Method for culturing in vitro tumor invasion evaluation model

Abstract

The invention relates to the field of medicine, in particular to a method for culturing an in-vitro tumor invasion evaluation model, the model obtained through the method and a medicine screening method. The method comprises the following steps: constructing a first cell model with high cell density and / or low cell density; the first cell model is cultured under the condition of high osmotic pressure and / or low osmotic pressure, the low osmotic pressure condition is a low osmotic medium composed of a complete medium and water according to the proportion of 3: 1 to 2: 1, and the high osmotic pressure condition is a high osmotic medium composed of a complete medium and a sucrose solution according to the proportion of 4: 1 to 3: 1. According to the method, the synergistic effect of solid stress and intracellular hydrostatic pressure is considered, the tumor invasion evaluation model and method based on the intracellular and intracellular pressure difference are provided, and an in-vitro model capable of truly and effectively simulating in-vivo tumor invasion can be provided.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a method for culturing an in vitro tumor invasion evaluation model, an in vitro model for evaluating tumor cell invasion obtained therefrom, and a drug screening method. Background Technology

[0002] Tumor cell invasion is a key driver of metastatic spread, and the development of secondary metastatic tumors is one of the leading causes of cancer-related deaths. During this process, the biomechanical microenvironment of tumor tissue undergoes significant changes, with marked differences in the biomechanical microenvironment and mechanical characteristics across different regions, including increased solid stress and hydrostatic pressure. Solid stress primarily stems from volume restriction caused by tumor cell proliferation. Intracellular hydrostatic pressure mainly originates from the osmotic pressure difference between the intracellular and extracellular spaces, ion pump activity, and changes in cell membrane permeability. Current research has found that both solid stress and hydrostatic pressure alter the proliferation, invasion, and apoptosis of cancer cells and are important factors in cancer progression.

[0003] In vitro methods used in related technologies to study and evaluate tumor cell invasion typically only investigate the effects of solid stress or hydrostatic pressure on invasive migration capabilities. How the intracellular and extracellular pressure differential caused by changes in intracellular hydrostatic pressure and solid stress affect tumor cell invasion remains unclear.

[0004] Therefore, there is an urgent need to provide an in vitro model that can realistically and effectively simulate tumor invasion in vivo, so as to study the molecular mechanisms of tumor cell invasion and screen anti-tumor drugs for treating tumor invasion. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the technical problems in the related art.

[0006] Therefore, an embodiment of the first aspect of the present invention proposes a method for culturing an in vitro tumor invasion evaluation model, comprising: constructing a first cell model with high and / or low cell density; culturing the first cell model under high and / or low osmotic pressure conditions to obtain the in vitro tumor invasion evaluation model, wherein the low osmotic pressure condition is a hypotonic culture medium composed of a complete culture medium and water in a ratio of 3:1 to 2:1, and the high osmotic pressure condition is a hypertonic culture medium composed of a complete culture medium and sucrose solution in a ratio of 4:1 to 3:1. This method takes into account the synergistic effect of solid stress and intracellular hydrostatic pressure, and for the first time provides a tumor invasion evaluation model and method based on the intracellular and extracellular pressure difference. It can realistically and effectively simulate an in vitro model of in vivo tumor invasion for studying the molecular mechanisms of tumor cell invasion and screening anti-tumor drugs for treating tumor invasion.

[0007] In some embodiments, constructing a first cell model with high and / or low cell density includes: setting a final concentration of tumor cells to 1 × 10⁻⁶. 5 A cell suspension of / mL was cultured at 37°C and 5% CO2 for 12-36 hours to obtain the first cell model with high cell density, wherein the tumor cells formed a monolayer; and / or the final concentration of tumor cells was 2×10⁶. 4 A cell suspension of / mL was cultured at 37°C and 5% CO2 for 12-36 hours to obtain the first cell model with low cell density, wherein the tumor cells were in a monolayer.

[0008] In some embodiments, the culture is carried out for 24 hours.

[0009] In some embodiments, culturing the first cell model under high and / or low osmotic pressure conditions includes: adding the hypotonic culture medium to the first cell model with high cell density and culturing it at 37°C and 5% CO2 for 12-48 hours; and / or adding the hypertonic culture medium to the first cell model with low cell density and culturing it at 37°C and 5% CO2 for 12-48 hours.

[0010] In some embodiments, the first cell model is rinsed after 12 hours of culture, and then the hypertonic culture medium and / or the hypotonic culture medium are added again to continue culturing for 48 hours.

[0011] In some embodiments, the in vitro tumor invasion evaluation model includes a high-invasive tumor model and / or a low-invasive tumor model, wherein the high-invasive tumor model is obtained by adding the hypotonic culture medium to the first cell model with high cell density; and / or the low-invasive tumor model is obtained by adding the hypertonic culture medium to the first cell model with low cell density.

[0012] In some embodiments, the intracellular and extracellular pressure difference of the high-invasive tumor model is 1.5 kPa to 1.8 kPa, preferably 1.65 kPa, and the intracellular and extracellular pressure difference of the low-invasive tumor model is 0.45 kPa to 0.65 kPa, preferably 0.55 kPa.

[0013] In some embodiments, the hypotonic culture medium consists of a complete culture medium and water in a ratio of 3:1, and the hypertonic culture medium consists of a complete culture medium and sucrose solution in a ratio of 4:1.

[0014] In some embodiments, the tumor cells are HeLa cell lines.

[0015] A second aspect of the present invention provides an in vitro tumor invasion evaluation model, obtained by the method described in any embodiment of the first aspect of the present invention. The model includes: a monoclonal cell population and a cell culture chamber, wherein the cell culture chamber includes a growth space and an invasion space, the growth space and the invasion space are connected by a biomembrane, the monoclonal cell population grows in the growth space and can penetrate the biomembrane to enter the invasion space, or wherein the cell culture chamber contains a gel matrix, the monoclonal cell population grows on the gel matrix and hydrolyzes the gel matrix, wherein the monoclonal cell population is derived from tumor cells.

[0016] In some embodiments, the model is used for non-diagnostic or non-therapeutic purposes.

[0017] In some embodiments, the cell culture chamber contains a hypotonic or hypertonic culture medium.

[0018] In some embodiments, the initial density of the monoclonal cell population is either high cell density or low cell density.

[0019] In some embodiments, the in vitro tumor invasion assessment model includes a high-invasive tumor model and / or a low-invasive tumor model.

[0020] An embodiment of the third aspect of the present invention provides a drug screening method, comprising: treating an in vitro evaluation model of tumor cell invasion as described in an embodiment of the second aspect of the present invention with the candidate drug.

[0021] In some embodiments, the method includes: contacting the candidate drug with a monoclonal cell population in a growth space; and comparing the change in the number of cells that cross the biomembrane from the growth space into an invasion space before and after the contact, wherein the reduction in the number of cells in the invasion space indicates that the contacted candidate drug is a drug that inhibits tumor cell invasion.

[0022] In some embodiments, the method includes: contacting the candidate drug with a monoclonal cell population in a cell culture chamber; and comparing the degree of hydrolysis of the gel matrix in the cell culture chamber before and after the contact, wherein a decrease in the degree of hydrolysis indicates that the contacted candidate drug is a drug that inhibits tumor cell invasion.

[0023] In some embodiments, the method includes: treating the high-invasive tumor model with the candidate drug based on the in vitro tumor invasion evaluation model, which includes a high-invasive tumor model and / or a low-invasive tumor model, and comparing the invasion ability evaluation indicators of the treated high-invasive tumor model and the low-invasive tumor model.

[0024] In some embodiments, the invasiveness evaluation index includes the number of cells in the invasive space, the degree of hydrolysis of the gel matrix, the intracellular and extracellular pressure difference, or other invasiveness indicators, wherein the invasiveness evaluation index of the treated high-invasive tumor model is close to, the same as, or better than the invasiveness evaluation index of the low-invasive tumor model, indicating that the contacted candidate drug is a drug that inhibits tumor cell invasion by affecting the intracellular and extracellular pressure difference.

[0025] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are as follows:

[0026] (1) The embodiments of the present invention take into account the synergistic effect of solid stress and intracellular hydrostatic pressure, and provide for the first time a tumor invasion evaluation model and method based on intracellular and extracellular pressure difference.

[0027] (2) By comparing relative indicators used as low-invasive tumor models and high-invasive tumor models, the embodiments of the present invention overcome the problem that absolute indicators are not reliable enough due to the susceptibility to other influencing factors in cell culture, and can more objectively and reliably indicate the effectiveness of drugs.

[0028] (3) The present invention provides a drug screening model for the first time using intracellular and extracellular pressure difference as a target, which provides a new idea for developing anti-tumor drugs that inhibit intracellular and extracellular pressure difference sensitive proteins and has important guiding significance for clinical treatment of tumor metastasis.

[0029] (4) The embodiments of the present invention can realistically and effectively simulate the in vitro model of tumor invasion in vivo, so as to study the molecular mechanism of tumor cell invasion process and screen anti-tumor drugs for treating tumor invasion. Attached Figure Description

[0030] Figure 1 This is an optical microscope image of a candidate in vitro tumor invasion evaluation model according to an embodiment of the present invention.

[0031] Figure 2 This is a diagram showing the intracellular and extracellular pressure difference after low-density and high-osmotic treatment of cells according to an embodiment of the present invention.

[0032] Figure 3 These are cell invasion diagrams after low-density and high-osmotic treatment of cells according to embodiments of the present invention.

[0033] Figure 4 This is a statistical chart showing the cell invasion pseudopodia and matrix degradation capabilities of cells after low-density and high-osmotic treatment in embodiments of the present invention. Detailed Implementation

[0034] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0035] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0036] Malignant metastasis of tumors and failure of clinical treatment are the leading causes of death in most patients with solid tumors. Metastasis is a complex, multi-step process, often referred to as the metastatic cascade, with key components including: local invasion of tumor cells, intravascular invasion, survival in the circulatory system, retention in distant organs, extravasation into distant tissue parenchyma, formation of micrometastases, and metastatic colonization. Among these, invasion is one of the early key steps for tumor cells to acquire the metastatic phenotype, involving a series of biological processes such as changes in cell morphology, loss of polarity, and cell migration, ultimately driving the metastatic spread of tumor cells. From a morphological perspective, invasion is the detachment of malignant cells from the primary tumor tissue, undergoing changes in cell morphology and polarity, and passing through the extracellular matrix. In-depth analysis of the dynamic mechanisms of cancer invasion and spread can not only help identify biomarkers that can predict the risk of metastasis in patients at an early stage, but also provide new targets for precision medicine. These targets may inhibit the progression of metastatic disease and even reverse the growth process of metastatic cancer by blocking key steps in the metastatic cascade.

[0037] Within the human body, cells are affected by various mechanical forces, including gravity, tension, compressive force, shear stress, and hydrostatic pressure. These mechanical signals are sensed by mechanoreceptors and transmitted into the cell via mechanotransduction. During tumor development, invasion, and metastasis, biomechanical factors significantly regulate tumor angiogenesis, the interaction between tumor cells and the extracellular matrix, the interaction between tumor cells and other cells, and the interaction between tumor cells and the circulatory and vascular systems. Studies have shown that physical abnormalities in the tumor microenvironment mainly include changes in the mechanical stiffness of the microenvironment and an increase in mechanical stress on tumor cells. Two types of mechanical stress are particularly important in influencing tumor tissue and cell function—intracellular hydrostatic pressure and solid stress. Intracellular hydrostatic pressure is an isotropic stress generated by the expansion of intracellular fluid, affecting cell behavior by directly compressing the cell nucleus and activating mechanosensitive channels; while solid stress is an anisotropic stress transmitted by the extracellular matrix and cellular components, driving malignant phenotypes by altering cell morphology and ECM topology. Studies have shown that these two stresses work synergistically: increased intracellular pressure enhances cell-matrix interactions, thus amplifying solid stress; conversely, increased ECM stiffness restricts cell expansion, further increasing intracellular pressure. This positive mechanical feedback loop is a crucial factor promoting cancer proliferation, invasion, and treatment resistance. Current research has found that both solid stress and hydrostatic pressure alter cancer cell proliferation, invasion, and apoptosis, and are significant factors in cancer progression. Oncogenes participate in many of the same signaling pathways activated under mechanical stress, thereby reprogramming the cell's intrinsic response to exogenous mechanical stimuli and enhancing cytoskeleton contractility. The active interaction of tumor cells with this mechanically evolved microenvironment leads to changes in cytoskeleton structure, cell morphology, differentiation, survival / death, proliferation, adhesion, and migration, ultimately driving tumor progression and invasiveness. However, research on how the intracellular and extracellular pressure differential caused by changes in intracellular hydrostatic pressure and solid stress affects tumor cell invasion is limited, and the specific mechanisms remain unclear.

[0038] Solid stress, also known as residual stress, is a mechanical force contained in and transmitted by the solid and elastic components of the extracellular matrix and cells. It mainly includes compressive, tensile, and shear forces. Solid stress primarily arises from the increase in tissue volume, caused by factors such as cell infiltration, proliferation, and matrix deposition. This increase in volume pushes and displaces viscoelastic structures inside and outside the tumor, thus creating solid stress in the tumor and surrounding tissues. Rapid tumor cell proliferation puts pressure on the tumor microenvironment, causing cells in the central region to typically experience compressive stress from all directions, while peripheral cells experience both circumferential tensile and radial compressive stresses. The growth pattern of a tumor significantly influences its solid stress distribution: some tumors tend to expand as well-defined nodular masses, generating significant solid stress by pushing surrounding tissues; while invasive rather than cohesive tumors grow by interpenetrating normal tissue, thus generating less solid stress. Furthermore, components in the extracellular matrix, such as hyaluronic acid, swell due to water absorption, further promoting solid stress generation. Within the tumor microenvironment, cell motility and repair activities generate contractile stress through an actomyosin-mediated mechanism. This cellular contractility produces tensile forces, causing the extracellular matrix components to contract, thereby creating tension within the tumor. These tensions are usually balanced by compression of other parts, ultimately manifesting as solid-state stress within the TME.

[0039] Intracellular hydrostatic pressure is a type of mechanical stress generated by the hydrostatic forces regulated by the intracellular fluid components (cytosol, organelle contents, etc.) and osmotic pressure. [5] Unlike solid stress, IHP primarily originates from the osmotic pressure difference between the inside and outside of cells, ion pump activity, and changes in cell membrane permeability, and plays a crucial role in mechanical regulation within the tumor microenvironment. [67,116]Ion pumps (IHPs) play a crucial role in cellular physiological and pathological processes, and their maintenance depends on the dynamic balance of osmotic pressure inside and outside the cell. Studies have shown that when extracellular osmotic pressure changes, water molecules are transported across the cell membrane, altering the intracellular fluid content and consequently affecting IHP. For example, in a hypertonic environment, the extracellular solute concentration is higher than intracellular, causing water molecules to flow from inside the cell to outside, reducing intracellular fluid content and lowering IHP. Conversely, in a hypotonic environment, water molecules flow into the cell, increasing intracellular fluid content and raising IHP. Furthermore, the activity of ion pumps is also critical to the regulation of IHP. The sodium-potassium pump (SNP), an important intracellular ion transport protein, pumps sodium ions out of the cell and potassium ions into the cell by consuming ATP. This transmembrane transport of ions maintains the intracellular ion concentration gradient, thus influencing IHP. When SNP activity increases, more sodium ions are pumped out of the cell, reducing intracellular ion concentration, lowering osmotic pressure, and consequently altering IHP. Changes in cell membrane permeability also significantly affect IHP. When ion channels on the cell membrane open or close, the entry and exit of ions alters the intracellular ion concentration, thereby indirectly affecting IHP. [117-121] For example, when calcium ion channels open, a large influx of calcium ions into the cell leads to an increase in intracellular ion concentration and osmotic pressure, resulting in an increase in IHP.

[0040] An embodiment of the first aspect of this invention provides a method for culturing an in vitro tumor invasion evaluation model, comprising: constructing a first cell model with high and / or low cell density; culturing the first cell model under high and / or low osmotic pressure conditions to obtain the in vitro tumor invasion evaluation model, wherein the low osmotic pressure condition is a hypotonic culture medium composed of a complete culture medium and water in a ratio of 3:1 to 2:1, and the high osmotic pressure condition is a hypertonic culture medium composed of a complete culture medium and sucrose solution in a ratio of 4:1 to 3:1. This method takes into account the synergistic effect of solid stress and intracellular hydrostatic pressure, and for the first time provides a tumor invasion evaluation model and method based on the intracellular and extracellular pressure difference, which can realistically and effectively simulate an in vitro model of in vivo tumor invasion for studying the molecular mechanisms of tumor cell invasion and screening anti-tumor drugs for treating tumor invasion.

[0041] This invention delves into the molecular mechanisms by which mechanical stress in the tumor microenvironment regulates tumor cell invasion from a mechanobiological perspective, possessing significant theoretical and clinical value. At the basic research level, it innovatively constructs an in vitro model simulating the mechanical characteristics of the tumor microenvironment, systematically studying the dynamic process by which solid stress caused by cell crowding and changes in osmotic pressure synergistically alter the intracellular hydrostatic pressure difference, promoting tumor invasion. This provides new insights for developing antitumor drugs that inhibit intracellular pressure difference-sensitive proteins, offering important guidance for the clinical treatment of tumor metastases.

[0042] In some embodiments, constructing a first cell model with high and / or low cell density includes: setting a final concentration of tumor cells to 1 × 10⁻⁶. 5 A cell suspension of / mL was cultured at 37°C and 5% CO2 for 12-36 hours to obtain the first cell model with high cell density, wherein the tumor cells formed a monolayer; and / or the final concentration of tumor cells was 2×10⁶. 4 A cell suspension of / mL was cultured at 37°C and 5% CO2 for 12-36 hours to obtain the first cell model with low cell density, wherein the tumor cells were in a monolayer.

[0043] In some embodiments, the culture is carried out for 24 hours.

[0044] In some embodiments, culturing the first cell model under high and / or low osmotic pressure conditions includes: adding the hypotonic culture medium to the first cell model with high cell density and culturing it at 37°C and 5% CO2 for 12-48 hours; and / or adding the hypertonic culture medium to the first cell model with low cell density and culturing it at 37°C and 5% CO2 for 12-48 hours.

[0045] In some embodiments, the first cell model is rinsed after 12 hours of culture, and then the hypertonic culture medium and / or the hypotonic culture medium are added again to continue culturing for 48 hours.

[0046] In some embodiments, the in vitro tumor invasion evaluation model includes a high-invasive tumor model and / or a low-invasive tumor model, wherein the high-invasive tumor model is obtained by adding the hypotonic culture medium to the first cell model with high cell density; and / or the low-invasive tumor model is obtained by adding the hypertonic culture medium to the first cell model with low cell density.

[0047] In some embodiments, the intracellular and extracellular pressure difference of the high-invasive tumor model is 1.5 kPa to 1.8 kPa, preferably 1.65 kPa, and the intracellular and extracellular pressure difference of the low-invasive tumor model is 0.45 kPa to 0.65 kPa, preferably 0.55 kPa.

[0048] In some embodiments, the hypotonic culture medium consists of a complete culture medium and water in a ratio of 3:1, and the hypertonic culture medium consists of a complete culture medium and sucrose solution in a ratio of 4:1.

[0049] In some embodiments, the tumor cells are HeLa cell lines.

[0050] A second aspect of the present invention provides an in vitro tumor invasion evaluation model, obtained by the method described in any embodiment of the first aspect of the present invention. The model includes: a monoclonal cell population and a cell culture chamber, wherein the cell culture chamber includes a growth space and an invasion space, the growth space and the invasion space are connected by a biomembrane, the monoclonal cell population grows in the growth space and can penetrate the biomembrane to enter the invasion space, or wherein the cell culture chamber contains a gel matrix, the monoclonal cell population grows on the gel matrix and hydrolyzes the gel matrix, wherein the monoclonal cell population is derived from tumor cells.

[0051] In some embodiments, the model is used for non-diagnostic or non-therapeutic purposes.

[0052] In some embodiments, the cell culture chamber contains a hypotonic or hypertonic culture medium.

[0053] In some embodiments, the initial density of the monoclonal cell population is either high cell density or low cell density.

[0054] In some embodiments, the in vitro tumor invasion assessment model includes a high-invasive tumor model and / or a low-invasive tumor model.

[0055] An embodiment of the third aspect of the present invention provides a drug screening method, comprising: treating an in vitro evaluation model of tumor cell invasion as described in an embodiment of the second aspect of the present invention with the candidate drug.

[0056] In some embodiments, the method includes: contacting the candidate drug with a monoclonal cell population in a growth space; and comparing the change in the number of cells that cross the biomembrane from the growth space into an invasion space before and after the contact, wherein the reduction in the number of cells in the invasion space indicates that the contacted candidate drug is a drug that inhibits tumor cell invasion.

[0057] In some embodiments, the method includes: contacting the candidate drug with a monoclonal cell population in a cell culture chamber; and comparing the degree of hydrolysis of the gel matrix in the cell culture chamber before and after the contact, wherein a decrease in the degree of hydrolysis indicates that the contacted candidate drug is a drug that inhibits tumor cell invasion.

[0058] In some embodiments, the method includes: treating the high-invasive tumor model with the candidate drug based on the in vitro tumor invasion evaluation model, which includes a high-invasive tumor model and / or a low-invasive tumor model, and comparing the invasion ability evaluation indicators of the treated high-invasive tumor model and the low-invasive tumor model.

[0059] In some embodiments, the invasiveness evaluation index includes the number of cells in the invasive space, the degree of hydrolysis of the gel matrix, the intracellular and extracellular pressure difference, or other invasiveness indicators, wherein the invasiveness evaluation index of the treated high-invasive tumor model is close to, the same as, or better than the invasiveness evaluation index of the low-invasive tumor model, indicating that the contacted candidate drug is a drug that inhibits tumor cell invasion by affecting the intracellular and extracellular pressure difference.

[0060] The embodiments of the present invention overcome the problem that absolute indicators are not reliable enough due to the susceptibility to other influencing factors in cell culture by comparing relative indicators used as low-invasive tumor models and high-invasive tumor models, and can more objectively and reliably indicate the effectiveness of drugs.

[0061] This invention provides, for the first time, a drug screening model that uses intracellular and extracellular pressure difference as a target, offering a new approach to developing antitumor drugs that inhibit intracellular and extracellular pressure difference-sensitive proteins, and has important guiding significance for the clinical treatment of tumor metastasis.

[0062] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0063] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0064] Example

[0065] Example 1: Culture of an in vitro tumor invasion evaluation model

[0066] 1.1 Culture of cell models for simulating solid stress

[0067] 1) Digest and centrifuge HeLa cells (catalog number: THu187, purchased from: Cell Bank of Chinese Academy of Sciences) that have proliferated to 80% of the bottom area of ​​the dish, and resuspend them in fresh DMEM high glucose medium (catalog number: 12491015, purchased from: Gibco).

[0068] 2) Use a pipette to continuously agitate the culture medium to ensure the cells are evenly distributed. Perform serial dilutions until the cell concentration is approximately 2 × 10⁶ cells per 500 μL of culture medium. 5 Left and right cells.

[0069] 3) Divide the sterile confocal dishes into two groups. Add 500 μL of cell suspension (final concentration approximately 1 × 10⁻⁶) to each dish in the high-density group. 5 For the low-density group, 100 μL of the same batch of cell suspension and 400 μL of fresh complete culture medium were added to each dish (final concentration approximately 2 × 10⁻⁶ cells / mL). 4 (cells / mL). After ensuring uniform cell distribution using the cross-hatching method, the uniformity of cell distribution was confirmed under a microscope, and then the small dishes were placed in an incubator at 37°C and 5% CO2 for incubation.

[0070] 4) Observe the cell adhesion under a microscope 12 hours after inoculation to confirm that the cells are evenly distributed in each experimental group. Continue culturing for 24 hours to form a stable monolayer of cells, thus obtaining the first cell model with high and / or low cell density.

[0071] 1.2 Culture of cell models for simulating intracellular hydrostatic pressure

[0072] 1) Three osmotic pressure media were prepared: hypotonic group: complete medium (product number: 12491015, purchased from Gibco) + 33.3% double distilled water and complete medium + 25% double distilled water; isotonic group: complete medium; hypertonic group: complete medium + 25% sucrose solution (0.9 mol / L) and complete medium + 20% sucrose solution (0.9 mol / L); the complete medium consisted of: 90% DMEM high sugar medium (product number: 12491015, purchased from Gibco) + 10% FBS (product number: A5256701, purchased from Gibco).

[0073] 2) Construct the first cell model in a six-well plate according to the experimental method in Example 1.1.

[0074] 3) Add the six prepared osmotic pressure media to the constructed first cell model, and then incubate them in a 37℃, 5% CO2 incubator. After 24h, rinse three times with 1×PBS buffer (product number: SH30256.01, purchased from HyClone), and change the medium according to the prepared osmotic pressure media.

[0075] 4) After 48 hours of culture, observe the cell morphology of the cell models cultured under low- and high-osmotic medium in each group under a microscope.

[0076] 5) Repeat steps 1)-4) above in a 96-well plate, with 6 wells per group. Dilute the CCK-8 reagent (product number: C0037, purchased from Beyotime) at a ratio of 1:10 using three different osmotic media to prepare the working solution. Discard the old medium and add 100 μL of medium containing the CCK-8 reagent to each well of the cultured cell model. Incubate in an incubator for 2 hours. After 2 hours of incubation, remove air bubbles from the wells and measure the absorbance at a wavelength of 450 nm using a microplate reader. Record and analyze the data.

[0077] Candidate in vitro tumor invasion evaluation models were obtained by subjecting first-cell models with high and / or low cell densities to low-osmotic and high-osmotic culture treatments, respectively. The low-osmotic group used a mixture of complete culture medium and double-distilled water at ratios of 3:1 (25% double-distilled water) and 4:1 (33.3% double-distilled water); the isotonic group used conventional complete culture medium; and the high-osmotic group used complete culture medium supplemented with 25% and 20% 0.9 mol / L sucrose solution, respectively.

[0078] Figure 1 This is an optical microscope image of a candidate in vitro tumor invasion evaluation model according to an embodiment of the present invention, where LC represents low density and HC represents high density. According to Figure 1 To assess cell morphology integrity, pseudopodia extension normality, and the presence of obvious apoptotic features, 3:1 (low osmotic pressure) and 4:1 (high osmotic pressure) were selected as further candidate in vitro tumor invasion evaluation models. Scale bar: 200 μm.

[0079] Example 2: Measurement of intracellular and extracellular pressure difference in an in vitro tumor invasion evaluation model

[0080] In this embodiment, the intracellular and extracellular pressure difference in an in vitro tumor invasion evaluation model was measured to study the regulatory effect of the synergistic effect of cell crowding and osmotic pressure on the mechanical microenvironment.

[0081] A high-precision micro-pressure measurement system was used to dynamically monitor the pressure difference between the inside and outside of cells. The specific steps are as follows:

[0082] The null-pressure servo method was used to measure the intracellular pressure of HeLa cells with different crowding levels after 24 hours of culture under different osmotic pressures. The specific procedure is briefly described below: Borosilicate glass capillaries with a diameter of less than 2 μm were prepared using a glass capillary puller (P-100, Narishige, Japan). A 1 mol / L NaCl solution was infused into the glass capillary. It was then connected to a micromanipulator (900A, WPI, USA) for initial pressure equilibration calibration. Using a micromanipulator (Longfubiotech, China), the glass microelectrode was inserted into the cytoplasm of the cell. At this point, the cell was compressed, and the pressure reading fluctuated slightly, showing a significant increase. The glass microelectrode was then slowly and slightly withdrawn to eliminate the influence of cell compression on the intracellular pressure measurement. The micromanipulator then stabilized the pressure reading. The micromanipulator read 100 data points per second. The average of these 100 data points per second was taken as the intracellular pressure value at that time point. During the pressure measurement process, the pressure reading should remain stable for at least 5 seconds, and the average pressure at each time point within this period should be taken.

[0083] Figure 2 This is a graph showing the intracellular and extracellular pressure difference of cells after low-density and high-osmotic treatment in an embodiment of the present invention, i.e., a statistical comparison graph of intracellular and extracellular pressure difference of candidate in vitro tumor invasion evaluation models in an embodiment of the present invention (**p<0.01, ****p<0.0001, n≥3), where HC represents high density, LC represents low density, HYPO represents low osmotic pressure, and Hyper represents high osmotic pressure. According to Figure 2 In the high-density, low-osmolarity group, the intracellular and extracellular pressure difference reached 1.65 ± 0.11 kPa, significantly higher than the isotonic control group's 1.2 ± 0.08 kPa by approximately 41.7%. Conversely, in the high-osmolarity group, the intracellular and extracellular pressure difference decreased to 1.0 ± 0.04 kPa, a reduction of approximately 16.7%. The ΔP in the high-density group was significantly higher than that in the low-density group under all three osmolarity conditions. Furthermore, the intracellular and extracellular pressure difference in the low-density, high-osmolarity group decreased to 0.55 ± 0.15 kPa. The high-density, low-osmolarity model simulated the mechanical stress changes in the tumor microenvironment within the range of 1.0–1.7 kPa through the synergistic effect of cell crowding and osmolarity changes, which is consistent with real clinical tumor tissue detection data (1.0–1.5 kPa).

[0084] Example 3: Detection of Tumor Cell Invasion Ability

[0085] 3.1 Gelatin Degradation Experiment

[0086] (1) First, accurately weigh 0.5g of Gelatin powder (product number: Y263031, purchased from: Beyotime) and dissolve it in 10mL of 1×PBS buffer. Heat in a 60℃ water bath for 20-30 minutes and shake intermittently until completely dissolved. After sterilization by filtration through a 0.22μm filter membrane, the solution is aliquoted and stored at -20℃.

[0087] (2) During the experiment, take an appropriate amount of the gelatin solution from step (1), melt it at 37°C, and add 500 μL evenly to the round glass slide of the 12-well plate. Let it stand at 4°C for 30 minutes to form a gel matrix. Then rinse three times with 1×PBS buffer for 5 minutes each time. After rinsing, sterilize with ultraviolet light for 30 minutes.

[0088] (3) Following the steps in Example 1, first cell models with high and low densities were constructed on gelatin gels. Three osmotic media were added: complete culture medium and double-distilled water at a ratio of 3:1 (25% double-distilled water), complete culture medium + 20% sucrose solution, and complete culture medium only. The cells were then incubated at 37°C in a 5% CO2 incubator. After 24 hours, the cells were washed three times with 1×PBS buffer, and the medium was changed according to the prepared osmotic media.

[0089] (4) After culturing for 48 hours, wash three times with 1×PBS buffer and fix with 4% PFA (product number: P0099, purchased from: Beyotime) at room temperature for 15 minutes. Then wash three times with 1×PBS buffer for 5 minutes each time.

[0090] (5) Use 1% Triton-X100 (product number: P0096, purchased from Beyotime) to break the membrane at room temperature for 10 min, and wash it 3 times with 1×PBS buffer for 5 min each time.

[0091] (6) The cytoskeleton was stained with phalloidin (product number: A22287, purchased from Thermo Fisher Scientific) diluted 1:200 in 1×PBS solution. The staining was carried out at room temperature for 1 h, and the cytoskeleton was washed 3 times with 1×PBS buffer for 5 min each time.

[0092] (7) DAPI (Catalog No.: C1002, purchased from: Beyotime) was diluted 1:1000 in 1×PBS solution to stain the cell nuclei. The staining was performed at room temperature for 15 min, and the cells were washed 3 times with 1×PBS buffer for 5 min each time.

[0093] (8) Use a confocal microscope to observe and obtain clear images, and use ImageJ software to count the degree of degradation of gelatin.

[0094] Figure 4This is a statistical diagram of cell invasive pseudopodia and matrix degradation capacity after low-density and high-osmotic treatment of cells according to an embodiment of the present invention. Specifically, it is a schematic diagram of the cell invasive pseudopodia and gelatin degradation results in the in vitro tumor invasion evaluation model of this embodiment of the present invention, where HC represents high density, LC represents low density, HYPO represents low osmotic pressure, and Hyper represents high osmotic pressure. (a) shows the gelatin degradation experiment (black: degraded area, green: undegraded area, scale bar: 50 μm); (b) shows the Cortactin immunofluorescence staining of cell invasive pseudopodia (scale bar: 20 μm); (c) shows the quantitative statistics of the degree of undegraded gelatin (p<0.0001, n≥20); and (d) shows the statistics of the number of invasive pseudopodia in a single cell (p<0.0001, n≥20). It should be noted that the gelatin degradation experiment simulates the ability of tumors to degrade the extracellular matrix (ECM) during development and invasion. Generally speaking, the stronger the ECM degradation capacity, the higher the tumor invasive capacity is considered to be.

[0095] Specifically, regarding ECM degradation capacity, cells in the low-osmolarity group exhibited stronger proteolytic activity. The proportion of undegraded area in the low-osmolarity group was only 9.9±1.2%, significantly lower than that in the isotonic group (30.1±2.5%) by 23.1%, indicating enhanced ECM degradation capacity of tumor cells in the low-osmolarity group. In contrast, the undegraded area in the high-osmolarity group was as high as 57.1±3.8%, suggesting that its ECM degradation capacity was significantly limited. Furthermore, the high-density low-osmolarity group had the lowest proportion of undegraded area, while the low-density high-osmolarity group had the highest proportion. This suggests that the high-density low-osmolarity group and the low-density high-osmolarity group can serve as in vitro tumor invasion evaluation models for high-invasiveness and low-invasiveness, respectively.

[0096] 3.2 Transwell Invasion Experiment

[0097] (1) Prepare a 24-well plate, add 500 μL of complete culture medium to each well, place it in a transwell chamber, and construct a first cell model with high density and low density in each chamber according to the method of Example 1. Then, treat it with three osmotic pressure culture media: complete culture medium and double distilled water at a ratio of 3:1 (25% double distilled water), complete culture medium + 20% sucrose solution, and complete culture medium only.

[0098] (2) Then it was placed in a 37°C, 5% CO2 incubator for incubation. After 24 hours, it was rinsed 3 times with 1×PBS buffer and the medium was changed accordingly according to the prepared osmotic medium.

[0099] (3) After 48 hours, the transwell chamber was removed, washed three times with 1×PBS buffer, and fixed with 4% PFA at room temperature for 15 minutes; the membrane was perforated with 1% Triton-X100 at room temperature for 10 minutes; the cytoskeleton was stained with phalloidin diluted 1:200 in 1×PBS solution at room temperature for 1 hour; and the nuclei were stained with DAPI diluted 1:1000 in 1×PBS solution at room temperature for 15 minutes. After each process, the cells were washed three times with 1×PBS buffer for 5 minutes each time. For details, refer to steps (4)-(7) in Example 3.1.

[0100] (4) Observation was performed using a confocal microscope to obtain clear images. The number of non-migrated cells and the number of cells that migrated to the lower layer of the chamber were counted using ImageJ software. Three biological replicates were set up for the experiment, with three random fields of view measured in each replicate. Data are expressed as mean ± SEM, and statistical analysis was performed using a t-test (p < 0.05 was considered significant). The migration rate was calculated using the following formula:

[0101] [Number of cells that migrated to the lower layer / (Number of cells that migrated to the lower layer + Number of cells that did not migrate)] × 100%.

[0102] The results of the Transwell invasion experiment are as follows: Figure 3 As shown. Figure 3 This is a cell invasion diagram of cells after low-density and high-osmolarity treatment in an embodiment of the present invention, i.e., the cell invasion ability measurement results of the in vitro tumor invasion evaluation model of the present invention. Among them, (a) shows the immunofluorescence staining (scale bar: 20μm) of cell nuclei (DAPI, blue) and cytoskeleton (Phalloidin, red) in the Transwell experiment; (b) shows the migration statistics of the Transwell experiment (**p<0.01, ****p<0.0001, n≥20).

[0103] 3.3 Cell Scratch Migration Assay

[0104] (1) A model with high density and low osmotic pressure and high density and high osmotic pressure was constructed in a six-well plate according to the method of Example 1.

[0105] (2) Using a 200μL sterile pipette tip perpendicular to the bottom of the well plate, apply constant pressure to make scratches of uniform width, approximately 0.5-1mm. After gently rinsing three times with PBS to remove detached cells, add three different osmotic pressure culture media respectively.

[0106] (3) At time points of 0h, 24h, 48h and 72h, the scratch area was photographed at a fixed position under a microscope, and the change in scratch area was measured using ImageJ software.

[0107] The results of the cell scratch migration assay are as follows Figure 3 As shown. Figure 3 This is a graph showing the cell invasion ability measurement results of the in vitro tumor invasion evaluation model according to an embodiment of the present invention. Part (c) shows the dynamic monitoring of the scratch healing experiment at time points of 0h, 24h, 48h, 72h, and 96h (scale bar: 200μm); Part (d) shows the dynamic changes in migration rate during the scratch healing experiment (**p<0.01, ***p<0.001, ****p<0.0001, n≥3).

[0108] Based on the results of Examples 3.2 and 3.3, regarding cell invasion ability, the low osmotic pressure environment under high-density culture conditions significantly promoted the invasive phenotype of tumor cells. Quantitative analysis of the Transwell invasion assay showed that the number of invasive cells in the low-osmotic group reached 47±5 cells / field, which was 1.03 times higher than that in the isotonic control group. Dynamic monitoring results of the scratch healing assay further confirmed this trend: at the three time points of 48h, 72h, and 96h, the migration rate of the low-osmotic group increased by 36.8%, 41.1%, and 21.0%, respectively. In stark contrast, the high osmotic pressure conditions showed a significant inhibitory effect, with an overall migration rate reduction of 47.3%, and migration rates at each time point decreasing by 28.2%, 32.7%, and 53.0%, respectively.

[0109] This indicates that the high-density low-osmolarity group and the low-density high-osmolarity group can be used as in vitro tumor invasion evaluation models for high-invasiveness and low-invasiveness, respectively.

[0110] In summary, this study found a positive correlation between enhanced cell invasiveness and increased intracellular and extracellular pressure gradient, strongly confirming that alterations in the biomechanical microenvironment are a key factor regulating tumor cell invasion behavior. The experimental results in this section revealed a significant synergistic effect between the hypotonic environment and cell crowding, suggesting that the combined effects of multiple biomechanical factors may more significantly promote tumor cell invasion and metastasis during solid tumor development. These findings provide important experimental evidence for understanding the biological effects of biomechanical factors in the tumor microenvironment and point the way for developing anti-metastatic strategies targeting the biomechanical microenvironment.

[0111] Meanwhile, this invention takes into account the synergistic effect of solid stress and intracellular hydrostatic pressure, and for the first time provides a tumor invasion evaluation model and method based on intracellular and extracellular pressure difference. By comparing relative indicators used as low-invasive and high-invasive tumor models, this invention overcomes the problem of unreliable absolute indicators due to susceptibility to other influencing factors in cell culture, and can more objectively and reliably indicate drug effectiveness.

[0112] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0113] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0114] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0115] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for culturing an in vitro tumor invasion evaluation model, characterized in that, include: Construct first-cell models with high and / or low cell densities; The first cell model is cultured under high and / or low osmotic pressure conditions to obtain the in vitro tumor invasion evaluation model. The low osmotic pressure condition refers to a low-osmotic medium composed of a complete culture medium and water in a ratio of 3:1 to 2:

1. The high osmotic pressure condition is a hypertonic medium composed of a complete culture medium and a sucrose solution in a ratio of 4:1 to 3:

1.

2. The method according to claim 1, characterized in that, The construction of the first cell model with high and / or low cell density includes: The final concentration derived from tumor cells was 1×10⁻⁶. 5 A cell suspension of / mL was cultured at 37°C and 5% CO2 for 12-36 hours to obtain the first cell model with high cell density, wherein the tumor cells formed a monolayer; and / or The final concentration derived from tumor cells was 2 × 10⁻⁶. 4 A cell suspension of / mL was cultured at 37°C and 5% CO2 for 12-36 hours to obtain the first cell model with low cell density, wherein the tumor cells formed a monolayer. Preferably, the culture time is 24 hours.

3. The method according to claim 2, characterized in that, The process of culturing the first cell model under high and / or low osmotic pressure conditions includes: Add the hypotonic culture medium to the first cell model with high cell density and culture at 37°C and 5% CO2 for 12-48 hours; and / or The hypertonic culture medium was added to the first cell model with low cell density and cultured at 37°C and 5% CO2 for 12-48 hours. Preferably, the first cell model is rinsed after 12 hours of culture, and then the hypertonic culture medium and / or the hypotonic culture medium are added again to continue culturing for 48 hours.

4. The method according to claim 3, characterized in that, The in vitro tumor invasion evaluation model includes a high-invasive tumor model and / or a low-invasive tumor model. The highly invasive tumor model was obtained by adding the hypotonic culture medium to the first cell model with high cell density; and / or The low-invasive tumor model is obtained by adding the hypertonic culture medium to the first cell model with low cell density.

5. The method according to claim 4, characterized in that, The intracellular and extracellular pressure difference in the tumor high-invasive model is 1.5 kPa to 1.8 kPa, preferably 1.65 kPa. The intracellular and extracellular pressure difference in the tumor low-invasive model is 0.45 kPa to 0.65 kPa, preferably 0.55 kPa.

6. The method according to claim 1, characterized in that, The hypotonic culture medium consists of a complete culture medium and water in a ratio of 3:

1. The hypertonic culture medium consists of a complete culture medium and a sucrose solution in a ratio of 4:

1.

7. The method according to claim 1, characterized in that, The tumor cells were HeLa cell lines.

8. An in vitro tumor invasion evaluation model, characterized in that, The model is obtained by the method of any one of claims 1 to 7, and comprises: Monoclonal cell populations and cell culture chambers The cell culture chamber includes a growth space and an invasion space, which are connected by a biomembrane. The monoclonal cell population grows in the growth space and can penetrate the biomembrane to enter the invasion space. The cell culture chamber contains a gel matrix, on which the monoclonal cell population grows and hydrolyzes the gel matrix. The monoclonal cell population mentioned above is derived from tumor cells.

9. The in vitro tumor invasion evaluation model according to claim 8, characterized in that, The model is intended for non-diagnostic or non-therapeutic purposes. Optionally, the cell culture chamber contains a hypotonic or hypertonic culture medium. Optionally, the initial density of the monoclonal cell population is either high or low. Optionally, the in vitro tumor invasion assessment model includes a high-invasive tumor model and / or a low-invasive tumor model.

10. A drug screening method, characterized in that, include: The candidate drug was used to treat the in vitro tumor cell invasion evaluation model as described in any one of claims 7 to 9. Optionally, the method includes: The candidate drug is brought into contact with a monoclonal cell population in a growth space; and Compare the changes in the number of cells that cross the biomembrane from the growth space into the invasion space before and after the contact. The reduction in the number of cells in the invasion space indicates that the candidate drug being contacted is a drug that inhibits tumor cell invasion; Optionally, the method includes: The candidate drug is contacted with a monoclonal cell population in a cell culture chamber; and Compare the degree of hydrolysis of the gel matrix in the cell culture chamber before and after the contact. The decrease in the degree of hydrolysis indicates that the candidate drug being contacted is a drug that inhibits tumor cell invasion; Optionally, the method includes: Based on the in vitro tumor invasion evaluation model, which includes a high-invasive tumor model and / or a low-invasive tumor model, the candidate drug is used to treat the high-invasive tumor model. The invasiveness evaluation indicators of the treated high-invasive tumor model and the low-invasive tumor model were compared. Optionally, the invasiveness evaluation indicators include the number of cells in the invasive space, the degree of hydrolysis of the gel matrix, the intracellular and extracellular pressure difference, or other invasiveness indicators. The invasiveness evaluation index of the treated high-invasive tumor model is close to, the same as or better than that of the low-invasive tumor model, indicating that the contacted candidate drug is a drug that inhibits tumor cell invasion by affecting the intracellular and extracellular pressure difference.

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