Antineoplastic drug metabolism evaluation and screening method based on intracellular redox probe

Abstract

The invention discloses an anti-tumor drug metabolism evaluation and screening method based on an intracellular oxidation-reduction probe. The method comprises the following steps: firstly, constructing a cancer cell line for stably expressing SoNar probe and iNapc probe fluorescence sensors; the SoNar probe is mainly used for rapidly responding to changes of NAD + and NADH in cells, and the iNapc probe is used for detecting changes of pH in the cells. Next, the cell line is cultured by 2D monolayer culture or 3D tumor sphere culture. The average cell fluorescence intensity of cells with the two probes in KO525 (405nm) and FITC (488nm) channels after dosing is detected by a flow cytometer, and the change condition of NAD < + > / NADH in the cells after pH correction is obtained through calculation and further serves as a basis for drug screening. The method provided by the invention can accurately and efficiently evaluate the influence of the drug on tumor cell metabolism, and is beneficial to more effective drug screening, thereby generating positive influence on frontier research of anti-tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of antitumor drug evaluation, and relates to an antitumor drug screening method and application, and in particular to an antitumor drug metabolism evaluation and screening method combining an intracellular redox probe with a three-dimensional tumor model. Background Art

[0002] Tumor in vitro models constructed from tumor cell lines are a key step in anti-tumor drug screening. Although 2D culture models are popular in drug development due to their ease of operation, high cost-effectiveness and good reproducibility, such models often fail to accurately simulate tumor growth. Therefore, 3D tumor models, especially tumor spheroid models, are gaining more and more attention. This is mainly because 3D models can better simulate the main characteristics of solid tumors in vivo, such as oxygen, pH, and nutrient gradients, and have good reproducibility and a shorter construction cycle.

[0003] Reprogramming of cancer cell metabolism enables cancer cells to meet their demands for energy and rapid synthesis of biomacromolecules. Redox reactions play a key role in cancer cell metabolism and are an important component of cell signaling and cell fate determination. In particular, pyridine nucleotides (NADPH / NADP + and NADH / NAD + ), thiols, and ROS form a complex interaction network that jointly regulates the redox state of cells. + NADH and its reduced form are the most important cofactors involved in cellular energy metabolism.

[0004] However, existing biochemical detection methods, such as enzyme cycling assays, chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, have certain limitations. These methods often require cell lysis, cannot monitor cell status in real time, and are not suitable for high-throughput drug screening. Therefore, the challenge we face is how to develop a method that can monitor the redox state of cells in real time and is suitable for high-throughput drug screening while maintaining a 3D tumor model.

[0005] The SoNar probe is a genetically encoded sensor with strong fluorescence, rapid response, resistance to pH changes, and a wide dynamic range, which can detect NAD in the cytoplasm of living cells. + The SoNar probe works by measuring the redox state of NAD + The changes in fluorescence spectrum caused by the ratio of NADH / NADH reflect the redox state of the cells. This allows the SoNar probe to detect the redox state of tumor cells in real time without cell lysis and be used for high-throughput drug screening, breaking through the limitations of existing biochemical detection methods.

[0006] Although existing anti-tumor drug screening methods have made some progress, there are still some shortcomings. For example, "Fluorescent cell model for anti-tumor drug screening and its labeling method and application" (CN102220284A) proposed a fluorescence-based screening method, but did not apply this method to 3D tumor models. "An anti-tumor drug screening method and application" (CN111893159A) uses a 3D model that is closer to the actual tumor environment, but relies on the CCK-8 kit to detect the viability of 3D cells, requires additional processing steps, and cannot obtain results in real time.

[0007] Therefore, our goal is to develop a new anti-tumor drug screening method that combines 3D tumor models and SoNar probes to achieve real-time, high-throughput metabolism-based anti-tumor drug screening. Specifically, we will introduce SoNar probes into 3D tumor models to monitor the redox state of cells in real time and evaluate and screen anti-tumor drugs based on this information.

[0008] Our approach is expected to provide more accurate and real-time information about the metabolic state of cells, thereby helping to identify more effective anti-tumor drugs. This approach aims to overcome the limitations of existing technologies, increase the success rate of drug development and reduce costs. Summary of the invention

[0009] A method for evaluating and screening anti-tumor drug metabolism based on intracellular redox probes

[0010] The technical solution provided by the present invention is as follows:

[0011] Step 1: construct a cell line that stably expresses the SoNar probe and the iNapc probe, and use the plasmid DNA of the SoNar probe and the iNapc probe to transfect cancer cells to construct a cancer cell line that stably expresses the SoNar probe and the iNapc probe fluorescent sensor.

[0012] Step 2: 2D monolayer cell culture: use T75 culture flasks to culture cancer cell lines carrying SoNar probes and iNapc probes in an incubator at 37° C., 5% CO 2 and 95% humidity for 2 days.

[0013] Step 3: Formation of 3D tumor spheroids. Cancer cell lines carrying SoNar probes and iNapc probes were inoculated in a "U-shaped" ultra-low adsorption 96-well plate at a certain cell density (2000 cells / well), and then the plate was centrifuged at 1000g for 10 minutes to form tumor spheroids, which were cultured in an incubator at 37°C, 5% CO2 and 95% humidity for 4 days.

[0014] Step 4: Use flow cytometry to detect the fluorescence of SoNar probe and iNapc probe after drug addition. For 2D monolayer cells, directly enzymatically dissociate into single cell suspension; for 3D tumor spheroids, tumor spheroids need to be collected and then enzymatically dissociated into single cell suspension. Use HBSS to resuspend cells and add drugs. After thorough mixing, use flow cytometry to detect immediately. Determine the average fluorescence intensity of HeLa cells carrying SoNar probe or iNapc probe in KO525 (405nm) and FITC (488nm) channels.

[0015] Step 5: Data processing: Process the results detected by the flow cytometer and calculate the ratio of the 405nm and 488nm channels after pH correction based on the average fluorescence intensity of the cells as a basis for drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Confocal imaging of a 96-well plate after 4 days of tumor spheroid culture. The top three rows are HeLa tumor spheroids carrying SoNar probes, and the bottom three rows are HeLa tumor spheroids carrying iNapc probes.

[0017] Figure 2 : The experimental results of using flow cytometry to detect cells carrying SoNar probes in KO525 (405nm) and FITC (488nm) channels after one drug addition.

[0018] Figure 3 : F obtained after treatment with metformin and plumbagin under 2D monolayer culture conditions 405nm / F 488nm value;

[0019] Figure A shows the F of the SoNar probe. 405nm / F 488nm Figure B is the F value of iNapc probe 405nm / F 488nm Figure C is the pH-corrected F 405nm / F 488nm value.

[0020] Figure 4 : Index values ​​calculated by the formula after treatment with metformin, plumbagin, and β-lapachone under 2D monolayer culture conditions

[0021] Figure 5 : F obtained after treatment with Fascaplysin and KP372-1 under 3D tumor spheroid culture conditions 405nm / F 488nm value;

[0022] Figure A shows the F of the SoNar probe. 405nm / F 488nm Figure B is the F value of iNapc probe 405nm / F 488nm Figure C is the pH-corrected F 405nm / F 488nm value.

[0023] Figure 6 : Index values ​​calculated by the formula after Fascaplysin and KP372-1 treatment under 3D tumor spheroid culture conditions DETAILED DESCRIPTION

[0024] The embodiment of the present invention takes cervical cancer as an example and provides an anti-tumor drug metabolism evaluation and screening method based on intracellular redox probe, comprising the following steps:

[0025] Step 1: Construct a HeLa cell line that stably expresses SoNar and iNapc probes.

[0026] (1) HeLa cell culture

[0027] HeLa cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM, 11995-06, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, 10099-141, Gibco, Grand Island, NY, USA) supplemented with 100 IU / mL penicillin and 100 mg / mL streptomycin (B540732, Sangon Biotech, Shanghai, CN) and passaged when the cell confluence reached 80%–90%.

[0028] (2) Construction of HeLa cell lines stably expressing SoNar and iNapc probes

[0029] HeLa cells were transfected with plasmid DNA of SoNar and iNapc, and positive cells with stable expression were selected using puromycin. SoNar probe was constructed to quantify intracellular NAD + / NADH ratio, while the iNapc probe was used to correct for pH perturbations caused by drug addition.

[0030] Step 2: Rapid Generation of Tumor Spheroids

[0031] The cells grown in monolayer were detached with trypsin to produce a cell suspension, and the cells were counted using Countstar (ALIT Life Science, Shanghai, CN) with automatic cell counting software. The cell suspension was diluted to 10 410 cells / mL, and then 200 μL of the cell suspension was added to a "U-shaped" ultra-low attachment 96-well plate (7007, Corning, NY, USA). The plate was centrifuged at 1000 g for 10 minutes, and then the plate was incubated in an incubator at 37°C, 5% CO2 and 95% humidity for 4 days. Figure 1 shown.

[0032] Step 3: Use flow cytometry to detect the fluorescence of SoNar probe and iNapc probe in cells after drug addition

[0033] (1) Preparation of cell samples

[0034] Tumor spheroids cultured in 96-well plates were collected into centrifuge tubes using a pipette, and trypsin-EDTA solution (T4049, Sigma-Aldrich) was used to disperse the tumor spheroids into individual cells. The cells were resuspended in HBSS, and the cell suspension was dispensed into 1.5 mL EP tubes, with 0.25 mL per tube.

[0035] (2) Prepare the drug library

[0036] The drug to be tested was prepared to the required concentration and diluted with HBSS buffer during the preparation process. For each drug screening, three samples of control group (HBSS), positive control group (pyruvate), negative control group (oxamate), low-concentration drug and high-concentration drug were required. The final treatment concentrations were 1mM pyruvate, 5mM oxamate, 1μM and 10μM drugs.

[0037] (3) Use flow cytometry to detect the fluorescence value of HeLa cells after drug addition.

[0038] The drug to be tested was added to the EP tube containing cells. After shaking, the fluorescence value was detected using a flow cytometer (Beckman Coulter). 20,000 cells were collected for each sample, and the detection channels were KO525 (405 nm) and FITC (488 nm). Figure 2 shown.

[0039] Step 4: Data Analysis

[0040] (1) Process the flow cytometry results and calculate the ratio value of each sample after drug treatment.

[0041] Since the SoNar probe will be affected by pH fluctuations when excited at 488nm, the iNapc probe is required for pH correction. The formula is as follows:

[0042]

[0043] Among them, SoNar F 405nm 、SoNar F 488nm represents the average fluorescence intensity of HeLa cells carrying SoNar probes in FITC and V525-KrO-A, iNapc F 405nm 、iNapc F 488nm The "ratio" value represents the average fluorescence intensity of HeLa cells carrying iNapc probe in FITC and V525-KrO-A. The "ratio" value represents the pH-corrected NAD after drug treatment. + / NADH ratio, which reflects the effect of drugs on intracellular NAD + The degree of influence of changes in the ratio of NADH.

[0044] (2) Normalize the ratio value

[0045]

[0046] Among them, ratio s Indicates the ratio value after a given drug treatment, ratio c Indicates the ratio value of the control group after treatment, ratio o Indicates the ratio value after oxamate treatment, ratio p Represents the ratio value after pyruvate treatment. "index" represents the normalized ratio value, which reflects the degree of influence of the drug on cell metabolism and can be used for drug screening and evaluation.

[0047] Example 1: Using the above method to screen drugs in 2D monolayer cells

[0048] Metformin (CAS NO: 657-24-9), plumbagin (CAS NO: 481-42-5), and β-lapachone (CAS NO: 4707-32-8) were selected as test drugs, and the above operation was performed using 2D monolayer cultured HeLa cells. Figure 3 As shown in Figure 1, Figure A shows the average fluorescence intensity of HeLa cells carrying SoNar probes in FITC and V525-KrO-A after drug treatment, which is the effect of drug treatment without pH correction; Figure B shows the average fluorescence intensity of HeLa cells carrying iNapc probes in FITC and V525-KrO-A, which is the pH disturbance effect caused by drug treatment; Figure C is the ratio value obtained after pH correction for the above two results, that is, the effect of drug on intracellular NAD + The degree of influence of changes in the ratio of NADH.

[0049] Through the above method, we can calculate the index value, which can quantify the effect of drugs on HeLa cell metabolism in the two-dimensional monolayer culture model, such as Figure 4 As shown in the figure. According to the data in the figure, we found that the effects of metformin, plumbagin and β-lapachone on HeLa cell metabolism were different at different drug concentrations. In the case of low-concentration drug treatment, the effects on HeLa cell metabolism were β-lapachone, plumbagin, and metformin from large to small; while in the case of high-concentration drug treatment, the effects were plumbagin, β-lapachone, and metformin from large to small. Therefore, we can screen out more effective drugs by comparing the index value of the degree of metabolic change.

[0050] Example 2: Using the above method to screen drugs in 3D tumor spheroids

[0051] Fascaplysin (CAS NO: 114719-57-2) and KP372-1 (CAS NO: 1374996-60-7) were selected as test drugs, and the above operations were performed using HeLa cells cultured in 3D spheroids. Figure 5 As shown in Figure 1, Figure A shows the average fluorescence intensity of HeLa cells carrying SoNar probes in FITC and V525-KrO-A after drug treatment, which is the effect of drug treatment without pH correction; Figure B shows the average fluorescence intensity of HeLa cells carrying iNapc probes in FITC and V525-KrO-A, which is the pH disturbance effect caused by drug treatment; Figure C is the ratio value obtained after pH correction for the above two results, that is, the effect of drug on intracellular NAD + The degree of influence of changes in the ratio of NADH.

[0052] Through the above method, we can calculate the index value, which can quantify the effect of drugs on HeLa cell metabolism in the three-dimensional tumor spheroid culture model, such as Figure 6 As shown in the figure. According to the data in the figure, we found that the degree of influence of Fascaplysin and KP372-1 on HeLa cell metabolism was different at different drug concentrations. In the case of low-concentration drug treatment and high-concentration drug treatment, the degree of influence of KP372-1 on HeLa cell metabolism was greater than that of Fascaplysin. Therefore, we can screen out more effective drugs by comparing the value of the metabolic change index.

Claims

1. A method for evaluating and screening anti-tumor drug metabolism, It is characterized in that The method uses 2D monolayer culture or 3D tumor spheroid culture and combines the genetically encoded sensor SoNar probe and iNapc probe to monitor the intracellular redox state of cells in real time and screen anti-tumor drugs. The specific steps include: Step 1: construct cancer cell lines that stably express SoNar probes and iNapc probes; Step 2: Cultivate the cell line in 2D monolayer culture or 3D tumor spheroid culture; Step 3: Detect the average fluorescence intensity of cells with these two probes in KO525 (405 nm) and TITC (488 nm) channels after drug addition; Step 4: Calculate the pH-corrected NAD in the cell + / The changes in NADH are used to evaluate the effect of drugs on cell metabolism.

2. The method for evaluating and screening anti-tumor drug metabolism according to claim 1, It is characterized in that The 2D monolayer culture is cultured in a T25 or T75 cell culture flask.

3. The method for evaluating and screening anti-tumor drug metabolism according to claim 1, It is characterized in that The 3D monolayer culture was cultured in a "U-shaped" ultra-low attachment 96-well plate.

4. The method for evaluating and screening anti-tumor drug metabolism according to claim 1, It is characterized in that The specific process of drug evaluation is as follows: (1) Drug stimulation; (2) Detect the average fluorescence intensity of cells with two probes in the KO525 (405 nm) and TITC (488 nm) channels; (3) Calculate the extent of the drug’s effect on cell metabolism.

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