) Silicon wafer cleaning: Immerse the silicon wafer in anhydrous ethanol and ultrasonically clean it for 10 minutes, then ultrasonically clean it in deionized water for 10 minutes, blow dry, bake it on a hot plate at 120°C for 10 minutes, and cool it to room temperature;

[0200] 3) Spin coating of photoresist: Treat the silicon wafer with plasma for 5 minutes, then drop the photoresist onto the treated silicon wafer. After removing any bubbles in the photoresist, spin coat at 1200 rpm for 20 seconds.

[0201] 4) Pre-baking: Bake on a hot plate at 120°C for 5 minutes to improve the adhesion between the photoresist and the silicon wafer, and then cool to room temperature;

[0202] 5) Exposure: Place the silicon wafer with photoresist on the UV exposure machine stage, fit the mask to the silicon wafer with the photoresist coating, and expose the silicon wafer under a light intensity of 11mW / cm 2 Expose for 45 seconds under the conditions of , at which point the designed model is transferred to a silicon wafer with photoresist;

[0203] 6) Post-baking: bake on a hot plate at 100°C for 5 minutes, then cool to room temperature;

[0204] 7) Development: First, use ethyl lactate to remove the uncured photoresist and develop for 15 minutes; then use isopropyl alcohol to wash away the ethyl lactate; after washing, use ddH2O to remove the residual isopropyl alcohol, blow dry and examine under a microscope;

[0205] 8) Hardening: Bake on a hot plate at 165°C for 20 minutes to ensure a firm bond between the photoresist and the silicon wafer, then cool to room temperature.

[0206] 9) Surface treatment: Trimethylchlorosilane is used for hydrophobic treatment to form a hydrophobic film, which facilitates the peeling of the cured PDMS from the silicon wafer without damaging the structure on the silicon wafer.

[0207] (2) Preparation of PDMS membrane

[0208] 1) Polydimethylsiloxane (PDMS) (Sylgard Siliconelastomer 184; Dow Corning Corp). A silicone polymer and a crosslinker were thoroughly mixed at a ratio of 10:1. The mixture was then applied to a designed silicon wafer to a thickness of approximately 5 mm and placed in an 80°C oven for 30 minutes to solidify.

[0209] 2) Peel the PDMS off the silicon wafer and cut it into 5 mm long and wide cubes, called PDMS membranes, and sterilize them with UV light for later use;

[0210] (3) Preparation of PDMS membrane containing bovine serum albumin:

[0211] 1) Add 1% bovine serum albumin dropwise to the surface of the PDMS membrane and block overnight at 4°C (to prevent adhesion of the added Matrigel to the PDMS membrane);

[0212] 2) 30 μL of Matrigel (3 mg / mL Collagen I containing 25% Matrigel) was added dropwise to the PDMS membrane and washed twice to remove excess bovine serum albumin (BSA), as the BSA solution is slightly acidic and would affect the solidification of the Matrigel.

[0213] (4) Preparation of Matrigel Microwells Containing CAFs:

[0214] 1) CAFs (CAF-L1 density included: 1×10 5 / mL, 2.5×10 5 / mL, 5×10 5 / mL and 1×10 6 100 μL of the matrix gel was digested and mixed with Matrigel, and 30 μL was dropped onto the cleaned PDMS membrane. The membrane was flipped over and the bottom of the Matrigel was placed on two 1 mm thick PDMS supports to prevent the PDMS membrane from sinking to the bottom of the culture dish. The membrane was placed in an incubator for 1 hour to solidify.

[0215] 2) Use tweezers to gently peel the PDMS membrane from the Matrigel to obtain Matrigel micropores containing CAFs. The integrity of the micropores was observed under a microscope. The spatial depth of the Matrigel micropores containing CAFs was 150 μm.

[0216] (5) Prepare digested tumor cell suspension with a density of approximately 1×10 7 / mL,

[0217] The adherent cells were washed twice with PBS buffer, digested with 0.25% trypsin for 3 min, and then digested with serum-containing medium. The cells were centrifuged to remove the supernatant and resuspended in medium for counting.

[0218] (6) Preparation of a 3D invasion model of CAFs and tumor cells co-cultured:

[0219] 1) 30 μL of tumor cell suspension was added dropwise to the surface of the Matrigel micropores containing CAFs. The suspension was allowed to stand for 3 minutes. After the tumor cells settled in the Matrigel micropores, the culture dish was tilted 45° and 400 μL of culture medium was taken to slowly flush away the tumor cells outside the Matrigel micropores.

[0220] 2) Place the culture dish in an incubator for 5 minutes to stabilize the tumor cells in the matrix gel micropores;

[0221] 3) Add 30 μL of Matrigel to a 15 mm diameter circular coverslip and place in an incubator for 30 min to obtain a Matrigel cover.

[0222] 4) Gently place the prepared Matrigel cover on the surface of the microwell to prevent the cells in the microwell from overflowing;

[0223] 5) Add 3 mL of culture medium to the culture dish and culture in a 37°C / 5% CO2 incubator to obtain a 3D invasion model of tumor cell nests induced by CAFs with different morphologies (e.g. Figure 6 shown).

[0224] like Figure 7 As shown in the figure, with the increase of CAF-L1 density, the number of tumor invasion increased. When the CAF-L1 density was 1×10 5 When the CAF-L1 density was 5×10 5 / mL and 1×10 6 When the density of CAF-L1 was 2.5×10 / mL, the matrix gel showed obvious shrinkage, which was not conducive to observing the interaction between CAFs and tumor cells. 5 cells / mL was used as the condition for subsequent experiments.

[0225] Example 3

[0226] This example provides an in vitro tumor cell 3D budding invasion model, in which normal fibroblasts GNF-4 are used to induce 3D invasion of tumor cell nests of different morphologies. In addition to the addition of GNF-4 in step (4) (GNF-4 density includes: 1×10 5 / mL, 2.5×10 5 / mL, 5×10 5 / mL and 1×10 6The cells / mL) were digested and mixed with Matrigel, and the rest of the construction method was the same as in Example 2.

[0227] like Figure 8 As shown, the bright field image of GNF-4 / CAF-L1-induced OSCC cell nest budding invasion. On the 5th day of co-culture, CAFs induced tumor cell nests to invade into the surrounding matrix gel in the form of budding.

[0228] like Figure 9 As shown, fluorescence confocal imaging of GNF-4 / CAF-L1 co-cultured with OSCC shows that GNF-4 / CAF-L1 induces OSCC to invade the surrounding area in a budding manner, and the front of the tumor invasion is guided by GNF-4 / CAF-L1 and contacts it;

[0229] like Figure 10 As shown, quantitative analysis of GNF-4 / CAF-L1-induced budding invasion of OSCC cell nests revealed that the number of budding invasions induced by CAF-L1 was higher than that induced by GNF-4 in three different tumor cell nest structures, but there was no statistically significant difference. Regarding the budding location, rod-shaped tumor cell nests budded more from the top than from the sides, while triangular tumor cell nests budded more from the top than from the sides.

[0230] like Figure 11 As shown in the figure, GNF / CAFs induces the budding invasion pattern of tumor cell nests. GNF / CAFs guides the budding invasion of OSCC cells by directly contacting them and shows position specificity. Invasion mostly occurs at the top of the rod-like structure and the tip of the triangle.

[0231] GNF / CAFs and tumor cell co-culture model: After five days of co-culture with tumors, GNF / CAFs were found to invade the surrounding areas by budding, with GNF / CAFs playing a leading role at the front of the invasion, indicating that GNF / CAFs play a significant role in promoting tumor invasion. Furthermore, more tumor cells invaded from the top or tip of the microstructure than from the sides, indicating that the invasive abilities of tumor cells in different locations vary, reflecting structural heterogeneity.

[0232] The tumor invasion model designed by the present invention recreates the state of tumor invasion in vivo, facilitating more accurate research on the mechanisms of tumor invasion, thereby providing a better research platform for clinically blocking tumor invasion and improving patient prognosis. Therefore, the in vitro 3D tumor cell budding invasion model designed by the present invention is highly universal and practical, allowing the study of the invasion of different tumors, providing an important experimental basis and theoretical support for studying tumor invasive behavior, and has good application prospects.

[0233] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for constructing an in vitro tumor cell 3D budding invasion model, characterized in that: The specific steps are as follows: S1, preparing PDMS membrane; S2. Preparation of PDMS membrane containing bovine serum albumin: Add bovine serum albumin dropwise onto the surface of the PDMS membrane obtained in step S1, and let it stand at low temperature to obtain a PDMS membrane containing bovine serum albumin; S3. Preparation of Matrigel Microwells Containing Induced Cells: Digesting the induced cells and mixing them with the matrix gel, dropping them onto the PDMS membrane containing bovine serum albumin obtained in step S2, solidifying them, and peeling the PDMS membrane off the solidified matrix gel to obtain matrix gel micropores containing the induced cells; S4, preparing tumor cell suspension; S5. Preparation of induced cells to induce 3D invasion model of tumor cell nests with different morphologies: The tumor cell suspension obtained in step S4 is added dropwise to the surface of the matrix gel micropores containing the induced cells obtained in step S3, and an in vitro tumor cell 3D budding invasion model is obtained after constant temperature culture.

2. The method for constructing an in vitro tumor cell 3D budding invasion model according to claim 1, characterized in that: In step S1, the method for preparing the PDMS membrane is as follows: S1-1, design silicon wafer; S1-2, mixing a silicon-based polymer and a cross-linking agent, coating the mixture on the silicon wafer obtained in step S1-1, and curing the mixture at high temperature to obtain a silicon wafer covered with PDMS; S1-3. Peel off the PDMS from the silicon wafer to obtain a PDMS membrane, which is sterilized by ultraviolet light for later use.

3. The method for constructing an in vitro tumor cell 3D budding invasion model according to claim 2, characterized in that: In step S1-1, the thickness of the silicon wafer is 4 to 6 mm; The silicon wafer is a SU8 silicon wafer template; In step S1-2, the silicon-based polymer and cross-linking agent are of the type Sylgard Silicone elastomer 184; The weight ratio of the silicon polymer to the crosslinking agent is (9-11):1; In step S1-3, the PDMS membrane has a length of 4 to 6 mm, a width of 4 to 6 mm, and a thickness of 4 to 6 mm.

4. The method for constructing an in vitro tumor cell 3D budding invasion model according to claim 1, characterized in that: In step S2, the concentration of bovine serum albumin is 0.8-1.2%. The matrix gel includes Collagen I and Matrigel, The concentration of the Collagen I is 2.5-3.5 mg / mL, and the volume percentage of the Matrigel in the Collagen I is 20-30%.

5. The method for constructing an in vitro tumor cell 3D budding invasion model according to claim 1, characterized in that: In step S3, the induced cells include normal fibroblasts GNFs and cancer-associated fibroblasts CAFs; The induced cells are digested and mixed with the matrix gel, and then dripped onto the PDMS membrane containing bovine serum albumin obtained in step S2, and then turned over, with the bottom surface of the matrix gel placed on the support, and solidified; The induced cell density was 2×10 5 ~4×10 5 Pieces / mL; The spatial depth of the matrix gel micropores containing the induced cells is 100-200 μm.

6. The method for constructing an in vitro tumor cell 3D budding invasion model according to claim 1, characterized in that: In step S4, the method for preparing the tumor cell suspension is as follows: The adherent cells were washed twice with PBS buffer, digested with trypsin for a period of time, and then digested with serum-containing medium to terminate the digestion. The cells were centrifuged to remove the supernatant, and then resuspended in medium for counting. The concentration of the tumor cell suspension was 5×10 6 ~1×10 7 Pieces / mL.

7. The method for constructing an in vitro tumor cell 3D budding invasion model according to claim 1, characterized in that: In step S5, the amount of the tumor cell suspension is 20-40 μL; In step S5, the method for preparing the 3D invasion model of tumor cells cultured alone is as follows: S5-1, dripping the tumor cell suspension obtained in step S4 onto the surface of the matrix gel micropores containing the induced cells obtained in step S3, and after standing for a period of time, tilting the matrix gel micropores at a certain angle, taking culture medium to flush out the tumor cells outside the matrix gel micropores, and culturing at a constant temperature to stabilize the tumor cells in the matrix gel micropores; S5-2. Place the matrix gel cover on the surface of the matrix gel micropores containing tumor cells, and then put it into the culture medium. After constant temperature culture, an in vitro tumor cell 3D budding invasion model is obtained.

8. The method for constructing an in vitro tumor cell 3D budding invasion model according to claim 7, characterized in that: In step S5-1, the tilt angle is 40° to 50°; In step S5-2, the method for preparing the matrix gel cover is: dropping matrix gel on the cover glass, placing it in an incubator for a period of time, and obtaining the matrix gel cover.

9. An in vitro tumor cell 3D budding invasion model, characterized in that: The in vitro tumor cell 3D budding invasion model is constructed by the construction method of any one of claims 1-8.

10. A use of the in vitro tumor cell 3D budding invasion model as claimed in claim 9, characterized in that: The in vitro tumor cell 3D budding invasion model is used in the preparation of drugs for evaluating the effect of induced cells on inducing tumor invasion.

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