Methods for screening autophagy modulators

Abstract

The present invention relates to a method for screening an autophagy modulator, which can accurately screen an autophagy modulator using only a single cell according to a screening method of one embodiment. Furthermore, screening can be performed without using a fluorescent label, and therefore cytotoxicity and photobleaching phenomena that occur when a fluorescent label analysis method is used can be prevented, and the limitation that only a fluorescent label marker can be monitored can be improved.

Description

Technical Field

[0001] The present invention relates to a method for screening autophagy regulators. Background Art

[0002] As is well known, autophagy is an essential process for clearing unnecessary components generated within cells and maintaining cellular homeostasis. However, if there are problems with this autophagy process, aggregates of various biological components such as undegraded intracellular proteins will accumulate, leading to various diseases.

[0003] Meanwhile, it has recently been found that inhibiting activated autophagy in specific cancer cells may be effective in cancer treatment. Therefore, it has become increasingly important to develop autophagy regulators that can artificially activate or inhibit these cellular processes.

[0004] However, in current drug development or drug mechanism research of autophagy regulators, it solely relies on the method of fluorescently observing overexpressed cells by genetically engineering techniques to label the autophagy biomarker LC3B with fluorescent proteins such as GFP or RFP. This invasive method has many drawbacks, such as cytotoxicity due to fluorescence during monitoring, reduced reliability of analysis results due to photobleaching of fluorescent molecules, low time efficiency in three-dimensional image acquisition, and monitoring being limited to the fluorescently labeled molecules. Summary of the Invention

[0005] Technical Problem

[0006] In one aspect, there is provided a method for screening autophagy regulators, which includes: a step of contacting a biological sample with a candidate substance; a step of measuring the refractive index (RI) distribution of the organelles of the biological sample contacted with the candidate substance; and a step of extracting the physical quantities of the organelles using the measured refractive index distribution.

[0007] Technical Solution

[0008] In one aspect, there is provided a method for screening autophagy regulators, which includes: a step of contacting a biological sample with a candidate substance; a step of measuring the refractive index distribution of the organelles of the biological sample contacted with the candidate substance, and a step of extracting the physical quantities of the organelles using the measured refractive index distribution.

[0009] The biological sample may be tissue or cells. The cells may be a cell population or a single cell, and the cell population may be composed of a single cell, or contain multiple cells of the same type, or contain multiple cells of different types.

[0010] In a specific example, the cells can be somatic cells or cancer cells.

[0011] In this specification, the term "somatic cell" can refer to all cells excluding germ cells. The somatic cells can be cells derived from mammals such as, for example, humans, horses, sheep, pigs, goats, camels, antelopes, and dogs, or cells isolated from mammals.

[0012] In a specific example, the somatic cells can be one or more selected from muscle cells, liver cells, fibroblasts, epithelial cells, nerve cells, adipocytes, bone cells, blood cells, mucosal cells, and stem cells, but are not limited thereto.

[0013] The cancer cells can be cells of liver cancer, squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, ovarian cancer, cholangiocarcinoma, or gallbladder cancer, but are not limited thereto.

[0014] In this specification, the term "organelle" can refer to the organs that make up a cell. The organelles can have nano- or micron-sized (diameter), for example, 0.1 to 100 μm, 0.1 to 50 μm, 0.1 to 25 μm, 0.1 to 15 μm, 0.1 to 10 μm, 0.1 to 5 μm, or 0.1 to 3 μm, but are not limited thereto.

[0015] According to a specific example, the organelles can be one or more selected from endoplasmic reticulum (ER), vesicles, lipid droplets, mitochondria, ribosomes, Golgi apparatus, endosomes, nucleoli, and lysosomes. The endoplasmic reticulum can be rough endoplasmic reticulum (rER) or smooth endoplasmic reticulum (sER).

[0016] The vesicles can have a structure composed of a lipid bilayer, and the vesicles (autophagosome) can be autophagosomes.

[0017] In this specification, "refraction index (RI)" may refer to an inherent optical physical quantity of a substance itself, indicating the degree of slowdown in the speed of light when passing through the substance. In addition, since the refractive index is inversely proportional to the density of the substance, it may refer to biological information reflecting the density of cells or organelles.

[0018] In a specific example, the step of measuring the refractive index distribution may include the step of generating a plurality of two-dimensional holograms using quantitative phase imaging (QPI) based on a diffraction interferometer; and the step of measuring the three-dimensional refractive index distribution by analyzing the plurality of two-dimensional holograms.

[0019] The quantitative phase imaging technique can measure the three-dimensional refractive index distribution by measuring the complex optical field of the amplitude and phase delay of diffracted light and transmitted light formed when light is diffracted by a biological sample using an interferometer. The complex optical field can be formed and measured at various incident angles, and based on the complex optical field, the three-dimensional refractive index distribution of the biological sample corresponding to the concentration of each organelle can be measured.

[0020] In a specific example, the quantitative phase imaging technique based on an interferometer can be optical diffraction tomography (ODT).

[0021] In a specific example, the screening method may include the step of generating an image for extracting the physical quantity of the organelles of the cells using the three-dimensional refractive index distribution.

[0022] The image may be a three-dimensional image. The three-dimensional image can be generated by generating a plurality of two-dimensional holograms from the two-dimensional refractive index distribution of the organelles based on the refractive index difference between the organelles and the cytoplasm, and using the three-dimensional refractive index distribution generated from the plurality of generated two-dimensional holograms. By generating the three-dimensional image, three-dimensional imaging of the organelles can be performed, and the physical quantity of the organelles can be quantified based on the imaged three-dimensional image.

[0023] In a specific example, the physical quantity may be at least one physical quantity selected from the volume of the organelle, the refractive index of the organelle, the density of the organelle, and the number of organelles.

[0024] In this specification, the term "autophagy" may refer to a catabolic process that removes cellular components containing unnecessary or denatured proteins within the cell. Therefore, autophagy regulators and regulatory methods can be effectively used for treating or preventing various diseases caused by abnormal autophagy regulation as described above.

[0025] In this specification, the term "modulation" may include activation, stimulation, or upregulation, or reduction or downregulation, or both, of a biological function, and may include modulation in vitro, in vivo, and ex vivo. Thus, the modulator may include an autophagy activator or enhancer, or an autophagy inhibitor.

[0026] In a specific example, the autophagy modulator may be an autophagy activator or an autophagy inhibitor. The autophagy activator or inhibitor may include a drug, nanoparticle, or nanoparticle encapsulating the drug having autophagy activation ability or inhibition ability. The drug having autophagy activation ability or inhibition ability may include Thapsigargin, Cyclopiazonic acid, Tunicamycin, Torin, Valproic acid, Verapamil hydrochloride, Carbamazepine, Dexamethasone, Niclosamide, Nimodipine, Nitrendipine, Rapamycin, or Colchicine.

[0027] In a specific example, the screening method may include the step of measuring the physical quantity of the organelles of the cells in contact with the candidate substance over time.

[0028] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy activator when the volume of the vesicles of the cells in contact with the candidate substance increases over time.

[0029] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy inhibitor when the volume of the vesicles of the cells in contact with the candidate substance remains unchanged or decreases over time.

[0030] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy activator when the number of vesicles of the cells in contact with the candidate substance decreases over time.

[0031] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy inhibitor when the number of vesicles of the cells in contact with the candidate substance remains unchanged or increases over time.

[0032] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy activator when the density of vesicles of cells in contact with the candidate substance decreases over time.

[0033] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy inhibitor when the density of vesicles of cells in contact with the candidate substance remains unchanged or increases over time.

[0034] In a specific example, the screening method may include the step of comparing the extracted physical quantity of the organelle with the physical quantity of the organelle of the biological sample not in contact with the candidate substance. At this time, the physical quantity of the organelle of the biological sample in contact with the candidate substance may be the physical quantity extracted immediately after contact with the candidate substance.

[0035] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy activator when the volume of vesicles of cells just after contact with the candidate substance increases compared to the volume of vesicles of cells not in contact with the candidate substance.

[0036] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy inhibitor when the volume of vesicles of cells just after contact with the candidate substance remains unchanged or decreases compared to the volume of vesicles of cells not in contact with the candidate substance.

[0037] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy activator when the number of vesicles of cells just after contact with the candidate substance increases compared to the number of vesicles of cells not in contact with the candidate substance.

[0038] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy inhibitor when the number of vesicles of cells just after contact with the candidate substance remains unchanged or decreases compared to the number of vesicles of cells not in contact with the candidate substance.

[0039] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy activator when the density of vesicles of cells just after contact with the candidate substance increases compared to the density of vesicles of cells not in contact with the candidate substance.

[0040] In a specific example, the screening method may include the step of determining that the candidate substance is an autophagy inhibitor when the density of vesicles of cells just after contact with the candidate substance remains unchanged or decreases compared to the density of vesicles of cells not in contact with the candidate substance.

[0041] The vesicles may include other organelles or organelle components. For example, the vesicles may include the endoplasmic reticulum or endoplasmic reticulum components, and the endoplasmic reticulum or endoplasmic reticulum components may be in a state encapsulated in the vesicles.

[0042] According to a specific embodiment, the screening method may include a step of measuring the biomarker expression of organelles. The measurement of the biomarker expression of the organelles may utilize immunofluorescence staining technique (immunofluorescence: IF), western blot, or fluorescence imaging, but is not limited thereto. The step of measuring the biomarker expression of the organelles may be performed simultaneously or non-simultaneously with the interferometer-based quantitative phase imaging technique. Thereby, the credibility of the interferometer-based quantitative phase imaging technique can be improved.

[0043] According to a specific embodiment, the biomarker of the organelles may be a biomarker for the autophagy process. The biomarker for the autophagy process may be LC3 (microtubule-associated protein 1 light chain 3alpha). In addition, the biomarker for the endoplasmic reticulum (ER)-specific autophagy process may be SEC62 (translocation protein SEC62), RTN3 (reticulon-3), FAM134B (reticulophagy regulator 1), CCPG1 (cell-cycle progression gene), but is not limited thereto.

[0044] In a specific embodiment, the step of measuring the biomarker expression of the organelles may include a step of treating a fluorescent tracker to the cells. The fluorescent tracker may be a fluorescent tracker specific to the organelles, and the changes of the organelles can be specifically confirmed through the fluorescent tracker. The fluorescent tracker may be, for example, an endoplasmic reticulum tracker (ER-tracker) specific to the endoplasmic reticulum, a lysosome tracker (lysosome-tracker) specific to the lysosomes, or a mitochondria tracker (mito-tracker) specific to the mitochondria (mitochondria), but is not limited thereto.

[0045] In a specific embodiment, the autophagy regulator may be used for preventing or treating cancer, neurodegenerative diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases, or hereditary muscle diseases.

[0046] Advantages of the Invention

[0047] According to the screening method of one embodiment, even a single cell can be used to accurately screen for autophagy regulators. In addition, screening can be performed without using fluorescent labels, thus preventing cytotoxicity and photobleaching that occur when using fluorescence-labeling analysis methods, and improving the limitations of only being able to monitor fluorescence-labeled markers. Brief Description of the Drawings

[0048] Figure 1 An optical diffraction tomography (ODT) image showing the process of generation and disappearance of high-density vesicles over time after treating SNU475 cells with thapsigargin.

[0049] Figure 2 is a graph showing the changes in the refractive index and radius of vesicles in SNU475 cells treated with thapsigargin over time.

[0050] Figure 2a A graph showing the change in the refractive index of vesicles in SNU475 cells treated with thapsigargin over time, Figure 2b A graph showing the change in the radius of vesicles in SNU475 cells treated with thapsigargin over time.

[0051] Figure 3 A graph showing the changes in the total number of vesicles and the volume of each vesicle over time in SNU475 cells treated with thapsigargin.

[0052] Figure 4 is an image and a graph classified according to the number and refractive index of vesicles generated in SNU475 cells treated with thapsigargin, and a graph showing the change in the refractive index of cell organelles before and after treating SNU475 cells with thapsigargin.

[0053] Figure 4a An image comparing the number of vesicles generated in SNU475 cells treated with thapsigargin and their refractive index, Figure 4b A graph showing the refractive index of vesicles generated in SNU475 cells treated with thapsigargin, Figure 4c A graph showing the refractive index of cell organelles in SNU475 cells treated with thapsigargin.

[0054] Figure 5 is an image showing the changes in the endoplasmic reticulum and lysosomes produced by staining with an endoplasmic reticulum tracer or a lysosome tracer after treating SNU475 cells with thapsigargin (10 μM).

[0055] Figure 5a An image showing the changes in the endoplasmic reticulum after treating with thapsigargin observed by staining with an endoplasmic reticulum tracer,Figure 5b Images showing the changes in lysosomes after treatment with thapsigargin as observed by lysosome tracer staining.

[0056] Figure 6 Images showing the changes in mitochondria after treatment with thapsigargin as observed by mitochondrial tracer staining.

[0057] Figure 7 Graphs for confirming whether the ER-phagy receptors CCPG1, FAM134B, RTN3, and SEC62 are activated upon treatment with thapsigargin.

[0058] Figure 8 is an optical diffraction tomography (ODT) image showing the vesicle changes observed after treating SNU475 cells with cyclopiazonic acid (CPA) or tunicamycin (TC).

[0059] Figure 8a An optical diffraction tomography (ODT) image of vesicle changes after treatment with cyclopiazonic acid (CPA), Figure 8b An optical diffraction tomography (ODT) image of vesicle changes after treatment with tunicamycin (TC). Detailed implementation mode

[0060] It will be described in more detail through the following examples. However, these examples are only for illustrative purposes, and the scope of the present invention is not limited to the following examples.

[0061] Example 1 Confirming the changes in vesicles in SNU475 cells treated with thapsigargin (TG)

[0062] To confirm the changes in vesicles caused by treatment with autophagy regulators, human hepatocellular carcinoma cell line SNU475 cells were treated with 10 μM of the autophagy activator thapsigargin, and the changes in vesicles in the cells were confirmed.

[0063] More specifically, the SNU475 cell line was prepared in a TomoDish cell culture dish and treated with 10 μM of thapsigargin, and then an optical diffraction tomography (ODT) image was generated using HT-2H fluorescence holographic tomography (Tomocube). The results are shown in Figure 1 .

[0064] Figure 1 An optical diffraction tomography (ODT) image showing the time-dependence of the changes in vesicles generated in SNU475 cells after treatment with thapsigargin.

[0065] AsFigure 1 As shown, it was confirmed that a high density of vesicles was generated after treatment with the autophagy activator thapsigargin.

[0066] Example 2 confirmed the changes in the refractive index and radius of vesicles in single SNU475 cells treated with thapsigargin.

[0067] After treating SNU475 cells with thapsigargin (10 μM) to induce endoplasmic reticulum stress, based on the images confirmed in Example 1, the changes in the refractive index and radius of the vesicles generated in the cells were measured, and the results are shown in Figure 2. In addition, SNU475 cells were treated with thapsigargin (10 μM), and the changes in the number and volume of vesicles in the SNU475 cells treated with the thapsigargin over 2 hours were observed, and the results are shown in Figure 3 .

[0068] Figure 2 is a graph showing the changes in the refractive index and radius of vesicles in SNU475 cells treated with thapsigargin over time.

[0069] Figure 2a is a graph showing the change in the refractive index of vesicles in SNU475 cells treated with thapsigargin over time, Figure 2b is a graph showing the change in the radius of vesicles in SNU475 cells treated with thapsigargin over time.

[0070] Figure 3 is a graph showing the changes in the total number of vesicles and the volume of each vesicle in SNU475 cells treated with thapsigargin over time.

[0071] As shown in Figure 2, it was confirmed that the refractive index of the vesicles generated after treatment with the autophagy activator thapsigargin decreased over time. In addition, it was confirmed that the radius of the vesicles generated after treatment with thapsigargin increased over time.

[0072] As Figure 3 shown, it was confirmed that the total number of vesicles generated after treatment with the autophagy activator thapsigargin decreased over time due to the fusion between vesicles. In addition, it was confirmed that the volume of the vesicles generated after treatment with thapsigargin increased over time.

[0073] These results indicate that when treating cells with a specific autophagy regulator candidate, if the density of vesicles in the cells decreases, or the radius or volume of the vesicles increases, or the total number of vesicles decreases, it can be determined that the specific autophagy regulator candidate is an autophagy activator.

[0074] Example 3 confirmed the statistical (or systematic) changes in the refractive index and radius of vesicles and organelles in SNU475 cells treated with thapsigargin.

[0075] To confirm the statistical changes in the density of vesicles and organelles treated with thapsigargin and the resulting refractive index, the cell density and refractive index were compared before and after treating SNU475 cells with thapsigargin (10 μM). In addition, to confirm the changes in the density of organelles treated with thapsigargin, the refractive indices of the nucleolus (No), endoplasmic reticulum (ER), cytosol (Cyt), and plasma membrane (PM) were measured. The results are shown in Fig. 4.

[0076] Fig. 4 is an image and graph classified according to the number and refractive index of vesicles generated in SNU475 cells treated with thapsigargin, and a graph of the refractive index changes of organelles before and after treating SNU475 cells with thapsigargin.

[0077] Figure 4a For comparing the image of the number and refractive index of vesicles generated in SNU475 cells treated with thapsigargin, Figure 4b For showing the graph of the refractive index of vesicles generated in SNU475 cells treated with thapsigargin, Figure 4c For showing the graph of the refractive index of the organelles of SNU475 cells treated with thapsigargin.

[0078] As shown in Fig. 4, it was confirmed that the refractive index was high when the number of vesicles was large. That is, it was confirmed that the number of vesicles and the refractive index were proportional. In addition, it was confirmed that the vesicles induced by thapsigargin had a higher density than the endoplasmic reticulum, and it was confirmed that the density of the endoplasmic reticulum decreased with thapsigargin treatment. From these results, it was confirmed that thapsigargin treatment induced autophagy of the endoplasmic reticulum, thereby generating vesicles containing high-density endoplasmic reticulum components.

[0079] Example 4 confirmed the specific changes in the endoplasmic reticulum using a fluorescent tracker

[0080] Example 4-1. Image analysis using an endoplasmic reticulum tracker and a lysosome tracker

[0081] Using a confocal microscope and a fluorescent tracker, the changes in the endoplasmic reticulum and lysosomes were measured. As the fluorescent tracker, an endoplasmic reticulum tracker that can specifically observe only the endoplasmic reticulum and a lysosome tracker that can specifically observe only the lysosomes were used.

[0082] Specifically, after treating SNU475 cells with thapsigargin (10 μM), they were stained with an endoplasmic reticulum tracker or a lysosome tracker, and the kinetic changes in the endoplasmic reticulum and lysosomes were analyzed using a super-resolution microscope (Airyscan). The results are shown in Fig. 5.

[0083] Figure 5 shows an image of the changes in the endoplasmic reticulum and lysosomes produced by staining with an endoplasmic reticulum tracer or a lysosome tracer after treating SNU475 cells with thapsigargin (10 μM).

[0084] Figure 5a An image showing the changes in the endoplasmic reticulum treated with thapsigargin observed by staining with an endoplasmic reticulum tracer. Figure 5a The arrow in the figure indicates the region where vesicles of endoplasmic reticulum components begin to be produced. Figure 5b An image showing the changes in lysosomes after treating with thapsigargin observed by staining with a lysosome tracer. Figure 5b The arrow in the figure indicates the region where large lysosomes are produced with a time lag (about 15 minutes) from the endoplasmic reticulum.

[0085] As shown in Figure 5, it was confirmed that treating SNU475 cells with thapsigargin produced vesicles containing endoplasmic reticulum components and lysosomes. This is the same as the result confirmed by optical diffraction tomography, which means that organelle physical quantities can be effectively screened for autophagy regulators by confirming them through optical diffraction tomography.

[0086] Furthermore, using an optical diffraction tomography setup that can connect three-dimensional optical diffraction tomography and two-dimensional fluorescence, the changes in the endoplasmic reticulum and lysosomes of SNU475 cells treated with thapsigargin were analyzed. The results confirmed that after treating with thapsigargin, the structure of the high-density vesicles produced in the cytoplasm was specifically co-localized with the endoplasmic reticulum and lysosomes. In addition, it was also confirmed that vesicles were produced in the endoplasmic reticulum region and then combined with lysosomes.

[0087] These results mean that after treating with thapsigargin, the vesicles of the cells contain endoplasmic reticulum components, and the vesicles containing endoplasmic reticulum components combine with lysosomes and are degraded, resulting in changes in the density of the vesicles.

[0088] 4-2. Image analysis using a mitochondrial tracer

[0089] To confirm that the high-density vesicles produced after treating with thapsigargin are specific to the endoplasmic reticulum, in the same method as described in 4.1, a mitochondrial-specific tracer, the mitochondrial tracer, was used to analyze the dynamics of mitochondria, and the results are shown in Figure 6 .

[0090] Figure 6 A figure showing the changes in mitochondria after treating with thapsigargin observed by staining with a mitochondrial tracer.

[0091] As Figure 6 shown, it was confirmed that the high-density vesicles produced after treating with thapsigargin do not contain mitochondrial components.

[0092] These results indicate that the high-density vesicles induced by thapsigargin treatment are specific to the endoplasmic reticulum.

[0093] 4-3. Measurement of the co-localization of the endoplasmic reticulum autophagy receptor

[0094] To measure the activation levels of autophagy markers and endoplasmic reticulum autophagy markers after thapsigargin treatment, the co-localization of the endoplasmic reticulum autophagy receptor was measured.

[0095] More specifically, immunofluorescence staining technique (immunofluorescence: IF) was used to analyze whether the endoplasmic reticulum autophagy receptors CCPG1, FAM134B, RTN3, and SEC62 were activated after treatment with thapsigargin (10 μM), and the results are shown in Figure 7 .

[0096] Figure 7 A graph for confirming whether the endoplasmic reticulum autophagy receptors CCPG1, FAM134B, RTN3, and SEC62 are activated according to thapsigargin treatment.

[0097] As Figure 7 shown, it was confirmed that the endoplasmic reticulum autophagy receptors CCPG1, FAM134B, RTN3, and SEC62 were activated by co-localizing with LC3 after thapsigargin treatment.

[0098] These results indicate that the high-density vesicles observed by optical diffraction tomography after thapsigargin treatment are caused by the endoplasmic reticulum autophagy receptor.

[0099] Example 5 confirmed the changes in the refractive index and radius of vesicles in SNU475 cells treated with Cyclopiazonic acid (CPA) or Tunicamycin (TC)

[0100] To confirm whether the same results as thapsigargin can be obtained when treating with autophagy regulators other than thapsigargin, SNU475 cells were treated with Cyclopiazonic acid (20 μM) and Tunicamycin (60 μg / mL) respectively to induce endoplasmic reticulum stress, and the vesicle changes in the cells were observed using HT-2H fluorescence holographic tomography (Tomocube). To conduct the observation, optical diffraction tomography images after 10 hours of treatment with Cyclopiazonic acid and after 2 hours of treatment with Tunicamycin were generated, and the results are shown in Figure 8.

[0101] Figure 8 is an optical diffraction tomography image of the vesicle changes observed after treating SNU475 cells with Cyclopiazonic acid or Tunicamycin.

[0102] Figure 8a Optical diffraction tomography image of vesicle changes after treatment with cyclopiazonic acid, Figure 8b Optical diffraction tomography image of vesicle changes treated with tunicamycin.

[0103] As shown in Fig. 8, it was confirmed that when treated with cyclopiazonic acid and tunicamycin, the same vesicle changes occurred as when treated with thapsigargin. This means that, as in the case of thapsigargin, autophagy regulators can be effectively screened by using the physical quantities of organelles extracted from the characteristics of vesicles generated in cells and the refractive index distribution of organelles.

Claims

1. A method for screening autophagy regulators, comprising: The step of contacting a biological sample with a candidate substance; The step of measuring the refractive index distribution of the cell organelles of the biological sample contacted with the candidate substance; And The step of extracting the physical quantity of the cell organelles using the measured refractive index distribution.

2. The method for screening autophagy regulators according to claim 1, wherein, The biological sample is a cell.

3. The method for screening autophagy regulators according to claim 2, wherein, The screening method includes the step of measuring the physical quantity of the cell organelles of the cells contacted with the candidate substance over time.

4. The method for screening autophagy regulators according to claim 1, wherein, The physical quantity is at least one of the volume of the cell organelles, the density of the cell organelles, and the number of the cell organelles.

5. The method for screening autophagy regulators according to claim 4, wherein, The autophagy regulator is an autophagy activator or an autophagy inhibitor.

6. The method for screening autophagy regulators according to claim 5, wherein, The cell organelles are one or more selected from the group consisting of endoplasmic reticulum, vesicles, lipid droplets, mitochondria, ribosomes, Golgi apparatus, endosomes, nucleoli, and lysosomes.

7. The method for screening autophagy regulators according to claim 1, wherein, The step of measuring the refractive index distribution includes the step of generating a plurality of two-dimensional holograms using an interferometer-based quantitative phase imaging technique; and The step of measuring the three-dimensional refractive index distribution by analyzing the plurality of two-dimensional holograms.

8. The method for screening autophagy regulators according to claim 7, wherein, The interferometer-based quantitative phase imaging technique is optical diffraction tomography.

9. The method for screening autophagy regulators according to claim 7, wherein, The screening method includes the step of generating an image for extracting the physical quantity of the cell organelles of the cell using the three-dimensional refractive index distribution.

10. The method for screening autophagy regulators according to claim 6, wherein, The cell organelle is a vesicle, and the screening method includes the step of determining that the candidate substance is an autophagy activator when the volume of the vesicle increases, the number of the vesicles decreases, or the density of the vesicles decreases over time.

11. The method for screening autophagy regulators according to claim 6, wherein, The cell organelle is a vesicle, and the screening method includes the step of determining that the candidate substance is an autophagy inhibitor when the volume of the vesicle remains unchanged or decreases, the number of the vesicles remains unchanged or increases, or the density of the vesicles remains unchanged or increases over time.

12. The method for screening autophagy regulators according to claim 1, wherein, The screening method includes the step of comparing the extracted physical quantity of the cell organelles with the physical quantity of the cell organelles of the biological sample not contacted with the candidate substance.

13. The method for screening autophagy regulators according to claim 12, wherein, The physical quantity of the cell organelles of the biological sample contacted with the candidate substance is the physical quantity extracted immediately after contact with the candidate substance.

14. The method for screening autophagy regulators according to claim 13, wherein, The screening method includes: When the volume of the vesicles of the cells just after contact with the candidate substance increases compared to the volume of the vesicles of the cells not contacted with the candidate substance, when the density of the vesicles of the cells just after contact with the candidate substance increases compared to the density of the vesicles of the cells not contacted with the candidate substance, or when the number of the vesicles of the cells just after contact with the candidate substance increases compared to the number of the vesicles of the cells not contacted with the candidate substance, the step of determining that the candidate substance is an autophagy activator.

15. The method for screening autophagy regulators according to claim 13, wherein, The screening method includes: When the volume of the vesicles of the cells just after contact with the candidate substance remains unchanged or decreases compared to the volume of the vesicles of the cells not contacted with the candidate substance, when the density of the vesicles of the cells just after contact with the candidate substance remains unchanged or decreases compared to the density of the vesicles of the cells not contacted with the candidate substance, or when the number of the vesicles of the cells just after contact with the candidate substance remains unchanged or decreases compared to the number of the vesicles of the cells not contacted with the candidate substance, the step of determining that the candidate substance is an autophagy inhibitor.

16. The method for screening autophagy regulators according to any one of claims 10, 11, 14 and 15, wherein, The vesicles include endoplasmic reticulum.

17. The method for screening autophagy regulators according to claim 1, wherein The autophagy regulator is for preventing or treating cancer, neurodegenerative diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases or genetic muscle diseases.

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