Method for predicting CAR-T killing effect in vitro based on NO probe cell imaging

Abstract

The invention discloses a method for predicting a CAR-T killing effect in vitro based on NO probe cell imaging, and relates to the field of immunotherapy curative effect evaluation. The method comprises the following steps: detecting the expression quantity of CD58 in cells through virus infection, a protein imprinting experiment and a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) experiment, then incubating tumor cells in vitro through an NO fluorescent probe, imaging to detect a fluorescent signal, finally co-culturing CAR-T and the tumor cells in vitro, and evaluating the killing effect through flow cytometry. The expression level of CD58 is reflected through the intensity of a fluorescence signal generated when the NO probe acts on tumor cells, and the killing effect of CAR-T is reflected through the expression level of CD58. According to the invention, the NO probe technology is utilized for simple, sensitive, rapid and visual detection, and the killing activity of the CAR-T is evaluated in vitro, so that a basis is provided for follow-up in-vivo killing treatment of the CAR-T.

Description

Technical Field

[0001] The present invention relates to the field of living cell imaging, and in particular to a method for predicting CAR-T killing activity in vitro. Background Art

[0002] Chimeric Antigen Receptor T Cells (CAR-T) are genetically engineered T cells that express chimeric antigen receptors (CARs) on their surface that specifically recognize tumor cell antigens, giving CAR-T cells the ability to kill tumor cells with antigen-specificity.

[0003] Factors influencing the efficacy of CAR-T therapy include the target selectivity of CAR-T cells, the tumor microenvironment, and the drug resistance genes of the tumor cells themselves. CD58, a key immune surveillance gene, is frequently deleted in various hematologic malignancies. Its loss can significantly affect the killing effect of CAR-T cells, leading to drug resistance in patients. Its gene expression status has become a key factor in evaluating CAR-T treatment outcomes.

[0004] Patent CN116745320A "Method for diagnosing or treating a health condition or optimizing the therapeutic efficacy of CAR-T cell therapy" discloses a method for diagnosing the therapeutic efficacy of CAR-T cell therapy, which measures the expression level of CD58 by immunohistochemistry (IHC), protein microarray, western blot, mass spectrometry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, multiple detection assays, etc. The expression level of CD58 in the sample is analyzed by comparing the expression level of CD58 in the sample with the reference expression level. Ultimately, the therapeutic effect of CAR-T cell therapy is determined by the expression level of CD58 in the sample.

[0005] Previous studies have found that the expression level of CD58 in tumor cells significantly affects metabolism-related pathways, including nitric oxide (NO) biosynthesis and metabolism-related signaling pathways. The intensity of the fluorescence signal generated when the NO probe acts on tumor cells is significantly positively correlated with the expression level of CD58 and can be significantly associated with the killing effect of CAR-T. Strong fluorescence signals reflect the high activity and efficient killing effect of CAR-T. In order to overcome the above problems, the present application creatively selects blood system tumor cells that express CAR-T therapeutic target antigens. Before the start of CAR-T treatment in vivo, these tumor cells are co-cultured with CAR-T cells, and the NO probe technology is used for simple, sensitive, rapid and intuitive detection to evaluate the killing activity of CAR-T in vitro, thereby providing a basis for subsequent CAR-T killing in vivo treatment. Summary of the Invention

[0006] The purpose of this study is to use NO probes to correlate CD58 expression levels on tumor cells and simultaneously reflect the anti-tumor efficacy of CAR-T cells. This allows for real-time dynamic assessment of the early activation state of CAR-T cells, study the interaction between CAR-T cells and the tumor microenvironment (TME), and evaluate the functional sustainability of CAR-T cells.

[0007] The specific steps of the present invention are: (1) Infect cells with lentivirus, centrifuge, and then culture the cells. After drug screening, construct diffuse large B-cell lymphoma cell lines that overexpress and knock down CD58 expression levels, and the target antigen expressed by the tumor cells is the target antigen specifically recognized by the CAR-T to be tested; (2) Western blotting and real-time fluorescence quantitative PCR were used to detect the expression of CD58 in cells overexpressing or knocking down CD58; (3) Incubate tumor cells with NO fluorescent probe in vitro and detect the fluorescence signal by imaging; (4) CAR-T cells were co-cultured with the above tumor cells in vitro, and the killing effect was evaluated by flow cytometry. At the same time, the correlation between the killing effect of CAR-T cells on tumor cells and the fluorescence signal intensity of the NO probe was evaluated.

[0008] Preferably, the CAR-T cells are immune cells that express chimeric antigen receptors on the cell surface that can specifically recognize tumor cell antigens and acquire the ability to kill tumor cells with antigen specificity.

[0009] CAR-T cells are genetically engineered T cells that express chimeric receptors that specifically recognize tumor antigens. They can be modified to target different types of tumors to suit specific needs.

[0010] Preferably, the immune cells are one or more of T cells, CIK cells, K cells, TIL cells or DC cells.

[0011] Immune cells can identify and eliminate pathogens and abnormal cells, and maintain the body's homeostasis, playing a huge role in human health.

[0012] Preferably, the culture conditions in step (1) are culture in a constant temperature incubator at 37°C and 5% CO2.

[0013] Under these conditions, cells can divide and proliferate rapidly.

[0014] Preferably, the time for the Chinese medicine screening process in step (1) is 48-72 hours.

[0015] A longer drug screening time can ensure that all screened tumor cells are drug-resistant, which is convenient for subsequent experiments.

[0016] Preferably, the lymphoma cells are hematological tumor cell lines.

[0017] Preferably, the tumor cells are selected from diffuse large B-cell lymphoma cells, T-lymphocytic leukemia, acute T-lymphocytic leukemia, etc.

[0018] Preferably, the final concentration of cDNA obtained in the real-time fluorescence quantitative PCR experiment in step (2) is 50 ng / μL.

[0019] Preferably, the imaging process is performed with 495 nm as the excitation wavelength and 515 nm as the emission wavelength.

[0020] Fluorescent probes have better imaging effects at this wavelength.

[0021] Preferably, the effector-target ratio during the verification of the CAR-T cell killing effect is 1:2.

[0022] Beneficial effects:

[0023] As an emerging technology, fluorescent probe monitoring directly observes the spatial distribution of NO signals through fluorescence imaging at specific excitation and emission wavelengths after specific binding to target molecules. Without the need to lyse cells or terminate the reaction, the same batch of cells can be monitored continuously for several days, making it suitable for evaluating CAR-T's persistent killing or tumor recurrence models. This avoids sample loss caused by multiple sampling in traditional methods. In 3D models such as organoids and tumor spheres, NO imaging can penetrate multiple layers of cells and more accurately reflect deep-layer killing effects than endpoint methods. Ultimately, highly selective, highly sensitive, and dynamic visual monitoring is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below with reference to the accompanying drawings.

[0025] Figure 1 This is the CD58 expression level detection diagram of the constructed CD58 overexpression, CD58 knockdown and control group OCI-LY1 cells (WB).

[0026] Figure 2 NO probe fluorescence imaging images of OCI-LY1 cells overexpressing CD58, knocking down CD58, and the control group (***, P < 0.001; ****, P < 0.0001).

[0027] Figure 3 Flow cytometry was used to detect the killing efficiency of CD19 CAR-T cells against OCI-LY1 cells (CD58 overexpression, CD58 knockdown, and control group) (representative graph of three repeated experiments) and statistical graph of three repeated experiments (****, P<0.0001).

[0028] Figure 4 Correlation diagram between the fluorescence intensity of OCI-LY1 cells incubated with NO probe and the efficiency of CD19 CAR-T cells in killing tumor cells (R=0.9344)

[0029] Figure 5 The CD58 expression level detection chart of the constructed U2932 cells overexpressing CD58, knocking down CD58 and control group (WB) (****, P<0.0001).

[0030] Figure 6 NO probe fluorescence imaging images of U2932 cells overexpressing CD58, knocking down CD58, and the control group (****, P<0.0001).

[0031] Figure 7 Flow cytometry was used to detect the killing efficiency of CD19 CAR-T cells against U2932 cells (CD58 overexpression, CD58 knockdown, and control group) (representative graph of three repeated experiments) and statistical graph of three repeated experiments (****, P<0.0001).

[0032] Figure 8 Correlation diagram between the fluorescence intensity of U2932 cells incubated with NO probe and the efficiency of CD19 CAR-T cells in killing tumor cells (R=0.8969) DETAILED DESCRIPTION

[0033] In order to better illustrate the technical effects of the present invention, the following is an analysis in conjunction with specific embodiments.

[0034] Overall embodiment:

[0035] 1. Virus-infected cells

[0036] Lentivirus-infected cells were used to construct CD58 overexpression and knockdown cell lines. The specific steps are as follows: (1) The cells to be infected were centrifuged, counted, and inoculated into 12-well plates and divided into 4 groups: ① control group (uninfected with virus and not screened with drugs), ② uninfected with virus and screened with drugs, ③ infected with control virus and screened with drugs, and ④ infected with target lentivirus and screened with drugs; (2) Prepare concentrated lentivirus and add it to cells infected with lentivirus or control virus in a gradient manner. At the same time, add infection enhancer; (3) Seal the 12-well plate and centrifuge; (4) After centrifugation, continue culturing in a constant temperature incubator. After a period of culturing, add the corresponding complete culture medium to each well and continue culturing; (5) After the cells are infected, puromycin (Puro) at a concentration corresponding to the cells used is added to each well to perform drug screening on the cells. The drug screening is carried out for 48-72 hours until the added Puro completely kills the cells in the control wells that are not infected with the virus. The cells in the wells infected with the lentivirus that are not killed by Puro continue to be cultured. The puromycin concentration is halved, the cells are expanded and cultured, and mixed clone cell lines with knockdown of the target gene are screened. Depending on the cell quantity, the cells can be transferred to T25 or T75 culture flasks for further culture.

[0037] 2. Verification of CD58 overexpression and knockdown (1) Protein imprinting experiment Western blot assay was performed to detect the expression of CD58 in OCI-LY1 cells with or without CD58 overexpression. The specific steps are as follows: After the cells were extracted, they were washed once with 1×PBS, and lysed on ice with RIPA lysis buffer. After quantification using BCA, the protein was qualitatively analyzed by SDS-Page gel electrophoresis, with the primary antibody being CD58 and the secondary antibody being the corresponding mouse or rabbit secondary antibody, and the results were obtained by development. (2) Real-time fluorescence quantitative PCR experiment The expression level of CD58 in OCI-LY1 cells with overexpression or knockdown of CD58 was detected by real-time fluorescence quantitative PCR experiment. The specific steps are as follows: After extraction, the cells were washed once with 1× PBS, and RNA was extracted. The concentration was measured using Nanodrop, and a reverse transcription system was configured to obtain a final cDNA concentration of 50 ng / μL. A real-time fluorescence quantitative PCR system was configured, and qPCR results were obtained using an Applied Biosystems QuantStudio 3 real-time fluorescence quantitative PCR instrument.

[0038] 3. NO probe fluorescence imaging of tumor cells

[0039] Use a live imaging device to perform NO probe fluorescence imaging on cells. The specific steps are as follows: (1) Collect the cells, count them, and wash them once with 1× PBS; (2) Prepare DAF-FM DA probe at a concentration of 5 μM / L; (3) Add DAF-FM DA probe to the ep tube, resuspend the cells, and image with an excitation wavelength of 495 nm and an emission wavelength of 515 nm.

[0040] 4. Verification of CAR-T cell killing effect

[0041] The CD19 CAR-T cell killing level was detected by flow cytometry. The specific steps are as follows: (1) Centrifuge, discard the supernatant, resuspend the cells in 1×PBS, and adjust the cell density to 1×10 6 / mL, add CFSE working solution (50mM) to the cell sample to stain the living cells, protect from light, incubate at room temperature, add an equal volume of serum, let it stand at room temperature for 1min, and terminate the reaction; (2) Count CD19 CAR-T cells and CFSE-labeled cells in each group separately. Plate CD19 CAR-T cells and CFSE-labeled OCI-LY1 cells in a 12-well plate at an effector-target ratio (E:T) of 1:2. Repeat 3 wells in each group. Add culture medium and culture for 48 hours. Negative control wells are CFSE-labeled OCI-LY1 cells in each group cultured alone. (3) After co-culture of CD19 CAR-T cells and CFSE-labeled OCI-LY1 cells for 48 h, cells from each group were collected, transferred to 1.5 mL EP tubes, and centrifuged; (4) Discard the supernatant, resuspend the cell pellet in 100 μL 1× PBS, and add Zombie NIR TM Dye FixableViability Dye dye, protect from light, incubate at room temperature; (5) After staining, add PBS to each tube, centrifuge, and wash the cells once; (6) Discard the supernatant and resuspend the cell pellet in 300 μL of 1× PBS. Transfer the cell suspension to a flow cytometer and analyze using a flow cytometer. CFSE- and Zombie NIR-positive cells are flow cytosed to identify the killed tumor cells. The percentage of tumor cells killed (%) = the percentage of tumor cells killed in the positive control (%) - the percentage of tumor cells killed in the negative control (%).

[0042] Example 1

[0043] 1. Experimental materials:

[0044] OCI-LY1 cells; virus; IMDM medium (containing 10% FBS); NO fluorescent probe; CAR-T cells; CFSE dye; Zombie NIRTM Dye Fixable Viability Dye dye; DAF-FM DA probe.

[0045] Instruments: flow cytometer; microscope.

[0046] 2. Experimental Methods

[0047] 1. Virus infection of OCI-LY1 cells

[0048] OCI-LY1 cells were infected with lentivirus to construct CD58 overexpression and knockdown cell lines. The specific steps are as follows: (1) Centrifuge the cells to be infected, count them, and calculate the number of cells to be infected. 5 / 1mL / well was inoculated into a 12-well plate and divided into 4 groups: ① control group (uninfected with virus and not screened with drugs), ② uninfected with virus and screened with drugs, ③ infected with control virus and screened with drugs, and ④ infected with target lentivirus and screened with drugs. (2) Prepare concentrated lentivirus and add it to cells infected with lentivirus or control virus in a gradient manner, adding 10 μL, 20 μL and 40 μL respectively. At the same time, add 1 μL of infection enhancer polybrene to make the final concentration 10 μg / mL; (3) Seal the 12-well plate with sealing film and centrifuge at 1000g for 2 hours; (4) After centrifugation, continue culturing in a 37°C, 5% CO2 incubator for 18 hours. After 18 hours, add 1 mL of complete culture medium corresponding to the cells to each well and continue culturing. (5) 48 hours after the cells were infected, puromycin (Puro) at a concentration corresponding to the cells used was added to each well to perform drug screening on the cells. The drug screening was performed for 48-72 hours until the added Puro completely killed the cells in the control wells that were not infected with the virus. The cells that were not killed by Puro in the cell wells infected with the lentivirus continued to be cultured. The puromycin concentration was halved, the cells were expanded and cultured, and mixed clone cell lines with knockdown of the target gene were screened. Depending on the cell quantity, the cells can be transferred to T25 or T75 culture flasks for further culture.

[0049] 2. Verification of CD58 overexpression and knockdown (1) Protein imprinting experiment Western blot assay was performed to detect the expression of CD58 in OCI-LY1 cells with or without CD58 overexpression. The specific steps are as follows: After the cells were extracted, they were washed once with 1×PBS, and 100 μL of RIPA lysis buffer was added to each 1 mL ep tube. The cells were lysed on ice for 30 min. After quantification using BCA, the proteins were qualitatively analyzed by SDS-Page gel electrophoresis, with the primary antibody being CD58 and the secondary antibody being the corresponding mouse or rabbit secondary antibody, and the results were obtained by development. (2) Real-time fluorescence quantitative PCR experiment Real-time fluorescence quantitative PCR experiments were performed to detect the expression of CD58 in OCI-LY1 cells that overexpressed or knocked down CD58. The specific steps are as follows: After extraction, the cells were washed once with 1× PBS, and RNA was extracted using the Easy Tissue & Cell RNA Rapid Extraction Kit. The concentration was measured using Nanodrop, and a reverse transcription system was configured to obtain a final cDNA concentration of 50 ng / μL. A real-time fluorescence quantitative PCR system was configured, and qPCR results were obtained using an Applied Biosystems QuantStudio 3 real-time fluorescence quantitative PCR instrument.

[0050] 3. NO probe fluorescence imaging of tumor cells

[0051] Use a live imaging device to perform NO probe fluorescence imaging on cells. The specific steps are as follows: (1) After collecting the cells, count them and calculate 1×10 6 -2×10 7 cells, and washed once with 1× PBS; (2) Prepare DAF-FM DA probe at a concentration of 5 μM / L; (3) Add 700 μL of DAF-FM DA probe to the eppendorf tube, resuspend the cells, add 200 μL to each well of a 96-well plate, and image with an excitation wavelength of 495 nm and an emission wavelength of 515 nm.

[0052] 4. Verification of CAR-T cell killing effect

[0053] The CD19 CAR-T cell killing level was detected by flow cytometry. The specific steps are as follows: (1) Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in 1× PBS, and adjust the cell density to 1×10 6 / mL, add 1 μL of CFSE working solution (50 mM) to each 1 mL of cell sample to stain the living cells, protect from light, incubate at room temperature for 15-30 min, add an equal volume of serum, let it stand at room temperature for 1 min, and terminate the reaction; (2) Count the CD19 CAR-T cells and CFSE-labeled OCI-LY1 cells in each group, and add 2.5×10 5 CD19CAR-T cells and 5×10 5 CFSE-labeled OCI-LY1 cells were plated in 12-well plates at an effector-target ratio (E:T) of 1:2. Three wells were plated in each group. 1 mL of culture medium was added to each well and cultured for 48 hours. Negative control wells were cultured with CFSE-labeled OCI-LY1 cells from each group alone. (3) After co-culture of CD19 CAR-T cells and CFSE-labeled OCI-LY1 cells for 48 h, cells from each group were collected, transferred to 1.5 mL EP tubes, and centrifuged at 300 g for 8 min; (4) Discard the supernatant, resuspend the cell pellet in 100 μL 1× PBS, and add Zombie NIR TM Dye FixableViability Dye dye, protect from light, incubate at room temperature for 20 minutes; (5) After staining, add 1 mL of PBS to each tube and centrifuge at 300 g for 8 min to wash the cells. (6) Discard the supernatant and resuspend the cell pellet in 300 μL of 1× PBS. Transfer the cell suspension to a flow cytometer and analyze using a flow cytometer. CFSE- and Zombie NIR-positive cells are flow cytosed to identify the killed tumor cells. The percentage of tumor cells killed (%) = the percentage of tumor cells killed in the positive control (%) - the percentage of tumor cells killed in the negative control (%).

[0054] Example 2

[0055] 1. Experimental materials:

[0056] U2932 cells; virus; IMDM medium (containing 10% FBS); NO fluorescent probe; CAR-T cells; CFSE dye; Zombie NIR™ Dye Fixable Viability Dye dye; DAF-FM DA probe

[0057] Instruments: flow cytometer; microscope

[0058] 2. Experimental Methods

[0059] 1. Virus infection of U2932 cells

[0060] U2932 cells were infected with lentivirus to construct CD58 overexpression and knockdown cell lines. The specific steps are as follows: (1) Centrifuge the cells to be infected, count them, and calculate the number of cells to be infected. 5 / mL / well were inoculated into 12-well plates and divided into 4 groups: ① control group (uninfected with virus and not screened with drugs), ② uninfected with virus and screened with drugs, ③ infected with control virus and screened with drugs, and ④ infected with target lentivirus and screened with drugs. (2) Prepare concentrated lentivirus and add it to cells infected with lentivirus or control virus in a gradient manner, adding 10 μL, 20 μL and 40 μL respectively. At the same time, add 1 μL of infection enhancer polybrene to make the final concentration 10 μg / mL; (3) Seal the 12-well plate with sealing film and centrifuge at 1000g for 2 hours; (4) After centrifugation, continue culturing in a 37°C, 5% CO2 incubator for 18 hours. After 18 hours, add 1 mL of complete culture medium corresponding to the cells to each well and continue culturing. (5) 48 hours after the cells were infected, puromycin (Puro) at a concentration corresponding to the cells used was added to each well to perform drug screening on the cells. The drug screening was performed for 48-72 hours until the added Puro completely killed the cells in the control wells that were not infected with the virus. The cells that were not killed by Puro in the cell wells infected with the lentivirus continued to be cultured. The puromycin concentration was halved, the cells were expanded and cultured, and mixed clone cell lines with knockdown of the target gene were screened. Depending on the cell quantity, the cells can be transferred to T25 or T75 culture flasks for further culture.

[0061] 2. Verification of CD58 overexpression and knockdown (1) Protein imprinting experiment Western blot experiments were performed to detect the expression of CD58 in U2932 cells that overexpressed or knocked down CD58. The specific steps are as follows: After the cells were extracted, they were washed once with 1×PBS, and 100 μL of RIPA lysis buffer was added to each 1 mL ep tube. The cells were lysed on ice for 30 min. After quantification using BCA, the proteins were qualitatively analyzed by SDS-Page gel electrophoresis, with the primary antibody being CD58 and the secondary antibody being the corresponding mouse or rabbit secondary antibody, and the results were obtained by development. (2) Real-time fluorescence quantitative PCR experiment Real-time fluorescence quantitative PCR experiments were performed to detect the expression of CD58 in U2932 cells that overexpressed or knocked down CD58. The specific steps are as follows: After extraction, the cells were washed once with 1× PBS, and RNA was extracted using the Easy Tissue & Cell RNA Rapid Extraction Kit. The concentration was measured using Nanodrop, and a reverse transcription system was configured to obtain a final cDNA concentration of 50 ng / μL. A real-time fluorescence quantitative PCR system was configured, and qPCR results were obtained using an Applied Biosystems QuantStudio 3 real-time fluorescence quantitative PCR instrument.

[0062] 3. NO probe fluorescence imaging of tumor cells

[0063] Use a live imaging device to perform NO probe fluorescence imaging on cells. The specific steps are as follows: (1) After collecting the cells, count them and calculate 1×10 6 -2×10 7 cells, and washed once with 1× PBS; (2) Prepare DAF-FM DA probe at a concentration of 5 μM / L; (3) Add 700 μL of DAF-FM DA probe to the eppendorf tube, resuspend the cells, add 200 μL to each well of a 96-well plate, and image with an excitation wavelength of 495 nm and an emission wavelength of 515 nm.

[0064] 4. Verification of CAR-T cell killing effect

[0065] The CD19 CAR-T cell killing level was detected by flow cytometry. The specific steps are as follows: (1) Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in 1× PBS, and adjust the cell density to 1×10 6 / 1mL, add 1μL of CFSE working solution (50mM) to each 1mL of cell sample to stain the living cells, protect from light, incubate at room temperature for 15-30min, add an equal volume of serum, let it stand at room temperature for 1min, and terminate the reaction; (2) Count the CD19 CAR-T cells and CFSE-labeled U2932 cells in each group respectively, and 2.5×10 5 CD19CAR-T cells and 5×10 5 CFSE-labeled U2932 cells were plated in 12-well plates at an effector-target ratio (E:T) of 1:2. Three wells were plated in each group. 1 mL of culture medium was added to each well and cultured for 48 hours. Negative control wells were CFSE-labeled U2932 cells from each group cultured alone. (3) After co-culture of CD19 CAR-T cells and CFSE-labeled U2932 cells for 48 h, cells from each group were collected, transferred to 1.5 mL EP tubes, and centrifuged at 300 g for 8 min; (4) Discard the supernatant, resuspend the cell pellet in 100 μL 1× PBS, add Zombie NIR™ Dye Fixable Viability Dye, protect from light, and incubate at room temperature for 20 min; (5) After staining, add 1 mL of PBS to each tube and centrifuge at 300 g for 8 min to wash the cells. (6) Discard the supernatant and resuspend the cell pellet in 300 μL of 1× PBS. Transfer the cell suspension to a flow cytometer and analyze using a flow cytometer. CFSE- and Zombie NIR-positive cells are flow cytosed to identify the killed tumor cells. The percentage of tumor cells killed (%) = the percentage of tumor cells killed in the positive control (%) - the percentage of tumor cells killed in the negative control (%).

[0066] Example 3

[0067] 1. Experimental materials:

[0068] NALM-6 cells; virus; IMDM medium (containing 10% FBS); NO fluorescent probe; CAR-T cells; CFSE dye; Zombie NIRTM Dye Fixable Viability Dye dye; DAF-FM DA probe.

[0069] Instruments: flow cytometer; microscope.

[0070] 2. Experimental Methods

[0071] 1. Virus infection of NALM-6 cells

[0072] NALM-6 cells were infected with lentivirus to construct CD58 overexpression and knockdown cell lines. The specific steps are as follows: (1) Centrifuge the cells to be infected, count them, and calculate the number of cells to be infected. 5 / 1mL / well was inoculated into a 12-well plate and divided into 4 groups: ① control group (uninfected with virus and not screened with drugs), ② uninfected with virus and screened with drugs, ③ infected with control virus and screened with drugs, and ④ infected with target lentivirus and screened with drugs. (2) Prepare concentrated lentivirus and add it to cells infected with lentivirus or control virus in a gradient manner, adding 10 μL, 20 μL and 40 μL respectively. At the same time, add 1 μL of infection enhancer polybrene to make the final concentration 10 μg / mL; (3) Seal the 12-well plate with sealing film and centrifuge at 1000g for 2 hours; (4) After centrifugation, continue culturing in a 37°C, 5% CO2 incubator for 18 hours. After 18 hours, add 1 mL of complete culture medium corresponding to the cells to each well and continue culturing. (5) 48 hours after the cells were infected, puromycin (Puro) at a concentration corresponding to the cells used was added to each well to perform drug screening on the cells. The drug screening was performed for 48-72 hours until the added Puro completely killed the cells in the control wells that were not infected with the virus. The cells that were not killed by Puro in the cell wells infected with the lentivirus continued to be cultured. The puromycin concentration was halved, the cells were expanded and cultured, and mixed clone cell lines with knockdown of the target gene were screened. Depending on the cell quantity, the cells can be transferred to T25 or T75 culture flasks for further culture.

[0073] 2. Verification of CD58 overexpression and knockdown (1) Protein imprinting experiment Western blot experiments were performed to detect the expression of CD58 in NALM-6 cells that overexpressed or knocked down CD58. The specific steps are as follows: After the cells were extracted, they were washed once with 1×PBS, and 100 μL of RIPA lysis buffer was added to each 1 mL ep tube. The cells were lysed on ice for 30 min. After quantification using BCA, the proteins were qualitatively analyzed by SDS-Page gel electrophoresis, with the primary antibody being CD58 and the secondary antibody being the corresponding mouse or rabbit secondary antibody, and the results were obtained by development. (2) Real-time fluorescence quantitative PCR experiment Real-time fluorescence quantitative PCR experiments were performed to detect the expression of CD58 in NALM-6 cells that overexpressed or knocked down CD58. The specific steps are as follows: After extraction, the cells were washed once with 1× PBS, and RNA was extracted using the Easy Tissue & Cell RNA Rapid Extraction Kit. The concentration was measured using Nanodrop, and a reverse transcription system was configured to obtain a final cDNA concentration of 50 ng / μL. A real-time fluorescence quantitative PCR system was configured, and qPCR results were obtained using an Applied Biosystems QuantStudio 3 real-time fluorescence quantitative PCR instrument.

[0074] 3. NO probe fluorescence imaging of tumor cells

[0075] Use a live imaging device to perform NO probe fluorescence imaging on cells. The specific steps are as follows: (1) After collecting the cells, count them and calculate 1×10 6 -2×10 7 cells, and washed once with 1× PBS; (2) Prepare DAF-FM DA probe at a concentration of 5 μM / L; (3) Add 700 μL of DAF-FM DA probe to the eppendorf tube, resuspend the cells, add 200 μL to each well of a 96-well plate, and image with an excitation wavelength of 495 nm and an emission wavelength of 515 nm.

[0076] 4. Verification of CAR-T cell killing effect

[0077] The CD19 CAR-T cell killing level was detected by flow cytometry. The specific steps are as follows: (1) Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in 1× PBS, and adjust the cell density to 1×10 6 / mL, add 1 μL of CFSE working solution (50 mM) to each 1 mL of cell sample to stain the living cells, protect from light, incubate at room temperature for 15-30 min, add an equal volume of serum, let it stand at room temperature for 1 min, and terminate the reaction; (2) Count the CD19 CAR-T cells and CFSE-labeled NALM-6 cells in each group, and add 2.5×10 5 CD19CAR-T cells and 5×10 5 CFSE-labeled NALM-6 cells were plated in 12-well plates at an effector-target ratio (E:T) of 1:2. Three wells were plated per group. 1 mL of culture medium was added to each well and cultured for 48 hours. Negative control wells contained CFSE-labeled NALM-6 cells from each group cultured alone. (3) After co-culture of CD19 CAR-T cells and CFSE-labeled NALM-6 cells for 48 h, cells from each group were collected, transferred to 1.5 mL EP tubes, and centrifuged at 300 g for 8 min; (4) Discard the supernatant, resuspend the cell pellet in 100 μL 1× PBS, and add Zombie NIR TM Dye FixableViability Dye dye, protect from light, incubate at room temperature for 20 minutes; (5) After staining, add 1 mL of PBS to each tube and centrifuge at 300 g for 8 min to wash the cells. (6) Discard the supernatant and resuspend the cell pellet in 300 μL of 1× PBS. Transfer the cell suspension to a flow cytometer and analyze using a flow cytometer. CFSE- and Zombie NIR-positive cells are flow cytosed to identify the killed tumor cells. The percentage of tumor cells killed (%) = the percentage of tumor cells killed in the positive control (%) - the percentage of tumor cells killed in the negative control (%).

[0078] Experimental results:

[0079] 1. CD58 virus infection of OCI-LY1 cells, U2932 cells and NALM-6 cells: Western blotting results showed that CD58 virus successfully infected OCI-LY1 cells, U2932 cells and NALM-6 cells.

[0080] 2. Real-time fluorescence quantitative PCR was used to detect the expression level of CD58 in OCI-LY1 cells, U2932 cells and NALM-6 cells that overexpressed CD58, verifying that the OCI-LY1 cell line, U2932 cell line and NALM-6 cell line with CD58 overexpression and knockdown were successfully constructed.

[0081] 3. NO probe fluorescence imaging was used to detect the CD58 expression levels in overexpressed and knocked-down OCI-LY1 cells, U2932 cells and NALM-6 cells.

[0082] 4. Verification of the killing effect of CAR-T cells shows that it is feasible to use the fluorescence intensity of the NO probe as a target cell to measure the killing function of CD19CAR-T cells.

[0083] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0084] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for predicting the killing effect of CAR-T cells in vitro based on NO probe cell imaging, comprising the following steps: (1) Infect cells with lentivirus, centrifuge, and then culture the cells. After drug screening, construct diffuse large B-cell lymphoma cell lines that overexpress and knock down CD58 expression levels, and the target antigen expressed by the tumor cells is the target antigen specifically recognized by the CAR-T to be tested; (2) Western blotting and real-time fluorescence quantitative PCR were used to detect the expression of CD58 in cells overexpressing or knocking down CD58; (3) Incubate tumor cells with NO fluorescent probe in vitro and detect the fluorescence signal by imaging; (4) CAR-T cells were co-cultured with the above tumor cells in vitro, and the killing effect was evaluated by flow cytometry. At the same time, the correlation between the killing effect of CAR-T cells on tumor cells and the fluorescence signal intensity of the NO probe was evaluated.

2. The method for predicting the killing effect of CAR-T cells in vitro based on NO probe cell imaging according to claim 1, characterized in that: The CAR-T cells are immune cells that express chimeric antigen receptors on the cell surface that can specifically recognize tumor cell antigens and acquire the ability to kill tumor cells with antigen specificity.

3. The method for predicting the killing effect of CAR-T cells in vitro based on NO probe cell imaging according to claim 2, characterized in that: The immune cells are one or more of T cells, CIK cells, K cells, TIL cells or DC cells.

4. The method of claim 1, wherein: The culture conditions in step (1) are culture in a constant temperature incubator at 37° C. and 5% CO 2 .

5. The method of predicting the killing effect of CAR-T cells in vitro based on NO probe cell imaging according to claim 1, characterized in that: The time for the Chinese medicine screening process in step (1) is 48-72 hours.

6. The method for predicting the killing effect of CAR-T cells in vitro based on NO probe cell imaging according to any one of claims 1 to 5, characterized in that: The lymphoma cells are blood tumor cell lines.

7. The method for predicting the killing effect of CAR-T cells in vitro based on NO probe cell imaging according to claim 6, characterized in that: The tumor cells are selected from diffuse large B-cell lymphoma cells, T-lymphocytic leukemia, and acute T-lymphocytic leukemia.

8. The method for predicting the killing effect of CAR-T cells in vitro based on NO probe cell imaging according to claim 1, characterized in that: The final concentration of cDNA obtained in the real-time fluorescence quantitative PCR experiment in step (2) is 50 ng / μL.

9. The method of claim 1, wherein: The imaging process is performed with 495 nm as the excitation wavelength and 515 nm as the emission wavelength.

10. The method for predicting the killing effect of CAR-T cells in vitro based on NO probe cell imaging according to any one of claims 8 to 9, characterized in that: During the verification of the CAR-T cell killing effect, the effector-target ratio was 1:2.

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