CN115855606A - Method for detecting CAR-T cell infiltration in solid tumor by using 3D model - Google Patents
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Abstract
The invention discloses a method for detecting the infiltration of CAR-T cells in solid tumors by using a 3D model, which is based on an in-vitro 3D tumor microsphere culture system and used for evaluating the infiltration capacity of the CAR-T cells to the solid tumors by using the technologies such as CAR-T fluorescent labeling, high content confocal photographing, 3D model analysis and the like. The method has the advantages of low cost, short time, large flux, high precision and the like, and the characteristics of clinical tumor microenvironment, physical barrier and the like are simulated in the whole culture process, the experimental result reflects the condition of CAR-T in clinical application more truly, and the method has great promotion effect on discovering CAR-T cell modification types with stronger infiltration capacity of solid tumors.
Description
Technical Field
The invention relates to a method for detecting cells, in particular to a method for detecting CAR-T cells.
Background
Currently, tumor immunotherapy has become the fastest growing aspect of the cancer field, and cell therapy techniques play an important role, mainly including CAR-T, CAR-NK, TCR-T, TIL, etc. types. Chimeric antigen receptor T cells (CAR-T) recognize tumor specific/associated antigens primarily through their surface chimeric antigen receptors, and then induce activation signals within T cells, initiating specific killing of tumor cells. CAR-T is already marketed in various products for hematological tumors, clinically shows a remarkable and lasting therapeutic effect, the application of CAR-T in solid tumors is widely researched, and various products are in preclinical and clinical research stages. However, from the results available, CAR-T is significantly less effective in solid tumors than in hematological tumors, mainly involving three factors: 1. solid tumors are heterogeneous, hematological tumors usually express a single, tumor-specific/associated antigen, the CAR-T aiming at the related antigen can efficiently and comprehensively kill tumor cells, and the solid tumor has obvious antigen heterogeneity; 2. the solid tumor has an immunosuppressive microenvironment, a plurality of immunosuppressive cells and higher-concentration immunosuppressive factors are distributed in a tumor mass, and the killing function of the CAR-T cells in the tumor is weakened; 3. the solid tumor has a physical barrier, and the components of tumor cells, fibroblasts, cell matrixes and the like prevent CAR-T cells in blood from effectively entering, so that the probability of recognizing tumor antigens is reduced.
Various research institutions and companies have made extensive alterations to CAR-T in relation to its problems encountered in the treatment of solid tumors, where increasing CAR-T is an important aspect of solid tumor infiltration. Research has shown that the over-expression of CCL19 on CAR-T cells can obviously increase the intratumoral infiltration level, and the CAR-T cells also show stronger tumor inhibition effect on animal models. In addition to CCL19, various chemokines or chemokine receptors such as CCL21, CXCR6, CXCR4, etc. can increase CAR-T infiltration into solid tumors, and researchers are also developing various new strategies to increase CAR-T infiltration capacity.
The ability of CAR-T cells to infiltrate solid tumors is currently evaluated, primarily by virtue of tumor immunohistochemical staining in animal models. Firstly, tumor-bearing mice are constructed, then the CAR-T cells are intravenously administered, tumor tissues are taken for T cell immunohistochemical staining after a period of time, and the infiltration capacity of different modified CAR-T cells on solid tumors is distinguished by observing the staining intensity of the T cells in the tumors. However, the method needs to construct a uniform animal model, has high cost, great difficulty and long period, and is difficult to evaluate the CAR-T modification strategy in a large-scale and high-throughput manner. Another evaluation method is to use in vitro cell migration Transwell assay, in which CAR-T cells 2D are cultured on one plane with microwells and after a period of time the number of CAR-T cells that pass through the microwells to the other side is measured to indirectly evaluate the infiltration capacity of different CAR-T cells for solid tumors. Although the cost of the method is lower than that of an animal experiment, the period is short, the CAR-T cells cultured by 2D alone are difficult to truly simulate the condition of solid tumor, no target cell exists, no tumor microenvironment exists, no physical barrier of the solid tumor exists, a cell culture plate with more than 24 holes is needed to reduce the error in the holes, the high-throughput operation difficulty is high, and the experimental result is difficult to reflect the clinical condition.
Based on the defects, a method for detecting the infiltration of CAR-T cells in solid tumors by using 3D tumor microspheres is developed. The method comprises the steps of co-culturing tumor cells and fibroblasts to form compact 3D tumor microspheres, constructing a tumor microenvironment and a solid tumor physical barrier, adding fluorescence-labeled CAR-T cells, performing cell transparentization after culturing for a period of time, and obtaining CAR-T cell fluorescence intensity information inside the tumor microspheres by using 3D high content scanning imaging and a specific analysis method so as to reflect infiltration capacity of different modified CAR-T cells. The method can be carried out in a low-adsorption 96-well plate, has high flux, low cost and short time, is favorable for efficiently evaluating the infiltration capacity of various CAR-T on solid tumors, and helps to find candidate CAR-T with the most clinical application potential for development.
Disclosure of Invention
The invention aims to overcome the defects that the traditional method has high cost and low flux, can not accurately reflect the clinical real condition and the like. According to the invention, the 3D tumor microspheres formed by mixing tumor cells into the fibroblasts are constructed, the tumor microenvironment and the physical barrier are established, and the condition met by CAR-T in clinic is simulated. And then rapidly, accurately and visually evaluating the infiltration level of CAR-T in the 3D microspheres by methods of fluorescence labeling, tumor microsphere transparentization treatment, high content confocal scanning, tomography analysis and the like, thereby providing a solid technical support for the development of CAR-T in solid tumors. The 3D tumor microsphere culture technology and the cell imaging analysis method are applied to detect the infiltration degree of the conventional and modified CAR-T cells to the tumor microspheres and reflect the infiltration capacity of different modified CAR-T cells in the solid tumor.
In order to realize the purpose, the invention adopts the following technical scheme: a method for detecting the infiltration of CAR-T cells in solid tumors by using a 3D model is based on an in-vitro 3D tumor microsphere culture system, and the infiltration capacity of the CAR-T cells to the solid tumors is evaluated by using technologies such as CAR-T fluorescent labeling, high content confocal photography, 3D model analysis and the like, and specifically comprises the following steps:
1. co-culturing tumor cells and fibroblasts in a low-adsorption U-shaped bottom plate to form 3D tumor microspheres and establish a tumor microenvironment and a physical barrier;
2. the CAR-T cells are subjected to harmless fluorescent staining in advance, and target cells are marked, so that subsequent fluorescent observation and quantitative analysis are facilitated;
3. fixing the 3D tumor microspheres by using paraformaldehyde, and performing transparentization treatment to ensure that CAR-T fluorescence can penetrate the microspheres to be accurately detected;
4. using a high content confocal tomography scanning mode to obtain CAR-T infiltration information of different layers of the microsphere;
5. and establishing an analysis method for a core layer, removing the interference of combining the CAR-T on the surface of the sphere, analyzing the CAR-T fluorescence intensity in different internal ranges, and more accurately evaluating the level of CAR-T cells infiltrated in the interior of the tumor microsphere.
Furthermore, the tumor cells used in the present invention are CAR-T target cells, and may be cell lines overexpressing fluorescent protein and target antigen or endogenously expressing target antigen, such as BxPc-3, NCI-N87, SHP-77, NCI-H82, MC38, LS174T and/or HepG 2.
Further, the fibroblasts used may be HDF α or MRC-5 cell lines expressing different fluorescent proteins.
Further, the CAR-T cell fluorescent labeling reagent may be a fluorescent dye that is not damaged to cells by Far infrared cell fluorescent dye (Far Red) or CFSE, etc., and can be maintained for a long time without being quenched by paraformaldehyde.
Further, the sensitivity of fluorescence detection can be increased by fixing paraformaldehyde and performing a transparentization treatment, and the transparentization reagent can be selected from commercial products such as Visikol HISTO-M or Cytovista 3D Cell.
Furthermore, the cell is subjected to a clearing treatment using a clearing reagent, which mainly dissolves a part of lipids, proteins, and the like in the cell that affect light transmission, and allows light to more efficiently penetrate the cell and be captured.
Further, fluorescence imaging of 3D tumor microspheres uses a confocal slice scan mode with 8um spacing to ensure that CAR-T cells of about 4um diameter are clearly captured by only one slice. In the analysis process, the image edge 20% range is removed, the influence of CAR-T cells bound to the surface of the microsphere on internal cell statistics is prevented, a more real and accurate experiment result is obtained, and the infiltration capacity of different types of CAR-T cells is distinguished.
The invention provides a method for evaluating the infiltration capacity of CAR-T cells to solid tumors based on an in-vitro 3D tumor microsphere culture system by combining technologies such as fluorescence labeling, high content confocal photography, 3D model analysis and the like. Compared with the traditional method, the method has the advantages of low cost, short time, large flux, high precision and the like, and the characteristics of a clinical tumor microenvironment, a physical barrier and the like are simulated in the whole culture process, so that the experimental result more truly reflects the condition of CAR-T in clinical application, and the method has a great promoting effect on the discovery of CAR-T cell modification types with stronger solid tumor infiltration capacity.
Drawings
Fig. 1 shows the construction of 3D tumor microspheres and the infiltration phenomenon after CAR-T co-culture for 24 hours-the results show that tumor cells and fibroblasts can form microspheres with a diameter of more than 500um, CAR-T co-culture has an obvious infiltration phenomenon, and CAR-T-4th group shows enhanced infiltration capacity due to the ability to secrete CCL19, consistent with literature reports.
Figure 2 is CAR expression levels of different types of CAR-T cells-results show that CAR-T cells used in infiltration experiments have higher CAR expression levels, which can meet experimental requirements after adjusted to the same CAR positive rate.
Figure 3 is CAR-T infiltration level indicated by Far infrared cell fluorescence (Far Red) before/after clearing treatment-results show that clearing treatment significantly increased CAR-T fluorescence signal intensity, increased signal sensitivity, and the overall tendency to infiltrate remained the same as before clearing treatment.
Figure 4 is a data processing and analysis of 80% microsphere interior region selected-results show that transparentization significantly increased CAR-T fluorescence signal intensity, and CAR-T-4th modification significantly increased infiltration levels; a shows that we have selected 80% of the area inside the microspheres for analysis; and B is the selection of CAR-T: CAR-T infiltration level profile of inner 80% area at target cell = 1:1; c is CAR-T: CAR-T infiltration level analysis plot of inner 80% area at target cell = 3:1.
FIG. 5 shows the selection of different infiltration ranges of the 3D microspheres from the center of the sphere by CAR-T cells and the corresponding analysis results, wherein A is the inner range of the tumor microspheres selected for analysis, which can be divided into an outer layer, a middle layer and an inner layer, each of which accounts for 20% of the total sphere; b is the level of fluorescently labeled CAR-T infiltrated by the outer layer of the sphere; c is the level of the layer-infiltrated fluorescently labeled CAR-T in the sphere; d is the level of fluorescently labeled CAR-T infiltrated by the inner layer of the sphere.
Figure 6 is an analysis of the extent of infiltration of different types of CAR-T cells over different ranges of 3D microspheres from the center of the sphere-the results show that CAR-T-4th engineered types significantly increased the level of infiltration and, from the center of the sphere outward, the signal was progressively enhanced, consistent with the animal level and clinical phenomenon reported publicly.
Detailed Description
The materials, instruments and reagents used in this example are shown in Table 1.
Example 1:
1.3D tumor microsphere culture
The method comprises the following steps: selecting a BxPC-3-GFP pancreatic cancer cell line with green fluorescence and a HDF alpha-mCherry fibroblast line with yellow fluorescence, mixing the two cells according to the number of 1.2 ten thousand/hole +1.2 ten thousand/hole, adding the mixture into a 96-hole low-adsorption U bottom plate, wherein the culture medium is a conventional RPMI1640 complete culture medium, and then placing the mixture in a 5% CO2 incubator at 37 ℃ for 24 or 48 hours to enable the two cells to form a 3D tumor microsphere.
As a result, as shown in FIG. 1, it can be seen that two kinds of cells form a sphere after 48 hours of culture.
CAR-T cells fluorescent staining
The method comprises the following steps: different types of CAR-T cells cultured for more than 14 days, including activated and untransfected T cells (ATC) were taken as a low-infiltration control group, 4 generations of CAR-T that can secrete CCL19 to promote CAR-T solid tumor infiltration (CAR-T-4 th) as a high-infiltration control group, the same 2 generations of CAR-T that do not secrete CCL19 (CAR-T-2 nd) as a potential intermediate-infiltration group, and another 2 generations of positive control CAR-T (CAR-T-PC) as a potential intermediate-infiltration group. The main purpose is to distinguish the infiltration capacity of different types of CAR-T cells and verify the reliability and sensitivity of the method by the invented method.
The results are shown in FIG. 1, where it can be seen that different kinds of CAR-T cells all have a considerable degree of red fluorescence.
CAR-T cells coculture with tumor 3D microspheres
The method comprises the following steps: sufficient CAR-T/ATC cells were removed and a portion was used for flow detection of CAR positivity to allow the stained CAR-T cells to be adjusted to the same positivity and added to the 3D tumor microspheres. During the staining process, the cells were washed once with serum-free medium, and then DPBS containing 400nM Cell Tracker Far Red fluorescent dye was added at a density of 1E6 cells/mL and placed in a 5% CO2 incubator at 37 ℃ for 20 minutes. After the staining was completed, 5 times the volume of the complete medium RPMI1640 was added, and the mixture was left at 37 ℃ in a 5% CO2 incubator to terminate the staining for 5 minutes. Followed by 350g,22 ℃, centrifugation for 8 minutes, removal of the supernatant, and addition of the same volume of RPMI1640 complete medium for another wash to completely remove the residual fluorescent dye. Finally, different types of CAR-T cells were adjusted to the same positive rate, added to 3D tumor microspheres at a ratio of CAR + T: tumor cells 3.
Results as shown in figure 2, different cells were flow-detected to have different CAR positivity rates and then adjusted to the same positivity rate and incubated with tumor microspheres for 24 hours. By confocal fluorescence imaging techniques, as shown in figures 1 and 3, it can be seen that different kinds of CAR-T have different levels of infiltration.
4. Fixing and transparentizing treatment of tumor microspheres
The method comprises the following steps: after 24 hours of co-culture, the microspheres were washed once with 200 ul/well DPBS, and then 4% paraformaldehyde was added and fixed for 30 minutes at room temperature in the dark. After fixation is finished, the microspheres are washed twice by 200 ul/hole DPBS, then 200 ul/hole cell clearing reagent Visikol HISTO-M is added, the mixture is incubated for 2 hours at room temperature in a dark place, and then the mixture is centrifuged for 8 minutes at 450g and 22 ℃ so that the microspheres are completely positioned at the bottom of a U-shaped bottom plate with 96 holes, and then photographing is carried out.
As a result, as shown in FIG. 3, more CAR-T cell infiltration (increased red fluorescence intensity) was observed after the fixation and clearing treatment than before the fixation and clearing treatment.
5. High content confocal photography and results analysis
The method comprises the following steps: after the 3D microspheres were clarified and centrifuged to the bottom, photographs were taken and data analyzed using a Perkinelmer Operetta CLS High Content. Adopting a confocal mode, scanning 25 layers from bottom to top at intervals of 8um, selecting 5 middle layers for analysis and calculation, removing 20% of the edge area, eliminating the interference of CAR-T combined on the surface of the microsphere on the result, obtaining the infiltration results in different ranges (far, medium and near) from the center of the sphere, and comprehensively judging the infiltration strength of different types of CAR-T. Table 1 shows the materials, instruments and reagents used in this example.
Results as shown in figure 4, if 80% of the area within the tumor microsphere was selected for analysis in its entirety, CAR-T target cells =1:1 and 3:1, both engineered CAR-T cells (CAR-T4 th) had enhanced levels of infiltration compared to pre-engineered CAR-T (CAR-T PC and CAR-T2 nd), consistent with the expected results. As shown in FIG. 5, if 80% of the area in the tumor microsphere was subdivided into 3 regions, i.e., outer, middle and inner, and analyzed separately, it was found that each layer was modified CAR-T (CAR-T4 th) with enhanced level of infiltration, consistent with the expected results. As shown in FIG. 6, comparing the results of the infiltration analysis of 3 regions of the outer layer, the middle layer and the inner layer, it can be seen that the infiltration intensity is outer layer > middle layer > inner layer for any CAR-T cell, which is consistent with the expected results and clinical phenomena, thus the detection and analysis method is reliable, can reflect the actual situations and has high application value.
Table 1.
Claims (9)
1. A method for detecting CAR-T cell infiltration in solid tumors using a 3D model, comprising the specific steps of:
1) Co-culturing tumor cells and fibroblasts in a low-adsorption U-shaped bottom plate to form 3D tumor microspheres and establish a tumor microenvironment and a physical barrier;
2) The CAR-T cells are subjected to harmless fluorescent staining in advance, and target cells are marked, so that subsequent fluorescent observation and quantitative analysis are facilitated;
3) Fixing the 3D tumor microspheres by using paraformaldehyde, and performing transparentization treatment to ensure that CAR-T fluorescence can penetrate the microspheres to be accurately detected;
4) Using a high content confocal tomography scanning mode to obtain CAR-T infiltration information of different layers of the microsphere;
5) And establishing an analysis method for a core layer, removing the interference of combining the CAR-T on the surface of the sphere, analyzing the CAR-T fluorescence intensity in different internal ranges, and more accurately evaluating the level of CAR-T cells infiltrated in the interior of the tumor microsphere.
2. The method of claim 1, wherein the tumor cell is a target cell of CAR-T, and the 3D model is used to detect CAR-T cell infiltration in a solid tumor.
3. Method for detecting CAR-T cell infiltration in solid tumors with 3D models according to claim 1 or 2, characterized in that said tumor cells are cell lines overexpressing fluorescent proteins or/and target antigens or endogenously expressing target antigens.
4. The method of claim 3, wherein the target antigen or the cell line endogenously expressing the target antigen is BxPC-3, NCI-N87, SHP-77, NCI-H82, MC38, LS174T and/or HepG 2.
5. Method for detecting CAR-T cell infiltration in solid tumors with 3D model according to claim 1 or 2, characterized in that said fibroblasts are hdfa or/and MRC-5 cell lines expressing different fluorescent proteins.
6. The method for detecting CAR-T cell infiltration in solid tumors using 3D model according to claim 1 or 2, wherein the reagents used in step 2) for harmless fluorescent staining of CAR-T cells are far infrared cytofluorescent dye or/and CFSE.
7. Method for the detection of CAR-T Cell infiltration in solid tumors using 3D models according to claim 1 or 2, characterized in that the reagent used for the clearing treatment in step 3) is Visikol HISTO-M or/and CytoVista 3D Cell.
8. The method for detecting CAR-T cell infiltration in solid tumors using 3D model as claimed in claim 1 or 2, wherein in step 4) fluorescence imaging of 3D tumor microspheres uses a confocal slice mode with 8um spacing to ensure that CAR-T cells of about 4um diameter size are clearly captured by only one slice.
9. The method for detecting CAR-T cell infiltration in solid tumors using 3D models as claimed in claim 1 or 2, wherein in step 5), the core layer analysis removes 20% of the image margin, preventing CAR-T cells bound to the microsphere surface from affecting the internal cell statistics.
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