A method for identifying apoptosis and necrosis and its use

Abstract

The application discloses a method for identifying cell apoptosis and necrosis and application thereof, and relates to the technical field of biological medicines. Through a two-photon microscope, fluorescence spectrum and a fluorescence lifetime imaging microscope, all cells in apoptosis and necrosis show an increase in red fluorescence intensity after excitation at 1060 nm, compared with living cells, the cells in apoptosis show a longer lipofuscin fluorescence lifetime in lysosomes, and the cells in necrosis show a shorter lipofuscin fluorescence lifetime. Based on the comparison of the fluorescence lifetime, the state change of the cells after drug treatment can be revealed, the technology can be applied to drug screening, and can have an influence on various fields of biomedical research and application, for example, drawing the heterogeneity of treatment response in a tumor microenvironment, marking single-cell drug resistance in spatial transcriptomics, and capturing the in-vivo dynamics of cell death in embryonic development.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method for identifying apoptosis and necrosis of cells and its application. Background Technology

[0002] Apoptosis is a programmed cell death process that regulates embryonic development, controls viral infections, eliminates cancer cells, and maintains tissue homeostasis. Its morphological characteristics include chromatin condensation, DNA breaks, cell contraction, and the formation of apoptotic bodies. Abnormalities in apoptosis can lead to various diseases, such as cancer, HIV / AIDS, Alzheimer's disease, and autoimmune diseases. This cellular aging mechanism is widely used in drug development, treatment, evaluation, and biosafety testing.

[0003] In contrast to apoptosis, necrosis is a passive, unregulated pattern of cell death. Most organelles, including lysosomes, are completely destroyed and their contents are released, leading to inflammatory tissue damage. In the context of tumor pathology, rapid tumor growth often induces cell necrosis and intratumoral hypoxia. The stress caused by cell necrosis has a high probability of leading to poor prognosis, such as chemotherapy resistance. Therefore, visualization of cell death and identification of death types can help assess the pharmacodynamics and therapeutic effects of cancer drugs.

[0004] Traditionally, cellular senescence is assessed by detecting β-galactosidases (e.g., X-GAL) with chromogenic substrates. With a deeper understanding of cell death mechanisms, several biochemical features, such as DNA breaks, changes in membrane permeability, and caspase 3 activity, can be used to detect dead cells. However, these methods all rely on endpoint observation, requiring cell fixation before staining and immunofluorescence labeling. This approach limits the development of dynamic, real-time assessment, while apoptosis kinetics are crucial for the rapid detection of cell death following drug treatment.

[0005] To visualize pharmacodynamics and apoptosis processes, scientists have designed biomimetic probes, such as titanium-customized gold nanoclusters and recombinant substrate proteins. However, currently reported visualization results rely on exogenous reagents, synthetic nanoprobes, or complex transfection methods. Some endogenous fluorophores in cells, such as tryptophan, NADH, and flavin, can rapidly and label-free assess cellular aging processes. However, these metabolic fluorophores do not show specificity for cellular aging and are easily influenced by other metabolic activities.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for identifying apoptosis and necrosis and its application.

[0008] This invention is implemented as follows:

[0009] In a first aspect, embodiments of the present invention provide a method for identifying apoptosis and / or necrosis, comprising: real-time detection of the fluorescence lifetime of lipofuscin and / or lipofuscin-adsorbed substances in the cells to be tested, and identifying apoptosis and / or necrosis by the length of the fluorescence lifetime.

[0010] The method described is not intended for the direct diagnosis or treatment of disease.

[0011] Secondly, embodiments of the present invention provide the application of the method for identifying apoptosis and / or necrosis as described in any of the foregoing embodiments in drug screening or efficacy evaluation.

[0012] The present invention has the following beneficial effects:

[0013] This invention discloses a label-free and non-invasive technique for detecting apoptosis and cell death. Using two-photon or single-photon microscopy, fluorescence spectroscopy, and fluorescence lifetime imaging microscopy, all apoptotic and necrotic cells showed increased red fluorescence intensity upon excitation at 1060 nm. Compared to living cells, apoptotic cells exhibited a longer lipofuscin fluorescence lifetime in lysosomes, while necrotic cells showed a shorter lipofuscin fluorescence lifetime. Based on the comparison of fluorescence lifetimes, the state changes of cells after drug treatment can be revealed. This technique can be applied to drug screening and can impact various fields of biomedical research and application, such as mapping the heterogeneity of treatment response in the tumor microenvironment, annotating single-cell drug resistance in spatial transcriptomics, and capturing the in vivo dynamics of cell death during embryonic development. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1Fluorescence lifetime imaging microscopy for cultured cells; SPC imaging software was used to obtain color images of lifetime components τm, τ1, τ2 in the range of 0-2000 ps and relative amplitudes α1 and α2 in the range of 0-100%; fluorescence photon counts were contributed by lipofuscin-like fluorescence (MDA-MB-231, 30M cisplatin); in each pixel, the fluorescence intensity decay curve was fitted by two exponential decay components with lifetimes of τ1 and τ2 (in picoseconds) and relative proportions of α1 and α2; the shift parameter represents the time delay between the fitted curve and the data; due to the low background photon count, the offset and scattering values ​​were zero; field of view: 128 × 128 μm;

[0016] Figure 2 (A) Flow cytometry of MDA-MB-231 cells in the control group (left) and the treatment group incubated with 300 μM H2O2; Labeling dye: Annexin V / PI staining, the percentage of apoptotic cells in quadrant 3 (Q3) increased from 3.39% to 16.5%; (B) Imaging of 300 μM H2O2-induced apoptosis of MDA-MB-231 cells under two-photon excitation at 820–1240 nm with the same excitation laser power of 15 mW; Detection wavelength range: 604–679 nm; Scale bar: 50 μm; (C) Fluorescence excitation spectrum of red autofluorescence in the treatment group in (B); (D) Mean red autofluorescence intensity in control cells excited at 1060 nm (N=30) and H2O2-treated cells; All data shown represent two or three independent experiments;

[0017] Figure 3For early detection of apoptosis signals; comparison of two-photon fluorescence imaging and intracellular fluorescence intensity (AC) and MDA-MB-231 cells excited at 1060 nm, apoptosis detection range 604-679 nm, treated with 300 mM H2O2 for 24 h; (D) Two-photon fluorescence spectrum of red autofluorescence excited at 1060 nm, cisplatin 30 mM, 72 h, H2O2. 300mM, 24h; (E) After 72h induction with 30μM cisplatin, lysosomes colocalized with accumulated red autofluorescent groups. Green channel: LysoTracker, excitation 488nm; red channel: lipofuscin, excitation 561nm; white indicates colocalization of the two channels; scale bar 50μm; (FH) MDA-MB-231 cells were excited at 1060nm, detection range 604-679nm, cisplatin 30μM, for 72h, scale bar 50μM; The statistical mean and standard deviation (error bars) of (C) and (H) were calculated based on data from three independent images acquired under the same excitation conditions, (N=30)***p<0.001, Student's t-test was performed between the treatment group and the control group; (I) Caspase in the control group and the cisplatin-treated group Confocal, bright-field, and merged images of 3-Green stained cells. Green represents the distribution of caspase-3, and red represents lipofuscin-like fluorescence. Scale bar: 50 μm. Before cisplatin treatment, cells stained with caspase-3 active dye showed low background green fluorescence signal (control group). In contrast, cisplatin-treated cells produced significant double-positive caspase-3 green fluorescence signal and enhanced red autofluorescence signal.

[0018] Figure 4 (A) Lysosomes in control and treatment cells were labeled with LysoTracker (excitation: 488 nm) and compared with red autofluorescence (cisplatin 30 μM, 72 h, excitation: 561 nm); white pixels indicate colocalization of green and red channels; scale bar: 50 m; (B) Gray values ​​of green and red fluorescence, representing colocalization of lysosomes and red autofluorescence; (C) Colocalization was quantified using Pearson and Mander coefficients; data represent three independent experiments.

[0019] Figure 5(A) Imaging of 30 μM cisplatin-induced apoptosis in MDA-MB-231 cells under two-photon excitation at 15 mW of the same excitation laser power at 970–1240 nm. Detection wavelength range: 604–679 nm; Scale bar: 50 μm; (B) Fluorescence excitation spectrum of red autofluorescence in (A); (C) Mean red autofluorescence intensity in control cells and cisplatin-treated cells excited at 1060 nm (N=30); (D) Flow cytometry of cisplatin-treated MDA-MB-231 cells; Labeling dye: Annexin V / PI staining; All data shown represent two or three independent experiments;

[0020] Figure 6 (A) Imaging of L929 cells induced by 30 μM cisplatin-induced apoptosis under two-photon excitation at 970–1240 nm and the same excitation laser power of 15 mW; Scale bar: 50 μm; (B) Fluorescence excitation spectrum of red autofluorescence in (A); (C) Mean red autofluorescence intensity in control cells and cisplatin-treated cells excited at 1060 nm (N = 30); All data shown represent two or three independent experiments;

[0021] Figure 7 (A) Flow cytometry analysis of MDA-MB-231 cells labeled with the Annexin V-FITC / PI assay kit before and after treatment with different doses of cisplatin (1, 5, 10, and 30 μM) (control); (B) The percentage of apoptotic cells increased with increasing dose, reaching approximately 50% at 72 hours; (C) Flow cytometry analysis of MDA-MB-231 cells labeled with the Annexin V-FITC / PI assay kit before and after treatment with 30 μM cisplatin for 12, 24, 48, and 72 hours (control); (D) The percentage of apoptotic cells increased over time, reaching approximately 50% at 72 hours; (E) Flow cytometry analysis of MDA-MB-231 cells after treatment with 30 μM cisplatin for 12 and 24 hours, followed by washing and further culture with fresh medium; All data shown represent two or three independent experiments.

[0022] Figure 8 Pharmacodynamic characteristics of apoptosis intensity and lifespan; (A) Time- and dose-dependent red autofluorescence images of MDA-MB-231 cells treated with 30 μM cisplatin, scale bar 100 μM; (B) Mean intensity of intracellular red autofluorescence after different incubation times; (C) Red autofluorescence lifespan of cells before treatment (0 h) and at 24 h, 48 h, and 72 h after treatment; Data are mean ± standard deviation and represent three independent experiments.

[0023] Figure 9 Time-series monitoring of the mean intensity of red autofluorescence in MDA-MB-231 cells (N=30) treated with different doses of cisplatin;

[0024] Figure 10 (A) Two-photon fluorescence imaging of red autofluorescence in cells before, 4, and 6 hours after treatment with 1 mM H2O2 (control) and after treatment; scale bar: 50 μm; (B) Mean red autofluorescence intensity in control cells excited at 1060 nm and H2O2-treated cells (N = 30); data are expressed as mean ± SD of three independent experiments; (C) Lifetime traces of red fluorescence in control cells (red curve) and H2O2-treated cells over 6 hours; (D) Flow cytometry analysis of MDA-MB-231 cells after treatment with 1 mM H2O2; labeling dye: Annexin V-FITC / PI;

[0025] Figure 11 (A) Two-photon fluorescence imaging of red autofluorescence in cells before (control) and after treatment with 1, 2, 2.5, 5, and 10 μg / mL shikonin; scale bar: 50 μm; (B) Mean red autofluorescence intensity in control cells and shikonin-treated cells excited at 1060 nm (N = 30); data are expressed as mean ± SD of three independent experiments; (C) Flow cytometry analysis of MDA-MB-231 cells after shikonin treatment; labeling dye: Annexin V-FITC / PI; (D) Percentage of live cells (orange bars) and necrotic cells (green bars) after 6 hours of shikonin treatment; (E) Fluorescence lifetime decay curves of the control group (black curve) and the treatment group (red and blue curves);

[0026] Figure 12(A) Fluorescence lifetime imaging of MDA-MB-231 cells before and after cisplatin treatment; the upper panel represents lipofuscin signal, and the lower panel represents color-coded imaging of the lifetime component τm in the range of 0-2000 ps; scale bar: 50 μm; (B) τ1-α2 scatter plot representing the separation and distribution of apoptotic cells and control cells; (C)(D) τ1, τ2, α1 and α2 parameters of control and experimental cells (N≥30); apoptotic cells have increased α2 and lifetime; (E) Two-photon fluorescence imaging of lipofuscin-like fluorophores in cells 6 hours before (control) and 6 hours after treatment with 1 mM H2O2 and 5 μg / mL shikonin; excitation wavelength: 1060 nm; scale bar: 50 μm; (F) Lifetime tracking of red fluorescence induced by H2O2 and shikonin within 6 hours; (G) 1 mM Flow cytometry analysis of MDA-MB-231 cells treated with H2O2 and 5 μg / mL and 10 μg / mL shikonin; Labeling dye: Annexin V-FITC / PI; (H) Confocal fluorescence imaging of cells labeled with lysosomes (λex = 488 nm) and lipofuscin fluorophores (λex = 561 nm) before and after H2O2 treatment; Scale bar: 50 μm; (I) Confocal fluorescence and bright-field imaging of cells after H2O2 treatment; Cell nuclei labeled with Hoechst 33342 (blue); Red represents lipofuscin fluorophores excited at 561 nm; Scale bar: 20 μm; Inset shows magnified images of areas highlighted with red circles;

[0027] Figure 13 (A) Color images of the lifetime component τm of L929 cells in the 0-2000ps range before and after cisplatin treatment, scale bar: 50μm; (B)(C) τ1, τ2, α1 and α2 parameters of single cells (N≥30) in the control and experimental groups; (D) Scatter plot showing cell distribution; (E) Flow cytometry analysis of the degree of apoptosis in L929 cells after cisplatin treatment; Labeling dye: Annexin V-FITC / PI assay kit;

[0028] Figure 14 (A) Flow cytometry analysis of MDA-MB-231 cells after treatment with different cell death inducers; labeling dye: Annexin V-FITC / PI assay kit. (B) Lifetime traces of red autofluorescence in cells in the control group, 30 μM cisplatin treatment group, and 5 μg / mL shikonin treatment group. Observation at fixed time points showed that, compared with the control cells, cisplatin-induced apoptosis had a higher photon count, while shikonin-induced necrosis had a lower photon count.

[0029] Figure 15The effects of different inducers and dosages on cell death types were determined by the lifespan parameters; (A) Two-photon red autofluorescence intensity (top) and lifespan imaging (bottom) after treatment with 30 μM cisplatin for 72 h, 200 nM TG for 48 h, and 1 mM H2O2 and 5 μg / mL shikonin for 6 h; scale bar is 50 μM; (B)(C) Comparison of cell lifespan parameters τ1, τ2, α1, and α2 after treatment with different inducers; (N≥30) ***p<0.001, Student's t-test when comparing H2O2, shikonin, and cisplatin; ###p<0.001, Student's t-test when comparing H2O2, shikonin, and TG. t-test; (B) and (C) blue and red boxes respectively classify apoptosis-inducing factors and necrosis-inducing factors; (D) OVCA429 cells were treated with 5 μM, 15 μM, and 30 μM cisplatin for 1 and 2 days respectively; (E) FLIM of OVCA429 cells at each dose; color scale: 0-2000ps; (F) Scatter plot of τ1-α2 of OVCA429 cells shows that most of the data points of the 5 μM (orange star) dose fall in the apoptosis area, while most of the data points of the 30 μM (purple triangle with cross) dose fall in the necrosis area. The coordinates of each point represent the τ1 and α2 values ​​of a single cell (N≥30); scale bar: 50 μm;

[0030] Figure 16 Comparison of fluorescence lifetime parameters (A) τ1 and τ2 and (B) α1 and α2 in OVCA429 cells (N≥30) after treatment with 5, 15, and 30 μM cisplatin; ***p<0.001, Student's t-test for cisplatin treatment group compared with control group, ###p<0.001; t-test for 15 μM and 30 μM cisplatin treatment group compared with 5 μM cisplatin treatment group; (C) Annexin V-FITC / PI analysis of OVCA429 cells by flow cytometry 2 days after treatment with cisplatin (0, 5, 15, or 30 μM);

[0031] Figure 17Imagery of stress-induced autofluorescence and lifetime of lipofuscin in 3D spheres and organoids; (A) Two-photon fluorescence images of red autofluorescence of lipofuscin in 3D spheres before and after 2 days of cisplatin (5, 15, and 30 μM) treatment, with an inset showing a 3D view of lipofuscin in the spheres; (B) Mean two-photon fluorescence intensity of cells (N=30) in the control and treatment groups, data are mean ± SD of three independent experiments; (C) Lifetime decay curves of red fluorescence of lipofuscin in the 30 μM cisplatin treatment group and the control group on day 2 post-treatment; (D) Two-photon fluorescence images of red autofluorescence of lipofuscin in organoids before and after 4 days of cisplatin (1, 5, and 30 μM) treatment, with an inset showing a 3D view of lipofuscin in the organoids; (E) Mean two-photon fluorescence intensity of cells (N=30) in the control and treatment groups, data are mean ± SD of three independent experiments; (F) Lifetime decay curves of red fluorescence of lipofuscin in the 30 μM cisplatin treatment group and the control group on day 4 post-treatment.

[0032] Figure 18 (A) Two-photon fluorescence images of lipofuscin-like red autofluorescence in 3D spheres before and after treatment with cisplatin (5, 15 or 30 μM) within 2 days; scale bar: 50 μm; (B) 3D view of red autofluorescence in cells within 3D spheres; (C) Flow cytometry Annexin V-FITC / PI analysis of spheres after 2 days of treatment;

[0033] Figure 19 (A) Two-photon fluorescence images of lipofuscin-like red autofluorescence in human breast cancer organoids before and after 4 days of treatment with cisplatin (1, 5 or 30 μM); Scale bar: 100 μm; (B) 3D view of red autofluorescence in cells of organoids; (C) Flow cytometry Annexin V-FITC / PI analysis of organoids after 4 days of treatment;

[0034] Figure 20 Time-pathway two-photon fluorescence image of lipofuscin-like red autofluorescence in a 3D organoid composed of cisplatin-resistant MDA-MB-231 cells; no increase in red autofluorescence was observed within 3 days before (control) and before treatment with 30 μM cisplatin; cell status was confirmed by flow cytometry Annexin-V / PI analysis;

[0035] Figure 21Imaging of stress-induced autofluorescence and lifetime of lipofuscin in 3D tumor sections; (A) Two-photon imaging of drug-induced lipofuscin fluorescence (red) (λex = 1060 nm) and second harmonic generation imaging of collagen network (green) in 3D-TSC; scale bar: 50 μm; (B) Mean two-photon fluorescence intensity of lipofuscin fluorescence in 3D-TSC treated with cisplatin, αPD-1, and αPD-L1 for four days; (C) Lifetime decay curves of lipofuscin fluorescence in the control group, chemotherapy (cisplatin) group, and immunotherapy group (αPD-1 and αPD-L1);

[0036] Figure 22 (A) MTT analysis of 3D tumor sections treated with cisplatin, αPD-1 and αPD-L1 on days 1, 2 and 4; (B) Cell viability of tumor sections on days 1, 2 and 4 after treatment compared with control;

[0037] Figure 23 (A) Human tumor sections treated with 10 μg / mL αPD-1 and αPD-L1; (B) Mean two-photon fluorescence intensity of lipofuscin-like fluorescence in 3D-TSCs treated with αPD-1 and αPD-L1; (C) GFP+ tumor sections 4 days after treatment with cisplatin, αPD-1 and αPD-L1. Green represents GFP+ breast tumor cells, and red represents two-photon lipofuscin-like fluorescence excited at 1060 nm. Scale bar: 50 μm. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0040] Although apoptosis induces an increase in lipofuscin-like fluorescence, an increase in its own red fluorescence does not necessarily indicate apoptosis. Other types of cell death, such as necrosis, may exhibit similar characteristics. Necrosis is an uncontrolled form of cell death that is harmful to surrounding cells in the tissue. It is commonly seen in acutely damaged cells. The distinction between apoptosis and necrosis is an important basis for evaluating the efficacy of anticancer drugs.

[0041] This invention provides a method for identifying apoptosis and / or necrosis, comprising: real-time detection of the fluorescence lifetime of lipofuscin and / or lipofuscin-adsorbed substances in the cells to be tested, and identifying apoptosis and / or necrosis by the length of the fluorescence lifetime; the method is not for the direct purpose of disease diagnosis or treatment.

[0042] This invention uses the fluorescence lifetime of lipofuscin as a parameter to sensitively display cell apoptosis and necrosis. It is a non-invasive method that can monitor in real time without the need for fixation, staining with exogenous substances, or destructive dissociation, representing a fundamental advancement.

[0043] The inventors discovered that when cells are in an apoptotic state, their fluorescence lifetime is longer than that of normal living cells, while when cells are in a necrotic state, their fluorescence lifetime is shorter than that of normal living cells. Therefore, the fluorescence lifetime of different cell states can be used to effectively identify the cell state.

[0044] Preferably, the method further includes setting an apoptosis threshold and / or a necrosis threshold for fluorescence lifetime;

[0045] If the fluorescence lifetime of the test cells is greater than or equal to the apoptosis threshold, then the test cells are determined to be apoptotic cells.

[0046] If the fluorescence lifetime of the test cell is less than or equal to the necrosis threshold, then the test cell is determined to be a necrotic cell.

[0047] Specifically, the fluorescence lifetime of cells under normal survival conditions is used as a threshold. Different cell types have varying fluorescence lifetimes under normal survival conditions due to their unique metabolic characteristics; therefore, a specific threshold can be established for different cell lines. For example, for MDA-MB-231 (… Figure 12 Cells with a t1 value higher than 400 ps and an α2 value higher than 20% can be considered apoptotic cells. For L929 cells (…),… Figure 13 When t1 is higher than 250 ps and α2 is higher than 15%, the cells can be identified as apoptotic cells.

[0048] In other embodiments, other statistical methods can be used to classify cell states based on the fluorescence lifetime of the cells to be tested; any technical solution that does so falls within the scope of protection of this application.

[0049] Preferably, the fluorescence lifetime refers to the decay time of fluorescence emission after lipofuscin is excited by a single photon or two photon.

[0050] More preferably, the fluorescence lifetime refers to the decay time of fluorescence emission after lipofuscin is excited by two photons. Using two-photon excitation can more effectively avoid co-excitation by other endogenous fluorophores and prevent interference.

[0051] There are two main analytical methods for calculating fluorescence lifetime: one is to directly fit the measurement curve in the time domain using an exponential decay model, and the other is to use a sine or cosine transform to convert the data to a phasor plot in the frequency domain for analysis.

[0052] Preferably, the detection conditions for the fluorescence lifetime of the lipofuscin are: single-photon excitation wavelength of 500-560 nm and two-photon excitation wavelength of 970-1140 nm, specifically 970 nm, 980 nm, 990 nm, 1000 nm, 1010 nm, 1020 nm, 1030 nm, 1040 nm, 1050 nm, 1060 nm, 1070 nm, 1080 nm, 1090 nm, 1100 nm, 1110 nm, etc. The range of any one or any two of 0nm, 1120nm, 1130nm and 1140nm; the emission wavelength of lipofuscin is 550-700nm, specifically the range of any one or any two of 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm and 770nm.

[0053] Preferably, the fluorescence lifetime parameters of the lipofuscin include at least one of fluorescence lifetime τ1, fluorescence lifetime τ2, the relative ratio of the initial intensity of τ1 to α1, and the relative ratio of the initial intensity of τ2 to α2. When identifying apoptosis and / or necrosis, it is necessary to make a judgment based on the relevant parameters.

[0054] The difference in fluorescence lifetime of fluorescent substances stems from the differences in the binding state of lipids and proteins, or the differences in the acidity and alkalinity of the organelles in which they are located. Therefore, there are two fluorescence lifetimes (τ1 and τ2) of different lengths, mainly due to the molecules being in two different extreme environmental states.

[0055] α1 is the relative proportion of the initial intensity of τ1. α2 is the relative proportion of the initial intensity of τ2.

[0056] Preferably, the cells to be tested are selected from any one of the following: cell spheroid models, organoids and 3D tumor slice models, primary tissue cultures, and embryonic cells.

[0057] More preferably, the cells to be tested are any one of 3D tumor slice models, primary tissue cultures, and embryonic cells. Compared with 2D cell cultures, 3D tumor models can better simulate the tumor microenvironment, primary tissue cultures can better simulate the organ microenvironment, and embryonic cells can reflect the responses of multiple organs, revealing more physiologically relevant targets in drug screening.

[0058] Preferably, the thickness of the 3D tumor slices and the primary tissue culture is 200-300 μm, specifically any one or any two of 200 μm, 220 μm, 240 μm, 260 μm, 280 μm and 300 μm.

[0059] Preferably, the method for obtaining the 3D tumor slice model and the primary tissue culture is: to encapsulate the tumor tissue and normal tissue with gel and then slice them.

[0060] Furthermore, embodiments of the present invention also provide the application of the methods for identifying apoptosis and / or necrosis as described in any of the foregoing embodiments in drug screening or efficacy evaluation.

[0061] Example 1

[0062] Experimental methods

[0063] Cell culture

[0064] MDA-MB-231 human breast cancer cells and L929 mouse fibroblasts were cultured for cell viability and drug sensitivity experiments. Cells were maintained in Dulbecco modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco) and incubated at 37°C in a 5% CO2 incubator. Cells were cultured at 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a concentration of 100 cells / mL in confocal culture dishes (NEST, 801006) and cultured to 60-80% confluence before drug treatment.

[0065] Drug treatment

[0066] To induce apoptosis, MDA-MB-231 cells were treated with 300 μM H2O2 for 6 hours or 30 μM cisplatin (Sigma) for 72 hours. L929 cells, due to their lower drug tolerance, were incubated with 30 μM cisplatin for 24 hours. For dose- and time-dependent experiments, MDA-MB-231 cells were treated with different concentrations of cisplatin (1, 5, 10, 30, and 60 μM).

[0067] To induce apoptosis associated with endoplasmic reticulum (ER) stress, MDA-MB-231 cells were treated with tocopherol (200 nM; TocrisBioscience) for 48 hours.

[0068] To induce cell necrosis, MDA-MB-231 cells were treated with a high concentration (1 mM) of H2O2 or 5 μg / mL of shikonin (Sigma) for 6 hours.

[0069] Cell spheroid culture

[0070] 24-well confocal plates (NEST, 801006) were coated with a 0.5% agarose scaffold to form cancer spheroids for microscopic observation. One hundred ovarian cancer OVCA429 cells were seeded into each pre-coated well and cultured in 500 μL of DMEM containing 10% FBS and 1% penicillin-streptomycin for 3–4 days to form multicellular cancer spheroids. Spheroids were collected on days 1, 2, and 4 after treatment with cisplatin (5 μM, 15 μM, and 30 μM) for TPF imaging and apoptosis / necrosis assays.

[0071] Preparation of three-dimensional (3D) tumor sections

[0072] All animal experiments were approved by the Animal Facility of the Faculty of Health Sciences, University of Macau (Approval No. UMARE-015-2019).

[0073] Tumors were obtained from genetically engineered mouse models and human primary tumors, stored in cold PBS, and sectioned into 200 μm thick sections using a vibratory microtome (Leica Biosystems Nussloch GmbH, VT1200S) within 6 hours post-surgery. Then, 100 μL of recombinant collagen solution was added to Millicell inserts (12 mm, Millipore, PIHP01250) and incubated at 37°C for 20 minutes until coagulation. Next, sections were pre-coated with 100 μL of recombinant collagen solution and incubated at 37°C for 20 minutes to solidify the tissue-containing gel top layer. Subsequently, 400 μL of culture medium (Ham's F12; Gibco containing 20% ​​FBS and 50 μg / ml gentamicin) was placed in the inserts. Finally, the sections were incubated in a 37°C incubator containing 5% CO2.

[0074] For the treatment group, culture sections were treated with 25 μM cisplatin, anti-mouse CD279 (αPD-1, Biolegend, 10 μg / mL), or anti-mouse CD274 (αPD-L1, Biolegend, 10 μg / mL) to evaluate the efficacy of the anticancer drugs. The treated tumor sections were imaged at each time point using single-photon and multiphoton microscopy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to measure cell viability in the tumor sections. Specifically, each well was stained with 400 μL of 25 μg / mL MTT solution and incubated at 37°C for 3 hours. The reagents were then washed away with phosphate-buffered saline (PBS), and 1 mL of isopropanol was added to dissolve the formed formazan. The absorbance at 570 nm was then measured using a microplate reader (PerkinElmer Victor X3, Waltham, MA, USA). MTT images of the sections were also taken using a fluorescence stereomicroscope (Leica, M165FC, Germany).

[0075] organoid culture

[0076] For human breast cancer organoid culture, tumor specimens were obtained from Jinghu Hospital (approval number 20180907T), transversely cut into small pieces, and completely minced with a scalpel. For cisplatin-resistant MDA-MB-231 cell organoid cultures, resistant clones were selected by gradually increasing the cisplatin concentration to obtain cell lines with high tolerance to 50 μM cisplatin. RPMI medium (Gibco) containing 5% FBS, collagenase I, and DNase was used for tumor digestion. After incubation at 37°C for 30 minutes to 1 hour on a shaker, the tumor tissue was filtered through a filter, and the remaining suspension was centrifuged at 2000 rpm for 5 minutes and washed with cold PBS. The cells were then mixed with Matrigel (Corning, 356234) and placed in preheated 6-well cell culture plates. Preheated medium 25 was added after 20 minutes, and the medium was changed every four days. For confocal microscopy, the organoids were digested with trypsin for 10 minutes, gently pipetting 4-6 times every 5 minutes, then terminated with serum, and washed twice with cold PBS. Then, the cells (6.5 × 10⁻⁶) 6 Mix ( / mL) with cold Matrigel (10 μL) in a 24-well confocal culture dish and incubate at 37°C in a 5% CO2 incubator. Add culture medium after 20 minutes. The next day, add normal culture medium or culture medium with different concentrations of cisplatin for drug sensitivity testing.

[0077] Flow cytometry analysis of cell death

[0078] Apoptosis / necrosis in 2D cell cultures and 3D tumor models (spheroids and organoids) was detected using the Annexin V-FITC / PI kit (Invitrogen). Harvested cells (5 × 10⁻⁶) 5 Wash gently with PBS, then resuspend in 195 μL of binding buffer. For Annexin V staining, add 5 μL of Annexin V-FITC to the sample solution and incubate at room temperature in the dark for 15 minutes. Wash cells with binding buffer and resuspend in 200 μL of buffer with 10 μL of propidium iodide (PI). Use BD Accurit with standard FITC and PI channels. TM Fluorescence signals were detected using a C6 cytometer (BDBiosciences, USA) and analyzed using FlowJo software (Tree Star). At least 10,000 cells were gated and analyzed using forward scatter (FSC) and side scatter (SSC) signals in each run. Single-label (PI or FITC) measurements were then obtained to compensate for background signals in the FITC and PI channels. Gating was performed on FITC and PI-labeled positive / negative populations after selecting appropriate intensity thresholds. Healthy cells were Annexin V / PI double-negative. Following treatment, apoptotic cells moved to both Annexin V-positive and PI-negative quadrants, while necrotic cells moved to the Annexin V / PI double-positive quadrant.

[0079] In situ imaging of apoptotic cells was performed using a caspase-3 detection kit: After imaging apoptotic cells with red autofluorescence, the initiation of apoptosis was confirmed using a caspase-3 detection kit (Invitrogen, Image-iT™ Live Green). Treated and control cells were mixed with 30X FLICA working solution in cell culture medium and incubated for 60 minutes under culture conditions. Cells were then washed twice with 1X wash buffer, and the distribution of caspase-3 was imaged using a confocal microscope at an excitation wavelength of 488 nm.

[0080] LysoTracker dye labeling of intracellular lysosomes

[0081] LysoTracker Green (Thermo Fisher, DND-26) was used to confirm the accumulation of pressure-induced red autofluorescence in lysosomes, to mark the location of lysosomes, and to observe the relative distribution of red autofluorescence. LysoTracker Green solution (75 nM) was added to the culture medium of the control and drug-treated groups. Cells were then incubated with the dye at 37°C for 45 minutes and washed twice with PBS.

[0082] Two-photon fluorescence image acquisition

[0083] For TPF imaging, cells were seeded in confocal culture dishes and cultured in a microscope-compatible miniature incubator system containing 5% CO2 and maintained at a constant temperature of 37°C (Nikon Instrument Inc., Japan). A wavelength-tunable (700-1300 nm) near-infrared femtosecond laser (InSight X3, Spectra-Physics) was used as the light source for two-photon fluorescence imaging, which extended the penetration depth and reduced photodamage to cells in 3D tumor cultures. Each image was captured using a Nikon Eclipse inverted multiphoton microscope (A1MP+Eclipse Ti-2E, Nikon Instruments Inc., Japan) with a 40×NA=1.15 water immersion objective. Excited TPF and second-harmonic generation (SHG) signals were collected by the same objective, reflected by a multiphoton dichroic beam splitter, and detected by four photomultiplier tubes (PMTs). Lipofuscin-like autofluorescence could be selectively excited at 1060 nm and detected by a PMT in channel 3 (λdet=604-679 nm). This excitation wavelength avoided two-photon co-excitation of flavin. PMT in channel 2 (λdet = 506–593 nm) detected a 520 nm SHG signal in the 3D tumor slices. In wavelength-dependent excitation experiments, the laser power at different excitation wavelengths was uniformly set to 15 mW behind the objective. For each experimental group, three images with a 317 × 317 μm field of view were acquired, and at least 30 cells were selected from them for fluorescence intensity analysis. Furthermore, a CCD-cooled spectrometer (iDus401plus shamrock 193i, ANDOR, Oxford Instruments) was connected to the back port of the inverted microscope. The corresponding two-photon emission spectrum was obtained from the integrated spectrometer each time a multiphoton image was acquired.

[0084] FLIM Image Acquisition and Data Analysis

[0085] Two photon counting PMTs (PMC-150-4, Becker & Hickl) sharing the same signal path as the fluorescence spectrometer were further mounted on the rear port of the same multiphoton microscope (Eclipse Ti-2E, Nikon). Fluorescence lifetime data were recorded using a time-correlated single-photon counting system (SPC-160, Becker & Hickl) synchronized with the scanning excitation of the Nikon A1 MP+ multiphoton microscope. To improve the quality of lifetime fitting, at least 120 seconds were spent acquiring a single FLIM image, ensuring that the lipofuscin fluorescence peak photon count for most pixels in the cell was above 200. A pixel dwell time of 25.21 μs was set to obtain a 256 × 256 pixel image.

[0086] Statistical analysis of fluorescence intensity and fluorescence lifetime of lipofuscin.

[0087] The mean fluorescence intensity within cells was assessed using Fiji software (ImageJ). For each group, three images were acquired, and at least 30 cells were analyzed. The intensity of lipofuscin in the cells was assessed by the mean fluorescence value in the cytoplasm.

[0088] For fluorescence lifetime statistics, SPC Imaged software (Beker & Hickl) was used to analyze FLIM data. The fluorescence lifetime of each pixel was fitted using a double exponential decay model. Here, IRF is the instrument response function measured from the SHG of urea. α1 and α2 represent the percentage amplitude contributions of photons from the two fluorescence lifetime components τ1 and τ2, respectively. Trajectory fitting is optimized to have low chi-square error, and the software generates amplitude-weighted lifetime histograms and color-coded lifetime images. For ease of comparison, analysis is sometimes performed using Tm = τ1 × α1 + τ2 × α2. Figure 1 Similar to fluorescence intensity studies, at least three images were acquired from some FLIM images to obtain more than 30 cells (image size: 512×512, 0.25μm pixel size, binning 4×4, 200 photons, acquisition time: 120s). Cell boundaries were selected from brightfield images, and the corresponding fluorescence lifetime characteristics of lipofuscin-like fluorescence were analyzed. Exported data were analyzed using GraphPad Prism software (version 6.0). Two-dimensional (2D) scatter plots were obtained using Origin software (version 8.6).

[0089] Experimental results

[0090] Lipofuscin autofluorescence is an early characteristic of apoptosis.

[0091] Common drugs and cytotoxic substances such as H2O2 can induce apoptosis or necrosis. The ROS (reactive oxygen species) generated by H2O2 can induce apoptosis and necrosis at different concentrations. At an appropriate concentration, such as 300 μM, H2O2 can induce apoptosis, and Annexin-V / PI staining assays confirmed that MDA-MB-231 cells tend towards apoptosis. Figure 2 (China A, 24-hour incubation).

[0092] The weak autofluorescence observed in the control group ( Figure 3 In contrast to A), apoptotic cells showed a significant red autofluorescence intensity in two-photon imaging (excitation wavelength Ex = 1060 nm). Figure 3 (B, C) and a fluorescence emission peak at approximately 600 nm. Figure 3 The red fluorescence (C) peak, similar to that of lipofuscin excited by a fundus camera, exhibits several effective excitation bands, such as 1020, 1060, and 1100 nm, by scanning the excitation wavelength from 820 nm to 1240 nm at equivalent laser power. Figure 2 (BD). These stress-induced autofluorescence clusters accumulate as multiple granules during apoptosis, while living cells show almost no red autofluorescence. It is well known that aging impairs mitochondrial function. Insufficient autophagy hinders the clearance of damaged mitochondria and leads to the formation of lipofuscin in lysosomes. LysoTracker labeling confirmed that most of these stress-induced autofluorescence clusters are located in lysosomes (Table 1 and BD). Figure 4 ).

[0093] Table 1. Brief Introduction to Pearson Coefficient and Mander Coefficient

[0094]

[0095] The distribution of red autofluorescent clusters within these cells is consistent with the metabolic pathway of lipofuscin. Cisplatin-induced MDA-MB-231 cells were also observed to exhibit this characteristic. Figure 3 (FH) and L929 fibroblasts ( Figure 6 During apoptosis, red autofluorescence increases and exhibits a similar excitation spectrum. Figure 5 ) and emission spectrum ( Figure 3 (D). In addition to Annexin-V / PI assay, apoptosis was further confirmed by labeling Caspase 3 in these red fluorescent cells. Figure 3 (I). These spectroscopic and imaging evidences support the idea that stress-induced lipofuscin-like red fluorescence is closely related to the apoptosis process and may originate from the acute accumulation of lysosomal lipofuscin.

[0096] Long-term monitoring of the pharmacodynamic characteristics of apoptosis.

[0097] The time- and dose-dependent relationship between red autofluorescence and apoptosis in cisplatin-treated MDA-MB-231 cells was analyzed. Annexin V / PI assay showed that apoptosis was time- and dose-dependent after stimulation with 30 μM cisplatin, with an apoptosis rate of 45.5% after 72 h of treatment. Figure 7 (A) In the control group (0 μM, 0–72 h), a small number of red fluorescent cells appeared in the field of view. This was consistent with the Annexin V / PI experiment; furthermore, the density of red fluorescent cells increased with increasing drug incubation time and concentration. Figure 8 AB in the middle Figure 7 (Chinese AD).

[0098] To further assess the extent of early apoptosis, the drug was washed off at 12 and 24 h post-treatment, and the cells were cultured in fresh medium for an additional 24 h. The apoptosis rates increased from 5.9% (12 h w / drug) and 12.0% (24 h w / drug) to 13.1% (12 h w / +24 h w / no drug) and 27.4% (24 h w / +24 h w / no drug), respectively. Figure 7 (E). They followed a trend that apoptosis increased with increasing drug incubation time. Figure 7 (C, D). This confirms that apoptosis begins as early as 12 hours after treatment, but traditional Annexin / PI assays are difficult to visualize the apoptotic trend at this early time point. In contrast, red fluorescence increased in the 10 μM and 30 μM dose groups at 12 hours post-treatment. Figure 8 (B) , 12 hours earlier than the 5μM dose group ( Figure 9 The 30 μM dose group reached the threshold of lipofuscin-like fluorescence (average intensity 8.968) 48 h after treatment, 12 h earlier than the 5 μM and 10 μM dose groups. Figure 9 Furthermore, the mean fluorescence lifetime also reported an early treatment response, increasing from 171 ps pretreatment to 287.7 ps at 12 h posttreatment. This lifetime increase ultimately reached 507 ps after 48–72 h of treatment, indicating that the chemical environment of the lipofuscin-like fluorophore remained unchanged after 48 h of treatment. Figure 8 (C). Therefore, if a threshold is set for the average lifetime of intracellular photon count and lipofuscin-like fluorescence, the treatment response can be measured 24 hours earlier than conventional methods, and even 12 hours earlier than possible with Annexin-V / PI staining.

[0099] Fluorescence lifetime imaging microscopy (FLIM) is used to distinguish between apoptosis and necrosis.

[0100] High concentrations of H2O2 and the traditional Chinese medicine anticancer agent shikonin can both induce cell necrosis. Treatment of MDA-MB-21 cells with 1 mM H2O2 and 5 μg / mL shikonin for 6 hours resulted in necrotic cells accounting for 46.9% and 59.5%, respectively. Under these conditions, a significant increase in red fluorescence was also observed in the necrotic cells. Figure 10 and Figure 11 Their spectral shapes are similar to those of apoptotic cells. This means that the intensity of lipofuscin-like red autofluorescence can indicate cellular senescence, but not the type of cell death.

[0101] To differentiate between necrosis and apoptosis, this embodiment introduces an additional parameter: the two-photon lifetime of lipofuscin-like fluorescence. Compared to intensity measurements, fluorescence lifetime measurements avoid imagery caused by photobleaching and do not require calibration under excitation conditions. Furthermore, fluorescence lifetime reflects the influence of environmental factors on fluorophore charge relaxation, including solution pH, the presence of quenchers, self-aggregation, and binding to macromolecules. Previous data have shown that lipofuscin-like fluorescence lifetime measurements can indicate the transition of cells from a normal state to apoptosis. If the increased apoptotic lifetime is due to the lysosomal environment of lipofuscin, it is reasonable to infer that lysosomal rupture during necrosis may significantly alter the fluorescence lifetime of lipofuscin. This could provide a label-free indicator to differentiate common necrosis from apoptosis.

[0102] Based on the analysis of lipofuscin FLIM ( Figure 12 In apoptotic cells (α1 and α2), two major lifespan components exist: τ1 = 400-700 ps and τ2 = 2200 ps, ​​accounting for 72% (α1) and 28% (α2), respectively. Figure 12 (C, D). The fluorescence lifetime of the control group was significantly shortened, τ1 = 100–300 ps, ​​τ2 = 1500 ps, ​​α1 = 88%. The α2-τ1 two-dimensional scatter plot showed good separation between data points from apoptotic cells and those from viable cells. Figure 12 (Middle B). Furthermore, similar measurements were performed on L929 fibroblasts. The red fluorescence lifetime of apoptotic L929 cells (τ1 = 200-400 ps; τ2 = 2000 ps) was also longer than that of control cells (τ1 = 150 ps; τ2 = 1750 ps), and apoptotic cells could also be separated from control cells by α2-τ1 scatter plots. Figure 13 (AD). The apoptotic status of L929 cells was also confirmed by Annexin V / PI staining and flow cytometry. Figure 13 (E in the text)

[0103] The understanding that FLIM can distinguish between apoptotic and normal cells further validates its role in differentiating between necrosis and apoptosis. Although both 1 mM H2O2 and shikonin can induce an increase in red two-photon fluorescence in necrotic cells, they shortened the fluorescence decay time compared to the increased fluorescence decay time observed during apoptosis. Figure 12 (medium EG). As expected, no lysosomes were found in the necrotic cells after LysoTracker labeling. Figure 12 (H). Therefore, it is speculated that the lysosomes were severely damaged during necrosis, leading to the release of accumulated lipofuscin into the cytoplasm. Figure 12 (I) altered the fluorescence lifetime of the lipofuscin-like fluorophore. Furthermore, it was hoped that FLIM could distinguish between necrosis and apoptosis at cellular resolution. Apoptosis was induced using the endoplasmic membrane stressor TG and cisplatin. Necrosis was induced using 1 mM H2O2 and 5 μg / mL shikonin. Cell status was confirmed by Annexin V / PI staining and flow cytometry. Figure 14 All treated cells showed a strong red lipofuscin-like TPF at the experimental endpoint, and the death type was clearly indicated by color-attached FLIM data of the mean lifespan τm. Figure 15 (A) Cells treated with TG showed a small amount of necrosis (uniformly yellow), while cells treated with H2O2 showed a small amount of apoptosis (blue granules). Compared with apoptotic cells, necrotic cells were much shorter at τ1 = 200 ps and τ2 = 1300 ps. Figure 15 (B) Short-lived components contribute more (α1 = 80%), while long-lived components (α2) contribute less ( Figure 15 (C). These results validate that FLIMs of lipofuscin-like TPFs can support marker-free differentiation of necrotic and apoptotic cells through their lifespan and subcellular structures. The method of this invention provides crucial information for assessing the types of cell death that occur after exposure to different doses of drugs.

[0104] To further elucidate the value of fluorescence lifetime in determining cell death types, ovarian cancer cell line OVCA429 was treated with 5 μM, 15 μM, and 30 μM cisplatin. Figure 15 As shown in Figure D, time- and dose-dependent studies revealed that the red autofluorescence of OVCA429 cells increased with increasing cisplatin dose and incubation time. Interestingly, FLIM data showed that different doses of treatment led to significantly different lifespan characteristics. Figure 15 E; Figure 16 The terms α2 and τ1 correspond to different types of cell death. Low-dose cisplatin (5 μM) induced apoptosis, while 15 μM and 30 μM induced necrosis. The separation of cells in different states can be easily observed in two-dimensional scatter plots of α2 and τ1. Figure 15(F). We confirmed this phenomenon through specific Annexin V / PI analysis. Cell apoptosis was observed after 2 days of treatment with 5 μM cisplatin. However, at doses of 15 μM and 30 μM, the necrosis rate increased to 16.2% and 29.2%, respectively. Figure 16 (C). In summary, combining the fluorescence intensity and fluorescence lifetime patterns of lipofuscin-like fluorophores can not only reveal drug sensitivity but also determine the effect of dosage on cell death types.

[0105] Label-free optical probes for therapeutic response in 3D tumor models.

[0106] Based on stress-induced autofluorescence of lipofuscin in OVCA429 cells ( Figure 17 Molecular imaging (using the red pigment in A) was used to observe the dynamics of OCVC429 cell spheroid treatment. Treatment with 5, 15, and 30 μM cisplatin significantly increased the lipofuscin fluorescence intensity in the OVCA429 spheroid model within 2 days. Figure 17 (A, B). On day 2, the lifetime of red fluorescence in cells was longer than that in the control group (A, B). Figure 17 (C). As confirmed by Annexin-V / PI staining, this stress-induced red fluorescence and increased lifespan are closely related to the degree of cellular senescence. Figure 18 (C)

[0107] However, while spheroidal cell models are widely recognized as a common research tool for drug screening, they cannot recreate the tumor microenvironment. Today, organoids are defined as 3D cell aggregates derived from primary tissues or stem cells, and can be classified as scaffolded or scaffoldless. They can self-renew and reveal organ function. Tumor organoids can maintain the organ cell type and genetic characteristics of the original tumor, ensuring the reproduction of the tumor microenvironment. To identify early markers that can determine apoptosis fate, this label-free optical method was used to achieve rapid and long-term monitoring of organoid models derived from human breast tumors. Based on imaging of lipofuscin fluorescence in 3D tumor organoids, pharmacodynamics dependent on cisplatin dose (1, 5, and 30 μM) and incubation time was observed. Figure 19 Following treatment, increased lipofuscin fluorescence intensity and lifetime were observed in organoids within four days. Figure 17 (middle DF). Annexin-V / PI staining confirmed that the red fluorescence in this cisplatin-induced human tumor organoid was closely related to the degree of cellular senescence. Figure 19 In contrast, organoids composed of cisplatin-resistant MDA-MB-231 cells did not show a significant increase in strength. Figure 20 This result indicates that if the tumor is resistant to treatment, the cells can cope with drug-induced stress and do not accumulate the red fluorescence of lipofuscin.

[0108] Application in 3D tumor slice culture model.

[0109] Furthermore, label-free metabolic imaging methods were applied to evaluate the efficacy of anticancer drugs in real time within a three-dimensional tumor slice culture (3D-TSC) model.

[0110] 3D-TSC can even preserve the tumor microenvironment and immune cells, which is crucial for evaluating immune checkpoint blockade therapies. This is achieved by targeting drug-induced lipofuscin (LFP) in cells. Figure 21 The red channels of A and collagen fibers ( Figure 21 Imaging was performed using the green color of αPD-L1 to investigate the dynamics of 3D-TSC treatment over 4 days. After treatment with 25 μM cisplatin, 2.5 μg / mL αPD-1, and 2.5 μg / mL αPD-L1, enhanced lipofuscin fluorescence intensity was observed in tumor sections. Furthermore, compared to the control group, the fluorescence lifetime increased from 573.4 ps to a longer 950 ps under αPD-L1 treatment. Figure 21 (B). However, for cisplatin treatment, the intensity and lifespan of lipofuscin fluorescence were slightly lower than in the immunotherapy group (B). Figure 21 (B). Meanwhile, stress-induced changes in red fluorescence intensity and lifetime are closely related to cell viability (B). Figure 22 This phenomenon may mean that immunotherapy eliminates more tumor cells compared to cisplatin treatment. The technique was applied to patient-derived tumor samples (PDTS), from colon and nasopharyngeal carcinoma samples from two patients. Seven days after αPD-L1 treatment, the lipofuscin fluorescence intensity in colon cancer significantly increased. For nasopharyngeal carcinoma, lipofuscin fluorescence remained almost stable after immunotherapy. Figure 23 (AB).

[0111] To further confirm that this method can support label-free detection of cell death in 3D-TSC, viable tumor cells were identified by GFP labeling in mouse GFP+ tumor sections. During treatment, the increase in lipofuscin fluorescence intensity was confirmed to be accompanied by the disappearance of GFP+ cells. Figure 23 (Green in C). Therefore, the results indicate that lipofuscin-like fluorescence can serve as a valuable optical probe for label-free and long-term monitoring of pharmacodynamics in 3D-TSC. This practical method could be translated into clinical practice.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for identifying apoptosis and / or necrosis, characterized in that, It comprises: Real-time detection of the fluorescence lifetime of lipofuscin in the test cell, to identify apoptosis and / or necrosis by the length of the fluorescence lifetime; The method is not directly aimed at the diagnosis or treatment of disease; The method further comprises setting an apoptosis threshold and / or a necrosis threshold for the fluorescence lifetime; If the fluorescence lifetime of the test cell is ≥ the apoptosis threshold, then the test cell is determined to be an apoptotic cell; If the fluorescence lifetime of the test cell is ≤ the necrosis threshold, then the test cell is determined to be a necrotic cell; The fluorescence lifetime refers to the decay time of fluorescence emission after single-photon or two-photon excitation of lipofuscin, and the detection conditions of the fluorescence lifetime of lipofuscin are: single-photon excitation wavelength of 500-560 nm, two-photon excitation wavelength of 970-1140 nm, and lipofuscin emission wavelength of 550-800 nm. The related parameters of the fluorescence lifetime of lipofuscin include at least one of the fluorescence lifetime τ1, the fluorescence lifetime τ2, the relative proportion of the initial intensity of τ1 α1, and the relative proportion of the initial intensity of τ2 α2. In identifying the apoptosis and / or necrosis state, the judgment is based on the related parameters.

2. The method of identifying apoptosis and / or necrosis according to claim 1, characterized in that, The fluorescence lifetime refers to the decay time of fluorescence emission after two-photon excitation of lipofuscin.

3. The method of identifying apoptosis and / or necrosis according to claim 1 or 2, characterized in that, The test cell is selected from any one of the cell spheroid model, organoid, 3D tumor section model, primary tissue culture, and embryonic cell.

4. The method of identifying apoptosis and / or necrosis according to claim 3, wherein, The test cell is any one of the 3D tumor section model, primary tissue culture, and embryonic cell.

5. The method of identifying apoptosis and / or necrosis according to claim 3, wherein, The thickness of the 3D tumor section and primary tissue culture is 200-300 μm.

6. The method of identifying apoptosis and / or necrosis according to claim 5, wherein, The method for obtaining the 3D tumor section model and primary tissue culture is to slice the tumor tissue or normal tissue after wrapping it with gel.

7. Use of any of the methods for identifying apoptosis and / or necrosis according to any one of claims 1-6 in drug screening or pharmacodynamic evaluation.

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