A method for evaluating migration and invasion ability of lung cancer organoids

Abstract

This invention proposes a method for evaluating the migration and invasion capabilities of lung cancer organoids, belonging to the technical field of in vitro functional evaluation of tumor organoids. This invention achieves decoupled evaluation of the migration and invasion capabilities of lung cancer organoids by establishing migration and invasion models. Images of the migration and invasion models are acquired before and after culture, respectively, and a standardized area calculation method is used to define the main organoid region as the core area A. core Define the migrating / invading cell region as the migration / invasion area A. out Based on A out With A core The normalized migration index (MI) and invasion index (II) are calculated, and the net invasiveness index is obtained by decoupling the score BIS = II / (MI+). This method can stably obtain the migration and invasion evaluation results of lung cancer organoids under a unified quantitative standard, and can be used for lung cancer organoid invasiveness assessment, drug screening, and in vitro functional studies.

Description

Technical Field

[0001] This invention belongs to the field of in vitro functional evaluation technology of tumor organoids, specifically relating to a method for evaluating the migration and invasion capabilities of lung cancer organoids. Background Technology

[0002] The metastatic and invasive capabilities of lung cancer are closely related to patient prognosis, treatment options, and drug sensitivity. Traditional methods such as two-dimensional scratch assays and Transwell migration / invasion assays, while technically mature, are insufficient to fully reflect the three-dimensional structure, cellular heterogeneity, and microenvironment-dependent behavioral differences of tumor tissues.

[0003] In recent years, patient-derived tumor organoids (PDOs) and three-dimensional tumor sphere models have become important in vitro models due to their ability to better preserve tumor heterogeneity. However, existing organoid-based "outgrowth" phenotypic evaluations often have the following shortcomings:

[0004] (1) Migration and invasion are often characterized in a mixed manner: The mechanisms of organoids expanding on the surface of the matrix gel and expanding across the matrix gel barrier are different, but they are often directly characterized by the same "change in extensional area", making it difficult to distinguish between "contribution of migration background" and "ability to cross the barrier".

[0005] (2) Inconsistent quantification standards and limited reproducibility: The initial size of organoids, the offset of landing points, and the changes in imaging field of view in different experiments can significantly affect the area results. Furthermore, the lack of a unified definition and normalization strategy for the core / extension region makes horizontal comparison difficult.

[0006] (3) Under the requirement of high-throughput screening, there is an urgent need for a method that can be stably implemented in multi-well plates and output migration and invasion capabilities with unified indicators.

[0007] Therefore, it is necessary to provide a method for evaluating the migration and invasion capabilities of lung cancer organoids that can achieve decoupled evaluation of migration and invasion in a three-dimensional organoid system and has a unified quantitative standard and good reproducibility. Summary of the Invention

[0008] This invention aims to address the problems of "difficulty in decoupling migration and invasion, inconsistent area caliber, and insufficient comparability of results" in the evaluation of the migration and invasion capabilities of lung cancer organoids. It provides a method for evaluating the migration and invasion capabilities of lung cancer organoids by establishing migration and invasion models in parallel and combining standardized area caliber and barrier correction scores.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for evaluating the migration and invasion capabilities of lung cancer organoids, based on in vitro evaluation, specifically includes the following steps:

[0011] (1) Lay Matrigel at the bottom of the culture container, which will gel to form the bottom layer;

[0012] (2) Establishment of migration model: Lung cancer organoids were seeded on the surface of Matrigel substrate and cultured in culture medium. Bright-field images were acquired at the start of culture time t0 and the evaluation time t1. The acquired images were divided into regions using image analysis software, and the core area of ​​the migrating organoid at time t0 was calculated. Area of ​​the extension region Area of ​​the core region of the migrated organoid at time t1 Area of ​​the extension region And calculate the migration index MI according to formula (1);

[0013] (1)

[0014] (3) Establishing an invasion model: Lung cancer organoids were embedded in Matrigel to form embedded bodies containing organoids. The embedded bodies were then placed on the surface of the Matrigel bottom layer and cultured. Bright-field images were acquired at the start of culture (t0) and the evaluation time (t1). Image analysis software was used to divide the acquired images into regions, and the area of ​​the core region of the invasive organoid at time (t0) was calculated. Area of ​​the extension region The area of ​​the organoid core region invaded at time t1 Area of ​​the extension region And calculate the invasion index II according to formula (2);

[0015] (2)

[0016] (4) Calculate the barrier-corrected invasion score (BIS) based on the migration index (MI) and invasion index (II). The barrier-corrected invasion score (BIS) is calculated according to formula (3):

[0017] (3)

[0018] In formula (3), For stabilization parameters;

[0019] (5) Output the calculated MI, II and BIS data as evaluation results of lung cancer organoid migration and invasion capabilities.

[0020] As a preferred embodiment of the present invention, the core area is defined as the area of ​​the organoid body mass in the image, and the extension area is defined as the area of ​​the extracellular cell coverage region located outside the core area and connected to the boundary of the core area in the image.

[0021] As a preferred embodiment of the present invention, the culture container is a 96-well plate, and the number of lung cancer organoids inoculated in each well is preferably 1 to 5, more preferably 1 to 5. During observation, only one lung cancer organoid is captured for bright-field image analysis and calculation. The original lung cancer organoid culture medium formula is used for culture during the experiment, and the medium is changed every 3 days. The evaluation time t1 is any time within 4 to 8 days after inoculation, preferably the 5th day after inoculation.

[0022] As a preferred embodiment of the present invention, the lung cancer organoid is placed in the central region of the bottom of the well during inoculation so that the same field of view can be imaged and the area measured at times t0 and t1.

[0023] As a preferred embodiment of the present invention, the Matrigel substrate in the migration model has a laying volume of 30-60 μL per well in the 96-well plate; the embedding body in the invasion model is a Matrigel droplet or block, with an embedding volume of 5-30 μL.

[0024] As a preferred technical solution of the present invention, in formula (3) To stabilize the parameters and prevent excessively small MI from causing abnormally amplified BIS scores, the lower limit of detection of the migration index MI from the same batch of experiments can be used.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) The migration and invasion were measured separately by establishing the “matrix surface migration model” and the “full glue embedding invasion model” in parallel.

[0027] (2) The introduction of the Barrier Corrected Invasion Score (BIS) can correct the migration background of the invasion readings, realize the decoupled evaluation of migration and invasion, and make the “ability to cross the ECM barrier” independently characterized.

[0028] (3) Adopt the standardized definition of the core area / extension area and normalize the extensional variation to reduce the bias caused by the difference in the initial size of organoids and improve the comparability between batches and samples.

[0029] (4) The method is adapted to 96-well plate high-throughput implementation. The organoid is placed in the center of the well and imaged in the same field of view at t0 and t1, which helps to reduce measurement errors caused by field of view offset and edge effect.

[0030] (5) The method does not require complex special equipment. It can obtain stable quantitative results by relying only on bright field imaging and outline analysis, and has the ability to be promoted and practical. Attached Figure Description

[0031] Figure 1The diagram shows the construction of migration and invasion models (comparison between surface seeding and full gel embedding) and the flowchart of the lung cancer organoid migration and invasion capability evaluation method proposed in this invention.

[0032] Figure 2 The image shows a bright field image of the observation field obtained in a specific experiment. (a) and (b) correspond to different transfer models. The image is divided into a core region and an outer region (out) by image analysis software. The area enclosed by the red line is the core region, and the area enclosed by the blue line minus the area enclosed by the red line is the outer region (out).

[0033] Figure 3 The image shows a bright field image of the observation field obtained in a specific experiment. (a) and (b) correspond to different invasion models. The image is divided into a core region and an outer region (out) by image analysis software. The area enclosed by the red line is the core region, and the area enclosed by the blue line minus the area enclosed by the red line is the outer region (out).

[0034] Figure 4 The images shown are bright-field images of the observation field obtained in Example 3. (a) and (b) are bright-field images of the same migration model at different times (0 and 5d), respectively.

[0035] Figure 5 The images shown are bright-field images of the observation field obtained in Example 4. (a) and (b) are bright-field images of the same invasion model at different times (0 and 5d), respectively. Detailed Implementation

[0036] Please see Figure 1 As shown, this invention proposes a method for evaluating the migration and invasion capabilities of lung cancer organoids. First, lung cancer organoids are constructed from patient-derived tumor tissue obtained from a hospital. These organoids are expanded and cultured from primary culture. Once the organoids are stable, organoids of appropriate size are seeded into the center of a 96-well plate. Then, the seeded organoids are tracked and photographed over a long period. Bright-field images are obtained, and image outline analysis is performed. Normalization is then used to calculate the scoring data, thereby obtaining a phenotypic evaluation of the migration and invasion capabilities of lung tumor organoids. Specifically, the method includes the following steps:

[0037] (1) Matrigel substrate: Matrigel is laid at the bottom of the 96-well plate and gelled to form the substrate.

[0038] (2) Migration model establishment and imaging: lung cancer PDO was inoculated onto the bottom surface, cultured in culture medium, and bright field images were acquired at the start time t0 and the evaluation time t1.

[0039] (3) Invasion model establishment and imaging: lung cancer PDO was embedded in Matrigel to form embedded droplets, and then the embedded droplets were placed on the bottom surface. Culture medium was added for incubation, and bright field images were acquired at the start time t0 and the evaluation time t1.

[0040] (4) Area standardization: The core area A is obtained by dividing the image into regions using image analysis software. core With the extension area A out ,like Figure 2 and 3 As shown.

[0041] (5) Calculation of migration index and invasion index: The migration index MI and invasion index II are calculated based on the standardized caliber.

[0042] (6) Barrier Corrected Invasion Score: The barrier corrected invasion score (BIS) is calculated based on MI and II, and the invasion capability index is output for decoupling evaluation.

[0043] (7) Output MI, II, and BIS as evaluation results of the migration and invasion ability of PDO in lung cancer.

[0044] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0045] Example 1: Organoid Culture Medium and Experimental Materials

[0046] Table 1. Organoid Culture Medium (Lung Cancer Organoid Culture Medium)

[0047] Element Final concentration S-Reduce® DMEM / F-12 (Yuanpei Biotechnology, L330KJ) 100% HEPES (Gibco, 15630080) 1% GlutaMAX (Gibco, 35050061) 1% Penicillin-streptomycin (ThermoFisher15140122) 1% N-acetylcysteine ​​(Sigma, A9165-25 g) 1.25 mM Human Noggin (Novoprotein, CB89) 100 ng / mL B27 (Gibco, 17504044) 2% Human EGF (PeproTech, AF-100-15) 50 ng / mL SAG (MCE, HY-12848) 100 nM Y27632 (APExBIO, A3008) 10 μM A8301 (MCE, HY-10432) 0.5 μM CHIR99021 (MCE, HY-10182) 250 nM FGF4 (Novoprotein, CR08) 100 ng / mL FGF10 (Novoprotein, CR11) 100 ng / mL

[0048] Table 2. Experimental Materials

[0049] name brand Item number 96 Well Cell Culture Plate Corning 3599 Matrigel matrix adhesive (standard type, phenol red free) Absin Abs9491 S-Reduce® DMEM / F-12 basal culture medium Yuanpei Biotechnology L330KJ

[0050] Example 2: Matrigel substrate application (1 mg / mL)

[0051] (1) Take Matrigel stock solution (10 mg / mL) and place it on ice for operation.

[0052] (2) Dilute with basal culture medium at a volume ratio of 1:9 to obtain 1 mg / mL Matrigel working solution.

[0053] (3) Add 30 μL of 1 mg / mL Matrigel working solution to each well of the 96-well plate to form the bottom layer, and place it in a 37℃ incubator for 3 h to form a stable bottom layer; after gelation, discard the ungelled liquid on the top layer, add 30 μL of basic culture medium to each well, and place it in a 37℃ incubator for 30 min to hydrate.

[0054] (4) After hydration is complete, discard the upper liquid and keep it for later use.

[0055] Example 3: Establishment of the migration model and calculation of the migration index MI

[0056] (1) Once the organoids are stable and mature, use AdDF. +++ Wash and pipette the culture medium (DMEM / F12, 10 mM HEPES, 1×GlutaMax, 1×Penicillin-streptomycin) into 15 mL centrifuge tubes. After centrifugation, discard the supernatant, resuspend in basal culture medium, select organoids with a diameter of about 100-200 μm, and gently transfer them to 96-well plates with a pipette tip. Inoculate 1-2 organoids into each well, placing them as centrally as possible in the well. After inoculation, gently add organoid culture medium along the well wall.

[0057] (2) Bright field imaging was recorded immediately after inoculation (starting time t0).

[0058] (3) Continue culturing until evaluation time t1 (select the 5th day after inoculation), during which lung cancer organoid culture medium is changed every 3 days; on the 5th day, the same well and the same field of view are imaged again in bright field.

[0059] (4) Image analysis: The images at t0 and t1 were processed and analyzed using image analysis software: the core region was obtained by outlining the boundary of the main organoid mass at the starting time t0 and the evaluation time t1, and the core region was calculated. and The extension region is obtained by calculating the area covered by extravasated cells outside the core region and connected to the core region boundary at the starting time t0 and the evaluation time t1. and .

[0060] Processing and analysis results as follows Figure 4 As shown, the calculation yields:

[0061] , , , .

[0062] (5) Calculate the migration index MI:

[0063]

[0064] After calculation, we can obtain MI = (15157-0) / 3870 ≈ 3.9165.

[0065] Example 4: Establishment of Invasion Model and Calculation of Invasion Index II (Full Glue Embedding)

[0066] (1) Take the screened organoids (same as in Example 3) and mix them with Matrigel stock solution to form embedding droplets. The volume of each embedding droplet is about 10 μL. The embedding droplets are fully embedded in gel (without mixing or dilution with culture medium).

[0067] (2) Place the embedding droplet on the surface of the gelled Matrigel bottom layer, with the embedding droplet located as close as possible to the center of the bottom of the well. After inoculation, place the well in a 37°C incubator for 30 min to gel. After the gel solidifies, add organoid culture medium.

[0068] (3) Bright field imaging was recorded immediately after inoculation (starting time t0).

[0069] (4) Continue culturing until evaluation time t1 (select the 5th day after inoculation), during which lung cancer organoid culture medium is changed every 3 days; on the 5th day, the same well and the same field of view are imaged again in bright field.

[0070] (5) Image analysis: The images at t0 and t1 were processed and analyzed using image analysis software: the core region was obtained by outlining the boundary of the main organoid mass at the starting time t0 and the evaluation time t1, and the core region was calculated. and The extension region is obtained by calculating the area covered by extravasated cells outside the core region and connected to the core region boundary at the starting time t0 and the evaluation time t1. and .

[0071] (6) Calculate the invasion index II:

[0072]

[0073] Processing and analysis results as follows Figure 5 As shown, the calculation yields:

[0074] , , , .

[0075] After calculation, we can obtain II = (6492-0) / 5526 ≈ 1.1748.

[0076] Example 5 Barrier Corrected Invasion Score (BIS)

[0077] Based on the MI and II obtained in Examples 3 and 4, the Barrier Corrected Invasion Score (BIS) is calculated:

[0078]

[0079] Since the detection limit for MI in this batch is 0, ε is 0.

[0080] After calculation, we can obtain BIS = 1.1748 / (3.9165+0) ≈ 0.3000.

[0081] This indicates that the lung cancer organoids in this case exhibited weak extravasation in the presence of the Matrigel barrier, with extravasation mainly driven by migration and exhibiting weak invasiveness.

[0082] Explanation: When the BIS is large (>1), it indicates that the intrusive ability is relatively strong and the outward expansion is relatively strong even in the presence of the Matrigel barrier; conversely, it is weak.

[0083] Example 6: Results Output and Application

[0084] (1) MI, II, and BIS are calculated for each well as organoid, and statistical summaries are made for multiple wells / multiple organoids under the same conditions.

[0085] (2) The results can be used for stratified assessment of lung cancer PDO invasiveness, evaluation of changes in migration / invasiveness before and after drug treatment, and in vitro mechanism research and screening applications.

Claims

1. A method for evaluating the migration and invasion capabilities of lung cancer organoids, based on in vitro evaluation, characterized in that, Specifically, the steps include the following: (1) Lay a substrate gel at the bottom of the culture container, which will gel to form the bottom layer; (2) Establishment of migration model: Lung cancer organoids were seeded on the surface of the matrix gel substrate and cultured in culture medium. Bright-field images were acquired at the start of culture time t0 and the evaluation time t1. The acquired images were divided into regions using image analysis software, and the core area of ​​the migrating organoid at time t0 was calculated. Area of ​​the extension region Area of ​​the core region of the migrated organoid at time t1 Area of ​​the extension region And calculate the migration index MI according to formula (1); （1） (3) Establishing an invasion model: Lung cancer organoids were embedded in matrix gel to form embedded bodies containing organoids. The embedded bodies were then placed on the surface of the matrix gel bottom layer and cultured with culture medium. Bright-field images were acquired at the start of culture time t0 and the evaluation time t1. The acquired images were divided into regions using image analysis software, and the area of ​​the core region of the invasive organoid at time t0 was calculated. Area of ​​the extension region The area of ​​the organoid core region invaded at time t1 Area of ​​the extension region And calculate the invasion index II according to formula (2); （2） (4) Calculate the barrier-corrected invasion score (BIS) based on the migration index (MI) and invasion index (II). The barrier-corrected invasion score (BIS) is calculated according to formula (3): （3） In formula (3), For stabilization parameters; (5) Output the calculated MI, II and BIS data as evaluation results of lung cancer organoid migration and invasion capabilities.

2. The method for evaluating the migration and invasion capabilities of lung cancer organoids as described in claim 1, characterized in that, The core area is defined as the area of ​​the organoid mass in the image, and the extension area is defined as the area of ​​the extracellular cell-covered region located outside the core area and connected to the boundary of the core area in the image.

3. The method for evaluating the migration and invasion capabilities of lung cancer organoids as described in claim 1, characterized in that, The culture container is a 96-well plate, and 1 to 5 lung cancer organoids are inoculated in each well. During observation, only one lung cancer organoid is captured in bright field image for analysis and calculation.

4. The method for evaluating the migration and invasion capabilities of lung cancer organoids as described in claim 3, characterized in that, The lung cancer organoids were placed in the central region of the bottom of the well during inoculation so that the same field of view could be imaged and the area measured at times t0 and t1.

5. The method for evaluating the migration and invasion capabilities of lung cancer organoids as described in claim 3, characterized in that, In the migration model, the substrate layer is laid in a 96-well plate with a volume of 30-60 μL per well; in the invasion model, the embedding body is a substrate droplet or block with a volume of 5-30 μL.

6. The method for evaluating the migration and invasion capabilities of lung cancer organoids as described in claim 1, characterized in that, In formula (3) The lower limit of detection of the migration index (MI) of the same batch of experiments was used.

7. The method for evaluating the migration and invasion capabilities of lung cancer organoids as described in claim 1, characterized in that, The evaluation time t1 is any time within 4 to 8 days after vaccination.

8. The application of the method as described in any one of claims 1 to 7 in in vitro lung cancer organoid invasiveness stratification assessment, drug inhibition of migration / invasion effect evaluation, or in vitro mechanism research.

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