Construction method of lung cancer cell model with pulse duct, lung cancer drug screening platform and use method thereof

Abstract

The invention provides a construction method of a lung cancer cell model with a pulse duct, the lung cancer cell model can be used for real-time dynamic image analysis of living cells or static image analysis of fixed cells, and the method comprises the following steps: planting a cell suspension between an upper 3D culture medium and a lower 3D culture medium, and hatching to obtain the lung cancer cell model, the upper 3D culture medium and the lower 3D culture medium are both culture media with less growth factors, and the volume of the upper 3D culture medium is larger than that of the lower 3D culture medium; the lung cancer cell model obtained by the method is further used for verifying the smoothness of a 3D-VM vascular structure by using fluorescent microspheres, and is put into establishment of a screening platform for anti-VM drug treatment and application of a screening method.

Description

Technical Field

[0001] The present invention relates to a three-dimensional cell culture technology, in particular to a three-dimensional culture method for simulating tumor formation with cancer cells, and further to an imaging method for verifying cancer cell endothelialization and duct formation; another aspect of the present invention relates to the establishment of a drug screening platform and screening method for a cell model simulating a tumor. Background Art

[0002] The invasiveness of malignant tumors depends on blood supply, which helps the growth of malignant tumors and promotes their metastasis through blood circulation. The theory of angiogenesis, first advocated by Folkman in 1971, has been studied for many years as a physiological mechanism and has made significant contributions to the progress of cancer treatment strategies. Despite the progress, the results of clinical trials of anti-angiogenic drugs, including bevacizumab (VEGF target antibody), are often disappointing. Most patients participating in the trials only experience short-term benefits and then develop drug resistance.

[0003] Vasculogenic mimicry (VM) was first reported in uveal melanoma and refers to the transdifferentiation of invasive tumor cells to endothelial cell characteristics, thereby forming a new vascular network that constitutes an autonomous microcirculatory system independent of benign cells. VM has been observed in many types of cancer and is often associated with poor prognosis. Several tumor cell characteristics have been hypothesized to be the cause of VM formation: tumor cell plasticity and hypoxic microenvironment; acquired characteristics of tumor cells include epithelial-to-mesenchymal transition (EMT) and cancer stemness, both of which may promote angiogenesis; on the other hand, the main signaling pathways involved in VM include VE-cadherin, Notch and hypoxia-inducible factor (HIF), which specifically promote VM generation and cancer cell invasiveness.

[0004] Lung cancer is one of the leading causes of cancer death worldwide. Among all lung cancer patients, up to 85% suffer from non-small cell lung cancer (NSCLS), which usually has a pessimistic prognosis and a 5-year survival rate of less than 15%. In Taiwan, epidermal growth factor receptor

[0005] Mutations in epidermal growth factor receptor (EGFR) are the most common cause of lung adenocarcinoma (LUAD), accounting for about 50%. This subgroup of lung cancer can prolong its survival by treatment with EGFR tyrosine kinase inhibitors (TKIs). However, the biochemical and molecular mechanisms of the complex vascular channels generated by lung cancer cells remain to be further studied. Recent studies have shown that VM can serve as an alternative blood supply channel when angiogenesis is inhibited, making VM a new target for cancer treatment.

[0006] Foslinanib is a novel small molecule anti-VM compound that has demonstrated excellent anti-cancer efficacy in many xenograft tumor models. It suppresses the production of VM in tumor cells by inducing cell cycle arrest and apoptosis.

[0007] In 1999, VM was first reported by Manitotis and sparked controversy. Two different types of VM were reported in the literature: tubular type and patterned matrix type, and the characteristics and corresponding indications of these VM types were also revealed. In determining the presence or absence of VM in vivo, including model animals or patient tumors, periodic acid-Schiff stain is usually used to show dense positive glycoprotein staining, but the results of this method are not widely accepted. In addition, early reports pointed out that monolayer cancer cells would form channels or channel structures on Matrigel, which may facilitate the movement of biological fluids. On the other hand, the intercellular connections of cancer cells cultured in Matrigel can also represent the generation of VM vascular structures. This interpretation has increased the attention of the cancer research community, and now a large number of scientific studies focus on intercellular connections rather than exploring the existence of biological fluid channels. Summary of the invention

[0008] Since the mainstream scientific research community has not paid attention to the verification of tube formation and patency in VM vascular structure, the screening effectiveness of existing cell models of VM vascular structure in anti-cancer drug screening remains to be discussed due to the lack of verification of functional VM vascular structure.

[0009] Therefore, in the present invention, the inventors attempt to propose a breakthrough drug testing platform for evaluating VM behavior and exploring new anti-VM drugs, especially for lung cancer; based on the preparation of a three-dimensional scaffold based on simulated basement membrane components to culture tumor cells, a cell model with a complex 3D-VM vascular structure is constructed, which can be used for real-time dynamic image analysis of living cells or static image analysis of fixed cells; in addition to revealing the complex architecture of the channels formed by the dynamics of tumor cells, the present invention also attempts to verify with a high-throughput microscope that the cell models have a functional 3D-VM vascular structure, and further put the cell model into the screening of anti-VM drug treatment and other applications.

[0010] One object of the present invention is to provide a method for constructing a lung cancer cell model with vascular channels, wherein the lung cancer cell model can be used for real-time dynamic image analysis of living cells or static image analysis of fixed cells, comprising: planting a cell suspension between an upper 3D culture matrix and a lower 3D culture matrix to obtain a pre-cell model, wherein the cell suspension includes lung cancer cells; and incubating the pre-cell membrane for an incubation time to obtain the lung cancer cell model, wherein both the upper 3D culture matrix and the lower 3D culture matrix are culture media with low growth factors, and the volume of the upper 3D culture matrix is ​​greater than the volume of the lower 3D culture matrix.

[0011] As described above, the step of obtaining the pre-cell model includes: solidifying the lower 3D culture pre-matrix of the lower volume on the culture surface for the solidification time to form the lower 3D culture matrix; incubating the cell suspension of the middle volume on the lower 3D culture matrix for the attachment time to allow the lung cancer cells to attach to the lower 3D culture matrix; coating the upper 3D culture pre-matrix of the upper volume on the lung cancer cells to form the upper 3D culture matrix to obtain the pre-cell model, wherein the upper volume is greater than the sum of the lower volume and the middle volume.

[0012] As described above, the cell suspension further comprises microspheres, which are labeled with a first fluorophore and contain negatively charged organic groups; the lung cancer cells are further labeled with a second fluorophore, which has a different emission light from the first fluorophore; the vascular structure comprises a lumen formed by the lung cancer cells and an outer membrane formed by the lung cancer cells surrounding the lumen, wherein the method further comprises: estimating the number of the microspheres located in the vascular structure to evaluate the patency of the vascular structure, wherein a first number of the microspheres in the lumen is estimated, and a second number of the microspheres on the outer membrane or outside the outer membrane is estimated, when the first number is greater than or equal to the second number, the vascular structure is judged to be a vascular structure with good patency, and when the first number is less than the second number, the vascular structure is judged to be a vascular structure with poor patency.

[0013] Another object of the present invention is to provide a lung cancer drug screening platform, which includes a lung cancer cell model prepared by the method described above.

[0014] Another object of the present invention is to provide a method for screening lung cancer drugs, comprising: providing a lung cancer cell model to be tested, which has a vascular structure with a first vascular density, wherein the cancer cell model to be tested is prepared as described above; co-culturing the lung cancer cell model and the anticancer drug for a screening time to obtain a lung cancer cell model to be tested, which has a vascular structure with a second vascular density; and evaluating the relationship between the first vascular density and the second vascular density to determine whether the anticancer drug has a therapeutic effect, wherein when the ratio of the first vascular density to the second vascular density is greater than 1, the anticancer drug is determined to have a therapeutic effect, and when the ratio of the first vascular density to the second vascular density is equal to or less than 1, the anticancer drug is determined to have no therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A The figure is a cross-sectional view to illustrate the layered structure of the lung cancer cell model;

[0016] Figure 1B is a flow chart for illustrating a first embodiment of a method for constructing a lung cancer cell model;

[0017] Figures 1C to 1F The flowchart and three-dimensional schematic diagram are used to explain in detail the construction method of the pre-cell model;

[0018] Figure 2A is a flow chart for illustrating a second embodiment of the method for constructing a lung cancer cell model;

[0019] Figure 2B The cross-sectional view is used to illustrate the specific structure of the vascular structure;

[0020] Figure 2C The cartoon diagram is used to illustrate the process of stereoscopic imaging of vascular structure under a fluorescence microscope;

[0021] Figure 2D A flow chart is used to illustrate the steps of the patency assessment process;

[0022] Figure 2E It is a cross-sectional diagram used to illustrate the criteria for judging the patency of the vascular structure;

[0023] Figure 3 The flowchart is used to illustrate the screening method of lung cancer drugs;

[0024] Figure 4 The cell morphology of stable cell lines under fluorescence microscope before and after FACS screening is presented;

[0025] Figure 5A The cartoon diagram schematically illustrates the observation angle of the network and cavity of the VM in Example 1;

[0026] Figure 5B Bright field images of optical microscope are used to show the generation of VM network structure of lung cancer cell lines. The images were taken before or after PAS staining.

[0027] Figure 5C Fluorescence microscopy images are superimposed to show the vascular structure of the cell model established by the stable cell line;

[0028] Figure 5D Comparison of vascular topological characteristics of cell models established by real-time dynamic fluorescence microscopy of living cells using EGF-transfected non-small cell lung cancer cell lines;

[0029] Figure 6 Overlay of real-time dynamic fluorescence microscopy images of living cells to show the VM vascular structures generated by H1299 and A549 cells under normal oxygen and hypoxic conditions;

[0030] Fig. 7A Overlay of real-time dynamic fluorescence microscopy images of living cells to show the localization of carboxyl-modified microspheres and amine-modified microspheres in the 3D-VM vascular structure;

[0031] Figures 7B to 7C Fluorescence microscopy images show the VM channel (xz) and VM channel (yz) cross sections of carboxyl-modified microspheres in the HCC827 lung cancer cell model;

[0032] Fig. 8A Live cell real-time dynamic fluorescence microscopy images show the changes in 3D-VM vascular structure of H1650 and HCC827 lung cancer cell models after treatment with different concentrations of Foslinanib;

[0033] Figure 8B The histogram and box-whisker plots are used to illustrate the quantitative analysis of the cell activity and 3D-VM vascular structure changes of H1650 and HCC827 lung cancer cell models after treatment with different concentrations of Foslinanib;

[0034] Fig.9A is a box-and-whisker plot to illustrate the difference in VM ssGSEA scores between non-small cell lung cancer and small cell lung cancer;

[0035] Fig. 9B is a box-and-whisker plot to illustrate the difference in VM ssGSEA scores between wild-type EGFR and mutant EGFR in lung adenocarcinoma cases;

[0036] Fig. 9CThe live cell real-time dynamic fluorescence microscopy image overlay and cross-section images are used to present the VM vascular structure of H1299-EGFRwt and H1299-EGFRmt;

[0037] Fig.9D Box-and-whisker plots are used to quantify the differences in the effective cavity structures of 3D-VMs of H1299-EGFRwt and H1299-EGFRmt;

[0038] Fig. 10A Fluorescence microscopy images and histograms are used to present the changes in the cell activity and 2D-VM network structure state of H1299-EGFRmt, H1299-EGFRwt and H1299 after Foslinanib treatment;

[0039] Fig. 10B The real-time dynamic fluorescence microscopy images of living cells are overlaid to show the effects of Foslinanib and Osimertinib alone and in combination on the generation of 3D-VM vascular structures.

[0040] Fig. 10C The histogram is used to quantitatively illustrate the effects of Foslinanib and Osimertinib alone and in combination on the generation of 3D-VM vascular structure;

[0041] Fig.11A Immunohistochemical staining images showing the formation of vascular structures and the expression of LAMC2 in EGFRmt human tumor tissues; and

[0042] Fig. 11B The bar graphs and histograms were used to quantitatively analyze the changes in the total expression of EGFR and LAMC2 and the degree of EGFR phosphorylation in H1299 cells treated with Foslinanib or Osimertinib alone or in combination. DETAILED DESCRIPTION

[0043] The first embodiment of the present invention provides a method for constructing a lung cancer cell model 100 with a vascular channel, wherein the lung cancer cell model 100 can provide real-time dynamic image analysis of living cells or static image analysis of fixed cells, see Figures 1A to 1B , the method comprising:

[0044] Pre-cell model acquisition step S1: planting a cell suspension 1 between the upper 3D culture matrix 2a and the lower 3D culture matrix 2b to obtain a pre-cell model 10, wherein the cell suspension 1 includes lung cancer cells; and

[0045] Incubation step S2: Incubate the pre-cell membrane type 100 for an incubation time to obtain the lung cancer cell model 100, wherein both the upper 3D culture matrix 2a and the lower 3D culture matrix 2b are culture media with less growth factors, and the volume of the upper 3D culture matrix 2a is greater than that of the lower 3D culture matrix 2b.

[0046] In many embodiments, in order to enable lung cancer cells to migrate, transfer and transdifferentiate in three-dimensional space to form a three-dimensional vascular structure, it can be understood that the upper 3D culture matrix 2a and the lower 3D culture matrix 2b are three-dimensional tissue scaffolds with porous properties, and in order to simulate the migration, transfer and transdifferentiation of cancer cells in normal tissues in vitro (in vivo), specifically simulate the three-dimensional form of the extracellular matrix, which can be a porous layer in the form of a gel, wherein the extracellular matrix can be listed as an interstitial matrix, a pericellular matrix, a cartilage matrix, a bone matrix, a tendon and ligament matrix, a dental matrix or a basement membrane according to the tissue morphology.

[0047] In terms of specific implementation, Figures 1C to 1F As shown, the pre-cell model 10 acquisition step S1 includes:

[0048] Matrix solidification treatment S1a: solidifying the lower 3D culture pre-matrix 2b' of the lower volume on the culture surface P for a solidification time to form the lower 3D culture matrix 2b;

[0049] Cell attachment treatment S1b: incubating the middle volume of the cell suspension 1 on the lower 3D culture matrix 2b for the attachment time, so that the lung cancer cells 1a attach to the lower 3D culture matrix 2;

[0050] Cell covering treatment S1c: coating the upper volume of the upper 3D culture pre-matrix 2a' on the lung cancer cells 1a to form the upper 3D culture matrix 2a, and obtaining the pre-cell model 10, wherein the upper volume is greater than the sum of the lower volume and the middle volume.

[0051] Please continue reading Figures 1E to 1FIt can be understood that the cell suspension 1 is a mixed liquid formed by fully breaking up the lung cancer cells 1a with a suspension 1b. The suspension 1b can be a physiological buffer solution with isotonic properties used for cell culture, such as DPBS, EBSS, HBSS, HEPES or PBS. In order to maintain the activity of the lung cancer cells 1a, the suspension 1b can also be a cell culture solution to which essential nutrients for the cells are added, such as DMEM, RPMI-1640, F12K or EMEM. In order to prevent the lung cancer cells from degenerating or transdifferentiating, other necessary components including fetal bovine serum, growth factors, etc. can also be added to the suspension 1b. In this embodiment, the suspension 1b is the cell culture medium required for the lung cancer cells 1a, preferably RPMI-1640.

[0052] In multiple embodiments, in order to simulate the gradual transdifferentiation of lung cancer cells during three-dimensional culture and acquire the cellular characteristics of epithelial cells and endothelial cells, trigger angiogenesis or vascular mimicry in a three-dimensional scaffold, and gradually develop observable vascular structures during the incubation time, the upper 3D culture pre-matrix 2a' and the lower 3D culture matrix 2b' specifically simulate the basement membrane.

[0053] In these embodiments, the upper 3D culture pre-matrix 2a' is prepared by mixing a first upper volume of an upper cell-like basement membrane material and a second upper volume of a cell culture medium, wherein the first upper volume is the same as or smaller than the second upper volume, and the sum of the first upper volume and the second upper volume is the same as the upper volume; the lower 3D culture pre-matrix 2b' includes a lower cell-like basement membrane material, and the upper cell-like basement membrane material is the same as or different from the lower cell-like basement membrane material; preferably, the upper cell-like basement membrane material is the same as the lower cell-like basement membrane material.

[0054] Specifically, the present embodiment utilizes the volume of the upper 3D culture pre-matrix 2a' to be larger than the volume of the lower 3D culture pre-matrix 2b', thereby providing the lung cancer cells 1a with a larger migration and growth space in the upper 3D culture pre-matrix 2a'; in addition, since the upper 3D culture pre-matrix 2a' and the cell culture medium can be mixed in equal volumes at a ratio of 1:1, or mixed in a smaller volume with the cell culture medium, for example, 1:(1.1 to 3), the concentration of the upper cell basement membrane-like substance contained therein is lower than the concentration of the lower cell basement membrane-like substance contained in the lower 3D culture pre-matrix 2b', thereby forming a loose basement membrane-like three-dimensional structure during the subsequent incubation process and generating a gradient difference.

[0055] During the migration and diffusion of cancer cells in vivo, in order to create an environment conducive to the growth and transdifferentiation of cancer cells, extracellular matrix remodeling (ECM remodeling) occurs, which involves the degradation and remanufacturing of the extracellular matrix; accordingly, by utilizing the concentration gradient difference of the cell-like basement membrane material between the upper 3D culture pre-matrix 2a' and the lower 3D culture pre-matrix 2b', a directional basement membrane remodeling microenvironment can be simulated, wherein the concentration change decreases gradually from the lower 3D culture pre-matrix 2b' toward the upper 3D culture pre-matrix 2a', thereby inducing lung cancer cells 1a to migrate toward the upper 3D culture pre-matrix 2a' with a looser structure and transdifferentiate into pseudo-epithelial cells or pseudo-endothelial cells, and form a vascular structure 3.

[0056] In these embodiments, the upper cell-like basement membrane material is a component of the basement membrane, which can be listed as fibronectin, collagen, laminin, vitronectin, polylysine, sulfated proteoglycan, entactin or any combination of two or more thereof; similarly, the lower cell-like basement membrane material is also a component of the basement membrane, which can be specifically listed as fibronectin, collagen, laminin, vitronectin, polylysine, sulfated proteoglycan, entactin or any combination of two or more thereof; in one example, Matrigel is used as the upper 3D culture pre-matrix 2a' and the lower 3D culture pre-matrix 2b', which is rich in laminin and type IV collagen.

[0057] In some specific embodiments, the ratio of the first upper volume, the second upper volume, the middle volume and the lower volume is 1:(1.00 to 3.00):(0.5 to 2.00):(0.3 to 1.00), preferably 1:(1.00 to 1.50):(0.8 to 1.5):(0.4 to 0.8), and more preferably 1:1:1:(0.6 to 0.7).

[0058] In these embodiments, the coating density of the upper 3D culture pre-matrix 2a' is 130 to 350 μL / cm 2 , preferably 135 to 180 μL / cm 2 The coating density of the lower 3D culture pre-matrix 2b' is 50 to 150 μL / cm 2 , preferably 80 to 120 μL / cm 2 , and the ratio of the coating density of the upper 3D culture pre-matrix 2a' to the coating density of the lower 3D culture pre-matrix 2b' is (0.80 to 2.60):1, preferably (1.00 to 1.50):1; the lung cancer cells 1a are cultured at a cell density of 4.0 x 10 4 Up to 7.0x10 4cells / cm 2 Planted on the lower 3D culture matrix 2b, preferably 5.0 x 10 4 Up to 6.0x 10 4 cells / cm 2 .

[0059] In one example, taking Trulive3D dish (Bruker) as an example, 50 to 150 μL of the lower 3D culture pre-matrix 2b' can be added to the culture well and wait for it to solidify into the lower 3D culture matrix 2b, preferably 100 μL or 150 μL; then, 100 to 300 μL of the cell suspension 1 is planted, and lung cancer cells 1a are attached, preferably 100 μL, 150 μL, 200 μL or 250 μL; 150 to 300 μL of cell culture medium is mixed with 150 μL of L to 450 μL of upper cell-like basement membrane material is used to prepare the upper 3D culture pre-matrix 2a', preferably 150 μL, 200 μL, 250 μL or 300 μL of cell culture medium, mixed with 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL or 450 μL of the upper cell-like basement membrane material; the upper 3D culture pre-matrix 2a' is coated on the attached lung cancer cells 1a to complete the pre-cell model 10.

[0060] In many embodiments, the lung cancer cells are derived from lung cancer tissue or lung cancer cell lines, wherein the lung cancer tissue can be obtained from frozen or fresh lung cancer tumors, which can be small cell lung carcinoma, large cell lung carcinoma, squamous cell carcinoma or lung adenocarcinoma; on the other hand, the type of the lung cancer cell line is not particularly limited, but only needs to be derived from the above types of lung cancer and established by immortalization treatment. In some preferred examples, the lung cancer cell lines include A549, HCC827, H460, H1299, H1650 or H1975.

[0061] In these embodiments, the incubation time can be determined according to the type of lung cancer cells, specifically within the range of 4 to 72 hours, preferably 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 24 to 36 hours, 36 to 48 hours, 48 ​​to 60 hours or 60 to 72 hours, more preferably 4 to 6 hours, 12 to 24 hours or 36 to 48; the solidification time can be adjusted with the type of the lower cell-like basement membrane material, specifically within the range of 5 to 60 minutes, preferably 10 to 40 minutes, more preferably 15 to 30 minutes; the cell attachment time also depends on the type of lung cancer cells, specifically within the range of 5 to 60 minutes, preferably 10 to 40 minutes, more preferably 15 to 30 minutes, wherein the cell attachment time is equal to the solidification time.

[0062] In these embodiments, the incubation treatment S2, the matrix solidification treatment S1a, and the cell attachment treatment S1b can be performed at room temperature or physiological temperature, specifically between 25 and 37°C; on the other hand, it can be understood that in order to simulate the physiological environment of cells and maintain the buffering capacity of the cell culture medium, the incubation treatment S2 and the cell attachment treatment S1b are both performed in an atmosphere of 5% CO2.

[0063] See also Figure 2A The second embodiment of the present invention provides a method for constructing a lung cancer cell model 100 with blood vessels. The method steps, cell types, materials, and condition parameters involved are substantially the same as those of the first embodiment, but further includes a blood vessel patency assessment process S3 to further verify whether the blood vessels of the lung cancer cell model 100 have a channel function, that is, a pipe structure with the function of assisting lung cancer cells in nutrient transport, exchange, and elimination of metabolites, specifically a vascular mimicry structure.

[0064] In this embodiment, the vessel patency assessment process S3 is performed based on live cell imaging, in particular, cell imaging is performed using a fluorescent microscope to observe and assess the cell morphology and vessel structure morphology in real time; see Figure 2B The vascular structure 3 includes a cavity 3a surrounded by the lung cancer cells 1a and an outer membrane 3b formed by the lung cancer cells 1a surrounding the cavity. In order to specifically distinguish the lung cancer cells 1a that form the outer membrane of the duct from the cavity 3a, based on the principle of high contrast, the lung cancer cells 1a are further marked with a first fluorescent group, which can be GFP, RFP, YFP or other fluorescent groups, without special restrictions.

[0065] It can be understood that the vascular structure 3 is a 3D structure. In this embodiment, the 3D imaging of the vascular structure 3 is used as the analysis object. A specific example is used to further illustrate. Figure 2C As shown, the three-dimensional imaging of the vascular structure 3 can be observed from the xz section (i surface) and the yz section (ii surface), wherein the portion indicated by the white dotted line frame is a cavity structure without cells, that is, the cavity 3a portion, and the fluorescent imaging portion surrounding the cavity 3a portion is the outer membrane 3b portion formed by the cells.

[0066] In some embodiments, in order to observe in real-time images that the cavity 3a does have the function of circulating biological fluid, it is necessary to further make the cavity 3a show the biological fluid flow under the field of view of a fluorescence microscope; accordingly, in order to mark the biological fluid flow, in the cell attachment process S1b, microspheres 4 are further added to the cell suspension 2, wherein the microspheres 4 are marked with a second fluorescent group, the emission light of which is different from the emission light of the first fluorescent group, and specifically can also be GFP, RFP, YFP or other fluorescent groups, without special limitation.

[0067] In these embodiments, please refer to Figures 2D to 2E , the patency assessment process S3 includes:

[0068] Real-time image capturing step S3a: capturing a real-time image of the vascular structure of the lung cancer cell model 100, wherein, on the real-time image, the microsphere 4 is imaged with a first color corresponding to the first fluorescent group, and the vascular structure 3 is imaged with a second color corresponding to the second fluorescent group; and

[0069] Microsphere positioning step S3b: positioning the microsphere 4 on the real-time image based on the relative positions of the first color and the second color;

[0070] Microsphere quantity estimation step S3c: estimate the quantity of the microspheres 4 in the vascular structure to evaluate the patency of the vascular structure 3, wherein a first quantity of the microspheres in the cavity 3a is estimated, and a second quantity of the microspheres 4 on the outer membrane 3b or outside the outer membrane 3b is estimated; when the first quantity is greater than or equal to the second quantity, the vascular structure 3 is judged to be a vascular structure 30 with good patency; and when the first quantity is less than the second quantity, the vascular structure 3 is judged to be a vascular structure 31 with poor patency.

[0071] As a matter of course, during the formation of the vascular structure 3, due to the migration and transdifferentiation of the lung cancer cells 1a, the relative distribution position of the microspheres 4 also continues to change. When the lung cancer cells 1a form the outer membrane 3b, the inner side of the cavity 3a surrounded by the lung cancer cells 1a will be covered with microspheres 4. If the biological fluid flows in the cavity 3a, the microspheres 4 will appear to be displaced in the cavity 3a in the real-time image along the flow direction of the biological fluid, but will not cross the boundary defined by the outer membrane 3b. Secondly, during the incubation process S2, in addition to the microspheres 4 covered in the cavity 3a due to the formation of the outer membrane 3b by the lung cancer cells 1a, since the lung cancer cells 1a are The place for biomass exchange between the cavity 3a and the outer membrane 3b is that, based on the hydrophilicity and electrical properties of the microspheres 4, some of the microspheres 4 on the outer membrane 3b will enter the cavity 3a through the outer membrane 3b, so the number of microspheres 4 in the cavity 3a should be greater than the number of microspheres 4 on the outer membrane 3b. Furthermore, if a large number of microspheres 4 are attached to the outer membrane 3b, it shows that although the lung cancer cell model 100 forms a vascular structure similar to VM, its biomass exchange efficiency is low or even no biomass exchange behavior across the outer membrane 3b occurs, or the observed hollow area similar to the cavity 3a is actually a cell gap, without a connected vascular structure, and is non-functional.

[0072] In some embodiments, in order to allow the microspheres 4 to pass through the cell membrane and participate in the biomass exchange across the outer membrane 3b, the microspheres 4 further include negatively charged organic groups, wherein the negatively charged organic groups can be exemplified by carboxyl groups (-COO - ), nitro (-NO2), sulfoxide (-SO - ), cyano (-CN - ) or sulfo group (-SO2), preferably carboxyl group (-COO - ).

[0073] Generally speaking, the vascular structure formed by VM can be specifically divided into microvascular channels or macrovascular channels, and their channel diameters are approximately 5 to 10 microns and 15 to 30 microns, respectively. The VM generated during the migration of cancer cells belongs to a dynamic vascular structure, and its channel diameter will change with the progression of the tumor, but it does not deviate from the range of the aforementioned channel diameter. Accordingly, in order to improve the reliability of the microsphere 4 entering the cavity 3a, if the size is too large, it is difficult to enter the cavity 3a, and if the size is too small, it may only enter the cell gap, resulting in misjudgment. Therefore, in these embodiments, the diameter of the microsphere 4 is selected to be 0.3 to 2.0 microns, preferably 0.5 to 1.0 microns.

[0074] The third embodiment of the present invention provides a method for constructing a lung cancer cell model 100 with vascular channels, and the specific method steps, cell types, materials, and condition parameters involved are substantially the same as those of the first embodiment or the second embodiment, except that the pre-cell membrane model 10 is incubated in a normal oxygen environment (normoxia), a physiological oxygen environment (physioxia), or an oxygen-deficient environment (hypoxia), wherein the atmospheric conditions of the normal oxygen environment are 5% CO2 and 18 to 22% O2 by mass, the atmospheric conditions of the physiological oxygen environment are 5% CO2 and 3.0 to 7.4% O2 by mass, and the atmospheric conditions of the oxygen-deficient environment are 5% CO2 and 0.01 to 2.0% O2 by mass; preferably, the pre-cell model 10 is incubated in an oxygen-deficient environment, which is closer to the tumor tissue in vivo, and enables the lung cancer cell model 100 to dynamically generate a VM vascular structure.

[0075] The fourth embodiment of the present invention provides a method for constructing a lung cancer cell model 100 with a vascular channel, and the specific method steps, cell types, materials, and condition parameters involved are generally the same as any one of the first to third embodiments, except that the lung cancer cell 1a includes: a first lung cancer cell that expresses a wild-type EGFR reaching a first baseline amount; or a second lung cancer cell that expresses a mutant EGFR reaching a second baseline amount.

[0076] The same as the first embodiment is that the first lung cancer cell or the second lung cancer cell is derived from a primitive cell (primitive cell) of lung cancer tissue or a lung cancer cell line, wherein the lung cancer tissue can be taken from a frozen or fresh lung cancer tumor, such as small cell lung carcinoma, large cell lung carcinoma, squamous cell carcinoma or lung adenocarcinoma; the lung cancer cell line can be an immortalized cell line derived from the above-mentioned types of lung cancer, such as A549, HCC827, H460, H1299, H1650 or H1975.

[0077] In some embodiments, the mutant EGFR expressed by the second lung cancer cell may be a mutant EGFR with amino acid residue substitution; taking non-small cell carcinoma as an example, the second lung cancer cell may express EGFR (p.Pro733Leu), EGFR (p.Pro753Ser), EGFR (p.Gly719Ala), EGFR (p.Glu734Lys), EGFR (p.Val742Ala), EGFR (p.Glu746Lys), EGFR (p.Ser752Tyr), EGFR (p.Ser768Val), EGFR (p.Ser768Ile), EGFR (p.Ser768Ile), EGFR (p.His773Arg), EGFR (p.Val834Leu), EGFR (p.Leu858Arg), g), EGFR(p.Leu858Arg), EGFR(p.Leu858Gln) or EGFR(p.Gly735Ser); taking squamous cell carcinoma as an example, the second lung cancer cell may express EGFR(p.Gly719Asp), EGFR(p.Leu861Gln), EGFR(p.Gly719Arg) or EGFR(p.Leu861Pro); taking lung adenocarcinoma as an example, the second lung cancer cell may express EGFR(p.Gly719Asp), EGFR(p.Ala237Phe), EGFR(p.His773Val), EGFR(p.Leu833Phe), EGFR(p.Lys852Thr), EGFR(p.Leu861Gln) or EGFR(p.Gly719Cys).

[0078] In some embodiments, the lung cancer cell 1a is derived from a lung cancer cell line, and an expression vector with a regulatory factor can be used to deliver exogenous genetic material encoding wild-type EGFR and mutant EGFR to the lung cancer cell 1a for overexpression; the first lung cancer cell further carries a first exogenous genetic material to express wild-type EGFR to a first expression level, wherein the first expression level is greater than the first reference level; or the second lung cancer cell further carries a second exogenous genetic material to express wild-type EGFR to a second expression level, wherein the second expression level is greater than the second reference level; in addition, it can be understood that in order to distinguish the overexpressed wild-type EGFR or mutant EGFR, the expression vector can be further configured with an appropriate tag sequence, such as a protein HA tag, a FLAG tag or a HIS tag, or a fluorescent protein tag such as GFP, YFP or RFP.

[0079] In these embodiments, the first lung cancer cell further expresses LAMC2 to a third expression level; and the second lung cancer cell further expresses LAMC2 to a fourth expression level, wherein the fourth expression level is greater than the third expression level, wherein upregulation of laminin subunit gamma-2 (LAMC2) in lung adenocarcinoma (LUAD) has been considered to be associated with tumor invasiveness, metastatic potential and poor prognosis; as a member of the extracellular matrix glycoprotein, it is a very important part of the epithelial basement membrane; LAMC2 is further involved in the regulatory mechanism of EGFR protein expression, and changes in its expression level may also indirectly regulate the generation of 3D-VM vascular structure and cell invasiveness of EGFRmt lung adenocarcinoma.

[0080] The fifth embodiment of the present invention provides a lung cancer drug screening platform, which includes a lung cancer cell model 100, which is prepared by the method described in any one of the first to fourth embodiments.

[0081] The sixth embodiment of the present invention provides a method for screening lung cancer drugs. Figure 3 , which includes:

[0082] Cell membrane type construction step A1: providing a lung cancer cell model 100 to be tested, which has a vascular structure with a first vascular density, wherein the cancer cell model 100 to be tested is prepared by the method described in any one of the first to fourth embodiments;

[0083] Drug co-culture step A2: co-culturing the lung cancer cell model 100 and the anticancer drug for a screening period to obtain a tested lung cancer cell model 100 having a vascular structure with a second vascular density; and

[0084] Efficacy evaluation step A3: Evaluate the relationship between the first vascular density and the second vascular density to determine whether the anticancer drug has efficacy, wherein when the ratio of the first vascular density to the second vascular density is greater than 1, the anticancer drug is determined to be effective, and when the ratio of the first vascular density to the second vascular density is equal to or less than 1, the anticancer drug is determined to be ineffective.

[0085] In this embodiment, the first vascular density and the second vascular density refer to the channel density parameters obtained by cell image analysis of 3D-VM vascular structure, specifically including cavity area, total channel length or total channel area; in order to facilitate the comparison of changes in vascular structure density before and after drug administration, the first vascular density can cover the first cavity area, the first total channel length or the first total channel area, and the second vascular density can cover the second cavity area, the second total channel length or the second total channel area.

[0086] In terms of specific implementation, the quantitative analysis of the lumen area of ​​the 3D-VM vascular structure is based on the calculation of the ROI average value (ROI) of at least three different areas of interest (AOI) on a single frame of real-time image, wherein the ROI represents the vascular tube or vascular lumen circled in each cell model to be tested; after circling the AOI, the ROI is delineated on the real image for quantification, and the ROI average value here is obtained by adding up the ROIs delineated on multiple AOIs and averaging them to reflect the average area of ​​the typical size of each cell model; in addition, the ROI average value is further divided by the average total channel area for standardization, so that the anticancer drug effects between test platforms of different drugs, experimental parameters or cell lines can be standardized for comparison, so as to truly present the destructive effect of anticancer drugs on 3D-VM; in some examples, the quantitative analysis can be performed using the angiogenesis analyzer of ImageJ.

[0087] In this embodiment, the anticancer drug used for testing can be EGFR tyrosine kinase inhibitors (TKIs), cell cycle blocking agents or apoptosis promoting agents, such as Foslinanib, Osimertinib, Palbociclib, Dinaciclib, Erlotinib, Ibrutinib, Imatinib, Venetoclax, Vorinostat, Bortezomib, Sorafenib, Gemcitabine or a combination of two or more thereof; preferably, the anticancer drug includes Foslinanib, Osimertinib or a combination thereof.

[0088] 1. Materials and Methods

[0089] 1.1 Cell culture and reagents

[0090] Cell lines A549, H1299, B16F10, HCC827, and H1650 were purchased from the American Type Culture Collection (ATCC); the H1299-EGFR-L858R mutant cell line was established by Professor Chien-Chung Hung of the Department of Biotechnology and Industrial Science at National Cheng Kung University; H1299, H1299-EGFR-wild type (hereinafter referred to as H1299-EGFRwt), H1299-EGFR-L858R (hereinafter referred to as H1299-EGFRmt), HCC827, and H1650 cell lines were cultured in RPMI-1640 medium, while the B16F10 cell line was cultured in DMEM medium, and the A549 cell line was cultured in F12K medium; all culture media were supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic drugs and cultured at 37°C and 5% CO2.

[0091] 1.2 Matrigel pipeline generation experiment

[0092] Matrigel was used to establish a vascular mimicry (VM) model to evaluate the ability of cancer cells to generate VM in vitro. A layer of 100 μL of growth factor-reduced Matrigel (Corning) was added to a chamber slide (TruLive3D Dishes, Burker), and then the Matrigel was solidified at 37°C for 30 minutes. After the Matrigel solidified, 150 μL of 1.25×10 5 A cell suspension of 10 cells was inoculated on the surface of Matrigel colloid, and then the cell-Matrigel colloid was placed in a CO2 incubator at 37°C for 30 minutes to allow the cells to adhere to the Matrigel surface; after the above-mentioned initial culture stage, the cell culture medium was mixed with an equal volume of 1-fold diluted Matrigel, and then added to the cell-Matrigel colloid to obtain a pre-cell model 10, and then the pre-cell membrane type 10 was continued to be cultured at 37°C for 5 to 24 hours to obtain a lung cancer cell model 100 for subsequent observation of the formation of 3D-VM vascular structure.

[0093] 1.3 3D hypoxic VM dynamic model

[0094] After the cell lines to be tested were planted in the culture chamber according to the method described in 1.2 above to obtain the pre-cell model 10, the culture chamber was arranged along a 10 cm culture dish filled with sterile water to ensure that the culture chamber could maintain appropriate humidity in the subsequent hypoxic environment; then, a mixed gas of 5% CO2, 1% O2 and 94% N2 was filled in the culture chamber and balanced for 5 minutes to create an hypoxic environment; after the hypoxic gasification stage was completed, the valve on the culture chamber was sealed to terminate the airflow exchange and maintain the hypoxic condition; after the hypoxic environment was formed in the culture chamber, it was transferred to a 37°C incubator, and the cancer cell lines were induced to grow in the hypoxic environment for 24, 48, and 72 hours, respectively, to further observe the 3D-VM vascular structure; the image data collected subsequently were analyzed by the Imaris system to comprehensively analyze the morphological changes of cancer cells and the 3D-VM vascular structure induced by hypoxic pressure.

[0095] 1.4 Pre-cell model - mammosphere 3D co-culture

[0096] First, take 10 μL of 1X Matrigel and evenly distribute it on the bottom of each culture plate, and then leave the culture plates at 37°C for 10 minutes to allow the Matrigel to solidify and form a uniform gel layer, which serves as a three-dimensional scaffold for cell attachment and growth. After the preparation of the Matrigel layer, place a 4x10 4 The cell suspension of each cell was mixed with carboxylate-modified microspheres (F8821, Invitrogen; 1 μm) or amine-modified microspheres (F8763, Invitrogen; 0.2 μm) at a dilution ratio of 1:1000; the cell suspension was then coated on the solidified Matrigel layer; in order to ensure the best conditions for cell attachment to the Matrigel surface, the cell culture plates were returned to the 37°C incubator for 15 minutes; after the completion of cell seeding, the cell nuclei were stained to assist in subsequent imaging and analysis; the cell nucleus staining was performed by diluting the cell nucleus-specific dye with 1X Matrigel to achieve an appropriate staining concentration and then administering it to the co-culture system; the cell culture plates were cultured for 24 hours to ensure that the dye was completely bound to the cell nucleus to assist in observing the interactive relationship between the lung cancer cell model and the microspheres in the 3D microenvironment.

[0097] 1.5 PAS staining of 3D cell culture

[0098] The cultured lung cancer cell model 100 was fixed in 4% formalin for 20 minutes, and then rinsed with PBS and 1xTBS for 15 minutes respectively; then, the lung cancer cell model 100 was immersed in 0.5% periodic acid for 3 minutes, rinsed with distilled water for 3 times, and treated in Schiff buffer for 10 minutes; after rinsing with distilled water, the lung cancer cells 1a were stained with hematoxylin for 5 minutes, and the aforementioned rinsing steps were repeated; finally, the stained lung cancer cell model 100 was observed with an optical microscope (Olympus BX50).

[0099] 1.6CCLE translational analysis

[0100] VM metagene signature was taken from the literature reported by Velez DO et al (Nat Commun 8, 1651, 2017); gene expression data of melanoma and lung cancer cells were downloaded from Cancer Cell Line Encyclopedia (CCLE) (data source: https: / / data.broadinstitute.org / ccle / CCLE_ DepMap_18Q2_RNAseq_RPKM_20180502.gct .); Population data and cell line mutation status were obtained from cBioPortal ( https: / / www.cbioportal.org / ); VM The gene activity score was calculated using the ssGSEA module (Single-Sample Gene Set Enrichment Analysis module) provided by GenePattern, and the ssGSEA scores were z-transformed. The box-whisker plots and the comparison of the mean values ​​of each group were calculated using the ggpubr component of the R software. The bioinformatics analysis was performed using the R programming language (version 4.2.1).

[0101] 1.7 Wild-type EGFR plasmid transfection

[0102] The lung cancer cell line H1299 was cultured at 37°C and 5% CO2 to 70 to 80% confluency to ensure optimal cell growth efficiency and activity; the plasmid DNA carrying wild-type EGFR and the empty vector pcDNA3.1(-) were prepared in serum-free culture medium and then mixed with an appropriate dose of Lipofectamine 3000 or a transfection reagent with equivalent effect to assist the transfection vectors to enter the cells; the diluted wild-type EGFR plasmid was mixed with the reagent and allowed to stand at room temperature for 15 to 20 minutes, then added to the H1299 cells and cultured in a 37°C, CO2 incubator for 24 hours; after stable transfection, G418 (500 μg / mL) was added to the culture medium, and the culture medium containing G418 was replaced every 2 to 3 days according to the cell growth rate; after the transfected cells were screened, the stable cell lines were continuously cultured in fresh complete medium for subsequent observation of EGFR expression; the transfection effect and the effect of EGFR expression were analyzed by various analytical methods including Western Blot. The cells were evaluated by blotting, qRT-PCR and immunofluorescence staining. In addition, changes in cell behaviors such as cell proliferation, migration and invasion were also further evaluated.

[0103] 1.8 Fluorescence activated cell screening (FACS) to isolate pEGFP-N1+ cells

[0104] After the above transfected cells were treated with trypsin, they were rinsed with PBS three times and suspended in PBS to maintain their activity and avoid cell aggregation. Then, the FACS screening system was used to further identify and separate the cell population expressing enhanced green fluorescent protein (EGFP); see Figure 4 , specifically presenting the morphology of the fluorescently-transfected stable cell lines B16F10, A549, H1299, H1299-EGFR-wild type, H1299-EGFR-L858R, HCC827, and H1650 under a fluorescence microscope before and after screening; after screening, these pEGFP-N1+ cells were preserved in appropriate culture medium for subsequent amplification culture or other experiments.

[0105] 1.9 Cell proliferation analysis

[0106] The cell viability was assessed by MTT assay, with 4 × 10 3The cells to be tested were seeded in a 96-well plate at a density of 10 cells / well. Then, the cells were exposed to different concentrations of anti-VM test drugs for multiple time points (0, 24, 48, and 72 hours). After treatment with the anti-VM test drugs, MTT reagent was added to each culture well and cultured at 37°C for 2 hours. Next, the culture plate was centrifuged at 1500 rpm for 10 minutes, and the cell supernatant was subsequently removed. Formazan crystals were dissolved in DMSO and added to each culture well, and the light absorbance of each culture well at a wavelength of 492 nm was measured using an ELISA reader as an indicator of cell activity.

[0107] 1.10 Analysis of 2D-VM network structure inhibition using high-content imaging

[0108] The cells to be tested were seeded in a 96-well plate and allowed to attach overnight under standard culture conditions. After cell attachment, they were treated with different concentrations of Foslinanib (ABEx BIO-Research Center) and monitored at different time points to record changes in the VM vascular structure for subsequent analysis. Furthermore, the disorder of the 2D-VM network structure was evaluated by high-resolution imaging of the 2D-VM network structure produced by the ImageXpress micro-conjugate focus high-content imaging system. The image data obtained were analyzed using MetaXpress software to quantitatively analyze the inhibitory effect of the test drug on the 2D-VM network structure.

[0109] 1.11 Quantitative analysis of the lumen of 3D-VM vascular structures

[0110] The quantitative analysis of the formation of the tube or cavity of the 3D-VM vascular structure is based on at least 8 different AOIs, of which 3 AOIs are designated as representative images; the ROI represents the tube or cavity defined in each cell to be tested; first, the AOI is identified and quantified; then, the ROI average is calculated by summing the ROIs of the 8 AOIs (area 1+area 2+…+area 8) and dividing by 8, which can reflect the average area of ​​the typical size of the analysis area in each lung cancer cell model 100; finally, the standard average is divided by the average ROI average by the average total tube area to provide a standardized measurement, which can be used to perform the effect of anti-3D-VM vascular structure generation across different experimental parameters and cell lines. The specific calculation formula is as follows:

[0111]

[0112] All the quantitative analysis processes mentioned above were performed using the angiogenesis analyzer of ImageJ.

[0113] 1.12 Protein sample preparation and Western blot analysis

[0114] 2x10 per culture plate 6 Cells were seeded at a density of 100 cells / mL in a 6 cm culture dish for protein sample preparation. After culturing, cells were rinsed twice with ice-cold 1x PBS and then lysed with RIPA buffer containing 25x protease inhibitor cocktail on ice. After centrifugation, the cell lysate was boiled with 6x sample buffer for 10 minutes. The protein samples were then resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was further blocked with TBST buffer containing 5% skim milk powder for 1 hour and then rinsed with TBST for 10 minutes and repeated rinsing 3 times. The membrane was then incubated with primary antibodies at 4°C, including LAMC2 (ab210959), EGFR (D38B1) (#4267), Phospho-EGFR (Tyr992) (4478G), and Actin (MAB1501). The membrane was then incubated with secondary antibodies at room temperature for 1 hour and then Western Lightning Plus Protein images on the transferred membrane were imaged by ECL, and protein images were captured by iBright FL1000 imaging system and analyzed by ImageJ.

[0115] 1.13PAS / CD31 double staining and immunohistochemical staining analysis

[0116] The paraffin-embedded tissue slices were dewaxed with xylene and rehydrated with a gradient alcohol aqueous solution. Then, the tissue slices were immersed in a pH 6.0 citric acid buffer and treated under high pressure for 5 minutes to present the antigen. The endogenous peroxidase activity was inhibited with 3% hydrogen peroxide for 10 minutes. The tissue slices were blocked with 5% fetal bovine serum albumin (BSA) for 1 hour at room temperature. After washing the tissue slices, they were co-incubated with HRP-conjugated secondary antibodies and then colored with DAB matrix. Then, the tissue slices were stained with PAS, which targets the polysaccharides and mucosal substances of the ECM and basement membrane and presents the foreign Red; the samples were incubated with LAMC2 primary antibody overnight at 4°C; finally, hematoxylin was used for background staining (counterstaining) before microscopic observation; the tissue slices were human tissue samples taken from lung adenocarcinoma tissue slices, and were classified into two groups based on EGFR status: wild-type EGFR and mutant EGFR; all tissue slices were diagnosed by histopathological examination, and the experiment was also approved by the IRB of the National Cheng Kung University Hospital, approval number B-ER-108-090.

[0117] 2. Results

[0118] Example 1: Establishment of a novel platform for observing dynamic 3D-VM vascular structures

[0119] A common problem in defining VM is misjudging the 2D-VM network structure as a 3D-VM vascular structure. It is currently known that cancer cell lines MDA-MB-231 and BxPC-3 can form a VM network with vertices and boundaries, which can be observed from a top-down perspective (observing the xy plane along the z-axis), such as Figure 5A As shown in the left half, vertices representing cell nodes and edges representing links between cell nodes (indicated by yellow dotted lines) can be seen.

[0120] Please see Figure 5B Compared with the lung cancer cell lines A549 and H1299 expressing wild-type EGFR, the EGFR mutant cell lines H1650 and HCC827 showed that there were cross-connections and stable boundaries (marked in pink) between the cell nodes (indicated by the yellow boxes).

[0121] In order to distinguish between 2D-VM network structure and 3D-VM vascular structure, an in vitro dynamic 3D-VM vascular structure model was first constructed using the mouse melanoma cell line B16F10, and the cells were fluorescently labeled to facilitate real-time dynamic observation.

[0122] In order to further confirm that these 2D-VM network structures correspond to the tubular and cavity structures of the 3D-VM vascular structure rather than a single-layer cell structure, a cell model with a dynamic 3D-VM vascular structure was established in Example 1 using fluorescently labeled cell lines; see Figure 5C Through LSFM microscopy imaging technology, Imaris software was used for z-stack reconstruction to present the cell models established after FACS screening of stable cell lines B16F10, A549, H1299, H1299-EGFR-wild type, H1299-EGFR-L858R, HCC827, and H1650. The 2D images can be used to observe part of the cross-section of the longitudinal channels and cavities in multiple fields of view.

[0123] See also Figure 5DAfter establishing a lung cancer cell model with dynamic 3D-VM vascular structure using four different EGF-transfected non-small cell lung cancer cell lines (H1650, HCC827, A549, and H1299), the topological characteristics of the vascular structures presented by each of them were also different; it is worth noting that H1650 and HCC827 formed a network-like pattern with cell nodes and boundaries, while A549 and H1299 cells aggregated to form cell clumps; in order to further examine whether these lung cancer cell lines generate unobstructed tubes and hollow cavities, after 24 hours of Matrigel culture, the average diameter of the tubes formed by H1650 and HCC827 cells was approximately 25 to 50um; in contrast, A549 and H1299 showed a single-layer cell structure without unobstructed tubes or hollow cavities.

[0124] Example 2: Hypoxic VM Dynamic Model

[0125] Some lung cancer cells lack 3D-VM vessel structures in a normal oxygen-containing environment (normoxia). In Example 2, it is further investigated whether a hypoxic environment (hypoxia) can induce these lung cancer cells to form 3D-VM vessel structures; Figure 6 As shown, H1299 cells generated significant 3D-VM vascular structure 48 hours after hypoxia induction, but at 72 hours after hypoxia induction, the 3D-VM vascular structure showed obvious collapse, indicating that the 3D-VM vascular structure can only maintain short-term structural stability in a continuous hypoxic environment.

[0126] Similarly, A549 cells generated significant 3D-VM vascular structures 24 hours after hypoxia induction, and the network complexity and number of vascular structures continued to increase between 24 and 48 hours; however, Figure 6 As shown, similar to H1299 cells, the 3D-VM vascular structure of A549 cells also showed obvious degradation after 72 hours of hypoxia induction.

[0127] Example 2 not only demonstrates that the lung cancer cell model can adapt to the hypoxic environment and acquire the characteristics of endothelial cells under hypoxia-induced conditions, and has the ability to form a 3D-VM vascular structure, but also highlights that the 3D-VM vascular structure has a short-term stability within 24 to 48 hours after hypoxia induction, which is consistent with the characteristics of forming a temporary VM vascular structure during tumor cell invasion.

[0128] Example 3: Patency analysis of 3D-VM conduit structure

[0129] In Example 3, lung cancer cells were co-cultured with fluorescent microspheres in 3D to further understand the changes in cell behavior in the VM pipeline structure; Fig. 7AAs shown on the left, the carboxyl-modified microspheres co-cultured with HCC827 showed amazing results. A large number of carboxyl-modified microspheres entered the VM channel structure and cavity, indicating that the 3D-VM vascular structure generated by the HCC827 cell line is indeed a smooth channel structure with channel properties for transporting nutrients or providing cell migration. On the other hand, Figure 7B As shown on the right, the amine-modified microspheres exhibit a completely different interaction pattern with the 3D-VM vascular structure. They are mainly attached to the outside of the cell membrane of HCC827 that forms the outer membrane, and most of them are retained in the range of Matrigel. Obviously, they only enter the channels or cavities of the 3D-VM vascular structure to a very low extent. Example 3 shows that the modification groups of the microspheres will significantly affect the interaction between the microspheres and the 3D-VM vascular structure, and can be used as a reference marker for evaluating the patency of the channels of the 3D-VM vascular structure.

[0130] Accordingly, please refer to Figure 7B , which is a partial enlargement of the VM channel (xz) section of the HCC827 lung cancer cell model, in which carboxyl-modified microspheres can be seen in the cavity; please refer to Figure 7C , which is a dynamic image screenshot of the VM conduit of the HCC827 lung cancer cell model presented by the (yz) cross-section. It can be seen that there are multiple carboxyl-modified microspheres in the cavity of the longitudinal conduit, indicating that the VM conduit is a functional and unobstructed conduit, and the HCC827 cell model has indeed formed a functional 3D-VM vascular structure.

[0131] Example 4: Foslinanib destroys 3D-VM vascular structure

[0132] In order to evaluate the reliability of the lung cancer cell model with dynamic 3D-VM vascular structure for anti-VM drug screening, the drug to be tested in Example 4 is Foslinanib, which is a derivative of phenylquinoline and has quite significant anti-VM properties.

[0133] H1650 and HCC827 cell lines were used as positive control groups in the anti-3D-VM vascular structure experiment, and LSFM was used to dynamically observe the changes in the generation of 3D-VM vascular structure in real time; Figures 8A to 8B As shown, it presents the changes in 3D-VM vascular structure and cell activity of H1650 and HCC827 lung cancer cell models after treatment with different concentrations of Foslinanib; in the H1650 lung cancer cell model, Foslinanib did not have a significant cytotoxic effect on H1650 cells, but the 3D-VM vascular structure was destroyed at a treatment concentration of only 62.5nM, and a stronger 3D-VM vascular structure inhibition effect was shown at a treatment concentration of 250nM to 500nM, as shown in FIG. Figure 8BAs shown in the box-and-whisker plot in the lower half, there was a significant reduction in the lumen area when the treatment concentration reached 250nM and 500nM; on the other hand, in the HCC827 lung cancer cell model, although Foslinanib showed a significant cytotoxic effect on HCC827 cells at a treatment concentration of 62.5nM, with cell activity inhibited to only 70%, its lumen area was significantly reduced after 24 hours of culture at a treatment concentration of 500nM, and also showed the effect of inhibiting 3D-VM vascular structure.

[0134] Accordingly, Example 4 verifies that the lung cancer cell model with a dynamic 3D-VM vascular structure is suitable for the screening of anti-VM drugs and can be used to evaluate the inhibitory and destructive effects of anti-VM drugs in the process of 3D-VM vascular structure generation.

[0135] Example 5: Interaction between VM and EGFR mutation

[0136] In Example 5, the CCLE public datasets of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) were further analyzed using bioinformatics analysis methods; Fig.9A As shown, in this analysis, the melanoma cell dataset was used as the reference data for VM positivity; Fig. 9B The figure shows the VMssGSEA scores of the lung adenocarcinoma (LUAD) dataset in the CCLE public dataset. The samples are divided into two groups based on EGFR mutant and EGFR wild-type classification. The EGFR mutant type presents a significantly higher VM ssGSEA score. This shows that compared with EGFR wild-type lung adenocarcinoma cases, EGFR mutant lung adenocarcinoma cases have a higher VM incidence.

[0137] Please see Fig. 9C After transfection, H1299-EGFRwt showed a unique tubular pattern, while H1299-EGFRmt had obvious 3D-VM vascular structure generation; it is worth noting that compared with the 3D-VM vascular structure generated by H1299-EGFRmt, the intercellular channels of H1299-EGFRwt showed a structure with lower cohesion and only sporadic hollow cavities; Fig.9D As shown in the figure, after quantitative analysis, the average cavity area of ​​H1299-EGFRmt is also significantly larger than that of H1299-EGFRwt. Obviously, overexpression of EGFRmt can indeed promote H1299 cells to form 3D-VM vascular structure with effective cavity structure.

[0138] like Figure 5DAs shown in Figure 2, the 2D-VM network structure generated by the H1299 lung cancer cell line lacks a visible cavity structure in the 3D model experiment, and no unobstructed pipeline structure is observed; in contrast, the stable cell line H1299-EGFRmt ( Fig. 9C (bottom) presents a hollow cavity with an unobstructed pipe structure.

[0139] Example 6: Combination of anti-VM drugs and EGFR tyrosine kinase inhibitors (TKI)

[0140] Please see Fig. 10A , which presents the changes in cell activity and 2D-VM network structure state of H1299-EGFRmt, H1299-EGFRwt and H1299 after Foslinanib treatment; in the action concentration range of 7.8125nM to 500nM, in the H1299 cell model that overexpresses EGFRmt, there was a significant decrease in cell activity when the concentration reached 31.25nM, and its 2D total network length and total network area showed a significant downward trend at any treatment concentration greater than 31.25nM; compared with H1299-EGFRwt, Foslinanib at high concentrations (greater than 31.25nM) had no obvious inhibitory effect on the generation of 2D-VM network structure of H1299-EGFRwt, showing a poor therapeutic response. On the other hand, the cells of the H1299 cell model had cell activity inhibition after being treated with high concentrations of Foslinanib (greater than 250 nM). Since H1299 cells do not have 3D-VM vascular channels under 3D-VM methodology observation, this example observed the inhibitory changes of Foslinanib on the 2D-VM network, and its 2D total network length and total network area also showed poorer inhibition compared with H1299-EGFRmt.

[0141] In order to improve the therapeutic response, Example 4 further combines EGFR TKI and anti-angiogenic drugs to simultaneously target multiple different regulatory pathways; although the third-generation EGFR TKI—Osimertinib—did not show additional benefits in anti-angiogenic therapy, in order to further understand the effect of combined treatment on dynamic 3D-VM vascular structure imaging, in this example, H1299 cells transfected with wild-type EGFR and mutant EGFR-L858R were used as test cell lines, and Osimertinib and Foslinanib were administered in combination.

[0142] like Fig. 10BAs shown in the control group, H1299 lung cancer cells did not develop 3D-VM vascular structures; in contrast, H1299-EGFRwt showed a unique tube pattern with loosely connected tubes and sporadic hollow cavities, while H1299-EGFR-L858R had significant 3D-VM unobstructed tube structure generation.

[0143] See also Figures 10B to 10C Foslinanib showed the effect of destroying the 3D-VM vascular structure of H1299-EGFRwt at a treatment concentration of 62.5nM. Although overexpression of EGFRmt can induce H1299 cells to generate 3D-VM vascular structure, the quantitative results showed that Foslinanib had significantly destroyed the 3D-VM vascular structure of H1299-EGFRmt. In contrast, the extremely high dose of 5μM Osimertinib used to test EGFR-resistant cell lines did not inhibit the generation of 3D-VM vascular structure, but this high dose had no inhibitory effect on H129 9. H1299-EGFRwt and H1299-EGFRmt are cytotoxic, and the quantification results still show that after treatment with Osimertinib, the cavity structure in the 3D-VM vascular structure is also significantly destroyed; further, when 5μM Osimertinib and 62.5nM Foslinanib are combined, in addition to the destruction of the 3D-VM vascular structure visible in the stereoscopic image, the quantification results show that the cavity structure in the 3D-VM vascular structure of both H1299-EGFRwt and H1299-EGFRmt has been almost completely inhibited.

[0144] Example 7: LAMC2 is overexpressed in an EGFRmt lung cancer cell model with 3D-VM vascular structure

[0145] Example 7: Immunohistochemical staining of LUAD EGFRmt human tumor tissue was used to evaluate the formation status of vascular structures in LUAD tumors; Fig.11A As shown, VM-like blood vessels and regions can be distinguished by PAS-CD31 double staining; on the one hand, the VM region is demarcated by PAS staining (indicated by red arrows); on the other hand, the VM region showing PAS-CD31 double staining also shows LAMC2 immunohistochemical staining (indicated by red asterisks); here, the immunohistochemical staining results show that LAMC2 may be involved in the generation mechanism of 3D-VM vascular structure in LUAD tumors.

[0146] In Example 7, the total expression of EGFR and LAMC2 and the degree of EGFR phosphorylation in H1299, H1299-EGFRwt, and H1299-EGFRmt were further quantitatively analyzed by Western Blot. The results are shown in FIG. Fig. 11BAs shown, Osimertinib significantly reduced the phosphorylation level of EGFR, while Foslinanib treatment significantly reduced the expression of LAMC2 protein per cell bead; in addition, the cell lines treated with Osimertinib and Foslinanib showed that the two had a synergistic effect, downregulating the expression of phosphorylated EGFR and LAMC2; Example 7 highlights that EGFRmt-EGFR(p.Leu858Arg)-has a potential interaction relationship with the generation mechanism of 3D-VM vascular structure and can be used as a drug target.

[0147]

Explanation of symbols

[0148] 100 lung cancer cell models

[0149] 1 Cell suspension

[0150] 1a Lung cancer cells

[0151] 1b Suspension

[0152] 10 Pre-cell model

[0153] 2a Upper 3D culture matrix

[0154] 2a' Upper 3D culture pre-matrix

[0155] 2b Lower 3D culture matrix

[0156] 2b' Lower 3D culture pre-matrix

[0157] P Culture surface

[0158] 3 Vascular structure

[0159] 3a cavity

[0160] 3b Outer membrane

[0161] 30 Good patency of vascular structure

[0162] 31 Poor patency of vascular structures

[0163] 4 microspheres

[0164] Steps S1 to S2

[0165] Steps S1 to S3

[0166] Steps S1a to S1c

[0167] Steps S3a to S3c

[0168] Steps A1 to A3

Claims

1. A method for constructing a lung cancer cell model with vascular ducts, characterized in that: include: Planting a cell suspension between the upper 3D culture matrix and the lower 3D culture matrix to obtain a pre-cell model, wherein the cell suspension includes lung cancer cells; and The pre-cell membrane model is incubated for an incubation time to obtain the lung cancer cell model, wherein both the upper 3D culture matrix and the lower 3D culture matrix contain growth factor-deficient culture medium, and the volume of the upper 3D culture matrix is ​​greater than that of the lower 3D culture matrix.

2. The method according to claim 1, characterized in that The pre-cell membrane forms are incubated in a normoxic environment, a physiological oxygenic environment, or an oxygen-deficient environment.

3. The method according to claim 1, characterized in that The lung cancer cells include: A first lung cancer cell, which expresses wild-type EGFR at a first reference amount, or the first lung cancer cell further carries a first exogenous gene material to express wild-type EGFR at a first expression amount, wherein the first expression amount is greater than the first reference amount; and The second lung cancer cell expresses mutant EGFR at a second reference amount, or the second lung cancer cell further carries a second exogenous gene material to express wild-type EGFR at a second expression amount, wherein the second expression amount is greater than the second reference amount.

4. The method according to claim 3, characterized in that: The lung cancer cell is derived from lung cancer tissue or lung cancer cell line, wherein the lung cancer tissue is derived from small cell lung carcinoma, large cell lung carcinoma, squamous cell carcinoma or lung adenocarcinoma; the lung cancer cell line is selected from the group consisting of A549, HCC827, H460, H1299, H1650 and H1975.

5. The method according to any one of claims 1 to 4, characterized in that: The steps of obtaining the pre-cell model include: Solidifying the lower volume of the lower 3D culture pre-matrix on the culture surface for a solidification time to form the lower 3D culture matrix; Incubating the middle volume of the cell suspension on the lower 3D culture matrix for the attachment time, so that the lung cancer cells attach to the lower 3D culture matrix; and The upper volume of the upper 3D culture pre-matrix is ​​coated on the lung cancer cells to form the upper 3D culture matrix to obtain the pre-cell model, wherein the upper volume is greater than the sum of the lower volume and the middle volume.

6. The method according to claim 5, characterized in that: The upper 3D culture pre-matrix comprises a first upper volume of an upper cell-like basement membrane material and a second upper volume of a cell culture medium, wherein the first upper volume is the same as the second upper volume, and the sum of the first upper volume and the second upper volume is the same as the upper volume; and The lower 3D culture pre-matrix includes a lower cell-like basement membrane material, and the upper cell-like basement membrane material is the same as or different from the lower cell-like basement membrane material, wherein the ratio of the first upper volume, the second upper volume, the middle volume and the lower volume is 1:(1.00 to 3.00):(1.05 to 1.65):(0.25 to 0.85).

7. The method according to claim 5, characterized in that The upper 3D culture pre-matrix is ​​Matrigel, and the lower 3D culture pre-matrix is ​​Matrigel.

8. The method according to claim 5, characterized in that: The cell suspension further includes microspheres labeled with a first fluorophore and containing negatively charged organic groups; The lung cancer cells are further labeled with a second fluorophore having a different emission from the first fluorophore; and The vascular structure includes a cavity formed by the lung cancer cells and an outer membrane formed by the lung cancer cells surrounding the cavity, wherein the method further includes: The number of the microspheres in the vascular structure is estimated to evaluate the patency of the vascular structure, wherein a first number of the microspheres in the cavity is estimated, and a second number of the microspheres on the outer membrane or outside the outer membrane is estimated, and when the first number is greater than or equal to the second number, the vascular structure is judged to be a vascular structure with good patency, and when the first number is less than the second number, the vascular structure is judged to be a vascular structure with poor patency.

9. A lung cancer drug screening platform, comprising a lung cancer cell model prepared by the method according to any one of claims 1 to 4.

10. A method for screening lung cancer drugs, comprising: Providing a lung cancer cell model to be tested, which has a vascular structure with a first vascular density, wherein the cancer cell model to be tested is prepared according to the method according to any one of claims 1 to 4; Co-culturing the lung cancer cell model and the anticancer drug for a screening period to obtain a tested lung cancer cell model having a vascular structure with a second vascular density; and The relationship between the first vascular density and the second vascular density is evaluated to determine whether the anticancer drug has therapeutic effect, wherein when the ratio of the first vascular density to the second vascular density is greater than 1, the anticancer drug is determined to be therapeutic, and when the ratio of the first vascular density to the second vascular density is equal to or less than 1, the anticancer drug is determined to be ineffective.

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