Breast cancer invasion and metastasis model based on anisotropic collagen hydrogel and construction method thereof

Abstract

The application specifically relates to a breast cancer invasion and metastasis model based on anisotropic collagen hydrogel and a construction method thereof, and belongs to the technical field of biomedical materials and tumor microenvironment. The anisotropic hydrogel with highly parallel arrangement of collagen fibers is prepared by adopting a PDMS mold pre-tensioning-stress rebound process, and the orientation index is 0.62+ / -0.05. The hydrogel has high matching of mechanical properties with the breast cancer microenvironment in vivo and good biocompatibility. The model can induce mature adipocytes to dedifferentiate and fibroblasts to transdifferentiate, and drive the breast cancer cells to have enhanced invasion and migration ability and EMT phenotype, and reproduce a pathological axis of "collagen orientation→adipocyte dedifferentiation→breast cancer invasion and metastasis". The process depends on the activation of a DDR1-integrin beta1-FAK-YAP signal path, and a DDR1 inhibitor can effectively reverse the above effects. The application can be used for breast cancer metastasis mechanism research and drug screening targeting the DDR1 path.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tumor microenvironment technology, specifically relating to a breast cancer invasion and metastasis model based on anisotropic collagen hydrogel and its construction method. Background Technology

[0002] Breast cancer is the leading cause of cancer-related morbidity and mortality among women worldwide, with over 90% of breast cancer-related deaths attributed to tumor invasion and distant metastasis. The dynamic remodeling of the tumor microenvironment is a core mechanism driving the malignant progression of breast cancer. Among these mechanisms, the structural remodeling of collagen fibers in the extracellular matrix, particularly the highly linear orientation of collagen fibers, is a typical pathological feature at the forefront of tumor invasion and is defined as tumor-associated collagen signature (TACS-3). This highly oriented collagen fiber can significantly reduce tumor cell migration resistance, activate mechanotransmission pathways, and accelerate the process of invasion and metastasis.

[0003] Adipocytes are the most abundant stromal cells in the breast tumor microenvironment. Under the stimulation of the tumor microenvironment, they can dedifferentiate and transform into a fibroblast-like phenotype, known as adipocyte-fibroblast transdifferentiation (AFT). Transdifferentiated adipocytes can significantly promote the migration, invasion, and epithelial-mesenchymal transition (EMT) of breast cancer cells through paracrine signaling, metabolic reprogramming, and stromal remodeling, making them a key factor driving malignant tumor progression. However, traditional research has focused on the regulation of adipocyte phenotype by chemical signals such as inflammatory factors and hypoxia, while the role of collagen orientation, a key physical and mechanical signal, has long been neglected.

[0004] Currently, in vitro tumor models mainly rely on two-dimensional culture and random collagen gels, which cannot accurately simulate the in vivo collagen orientation structure, make it difficult to isolate collagen orientation physical signals for mechanism research, and cannot stably reproduce the regulatory axis of "collagen orientation → adipocyte dedifferentiation → breast cancer invasion and metastasis," thus becoming a technical bottleneck for mechanism analysis and targeted drug development.

[0005] Existing methods for preparing anisotropic hydrogels have significant drawbacks: cell-induced methods offer poor orientation tunability; microfluidic and extrusion methods are complex and costly; spin-coating can only prepare two-dimensional structures; and mechanical stretching methods suffer from uneven force fields and poor repeatability. Therefore, developing a three-dimensional anisotropic collagen hydrogel model that is easy to prepare, has controllable orientation, good biocompatibility, and mechanical properties that match the in vivo tumor microenvironment is of great significance for elucidating the mechanisms of breast cancer metastasis and developing anti-metastatic drugs. Summary of the Invention

[0006] To address the technical challenges of existing technologies, such as the inability to accurately simulate the physical signals of collagen orientation in the breast cancer microenvironment, the difficulty in stably inducing adipocyte dedifferentiation, and the lack of a three-dimensional biomimetic model capable of reproducing the pathological axis of "collagen orientation → adipocyte dedifferentiation → breast cancer invasion and metastasis," this invention provides a breast cancer invasion and metastasis model based on anisotropic collagen hydrogel and its construction method. This method achieves precise control of collagen fiber orientation through a PDMS mold pre-stretching-stress rebound process, constructing a three-dimensional anisotropic hydrogel platform that highly matches the in vivo tumor microenvironment.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for constructing a breast cancer invasion and metastasis model based on anisotropic collagen hydrogel, comprising the following steps:

[0009] (1) Prepare type I collagen solution under ice bath conditions, with a final concentration of 2.5-3.5 mg / mL;

[0010] (2) Stretch the PDMS mold laterally to 0.4-0.5 times its original length and fix it;

[0011] (3) Inject the collagen solution prepared in step (1) into the stretched PDMS mold and incubate at 37°C for 4-5 min to allow the collagen to undergo initial cross-linking.

[0012] (4) Quickly release the lateral stress of the PDMS mold to make it rebound, and the collagen fibers align in the direction of the stress rebound;

[0013] (5) Let stand until the collagen is completely gelled to obtain anisotropic collagen hydrogel.

[0014] Preferably, the final concentration of the collagen solution in step (1) is 3.0 mg / mL. The specific components of the collagen solution include 191 μL of 10×PBS, 14 μL of 1 mol / L NaOH, 434 μL of 1×PBS, and 375 μL of 8 mg / mL collagen stock solution (taking a 1 mL system as an example).

[0015] Preferably, the overall dimensions of the PDMS mold are 75 mm × 40 mm × 5 mm, the dimensions of the middle groove are 30 mm × 5 mm × 0.4 mm, and the lateral stretching distance is 2.5 mm.

[0016] Preferably, the complete gelation time in step (5) is 30 min, and the environment is a magnetic field-free, room temperature condition.

[0017] Secondly, this invention also provides an anisotropic collagen hydrogel model prepared by the above-described construction method. This model has a three-dimensional porous structure with highly parallel linear arrangement of internal collagen fibers and an orientation index of 0.5-0.7 (preferably 0.62±0.05). The storage modulus G' of this model is significantly higher than that of random collagen hydrogels, its mechanical properties are highly matched to the extracellular matrix of the breast cancer microenvironment in vivo, and it exhibits good biocompatibility (cell viability >92%).

[0018] This model supports the embedding and culture of mature adipocytes, inducing their directional extension along collagen fibers, loss of lipid droplets, dedifferentiation, and fibroblast transdifferentiation (AFT). When this model is used to form an indirect co-culture system with breast cancer cells, it can drive breast cancer cells to exhibit an epithelial-mesenchymal transition (EMT) phenotype characterized by downregulated E-cadherin and upregulated Vimentin, while simultaneously inhibiting proliferation and enhancing invasive and migratory abilities, thus fully replicating the pathological axis of "collagen orientation → adipocyte dedifferentiation → breast cancer invasion and metastasis".

[0019] Mechanistic studies have shown that adipocyte dedifferentiation in this model depends on the activation of the DDR1-integrin β1-FAK-YAP mechanotransmitter pathway; the addition of a DDR1-specific inhibitor significantly reversed adipocyte dedifferentiation, and the invasive ability of breast cancer cells also decreased.

[0020] Thirdly, the present invention also provides the application of the anisotropic collagen hydrogel model in the preparation of kits for studying the invasion and metastasis mechanisms of breast cancer, analyzing physical signals of the tumor microenvironment, or screening anti-breast cancer metastasis drugs.

[0021] Specifically, the applications include: simulating tumor-associated collagen orientation microenvironment in vitro, studying the mechanism of collagen orientation-induced adipocyte phenotypic transformation, evaluating the reversal effect of DDR1 inhibitors on adipocyte dedifferentiation and breast cancer invasion, and high-throughput screening of anti-metastatic drugs targeting the tumor physical microenvironment.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention adopts the PDMS mold pre-stretching-stress rebound process, which is simple to operate and mild. The collagen fiber orientation index of the anisotropic collagen hydrogel can reach 0.62±0.05. The orientation degree is controllable and the reproducibility is good. It can quickly prepare three-dimensional biomimetic matrix in batches.

[0024] (2) The storage modulus G' of the hydrogel of the present invention is significantly higher than that of random collagen hydrogel. Its mechanical properties are highly matched with the in vivo breast cancer microenvironment and it has excellent biocompatibility (cell survival rate >92%). It can separate the physical signal of collagen orientation and provide a standardized platform for in vitro tumor microenvironment research.

[0025] (3) Compared with the prior art, the present invention does not require the addition of other substances, the gelation process is mild and controllable, and collagen molecules can be gelled in situ around the cells, which has a better cell matrix simulation effect.

[0026] (4) The hydrogel of the present invention can stably induce mature adipocytes to undergo AFT transdifferentiation (lipid droplet content decreased by 62.3%), and drive breast cancer cells to exhibit the EMT phenotype with E-cadherin downregulated by 70% and Vimentin upregulated by 3 times, increasing the invasive ability by more than 4 times, and completely reproducing the pathological axis of "collagen orientation → adipocyte dedifferentiation → breast cancer invasion and metastasis".

[0027] (5) The model of the present invention can activate the DDR1-integrin β1-FAK-YAP signaling pathway. DDR1 inhibitors can effectively reverse adipocyte dedifferentiation and breast cancer invasion. It is suitable for the analysis of mechanical signal axis mechanism and the screening of targeted anti-metastasis drugs, and has high clinical translational value. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the preparation process of the anisotropic collagen hydrogel of the present invention;

[0029] Figure 2 In Figure A, the SDS-PAGE result of rat tail type I collagen prepared in Example 1 is shown, and in Figure B, the circular dichroism chromatogram result of rat tail type I collagen prepared in Example 1 is shown.

[0030] Figure 3 Image A shows a comparison of the SEM structures and quantitative statistics of oriented and random hydrogels, while image B shows a comparison of the fluorescence staining and quantitative statistics of oriented and random hydrogels, with FITC-labeled cytoskeleton.

[0031] Figure 4 The rheological properties of the anisotropic collagen hydrogel (oriented group) in Example 1 of the present invention and the random collagen hydrogel (disordered group) in Comparative Example 1 are compared. In this paper, A is the amplitude scan result, B is the stress relaxation result, and C is the frequency scan result.

[0032] Figure 5 In the figure, A is a representative image of Calcein-AM / PI staining of 3T3-L1 adipocytes in anisotropic collagen hydrogel, B is a statistical graph of cell survival rate in hydrogels of different concentrations (48 h), and C is a graph of diffusion coefficient over time.

[0033] Figure 6 The results of staining and quantitative analysis of lipid droplets in oriented and disordered hydrogels are shown. In the figure, A is the Nile Red staining result of lipid droplets, B is the statistical analysis of the average fluorescence intensity on day 0 and day 7 in Figure A, and C is the BODIPY staining result of lipid droplets.

[0034] Figure 7 The images show the results of fibroblast marker expression and gene expression analysis of mature adipocytes embedded in anisotropic collagen hydrogels (oriented group) and random hydrogels (disordered group) in Example 1 of this invention. In the images, A is a representative image of α-SMA (green) and DAPI (blue) immunofluorescence staining, B is the qRT-PCR quantitative results of fibroblast-related genes (Col1a1, FN, LAM, COL6) and stemness-related genes (OCT4, SOX2, Nanog), and C is the qRT-PCR quantitative results of adipocyte-related genes (C / EBPα, PPARγ, FABP4).

[0035] Figure 8 To characterize the invasion, migration, and EMT phenotype of breast cancer cells, A shows representative images of the invasion assay (Transwell) and migration assay (scratch assay), B shows the statistical graph of the number of invaded and migrated cells, C shows the results of immunofluorescence staining (E-cadherin, Vimentin, DAPI), and D shows the statistical results of the average fluorescence intensity of E-cadherin, the average fluorescence intensity of Vimentin, the number of cells, the cell area, and the roundness of cells in graph C.

[0036] Figure 9 To verify the DDR1-integrin β1-FAK-YAP mechanotransmission pathway and the effect of inhibitor intervention, A represents the immunofluorescence staining results of different groups, B represents the CCK-8 cell proliferation detection results, C represents the BODIPY staining results of lipid droplets, D represents the statistical count of lipid droplets in different groups, and E represents the qPCR gene expression detection results of different groups.

[0037] In the attached figure, "*" indicates P < 0.05, meaning the result is statistically significant; "**" indicates P < 0.01, meaning the result is highly statistically significant; "***" indicates P < 0.001, meaning the result is extremely statistically significant; "****" indicates P < 0.0001, meaning the result is extremely statistically significant; and "ns" indicates that the result is not statistically significant. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0039] After reading the contents disclosed in this invention, those skilled in the art can make appropriate adjustments or substitutions to the process parameters of the methods and applications described in this invention without departing from the spirit and scope of this invention. Such obvious adjustments, substitutions or combinations should be included within the protection scope of this invention.

[0040] Unless otherwise specified, the materials, reagents, instruments and testing methods used in the following embodiments can be obtained commercially or prepared, operated and implemented with reference to conventional methods disclosed in the art.

[0041] It should be noted that all technical parameters described in this document as numerical ranges (such as temperature, ratio, time, content, etc.) should be understood as encompassing all possible sub-ranges and specific numerical points within that range, regardless of whether the specific numerical value or sub-range is explicitly listed. Unless otherwise specified, the technical terms used in this document have the meanings commonly understood by those skilled in the art.

[0042] Experimental materials:

[0043] SD rats used for type I collagen extraction from rat tails were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF grade, 8-10 weeks old; 3T3-L1 adipocytes and MDA-MB-231 breast cancer cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee; DMEM high glucose medium, RPMI-1640 medium, fetal bovine serum, and penicillin-streptomycin antibody were purchased from Gibco; BODIPY, Nile red, Calcein-AM / PI staining kit, CCK-8 kit, E-cadherin antibody, Vimentin antibody, integrin β1 antibody, p-FAK antibody, YAP antibody, and p-YAP antibody were purchased from Abcam; DDR1 inhibitor (DDR1-IN-1) was purchased from Selleck; other routine chemical reagents were of analytical grade and purchased from Sinopharm Group.

[0044] Experimental consumables and instruments:

[0045] PDMS mold (customized), CO2 incubator (Thermo Fisher), environmental field emission scanning electron microscope (JEOL), modular rheometer (Anton Paar), laser confocal microscope (Leica), real-time fluorescence quantitative PCR instrument (Bio-Rad), Western blotting electrophoresis system (Bio-Rad), and ultra-micro spectrophotometer (Nanodrop).

[0046] Example 1: Method for constructing a breast cancer invasion and metastasis model using anisotropic collagen hydrogels

[0047] This embodiment aims to construct a three-dimensional anisotropic hydrogel model that can accurately simulate the physical signals of collagen orientation in the breast cancer microenvironment, for subsequent research on adipocyte dedifferentiation and malignant phenotypes of breast cancer cells. The specific steps are as follows:

[0048] (1) Extraction and purification of type I collagen from rat tail

[0049] To obtain high-purity type I collagen with a complete triple helix conformation, the following methods are employed: Figure 1 The “acid dissolution-neutralization-dialysis-lyophilization” process shown is used to extract collagen from the tail tendon of SD rats. The specific steps are as follows:

[0050] S1 rat tail tendon pretreatment: Take the tail tendon of SPF grade SD rat, carefully peel off the attached fascia with a scalpel blade, cut it into a small segment of about 1 cm, and disinfect it in 75% ethanol for 10 min.

[0051] S2 acid extraction: The sterilized tail tendon segment was placed in a 0.5 mol / L acetic acid solution (the ratio of tail tendon to acetic acid solution was 1 g: 50 mL) and magnetically stirred at 4 ℃ for 12 h to fully dissolve the collagen;

[0052] S3 Centrifugation to remove residue: Centrifuge the acid hydrolysate at 12000 r / min and 4 ℃ for 10 min, discard the insoluble residue, and collect the supernatant;

[0053] S4 Neutralization Precipitation: Under ice bath conditions, slowly titrate the supernatant with 1 mol / L NaOH to pH 7.0 while gently stirring. A white flocculent collagen precipitate will be visible. Let stand for 30 min to allow the precipitation to complete.

[0054] S5 dialysis purification: Centrifuge at 12000 r / min, 4 ℃ for 10 min to collect the precipitate, put the precipitate into a dialysis bag (molecular weight cutoff 8000-14000 Da), and dialyze in ultrapure water for 48 h. Change the dialysis solution every 12 h to remove residual acetic acid and salt ions.

[0055] S6 Freeze-drying preservation: The dialyzed collagen solution was aliquoted into freeze-drying bottles, placed in a freeze dryer, and freeze-dried under vacuum at -50 ℃ for 48 h to obtain white spongy type I collagen.

[0056] S7 purity identification: A small amount of lyophilized collagen was subjected to SDS-PAGE electrophoresis, and the triple helix conformation of collagen was detected by circular dichroism spectroscopy.

[0057] SDS-PAGE results ( Figure 2 As shown in Figure A, the collagen sample prepared in Example 1 of this invention exhibits a clear type I collagen characteristic band at a molecular weight of approximately 120 kDa, and the band purity is superior to that of Corning commercial collagen. Circular dichroism chromatogram results ( Figure 2 As shown in Figure B), the collagen sample exhibits a characteristic positive peak at 221 nm and a characteristic negative peak at 198 nm, consistent with a typical triple helix conformation. These results indicate that the collagen prepared in Example 1 has good purity and a complete conformation, meeting the requirements for subsequent experiments.

[0058] (2) Preparation of anisotropic collagen hydrogels

[0059] To construct a three-dimensional hydrogel with highly oriented collagen fibers, a PDMS mold pre-stretching-stress rebound process was used. The specific steps are as follows:

[0060] S1. Prepare collagen solution in an ice bath: Use an ice bath throughout the process to prevent premature gelation of collagen. Taking a 1 mL system as an example, add 191 μL of 10×PBS, 14 μL of 1 mol / L NaOH, and 434 μL of 1×PBS to the centrifuge tube in sequence, mix gently, and finally add 375 μL of the collagen stock solution prepared in step (1) with a final concentration of 3.0 mg / mL. Gently pipette to mix, avoiding the generation of bubbles.

[0061] S2 PDMS mold pre-stretching: Take a PDMS mold (overall size 75 mm × 40 mm × 5 mm, middle groove size 30 mm × 5 mm × 0.4 mm), use a micro stretching device to stretch the mold laterally to 0.5 times the original length of the groove (i.e. stretch 2.5 mm), and fix it in the stretched state with a clamp.

[0062] S3 Collagen Partial Gelification: Use a microsyringe to evenly inject the collagen solution into the groove of the stretched PDMS mold, taking care to avoid generating air bubbles; transfer the mold to a 37 ℃ constant temperature incubator and let it stand for 4-5 min to allow the collagen to initially cross-link but remain in a reconfigurable state.

[0063] S4 Stress Release and Complete Gelation: Rapidly release the lateral stress of the PDMS mold, allowing it to elastically rebound to its original size; during the stress rebound process, collagen fibers align in an orientation as the mold shrinks; place the mold in a non-magnetic environment at room temperature for 30 minutes to allow the collagen to completely gel, forming anisotropic collagen hydrogel.

[0064] Post-processing and storage of S5 hydrogel: Carefully remove the molded hydrogel from the mold with sterile tweezers and transfer it to a sterile culture dish. Add PBS buffer (pH 7.4) and wash three times for 5 minutes each time to remove residual uncrosslinked collagen and buffer salts. Store the washed hydrogel at 4 ℃ for later use.

[0065] Comparative Example 1

[0066] This comparative example aims to construct a three-dimensional hydrogel with a random network arrangement of collagen fibers, serving as a control sample for the anisotropic hydrogel prepared in Example 1, to verify the specific regulatory effect of collagen fiber orientation structure on adipocyte phenotype and breast cancer cell behavior. The specific steps are as follows:

[0067] Compared to Example 1, the difference lies in that: after preparing the collagen solution using the same formula, the stretching and stress rebound operations in the PDMS mold were not performed. Specifically, the collagen solution was directly injected into the unstretched PDMS mold groove, allowed to stand at 37 ℃ for 30 minutes to gel, washed with PBS, and stored at 4 ℃ to obtain random collagen hydrogels.

[0068] Example 1: Characterization of the microstructure and orientation of hydrogels

[0069] To verify whether the collagen fibers in the anisotropic hydrogel constructed in this invention are highly oriented and to quantitatively assess their orientation, the microstructure of the hydrogel was observed using scanning electron microscopy (SEM), and the orientation degree was quantitatively analyzed using the OrientationJ plugin of ImageJ software. Simultaneously, the orientation of the skeleton of embedded adipocytes was observed by fluorescence staining, and random collagen hydrogels (disordered group) were used as controls.

[0070] The specific steps are as follows:

[0071] S1 Sample Fixation: The anisotropic collagen hydrogel prepared in Example 1 (referred to as "Orientation Group" in the figure) and the random collagen hydrogel prepared in Comparative Example 1 (referred to as "Disorder Group" in the figure) were cut into 5 mm × 5 mm × 2 mm pieces respectively, placed in 2.5% glutaraldehyde solution, and fixed overnight at 4 ℃.

[0072] S2 gradient dehydration: After washing three times with PBS buffer, dehydrate with 30%, 50%, 70%, 90%, and 100% ethanol in a gradient, 15 min each time.

[0073] S3 Drying and Gold Spraying: The sample was dried using the critical point drying method. After drying, the sample was attached to the sample stage and platinum was sprayed for 120 seconds using an ion sputtering instrument to enhance conductivity.

[0074] S4 SEM observation: Use an ambient field emission scanning electron microscope (accelerating voltage 5-10 kV) to observe and photograph the surface and cross-sectional fiber structure of the hydrogel;

[0075] S5 Orientation Quantitative Analysis: SEM images were opened using ImageJ software, and the OrientationJ plugin was used to calculate the orientation index (a value between 0 and 1, with a higher value indicating higher orientation) and angle distribution histogram of collagen fibers. Five different fields of view (2000x magnification) were randomly selected for each sample group, and the mean orientation index and standard deviation were calculated.

[0076] SEM results are as follows Figure 3In Figure A, the oriented collagen fibers exhibited a highly parallel linear arrangement and were densely distributed along the stress release direction; the disordered collagen fibers showed a disordered network distribution. The orientation index was 0.62 ± 0.05, with angles concentrated within ±15° of the principal direction; the orientation index of the disordered group was less than 0.1, with a uniformly scattered angle distribution (P < 0.001). Fluorescent staining was as follows. Figure 3 The results showed that the adipocyte cytoskeleton in the oriented group extended in a spindle shape along the collagen direction, and the orientation factor was significantly higher than that in the disordered group (P<0.0001); the cells in the disordered group remained round or oval.

[0077] The above results demonstrate that the present invention successfully constructed a three-dimensional anisotropic hydrogel with highly oriented collagen fibers using a PDMS mold pre-stretching-stress rebound process, and the oriented structure can guide the directional alignment of cells.

[0078] Example 2: Hydrogel Rheological and Mechanical Property Testing

[0079] To evaluate whether the mechanical properties of the anisotropic collagen hydrogel constructed in this invention match the extracellular matrix in the in vivo breast cancer microenvironment, and to investigate the influence of collagen fiber orientation structure on the mechanical strength of the hydrogel, a modular rheometer was used to measure the storage modulus, loss modulus, and stress relaxation characteristics of the anisotropic and random hydrogels, respectively. The specific steps are as follows:

[0080] S1 Sample preparation: The anisotropic collagen hydrogel prepared in Example 1 and the random collagen hydrogel prepared in Comparative Example 1 were respectively made into circular samples with a diameter of 30 mm and a thickness of 2 mm. Three parallel samples were prepared for each group.

[0081] S2 Amplitude Scan: Place the sample on the parallel plate fixture of the rheometer, set the angular frequency to 10 rad / s, the temperature to 37℃, and the strain to 0.1%-10%, and measure the linear viscoelastic region to determine the strain parameters for subsequent frequency scans;

[0082] S3 Frequency Scan: The strain is set to 1% (within the linear viscoelastic region), the angular frequency scan range is 0.1-100 rad / s, the temperature is 37 ℃, and the changes of storage modulus G' (reflecting material elasticity) and loss modulus G'' (reflecting material viscosity) with frequency are measured.

[0083] S4 stress relaxation test: Apply a constant strain of 1%, record the stress decay curve over time within 300 s, and calculate the stress relaxation half-life;

[0084] S5 Data Processing: Data was collected using the rheometer's accompanying software. Each sample was measured three times, and the results are expressed as mean ± standard deviation.

[0085] like Figure 4As shown, compared with random hydrogels (disordered group), the anisotropic collagen hydrogels (oriented group) constructed in this invention exhibit superior mechanical response characteristics in terms of rheological parameters such as shear strain, shear stress, and shear rate. The relationship between shear stress and shear rate further verifies the high degree of matching between the anisotropic hydrogel and the mechanical characteristics of the extracellular matrix (ECM) of the breast cancer microenvironment in vivo. Statistical results show that the anisotropic collagen hydrogel constructed in this invention is significantly superior to random collagen hydrogels in terms of storage modulus and stress relaxation half-life (p<0.01), and its mechanical properties are highly matched with the ECM of the breast cancer microenvironment in vivo, providing a biomimetic physicomechanical microenvironment for adipocytes and breast cancer cells.

[0086] Example 3: Hydrogel Biocompatibility Test

[0087] To evaluate whether the anisotropic collagen hydrogel constructed in this invention has good biocompatibility and is suitable as a scaffold material for three-dimensional cell culture, the cell viability and proliferation activity embedded in the hydrogel were detected using Calcein-AM / PI live / dead cell staining and the CCK-8 assay. The specific steps are as follows:

[0088] Cell culture conditions: 3T3-L1 adipocytes were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics, and MDA-MB-231 breast cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin antibiotics. Both were cultured routinely in an incubator at 37 ℃ and 5% CO2.

[0089] (1) Cell embedding: 3T3-L1 cells and MDA-MB-231 cells were collected respectively. Following the method in step (2) of Example 1, the cell suspension was added after the collagen solution was prepared and before it was injected into the mold, so that the final density was 1×10⁻⁶. 6 The cells / mL were gently mixed and then the cell-containing hydrogel was prepared according to the methods of Example 1 and Comparative Example 1.

[0090] (2) Culture and detection: After culturing the cell-containing hydrogel at 37 ℃ and 5% CO2 for 48 h, Calcein-AM / PI live / dead staining was performed, and cell viability was observed and counted using laser confocal microscopy. CCK-8 reagent was added at 24 h, 48 h, and 72 h of culture, and the absorbance at 450 nm was measured to calculate cell viability. Each group was set up with 3 replicates, and the experiment was repeated 3 times.

[0091] like Figure 5As shown in Figure A, the Calcein-AM / PI live and dead cell staining results indicate that the 3T3-L1 adipocytes in the anisotropic collagen hydrogel prepared in Example 1 of this invention exhibit a large amount of green fluorescence (live cells) and only a very small amount of red fluorescence (dead cells). Figure 5 The statistical results of B showed that the cell survival rate in the hydrogel was higher than 92%. Figure 5 The results from C show that the anisotropic collagen hydrogel constructed in this invention has a higher diffusion coefficient and better mass transfer performance.

[0092] The above results indicate that the anisotropic collagen hydrogel constructed in this invention has good biocompatibility and is suitable as a scaffold material for three-dimensional culture of adipocytes and breast cancer cells.

[0093] Example 4: Characterization of collagen orientation-induced adipocyte dedifferentiation

[0094] To verify whether the physical signals of collagen orientation in anisotropic collagen hydrogels can induce dedifferentiation of mature adipocytes and transdifferentiation of fibroblasts (AFT), a systematic characterization was performed at four levels: lipid droplet morphology, lipid droplet content, expression of fibroblast marker proteins, and expression of fibroblast / stem / adipocyte-related genes. The specific steps are as follows:

[0095] (1) Preparation of mature adipocytes: 3T3-L1 preadipocytes were cultured in differentiation induction medium (containing 10 μg / mL insulin, 0.5 mM IBMX, and 1 μM dexamethasone) for 2 days, and then cultured in maintenance medium (containing 10 μg / mL insulin) for 6-8 days to obtain mature adipocytes;

[0096] (2) Cell embedding and culture: Mature adipocytes were embedded at a concentration of 1×10⁻⁶ cells / mL. 6 Cells / mL density were embedded in anisotropic hydrogels (oriented group) and random hydrogels (disordered group), respectively, and cultured for 7 days, with the medium changed every 2 days;

[0097] (3) Lipid droplet staining: After culturing for 7 days, the samples were fixed and double-stained with BODIPY and Nile Red. The morphology and distribution of lipid droplets were observed by confocal microscopy.

[0098] (4) Immunofluorescence staining: Immunofluorescence staining was performed on α-smooth muscle actin (α-SMA) to observe the expression of fibroblast markers;

[0099] (5) Gene expression detection: qRT-PCR was used to detect the expression levels of fibroblast-related genes (Col1a1, FN, LAM, COL6), stemness-related genes (OCT4, SOX2, Nanog), and adipocyte-related genes (C / EBPα, PPARγ, FABP4).

[0100] Staining results as follows Figure 6 In Figures A and C, the lipid droplet content of adipocytes in the oriented group was significantly reduced, and the cells extended in a spindle shape; while the lipid droplets in the disordered group were abundant, and the cells remained round / elliptical. Figure 6 Quantitative analysis of B showed that the average fluorescence intensity of Nile Red staining in the oriented group was significantly lower than that in the disordered group, the relative content of lipid droplets decreased by 62.3%, and there was no significant difference in DAPI fluorescence intensity between the two groups.

[0101] Immunofluorescence results as follows Figure 7 As shown in Figure A, the fluorescence intensity of α-SMA (fibroblast marker) in the oriented group was significantly higher than that in the disordered group; Figure 7 The results in sections B and C showed that the expression levels of fibroblast-related genes (Col1a1, FN, LAM, COL6) and stemness-related genes (OCT4, SOX2, Nanog) in the oriented group were significantly higher than those in the disordered group, while the expression levels of mature adipocyte markers (C / EBPα, PPARγ, FABP4) in the oriented group were significantly lower than those in the control group. These results indicate that the physical signaling of collagen orientation in anisotropic collagen hydrogels can significantly induce dedifferentiation and lipid droplet loss in mature adipocytes, successfully inducing fibroblast transdifferentiation (AFT).

[0102] Example 5: Co-culture of adipocytes and breast cancer cells and detection of malignant phenotypes

[0103] To verify whether anisotropic hydrogel-induced dedifferentiation of adipocytes can regulate the malignant phenotype of breast cancer cells, a Transwell indirect co-culture system was used. Adipocytes embedded in hydrogels were co-cultured with breast cancer cells, and changes in the invasion, migration ability, and expression of epithelial-mesenchymal transition (EMT) markers of breast cancer cells were detected. Results are as follows: Figure 8 As shown. The specific steps are as follows:

[0104] (1) Establishment of co-culture model: Mature adipocytes were embedded in anisotropic hydrogels (oriented group) or random hydrogels (disordered group) (1×10 6 Cells / mL were placed in the upper chamber of the Transwell (0.4 μm pore size); MDA-MB-231 cells were seeded in the lower chamber (5 × 10⁶ cells / mL). 4 (cells / well). The control group consisted of a blank upper chamber without hydrogel and adipocytes. Co-cultured for 72 h.

[0105] (2) Invasive ability test (Transwell invasion assay): Matrigel coated the upper chamber, and breast cancer cells (1×10⁻⁶) were added. 5 (cells / well), the lower chamber is a culture medium containing adipocyte hydrogel, and the cells that have passed through the membrane are counted after co-culturing for 24 h.

[0106] (3) Migration ability test (scratch test): After scratching breast cancer cells, they were co-cultured in an upper chamber containing adipocyte hydrogel for 24 h, and the scratch closure rate was calculated.

[0107] (4) Detection of EMT markers: Immunofluorescence was used to detect the expression of E-cadherin and Vimentin.

[0108] Figure 8 The results in A and B showed that the number of invasive and migrating breast cancer cells in the orientation group was significantly higher than that in the disordered group and the control group, the number of transmembrane cells was significantly increased, and the scratch closure rate was significantly improved. Figure 8 The results of C and D showed that the orientation group had decreased E-cadherin expression and increased Vimentin expression, exhibiting a typical EMT phenotype, while the disordered group showed the opposite. Quantitative analysis further confirmed that the average fluorescence intensity of E-cadherin was significantly decreased and the average fluorescence intensity of Vimentin was significantly increased in the orientation group, and the number of cells was significantly higher than that in the control group, while there were no significant differences in cell area, roundness and cell viability between the two groups.

[0109] The above results indicate that anisotropic collagen hydrogel-induced dedifferentiated adipocytes can drive breast cancer cells to exhibit a characteristic malignant phenotype of "inhibiting proliferation, promoting invasion and migration, and inducing EMT," fully replicating the pathological axis of "collagen orientation → adipocyte dedifferentiation → breast cancer invasion and metastasis."

[0110] Example 6: Verification of Mechanical Signal Path and DDR1 Target

[0111] To elucidate the molecular mechanism by which anisotropic collagen hydrogels induce adipocyte dedifferentiation and to verify whether this model can be used for the screening and evaluation of targeted drugs, the expression levels of proteins related to the DDR1-integrin β1-FAK-YAP mechanotransmission pathway were detected, and functional intervention experiments were conducted using DDR1-specific inhibitors. The specific steps are as follows:

[0112] (1) Signal protein detection (Western Blot): Total protein was extracted from adipocytes cultured for 7 days embedded in anisotropic or random hydrogels, and the expression of DDR1, integrin β1, FAK, p-FAK (Tyr397), YAP, p-YAP (Ser127) and GAPDH was detected. The random group was used as the control (set to 1.0). The experiment was repeated 3 times.

[0113] (2) Immunofluorescence staining: Adipocytes in hydrogel were stained with DDR1, integrin β1 and YAP immunofluorescence to observe protein localization and expression intensity.

[0114] (3) DDR1 inhibition experiment: A DDR1-specific inhibitor (DDR1-IN-1, 1 μM) was added during the construction of the anisotropic hydrogel, with DMSO as a control. After 7 days of culture, the lipid droplet content and the expression of α-SMA and PPARγ were detected; and the invasive ability of breast cancer cells in the co-culture system was detected according to the method in Example 5.

[0115] Depend on Figure 9 It can be seen that anisotropic collagen hydrogels can enhance the expression of integrin β1 and YAP in adipocytes and promote YAP nuclear translocation. Figure 9 (A), enhances cell proliferation activity ( Figure 9 (B), inducing lipid droplet loss and dedifferentiation phenotype ( Figure 9 In C and D), and significantly downregulated the expression of PPARγ and C / EBPα genes ( Figure 9 (D) DDR1 inhibitors can effectively reverse the above effects. These results indicate that anisotropic collagen hydrogels induce adipocyte dedifferentiation by activating the DDR1-integrin β1-FAK-YAP mechanotransmission pathway, and DDR1 inhibitors can significantly reverse this process and block its pro-invasive effect on breast cancer cells. Therefore, this model can be used for screening anti-metastatic drugs targeting the DDR1 signaling pathway.

[0116] In summary, the anisotropic collagen hydrogel model provided by this invention can accurately simulate the physical signals of collagen orientation in the tumor microenvironment, and completely reproduce the pathological axis of "collagen orientation → adipocyte dedifferentiation → breast cancer invasion and metastasis". It is suitable for the study of the mechanism of breast cancer invasion and metastasis and the screening of anti-metastatic drugs targeting the DDR1 signaling pathway, and has important scientific research and clinical translational value.

Claims

1. A breast cancer invasion and metastasis model based on anisotropic collagen hydrogel, characterized in that, The model is composed of collagen hydrogel, which has a three-dimensional porous structure with highly parallel linear arrangement of collagen fibers inside, and an orientation index of 0.5-0.

7.

2. A method for constructing a breast cancer invasion and metastasis model as described in claim 1, characterized in that, Includes the following steps: (1) Prepare collagen solution under ice bath conditions; (2) Stretch the PDMS mold laterally to 0.4-0.5 times its original length and fix it; (3) Inject the collagen solution prepared in step (1) into the stretched PDMS mold and incubate at 37°C for 4-5 min to allow the collagen to undergo initial cross-linking. (4) Quickly release the lateral stress of the PDMS mold to make it rebound, and the collagen fibers align in the direction of the stress rebound; (5) Let stand until the collagen is completely gelled to obtain anisotropic collagen hydrogel.

3. The construction method according to claim 2, characterized in that, The final concentration of the collagen solution in step (1) is 2.5-3.5 mg / mL.

4. The construction method according to claim 2, characterized in that, The complete gelation time in step (5) is 30 min, and the environment is a magnetic field-free, room temperature condition.

5. The breast cancer invasion and metastasis model according to claim 1, characterized in that, The hydrogel can induce embedded mature adipocytes to extend directionally along the collagen fiber direction, lose lipid droplets, and undergo dedifferentiation and fibroblast transdifferentiation.

6. The breast cancer invasion and metastasis model according to claim 1, characterized in that, The model can be used to form an indirect co-culture system with breast cancer cells and induce the breast cancer cells to exhibit a phenotype of E-cadherin downregulation and Vimentin upregulation, and enhance their invasive and migration abilities.

7. The breast cancer invasion and metastasis model according to claim 1, characterized in that, In the model, dedifferentiated adipocytes showed upregulated expression levels of DDR1, integrin β1, p-FAK, and YAP, and downregulated expression levels of p-YAP.

8. The application of the breast cancer invasion and metastasis model of claim 1 in the preparation of kits for studying the mechanisms of breast cancer invasion and metastasis or screening anti-breast cancer metastasis drugs.

9. The application according to claim 8, characterized in that, The applications are: to evaluate the reversal effect of DDR1 inhibitors on adipocyte dedifferentiation, or to screen anti-metastatic drugs targeting the DDR1 signaling pathway.

Images