CN114414541A - Method for detecting killing effect of T cells by applying 3D cell imaging analysis system - Google Patents
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Abstract
The invention discloses a method for detecting killing effect of T cells by applying a 3D cell imaging analysis system, which comprises the following steps: constructing a three-dimensional tumor sphere; co-culturing target cells, effector cells and TCE antibody and performing staining and marking; carrying out image acquisition by using a high content imaging analysis system; and the application software analysis system performs single or superposition analysis on the obtained images. The method has the advantages of good stability, high accuracy, intuition and visibility, and capability of evaluating the killing effect of the T cells closer to the physiological environment in vivo.
Description
Technical Field
The invention relates to a method for detecting killing effect of T cells, in particular to a method for detecting the killing effect of T cells on tumor cells by applying a 3D cell imaging analysis system.
Background
At present, tumor immunotherapy has become the fourth major tumor therapy after surgery, radiotherapy and chemotherapy drug therapy, and is the hottest tumor therapy at present. Tumor immunotherapy is the technical means to reactivate the immune system so that it can recognize and eliminate cancer cells. In the field of tumor immunotherapy, the development of antibody drugs has been a hot direction. Bispecific antibodies targeting the CD3 antigen are a class of antibody therapies that can spatially draw T cells closer to tumor cells and treat hematologic and solid tumors using the killing effect of T cells on tumor cells, which are called T cell-engaging bsAb (TCE dual antibody). The action mechanism of the TCE double-antibody medicament is that immune synapse is formed by simultaneously combining T cell surface antigen and tumor cell surface antigen, so that the T cell is directly activated and proliferated, and then cytotoxin or cytokine is released to kill the tumor cell.
Cell killing experiments, i.e., cytotoxicity assays, are one type of experiment used to evaluate the killing efficacy of TCE dual-antibody mediated T cells on tumor cells. Common cytotoxicity detection methods include a 51Cr release experiment, a Lactate Dehydrogenase (LDH) release method, a BATDA method, a CytoTox-Glo method and the like, and the classical detection method is the 51Cr release method, but because radioactive isotopes are not beneficial to safe operation and waste disposal, the method has great threat to the environment and human health, and the application of the method is limited due to high spontaneous release rate. The LDH and CytoTox-Glo methods are simple and convenient to operate, but have the defects of short half-life, low sensitivity and incapability of distinguishing release of target cells and effector cells. The BATDA method needs to label target cells, is complex to operate, has high spontaneous release rate, and is not suitable for long-time cell killing experiments.
The current method for detecting cytotoxicity is carried out in the environment of two-dimensional planar culture of tumor cells, and the difference between the planar culture and the growth mode and the in-vivo three-dimensional environment is large, so that the cell morphology, differentiation, interaction between cells and matrixes, and interaction between cells and behaviors of cells under in-vivo physiological conditions are obviously different. Unlike traditional cell culture, three-dimensional (3D) cell culture reproduces the in vivo environment of cells. The gradient of oxygen, nutrient substances, metabolites and soluble signals can be formed, a microenvironment which is closer to the in vivo survival condition is provided for cells, and the physiological state can be better simulated, so that the experimental result which is more consistent with the in vivo experiment is obtained.
Disclosure of Invention
The invention aims to overcome the defects that the traditional detection method is complex to operate, low in stability, incapable of accurately reflecting the real physiological state in vivo and the like, and establishes a method for evaluating the killing effect of T cells, which is stable, high in accuracy, visual and closer to the physiological environment in vivo, by constructing a 3D tumor sphere and using a high content imaging analysis system.
In order to achieve the purpose, the invention adopts the following technical scheme,
a method for detecting killing effect of T cells by using a 3D cell imaging analysis system comprises the following steps:
1. constructing a three-dimensional tumor sphere by using a 3D cell culture technology;
2. co-culturing target cells, effector cells and TCE antibodies, performing dyeing marking on the target cells by using a fluorescent marker A, performing dyeing marking on the effector cells by using a fluorescent marker B, and performing dyeing marking on dead cells generated after co-culturing by using a fluorescent marker C;
3. using a high content imaging analysis system, and respectively using an excitation light channel corresponding to the fluorescent marker A, an excitation light channel corresponding to the fluorescent marker B and an excitation light channel corresponding to the fluorescent marker C to acquire images of the same visual field;
4. and (3) performing single or superimposed analysis on the obtained image by using a software analysis system, and calculating the killing effect of the T cells on the target cells according to the size of the tumor sphere.
Preferably, the excitation wavelengths of the fluorescence label a, the fluorescence label B and the fluorescence label C are different. Preferably, the fluorescent marker A comprises a fluorescent protein and/or a cell dye, the fluorescent protein comprises a Green Fluorescent Protein (GFP) and/or a Red Fluorescent Protein (RFP) and the like, and the target cells express the fluorescent protein by means of transfection or lentivirus infection. Target cells can also be labeled with a cell dye, such as one or more of Hoechst 33342 (hurst), carboxyfluorescein diacetate succinimidyl ester, a far-red dye, a calcein green dye, and the like.
Preferably, the fluorescent marker B is a living cell staining reagent comprising one or more of carboxyfluorescein diacetate succinimidyl ester, a far-red dye, a calcein green dye, a calcein purple dye, a calcein blue dye and a calcein orange red dye.
Preferably, the fluorescent marker C is a dead cell dye, such as one or more of PI (propidium iodide), Caspase 3/7 (cysteine protease), SYTOX Green (chlorocyanine), 7-AAD (7-amino-actinomycin D), Annexin V (Annexin-V), and the like.
The target cell in the invention refers to various tumor cells or various engineered cells, including but not limited to LS174T, HPAF-II, T84, SKBR-3, MCF-7, HepG2, ASPC-1, PC-3, etc. The T cells are derived from human PBMC (peripheral blood mononuclear cells), and can be directly used as the human PBMC, or T cell components obtained by a magnetic bead sorting method, or T cells amplified in vitro.
In the process of superposition analysis, only the cells with the fluorescence markers A are live target cells, and the cells with the fluorescence markers A and the fluorescence markers C are dead target cells; only those showing fluorescence label B are live effector cells, while those showing fluorescence label B and fluorescence label C are dead effector cells. 3D image analysis was performed using high content analysis software, and tumor sphere volumes were calculated by analyzing the recognition spheres using fluorescence labeled A stained images, the target cell specific killing efficiency being tumor sphere volume of antibody-free group tumor sphere volume-antibody treatment group tumor sphere volume/antibody-free group tumor sphere volume 100.
The invention has the advantages that:
1. the invention provides a method for evaluating cell killing effect based on change of 3D tumor sphere volume, wherein a fluorescence-labeled target cell forms a 3D tumor sphere under a specific culture condition, a fluorescence-labeled T cell is added as an effector cell according to a proper effective target ratio, a TCE antibody diluted in a certain concentration gradient is added for co-culture, and PI is added for carrying out labeling and dyeing on dead cells in a population. By utilizing a high content imaging system, corresponding target cells, dead target cells, effector cells and dead effector cells can be clearly and visually observed, the corresponding size of the tumor sphere can be obtained through software analysis and calculation, and the cell killing effect can be obtained through calculation according to the change ratio of the volume of the tumor sphere to a control group.
2. According to the method, the 3D cell culture technology is combined with high content imaging, image information and a data processing result can be obtained at the same time, the result is more visual and vivid, and compared with the traditional 2D plane cell culture technology, the 3D cell culture can better simulate the physiological state, so that an experiment result more consistent with an in vivo experiment is obtained.
Drawings
FIG. 1 shows the killing potency of T cells on tumor spheres mediated by TCE antibodies at different concentrations in the stack of three fluorescence channels;
FIG. 2 shows the T cell spheroid infiltration by TCE antibody at different concentrations in the excitation light channel of 494 nm;
FIG. 3 shows the death counts of target cells by different concentrations of TCE antibody in the 535nm excitation light channel;
figure 4 is a graph of the killing efficacy of T cells on tumor spheres mediated by TCE antibodies at various concentrations.
Detailed Description
Example 1: target cell staining and construction of 3D tumor spheres
Target cells LS174T were fluorescently labeled with Hoechst 33342 (Herster), washed by centrifugation and counted with a cytometer with cell density adjusted to 5X 104Inoculating 100ul cell suspension to Prime/mL
3D Culture spherical plates (S-bio, # MS-9096UZ) were cultured in a 5% CO2 incubator at 37 ℃ for 48 hours per well to form 3D tumor spheres.
Example 2: effector cell staining
After 48 hours, the T cells were fluorescently stained with Calcein Green AM (Calcein acetoxymethyl ester), washed by centrifugation, counted with a cytometer, and adjusted to a cell density of 5X 105Per mL50ul of cell suspension was added to each well of a 96-well plate in which tumor spheres had formed.
Example 3: co-culture of target cells, effector cells and test antibody
Preparing antibodies to be detected with different concentrations by using EMEM complete culture medium, starting from 357nM, diluting with 3-fold gradient to 0.1nM, adding 50ul of diluted antibody solution into each well of 96-well plate with formed tumor spheres, respectively, and adding 5% CO at 37 deg.C2The culture was carried out in an incubator for 48 hours.
Example 4: fluorescent dye PI labeling dead cells
After 48 hours of co-culture, the plates were removed and 5ul of PI staining solution at 40 Xworking concentration was added to each well and gently pipetted and mixed.
Example 5: high content imaging analysis
And (3) using an Operetta CLS PreciScan function, firstly, scanning the sample by using a low power mirror, finding out a target object by using an editing algorithm, and then, shooting the target object by using a high power mirror. And respectively taking a 350nm excitation light channel in the same visual field to collect Hoechst 33342 fluorescence, collecting Calcein Green fluorescence in a 494nm excitation light channel, collecting PI fluorescence in a 535nm excitation light channel, and respectively obtaining a single or multiple fluorescence superposition imaging picture. The same field of view is scanned in multiple slices, and a Z-stack image set is acquired, wherein the step of Z slice is 5 mu m and covers at least half of the cell sphere volume. The high content imaging results are shown in fig. 1-3, and fig. 1 is an image showing the killing efficacy of T cells on tumor spheres mediated by TCE antibodies at different concentrations under the superposition of three fluorescence channels. The concentration of antibody from left to right was sequentially reduced from 357nM to 0.1nM by a factor of three, showing blue fluorescence as tumor spheres, and as the antibody concentration increased, a decrease in tumor volume was observed. The red color indicates the imaging of the dye PI of dead cells, and compared with an isotype control antibody, the red fluorescent signal of the TCE antibody group is obviously enhanced, which indicates that the TCE antibody group has the specific killing of target cells. FIG. 2 shows the T cell spheroid infiltration by TCE antibody at different concentrations in the excitation light channel at 494 nm. From left to right the concentration of antibody was sequentially reduced from 357nM to 0.1nM, and a higher degree of T cell infiltration was observed in the TCE antibody group compared to the isotype control group. FIG. 3 shows the number of target cell deaths from different concentrations of TCE antibody in the 535nm excitation light channel.
When the 3D tumor sphere volume was analyzed using the Harmony 4.9 high content imaging and analysis software, the TCE antibody-mediated tumor specific killing rate was ═ tumor sphere volume in the no antibody group-tumor sphere volume in the test antibody-treated group/tumor sphere volume in the no antibody group 100, and the results are shown in fig. 4, where the TCE antibody showed specific killing of tumor spheres and the killing efficacy was concentration-dependent.
Claims (8)
1. A method for detecting killing effect of T cells by using a 3D cell imaging analysis system is characterized by comprising the following steps:
1) constructing a three-dimensional tumor sphere by applying a 3D cell culture technology;
2) co-culturing the target cells, the effector cells and the TCE antibody, performing dyeing marking on the target cells by using a fluorescent marker A, performing dyeing marking on the effector cells by using a fluorescent marker B, and performing dyeing marking on dead cells generated after co-culturing by using a fluorescent marker C;
3) applying a high content imaging analysis system, and respectively using an excitation light channel corresponding to the fluorescent marker A, an excitation light channel corresponding to the fluorescent marker B and an excitation light channel corresponding to the fluorescent marker C to acquire images of the same visual field;
4) performing single or superposition analysis on the obtained image by using a software analysis system, and calculating the killing effect of the T cells on the target cells according to the size of the tumor sphere;
the effector cell is a human T lymphocyte or a human peripheral blood mononuclear cell; the excitation light wavelengths corresponding to the fluorescent marker A, the fluorescent marker B and the fluorescent marker C are different.
2. The method of claim 1, wherein the fluorescent marker a is a fluorescent protein or/and a cellular dye.
3. The method of claim 2, wherein the fluorescent protein is green fluorescent protein or/and red fluorescent protein.
4. The method of any one of claims 1-3, wherein the fluorescent marker B is a live cell staining reagent.
5. The method of claim 4, wherein the fluorescent marker B is one or more of carboxyfluorescein diacetate succinimidyl ester, a far-red dye, a calcein green dye, a calcein violet dye, a calcein blue dye, and a calcein orange-red dye.
6. The method of any one of claims 1-3, wherein the fluorescent label C is one or more of propidium iodide, cysteine protease, cyanine, 7-amino-actinomycin D, and annexin-V.
7. The method of any one of claims 1-3, wherein the image acquisition comprises one or more of fluorescent marker A, fluorescent marker B, and fluorescent marker C channel.
8. A method according to any one of claims 1 to 3, wherein the analysis method in step 4) comprises the steps of: obtaining the specific killing efficiency of the target cells by analyzing the volume of the 3D tumor sphere; tumor sphere volume in antibody-free group-tumor sphere volume in test antibody-treated group/tumor sphere volume in antibody-free group 100.
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| CN115855606A (en) * | 2022-12-07 | 2023-03-28 | 上海药明生物技术有限公司 | Method for detecting CAR-T cell infiltration in solid tumor by using 3D model |
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