CN116593426A - Method for identifying apoptosis and necrosis and application thereof - Google Patents

Method for identifying apoptosis and necrosis and application thereof Download PDF

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CN116593426A
CN116593426A CN202210116490.6A CN202210116490A CN116593426A CN 116593426 A CN116593426 A CN 116593426A CN 202210116490 A CN202210116490 A CN 202210116490A CN 116593426 A CN116593426 A CN 116593426A
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lipofuscin
apoptosis
necrosis
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闫映寒
刘子铭
邓初夏
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University of Macau
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Abstract

The invention discloses a method for identifying apoptosis and necrosis and application thereof, and relates to the technical field of biological medicines. By two-photon microscopy, fluorescence spectroscopy and fluorescence lifetime imaging microscopy, all apoptotic and necrotic cells showed an increase in red fluorescence intensity after 1060nm excitation, whereas apoptotic cells showed a longer lipofuscin fluorescence lifetime in lysosomes compared to living cells, whereas necrotic cells showed a shorter lipofuscin fluorescence lifetime. Based on comparison of fluorescence lifetime, the state change of cells after drug treatment can be revealed, the technology can be applied to drug screening, and can have influence on various fields of biomedical research and application, such as drawing heterogeneity of therapeutic response in tumor microenvironment, marking single cell drug resistance in space transcriptome, capturing in vivo dynamics of cell death in embryo development, and the like.

Description

Method for identifying apoptosis and necrosis and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for identifying apoptosis and necrosis and application thereof.
Background
Apoptosis is a programmed cell death process that regulates embryonic development, controls viral infection, kills cancer cells, and maintains homeostasis in tissues. Its morphological features include chromatin condensation, DNA fragmentation, cell contraction and apoptotic body formation. Abnormalities in apoptosis can lead to the development of various diseases such as cancer, aids, alzheimer's disease, and autoimmune diseases. This mechanism of aging of cells is widely used for drug development, treatment and evaluation, as well as biosafety testing.
In contrast to apoptosis, necrosis is a passive unregulated pattern of cell death. Most organelles, including lysosomes, are completely destroyed and release the contents, resulting in inflammatory tissue damage. In the context of oncological pathology, rapid growth of tumors usually induces cell necrosis and intratumoral hypoxia, with a high probability of developing stress that results in poor prognosis, such as resistance to chemotherapy. Thus, visualization of cell death and identification of the type of death can help assess the pharmacodynamics and therapeutic effects of cancer drugs.
Traditionally, cell aging is assessed by detecting beta-galactosidase (e.g., X-GAL) with a chromogenic substrate. With the increasing understanding of the mechanism of cell death, some biochemical features, such as DNA fragmentation, membrane permeability changes, and caspase 3 activity, can be used to detect dead cells. However, the above methods all resort to end point observations, where the cells need to be fixed before staining and immunofluorescent labelling, which limits the development of dynamic real-time assessment, whereas the kinetics of apoptosis are essential for achieving rapid detection of cell death following drug treatment.
To visualize pharmacodynamics and the progress of the apoptosis reaction, scientists designed biomimetic probes such as titanium-tailored gold nanoclusters and recombination substrate proteins. However, the currently reported visual detection results all rely on exogenous reagents, synthetic nanoprobes or complex transfection methods to achieve the objective. Some endogenous fluorophores in the cell, such as tryptophan, NADH and flavin, can rapidly and label-free assess the progression of cell aging. However, these metabolic fluorophores do not show specificity for the process of cell aging and are susceptible to other metabolic activities.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for identifying apoptosis and necrosis and application thereof.
The invention is realized in the following way:
in a first aspect, embodiments of the present invention provide a method of identifying apoptosis and/or necrosis comprising: detecting fluorescence life of lipofuscin and/or lipofuscin adsorption material in the cell to be detected in real time, and identifying apoptosis and/or necrosis according to the length of fluorescence life;
the method is not directed to the diagnosis or treatment of a disease.
In a second aspect, embodiments of the present invention provide the use of a method for identifying apoptosis and/or necrosis according to any of the preceding embodiments in the screening of a medicament or evaluation of the efficacy of a medicament.
The invention has the following beneficial effects:
the present invention discloses a technique that does not require labeling and is non-invasive in order to detect apoptosis and death. By two-photon or single-photon microscopy, fluorescence spectroscopy, and fluorescence lifetime imaging microscopy, all apoptotic and necrotic cells showed an increase in red fluorescence intensity after 1060nm excitation, whereas apoptotic cells showed longer lipofuscin fluorescence lifetime in lysosomes compared to living cells, whereas necrotic cells showed shorter lipofuscin fluorescence lifetime. Based on comparison of fluorescence lifetime, the state change of cells after drug treatment can be revealed, the technology can be applied to drug screening, and can have influence on various fields of biomedical research and application, such as drawing heterogeneity of therapeutic response in tumor microenvironment, marking single cell drug resistance in space transcriptome, capturing in vivo dynamics of cell death in embryo development, and the like.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and that other related drawings may be obtained according to these drawings without paying inventive effort to a person of ordinary skill in the art.
FIG. 1 is a fluorescence lifetime imaging microscope of cultured cells; SPC imaging software is used to obtain color images of lifetime components τm, τ1, τ2, and relative amplitudes α1 and α2 in the range of 0-2000ps and 0-100%; fluorescence photon counting is contributed by lipofuscin-like fluorescence (MDA-MB-231, 30M cisplatin); in each pixel, the decay curve of the fluorescence intensity is fitted by two exponential decay components, the lifetimes of which are τ1 and τ2 (in picoseconds), the relative duty cycles of which are α1 and α2; the shift parameter represents the time delay between fitting the curve and the data; due to low background photon counts, the offset and scatter values are zero; the field of view: 128X 128 μm;
control (left) and 300. Mu. M H in FIG. 2 (A) 2 O 2 Flow cytometry of MDA-MB-231 cells in the incubated treatment groups; marking dye: annexin V/PI staining, the percentage of apoptotic cells in quadrant 3 (Q3) increased from 3.39% to 16.5%; (B) 300 mu M H with the same excitation laser power of 15mW under the excitation of 820-1240nm of two photons 2 O 2 Imaging induced MDA-MB-231 apoptosis; detection wavelength range: 604-679nm; scale bar: 50 μm; (C) Fluorescence excitation spectrum of red autofluorescence of the treatment group in (B); (D) Control cells and H stimulated at 1060nm (n=30) 2 O 2 Average red autofluorescence intensity in treated cells; all data shown represent two or three independent experiments;
FIG. 3 is early apoptosis signal detection; contrast of two-photon fluorescence imaging and intracellular fluorescence intensity (A-C) and MDA-MB-231 cells were stimulated at 1060nm, apoptosis detection range 604-679nm,300mM H 2 O 2 Treating for 24 hours; (D) Two-photon fluorescence spectrum excited by red autofluorescence at 1060nm, cisplatin 30mM,72h, H 2 O 2 300mM,24h; (E) After 72h induction with 30 μm cisplatin, lysosomes co-localize with the accumulated red autofluorophores, green channel: lysoTracker, excitation 488nm, red channel: lipofuscin, laser561nm, white, indicates co-localization of the two channels; scale bar 50 μm; (F-H) MDA-MB-231 cells are excited at 1060nm, the detection range is 604-679nm, cisplatin is 30 mu M, the duration is 72H, and the proportion is 50 mu M; (C) And (H) the statistical mean and standard deviation (error bars) are calculated from the data of three independent images acquired under the same excitation conditions, (n=30) ×p<0.001, student t-test was performed in the treatment group and the control group; (I) Caspase 3-Green stained cells of control and cisplatin-treated groups were confocal, brightfield and pooled, green represented Caspase-3 distribution, red represented lipofuscin-like fluorescence, scale bar 50 μm, cells stained with Caspase 3 reactive dye showed low background Green fluorescence signal (control) prior to cisplatin treatment, whereas cisplatin-treated cells produced significant double-positive Caspase 3 Green fluorescence signal and enhanced red autofluorescence signal;
Lysosomes in the cells of the control and treatment groups in FIG. 4 (A) were labeled with LysoTracker (excitation: 488 nm) and compared to red autofluorescence (cisplatin 30. Mu.M, 72 hours, excitation: 561 nm); white pixels represent co-located pixels of the green and red channels coincident; scale bar: 50m; (B) Gray values of green and red fluorescence, representing co-localization of lysosomes and red autofluorescence; (C) quantifying co-localization using Pearson and Mander coefficients; data represent three independent experiments;
in FIG. 5 (A) MDA-MB-231 cells were imaged for 30. Mu.M cisplatin-induced apoptosis at the same excitation laser power of 15mW with two-photon excitation of 970-1240 nm. Detection wavelength range: 604-679nm; scale bar: 50 μm; (B) fluorescence excitation spectrum of red autofluorescence in (A); (C) Average red autofluorescence intensity (n=30) in 1060 nm-stimulated control cells and cisplatin-treated cells; (D) Flow cytometry of cisplatin-treated MDA-MB-231 cells; marking dye: annexin V/PI staining; all data shown represent two or three independent experiments;
FIG. 6 (A) images L929 cells that were subject to 30. Mu.M cisplatin-induced apoptosis under two-photon excitation at 970-1240nm at the same excitation laser power of 15 mW; scale bar: 50 μm; (B) fluorescence excitation spectrum of red autofluorescence in (A); (C) Average red autofluorescence intensity (n=30) in 1060 nm-stimulated control cells and cisplatin-treated cells; all data shown represent two or three independent experiments;
FIG. 7 (A) flow cytometry analysis of MDA-MB-231 cells labeled with an Annexin V-FITC/PI detection kit before (control) and after treatment with different doses of cisplatin (1, 5, 10, and 30. Mu.M); (B) The percentage of apoptotic cells increased with increasing dose and was near 50% level at 72 hours; (C) Flow cytometry analysis was performed on MDA-MB-231 cells labeled with Annexin V-FITC/PI detection kit before (control) and after treatment with 30. Mu.M cisplatin for 12, 24, 48, and 72 hours; (D) The percentage of apoptotic cells increased over time, approaching 50% levels at 72 hours; (E) Flow cytometry analysis of MDA-MB-231 cells after 12 and 24 hours of treatment with 30. Mu.M cisplatin, followed by washing and further culture with fresh medium; all data shown represent two or three independent experiments;
FIG. 8 is a pharmacodynamic profile of apoptosis intensity and longevity; wherein, (A) 30. Mu.M cisplatin treatment MDA-MB-231 cells were time-and dose-dependent red autofluorescence images, scale bar 100. Mu.M; (B) Average intensity of red autofluorescence in cells after different incubation times; (C) Red autofluorescence lifetime of cells before (0 h) and 24h, 48h and 72h after treatment; data are mean ± standard deviation, representing three independent experiments;
Fig. 9 is a time course monitoring of mean intensity of red autofluorescence in MDA-MB-231 cells (n=30) treated with different doses of cisplatin;
FIG. 10 (A) shows the reaction with 1mM H 2 O 2 Two-photon fluorescence imaging of red autofluorescence in cells before (control) and after 2, 4, and 6 hours of treatment; scale bar: 50 μm; (B) Control cells stimulated at 1060nm and H 2 O 2 Average red autofluorescence intensity in treated cells, (n=30); data are expressed as mean ± SD of three independent experiments; (C) Control cells (red curve) and H within 6 hours 2 O 2 Life trace of red fluorescence in treated cells; (D) With 1mM H 2 O 2 Flow cytometry of post-treatment MDA-MB-231 cellsPerforming surgical analysis; marking dye: annexin V-FITC/PI;
two-photon fluorescence imaging of red autofluorescence in cells before (control) and after (A) treatment with 1, 2, 2.5, 5 and 10 μg/mL shikonin in FIG. 11; scale bar: 50 μm; (B) Average red autofluorescence intensity in 1060 nm-stimulated control cells and shikonin-treated cells, (n=30); data are expressed as mean ± SD of three independent experiments; (C) Flow cytometry analysis of MDA-MB-231 cells after shikonin treatment; marking dye: annexin V-FITC/PI; (D) Percentage of living cells (orange bars) and necrotic cells (green bars) after 6 hours of shikonin treatment; (E) Fluorescence lifetime decay curves for control (black curve) and treatment (red and blue curve);
FIG. 12 is a lifetime parameter for apoptosis and necrosis; (A) Fluorescence lifetime imaging of MDA-MB-231 cells before and after cisplatin treatment; the upper panel block represents lipofuscin signals and the lower panel block represents color coded imaging of a lifetime component τm in the range of 0-2000 ps; scale bar: 50um; (B) The τ1- α2 scatter plot represents the separation profile of apoptotic cells from control cells; (C) (D) control and test group cells τ1, τ2, α1 and α2 parameters (N.gtoreq.30); apoptotic cells possess increased α2 and longevity; (E) 1mM H 2 O 2 And 5 μg/mL shikonin before 6 hours of treatment (control) and after 6 hours of treatment, two-photon fluorescence imaging of the cell lipofuscin-like fluorophores; excitation wavelength: 1060nm; scale bar: 50 μm; (F) H 2 O 2 And life tracking of shikonin induced red fluorescence within 6 hours; (G) 1mM H 2 O 2 And flow cytometry analysis of MDA-MB-231 cells after 5 μg/mL, 10 μg/mL shikonin treatment; labeling dye, annexin V-FITC/PI; (H) H 2 O 2 Confocal fluorescence imaging of cell-labeled lysosomes (λex=488 nm) and lipofuscin fluorophores (λex=561 nm) before and after treatment; scale bar: 50 μm; (I) H 2 O 2 Confocal fluorescence and bright field imaging of the treated cells; nuclei were labeled with Hoechst 33342 (blue); red represents lipofuscin fluorophore excited at 561 nm; scale bar: 20 μm; the inset shows an enlarged image of the area highlighted with the red circle;
FIG. 13 is a color image of the lifetime component τm of L929 cells in the range of 0 to 2000ps before and after cisplatin treatment, scale bar: 50 μm; (B) (C) parameters of τ1, τ2, α1 and α2 of single cells (N.gtoreq.30) in the control group and the experimental group; (D) a scatter plot represents the distribution of cells; (E) Analyzing apoptosis degree of L929 cells after cisplatin treatment by using flow cytometry; marking dye: annexin V-FITC/PI detection kit;
FIG. 14 (A) is a flow cytometry analysis of MDA-MB-231 cells after treatment with different cell death inducers; marking dye: annexin V-FITC/PI detection kit. (B) Life trace of red autofluorescence of cells in control, 30. Mu.M cisplatin-treated and 5. Mu.g/mL shikonin-treated groups. Observing at fixed time points, we can see that the photon count of cisplatin-induced apoptosis is higher and the photon count of shikonin-induced death is lower relative to the signal of control cells;
FIG. 15 is a graph showing that life span parameters determine the effect of different inducers and doses on cell death type; (A) 30. Mu.M cisplatin action 72H,200nM TG action 48H,1mM H 2 O 2 And 5 μg/mL shikonin for 6 hours, two-photon red autofluorescence intensity (upper) and lifetime imaging (lower); the scale is 50. Mu.M; (B) (C) comparison of cell life parameters τ1, τ2, α1 and α2 after treatment with different inducers; (N.gtoreq.30). Times.p <0.001,H 2 O 2 Student's t examined when shikonin was compared with cisplatin, # # p<0.001,H 2 O 2 Checking Student's t when shikonin is compared with TG; (B) And (C) classifying the apoptosis-inducing factor and the necrosis-inducing factor by blue and red boxes, respectively; (D) OVCA429 cells were treated with 5. Mu.M, 15. Mu.M, 30. Mu.M cisplatin for 1 and 2 days, respectively; (E) individual doses of FLIM by OVCA429 cells; color code: 0-2000ps; (F) The OVCA429 cell τ1- α2 scatter plot shows that the data points for the 5 μm (orange star) dose fall mostly in the apoptotic zone, while the data points for the 30 μm (purple triangle with cross) dose fall mostly in the necrotic zone, the coordinates of each point represent the τ1, α2 values of an individual cell (n.gtoreq.30); the scale bar is 50 mu m;
FIG. 16 is a comparison of fluorescence lifetime parameters (A) τ1 and τ2 and (B) α1 and α2 of OVCA429 cells (N.gtoreq.30) after treatment with 5, 15 and 30. Mu.M cisplatin; * P <0.001, # # p <0.001, student's t test for cisplatin treated group compared to control group; t-test of 15 μm and 30 μm cisplatin-treated groups compared to 5 μm cisplatin-treated groups; (C) Flow cytometry Annexin V-FITC/PI analysis was performed on OVCA429 cells after 2 days of treatment with cisplatin (0, 5, 15, or 30 μM);
FIG. 17 is an image of stress-induced lipofuscin autofluorescence and lifetime in 3D spheres and organoids; (A) Cisplatin (5, 15 and 30 μm) treatment for 2 days was followed by two-photon fluorescence images of lipofuscin red autofluorescence in 3D spheres, with the inset showing a 3D view of lipofuscin in spheres; (B) Average two-photon fluorescence intensity of cells (n=30) in control and treatment groups, data are the average ± SD of three independent experiments; (C) Lifetime decay curve of lipofuscin red fluorescence at day 2 post-treatment for 30 μm cisplatin-treated and control groups; (D) Two-photon fluorescence images of lipofuscin red autofluorescence in organoids before and after 4 days of cisplatin (1, 5, and 30 μm) treatment, the inset shows a 3D view of lipofuscin in organoids; (E) Mean two-photon fluorescence intensity of cells (n=30) in control and treatment groups, data are mean ± SD of three independent experiments, (F) lifetime decay curve of lipofuscin red fluorescence at day 4 post-treatment for 30 μm cisplatin-treated and control groups;
FIG. 18 (A) is a two-photon fluorescence image of lipofuscin-like red autofluorescence in 3D spheres before and after treatment with cisplatin (5, 15, or 30. Mu.M) over 2 days; scale bar: 50 μm; (B) a 3D view of cellular red autofluorescence in a 3D sphere; (C) Flow cytometry Annexin V-FITC/PI analysis of spheres after 2 days of treatment;
FIG. 19 (A) is a two-photon fluorescence image of lipofuscin-like red autofluorescence in 3D human breast cancer organoids before and after treatment with cisplatin (1, 5, or 30 μM) over 4 days; scale bar: 100 μm; (B) 3D view of cellular red autofluorescence in organoids; (C) Flow cytometry Annexin V-FITC/PI analysis of organoids after 4 days of treatment;
FIG. 20 is a time-course two-photon fluorescence image of lipofuscin-like red autofluorescence in 3D organoids composed of cisplatin-resistant MDA-MB-231 cells; no increase in red autofluorescence was observed for 3 days before (control) and before treatment with 30 μm cisplatin; confirming the cell status by flow cytometry Annexin-V/PI analysis;
FIG. 21 is an image of stress-induced lipofuscin autofluorescence and lifetime in 3D tumor sections; (A) Drug-induced lipofuscin fluorescence (red) bi-photon (λex=1060 nm) and second harmonic generation imaging of collagen network (green) in 3D-TSC; scale bar: 50 μm; (B) Average two-photon fluorescence intensity of lipofuscin fluorescence in 3D-TSC for four days with cisplatin, αpd-1, and αpd-L1 treatment; (C) Life decay curves of lipofuscin fluorescence in control, chemotherapy (cisplatin) and immunotherapy (αpd-1 and αpd-L1);
MTT analysis of 3D tumor sections of cisplatin, αpd-1 and αpd-L1 treatment for 1, 2 and 4 days in fig. 22 (a); (B) Cell viability of tumor sections at days 1, 2 and 4 after treatment compared to control;
FIG. 23 (A) human tumor sections after treatment with 10 μg/mL of alpha PD-1 and alpha PD-1; (B) Average two-photon fluorescence intensity of lipofuscin-like fluorescence in αpd-1 and αpd-L1 treated 3D-TSCs; (C) Cisplatin, αpd-1 and αpd-L1 treatment for 4 days, green represents gfp+ breast tumor cells, red represents two-photon lipofuscin-like fluorescence stimulated at 1060nm, scale bar: 50 μm.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Although the apoptosis process induces an increase in lipofuscin-like fluorescence, an increase in its own red fluorescence does not necessarily represent apoptosis. Other types of cell death, such as necrosis, may have the same characteristics. Cell necrosis is an uncontrolled cell death that is detrimental to surrounding cells in tissue. It is common in acutely damaged cells. The differentiation between apoptosis and necrosis is an important basis for evaluating the efficacy of anticancer drugs.
Embodiments of the present invention provide a method for identifying apoptosis and/or necrosis comprising: detecting fluorescence life of lipofuscin and/or lipofuscin adsorption material in the cell to be detected in real time, and identifying apoptosis and/or necrosis according to the length of fluorescence life; the method is not directed to the diagnosis or treatment of a disease.
The invention adopts the fluorescence lifetime of lipofuscin as a parameter, sensitively displays apoptosis and necrosis of cells, does not need to fix, does not need to use an analysis method of exogenous substance staining or destructive dissociation, is a non-invasive method and can be monitored in real time, and achieves fundamental progress.
The inventors found that when cells are in an apoptotic state, their fluorescence lifetime is prolonged relative to that of normal living cells, and when cells are in a necrotic state, their fluorescence lifetime is shortened relative to that of normal living cells, and therefore, the states of cells can be effectively identified between those of different cell states.
Preferably, the method further comprises setting an apoptosis threshold and/or a necrosis threshold of fluorescence lifetime;
if the fluorescence lifetime of the cells to be detected is more than or equal to the apoptosis threshold value, judging the cells to be detected as apoptotic cells;
And if the fluorescence lifetime of the cell to be detected is less than or equal to the necrosis threshold value, judging the cell to be detected as necrotic cell.
Specifically, the fluorescence lifetime of the cells in the normal survival state is taken as a threshold value, and the fluorescence lifetime of the normal survival of different types of cells is different due to the specificity of metabolism, so that a definite threshold value can be established according to different cell lines. For MDA-MB-231 (FIG. 12), t1 above 400 ps.alpha.2 above 20% can be interpreted as apoptotic cells. For L929 (fig. 13), t1 is higher than 250ps, while α2 is higher than 15% is interpreted as apoptotic cells.
In other embodiments, any other statistical method may be used, so long as the technical scheme of dividing the cell states based on the fluorescence lifetime of the cells to be tested is within the scope of the present application.
Preferably, the fluorescence lifetime refers to the decay time of fluorescence emission after single photon or two photon excitation of lipofuscin.
More preferably, the fluorescence lifetime refers to the decay time of fluorescence emission after lipofuscin is excited by two photons. The use of two-photon excitation can more effectively avoid co-excitation of other endogenous fluorophores and avoid interference.
The calculation of fluorescence lifetime mainly has two analysis modes, one is to directly fit a measurement curve in the time domain through an exponential decay model, and the other is to convert data into a phasor plot in the frequency domain for analysis by using sine or cosine transform.
Preferably, the detection conditions of fluorescence lifetime of the lipofuscin are: the single photon excitation wave band is 500-560nm, the excitation wavelength of two photons is 970-1140 nm, and the two photons can be any one or any two ranges of 970nm, 980nm, 990nm, 1000nm, 1010nm, 1020nm, 1030nm, 1040nm, 1050nm, 1060nm, 1070 nm, 1080nm, 1090nm, 1100nm, 111 0nm, 1120nm, 1130nm and 1140 nm; the lipofuscin has an emission wavelength of 550-700 nm, specifically 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, and 770 nm.
Preferably, the relevant parameters of fluorescence lifetime of lipofuscin include at least one of fluorescence lifetime τ1, fluorescence lifetime τ2, relative proportion of initial intensity of τ1 α1 and relative proportion of initial intensity of τ2 α2, and a judgment based on the relevant parameters is required in identifying apoptosis and/or necrosis status.
The difference in fluorescence lifetime of fluorescent substances is due to the difference in the binding state of lipids and proteins or the difference in the pH value of the organelles, so that two kinds of fluorescence lifetimes (τ1 and τ2) exist, mainly due to two different extreme environmental conditions of the molecules.
α1 is the relative proportion of the onset intensity of τ1. α2 is the relative proportion of the onset intensity of τ2.
Preferably, the test cell is selected from any one of a cell spheroid model, an organoid and 3D tumor section model, primary tissue culture and embryonic cells.
More preferably, the test cell is any one of a 3D tumor section model, primary tissue culture and embryonic cells. Compared with 2D cell culture, the 3D tumor model can better simulate tumor microenvironment, primary tissue culture can better simulate organ microenvironment, embryo can reflect multi-organ response, and more physiologically relevant targets are revealed in drug screening.
Preferably, the thickness of the 3D tumor section and the primary tissue culture is 200-300 μm, and specifically can be any one or any range between two of 200 μm, 220 μm, 240 μm, 260 μm, 280 μm and 300 μm.
Preferably, the 3D tumor section model and the primary tissue culture obtaining method are: the tumor tissue and the normal tissue are wrapped by gel and then cut into slices.
In addition, the embodiment of the invention also provides the application of the method for identifying apoptosis and/or necrosis in screening medicines or evaluating the efficacy of medicines.
Example 1
Experimental method
Cell culture
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's Modified Eagle's Medium (DMEM) (Gibco) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin (Gibco) and in a culture medium containing 5% CO 2 Is incubated at 37℃in an incubator. At 5X 10 5 The individual cells/mL concentration was seeded in confocal dishes (NEST, 801006) and incubated to 60-80% confluency for drug treatment.
Drug treatment
To induce apoptosis, MDA-MB-231 cells were treated with 300. Mu. M H 2 O 2 Treatment for 6 hours or 30. Mu.M cisPlatinum (Sigma) treatment was performed for 72 hours. Since L929 cells have lower drug tolerance, they were incubated with 30 μm cisplatin for 24 hours. MDA-MB-231 cells were treated with different concentrations (1, 5, 10, 30 and 60. Mu.M) of cisplatin for dose and time dependent experiments.
To induce apoptosis associated with Endoplasmic Reticulum (ER) stress, MDA-MB-231 cells were treated with thapsigargin (200nM;Tocris Bioscience) for 48 hours.
MDA-MB-231 cells were treated with high concentration (1 mM) of H in order to induce cell necrosis 2 O 2 Or 5 μg/mL shikonin (Sigma) for 6 hours.
Cell sphere culture
24 Kong Gongju coke plates (NEST, 801006) were coated with 0.5% agarose scaffolds to form cancer spheres for microscopic observation. One hundred ovarian cancer OVCA429 cells were inoculated into each pre-coated well and cultured in 500 μl DMEM containing 10% fbs and 1% penicillin-streptomycin for 3-4 days to form multicellular carcinoma spheres. Spheres were collected on days 1, 2 and 4 after treatment with cisplatin (5. Mu.M, 15. Mu.M and 30. Mu.M) for TPF imaging and apoptosis/necrosis assay.
Three-dimensional (3D) tumor section preparation
All animal experiments were approved by the animal facility of the university of Australian university, health sciences institute (approval number UMARE-015-2019).
Tumors were obtained from genetically engineered mouse models and human primary tumors, respectively, stored in cold PBS and cut into 200 μm thick sections within 6 hours after surgery using a vibrating microtome (Leica Biosystems Nussloch GmbH, VT 1200S). Then, 100. Mu.L of the recombinant collagen solution was added to the Millicell insert (12 mm, millipore, PIHP01250) and incubated at 37℃for 20 minutes until clotting. Next, the sections were pre-coated with 100 μl of recombinant collagen solution and incubated at 37 ℃ for 20 minutes to solidify the top layer of the gel containing the tissue. Subsequently, 400. Mu.L of medium (Ham's F; gibco containing 20% FBS and 50. Mu.g/ml gentamicin) was placed in the outer insert. Finally, the slices are placed in a mixture containing 5% CO 2 Is cultured in an incubator at 37 ℃.
For the treatment group, the culture sections were treated with 25. Mu.M cisplatin, anti-mouse-CD 279 (αPD-1, biolegend, 10. Mu.g/mL) or anti-mouse-CD 274 (αPD-L1, biolegend, 10. Mu.g/mL) to evaluate the efficacy of the anticancer drug. The treated tumor sections were imaged at each time point using single photon and multiphoton microscopy. 3- (4, 5-Dimethylhizol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) was used to measure cell viability of tumor sections. Specifically, tissue sections were stained with 400. Mu.L of 25. Mu.g/mL MTT solution per well and incubated for 3 hours at 37 ℃. Subsequently, the reagent is washed out with Phosphate Buffered Saline (PBS) buffer, 1ml of isopropanol is added to dissolve the formazan formed, and the absorbance at 570nm is measured using a microplate reader (Perkin Elmer Victor X, waltham, MA, USA). MTT images of the sections were also taken with a fluorescence stereo microscope (Leica, M165FC, germany).
Organoid culture
For human breast cancer organoids, tumor specimens were taken from the mirror lake hospital (approval document 20180907T), transected into small pieces, and minced completely with a scalpel. For cisplatin-resistant MDA-MB-231 cell organoid cultures, resistant clones were selected by gradually increasing cisplatin concentration, resulting in cell lines with high tolerance to 50. Mu.M cisplatin. RPMI medium (Gibco) containing 5% fbs, collagenase I and DNase was used for tumor digestion. After incubation for 30 min to 1 hr at 37 ℃ with shaker, the tumor tissue was filtered with filter screen and the remaining suspension was centrifuged at 2000rpm for 5 min and washed with cold PBS. The cells were then mixed with Matrigel (Corning, 356234) and placed on a pre-warmed 6-well cell culture plate. After 20 minutes, the pre-heated medium 25 was added and replaced every four days. For the confocal experiments, organoids were digested with trypsin for 10 min, gently swirled 4-6 times every 5 min, then terminated with serum, and washed twice with cold PBS. Then, the cells (6.5X10) 6 Per mL) was mixed with cold Matrigel (10. Mu.L) in 24 Kong Gongjiao petri dishes and mixed with 5% CO 2 Is cultivated in an incubator at 37 ℃. After 20 minutes the medium was added. The following day, normal medium or cisplatin medium at different concentrations was added for drug sensitivity test.
Flow cytometry analysis of cell death
2D cell cultures and 3D tumor models (spheres andorganoids) were tested using the Annexin V-FITC/PI kit (Invitrogen). Harvested cells (5X 10) 5 ) Wash with PBS gently shake and then re-suspend in 195 μl binding buffer. For Annexin V staining, 5. Mu.L of Annexin V-FITC was added to the sample solution and incubated at room temperature for 15 minutes in the absence of light. Cells were washed with binding buffer and resuspended in 200 μl buffer with the addition of 10 μl Propidium Iodide (PI). BD Accuri with Standard FITC and PI channels TM The fluorescence signal was detected by a C6 cytometer (BD Biosciences, usa) and analyzed using FlowJo software (Tree Star). At least 10000 cells were gated and analyzed using Forward Scatter (FSC) and Side Scatter (SSC) signals in each run. Then, single tag (PI or FITC) measurements were obtained to compensate for background signals in FITC and PI channels. After selection of the appropriate intensity threshold, FITC and PI-labeled positive/negative populations were gated. Healthy cells were Annexin V/PI double negative. After treatment, apoptotic cells moved to the Annexin V positive and PI negative quadrants, while necrotic cells moved to the Annexin V/PI double positive quadrants.
Apoptotic cells were imaged in situ using a caspase-3 detection kit: after imaging red autofluorescence in apoptotic cells, caspase-3 detection kit (Invitrogen, image-ITTM LIVE Green) was used to confirm the onset of apoptosis. The treated cells and control cells were mixed with 30X FLICA working solution in cell culture medium and incubated under culture conditions for 60 minutes. Thereafter, the cells were washed twice with 1X wash buffer and caspase-3 distribution was imaged by confocal microscopy at excitation wavelength of 488 nm.
Lysotecker dye labeled intracellular lysosomes
LysoTracker Green (Thermo Fisher, DND-26) was used to confirm whether pressure-induced red autofluorescence accumulated in lysosomes, to label the position of lysosomes, and to observe the relative distribution of red autofluorescence. LysoTracker Green solution (75 nM) was added to the medium of the control and drug-treated groups. Then, the cells were incubated with the dye at 37℃for 45 minutes and washed twice with PBS.
Two-photon fluorescence image acquisition
For TPF imaging, cells were seeded in confocal dishes and incubated in the presence of 5% CO 2 And a microscope compatible mini incubator system at 37℃constant temperature (Nikon Instrument Inc., japan). A near infrared femtosecond laser (InSight X3, spectra-Physics) with adjustable wavelength (700-1300 nm) is used as a light source for two-photon fluorescence imaging, so that the penetration depth can be prolonged and the photodamage to cells can be reduced in 3D tumor culture. Each image was taken using a Nikon Eclipse inverted multiphoton microscope (a1mp+eclipse Ti-2E,Nikon Instruments Inc, japan) with a 40 xna=1.15 water immersion objective. The excited TPF and double frequency (SHG) signals are collected by the same objective lens, reflected by a multiphoton dichroic beam splitter, and detected by four photomultiplier tubes (PMTs). Lipofuscin-like autofluorescence can be selectively excited at 1060nm and detected by PMT in channel 3 (λdet=604 679 nm). This excitation wavelength avoids two-photon co-excitation of flavins. PMT in channel 2 (λdet=506-593 nm) detected a 520nm SHG signal of the 3D tumor slice. In the wavelength-dependent excitation experiments, the laser power at different excitation wavelengths was equalized to 15mW after the objective lens. For each experimental group, 3 images with 317×317 μm field of view were taken and at least 30 cells were selected therefrom for analysis of fluorescence intensity. In addition, a CCD cooling spectrometer (iDus 401plus shamrock 193i,ANDOR,Oxford Instruments) was connected to the back port of the inverted microscope. Each time a multiphoton image is taken, a corresponding two-photon emission spectrum is obtained from the integrated spectrometer.
FLIM image acquisition and data analysis
Two photon counting PMTs (PMC-150-4, becker & Hickl) sharing the same signal light path as the fluorescence spectrometer are further mounted on the back port of the same multiphoton microscope (Eclipse Ti-2E, nikon). Fluorescence lifetime data were recorded by a time-dependent single photon counting system (SPC-160, becker & Hickl) synchronized with the scanning excitation of a Nikon A1MP+ multiphoton microscope. To improve the quality of the lifetime fit, it takes at least 120 seconds to acquire one FLIM image, such that the lipofuscin fluorescence peak photon count for most pixels in the cell is above 200. The pixel dwell time of 25.21 mus is set to obtain a 256 x 256 pixel image.
Statistical analysis of lipofuscin fluorescence intensity and fluorescence lifetime.
The mean fluorescence intensity in the 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 mean fluorescence values within the cytoplasm.
For fluorescence lifetime statistics, SPC image software (Beker&Hickl) was used to analyze FLIM data. The fluorescence lifetime of each pixel is fitted by a double exponential decay modelWherein IRF is the instrument response function measured from SHG of urea. α1 and α2 represent the percentage of the magnitude of the photon contribution from the two fluorescence lifetime components τ1 and τ2, respectively. The trajectory fit is optimized for low chi-square error and the software generates an amplitude weighted lifetime histogram and a color coded lifetime image. For ease of comparison, analysis was sometimes performed with tm=τ1×α1+τ2×α2 (fig. 1). Similar to the study of fluorescence intensity, in some FLIM images, at least 3 images were acquired to obtain more than 30 cells (image size: 512 x 512,0.25 μm pixel size, binding 4 x 4, 200 photons, acquisition time: 120 s). The boundaries of the cells were selected from the bright field image and the corresponding fluorescence lifetime characteristics of lipofuscin-like fluorescence were analyzed. The exported data was analyzed using GraphPad Prism software (version 6.0). Two-dimensional (2D) scatter plots were obtained by Origin software (version 8.6).
Experimental results
Lipofuscin autofluorescence is an early feature of apoptosis.
Common drugs and cytotoxic substances H 2 O 2 Can induce apoptosis or necrosis, H 2 O 2 Induction of ROS (reactive oxygen species) produced can induce apoptosis and necrosis at various concentrations. While at an appropriate concentration, e.g., 300. Mu.M, H 2 O 2 Apoptosis was triggered and it was confirmed by an Annexin-V/PI staining assay that MDA-MB-231 cells tended to apoptosis (FIG. 2Medium a,24 hour incubation).
In contrast to the weak autofluorescence exhibited by the control group (a in fig. 3), apoptotic cells showed a pronounced red autofluorescence intensity in two-photon imaging (excitation wavelength ex=1060 nm, B, C in fig. 3) and a fluorescence emission peak at about 600nm (D in fig. 3), similar to that of lipofuscin excited with fundus cameras. By scanning the excitation wavelength from 820nm to 1240nm at equivalent laser power, the red fluorescence exhibits several effective excitation bands, e.g., 1020, 1060 and 1100nm (B-D in fig. 2). These stress-induced autofluorophores accumulate in the form of a plurality of particles during apoptosis, whereas living cells show little red autofluorescence. It is well known that aging can impair mitochondrial function. Insufficient autophagy can hinder clearance of damaged line granulocytes and lead to formation of lipofuscin in lysosomes. Most of these stress-induced autofluorophores were located in lysosomes as demonstrated by LysoTracker labeling (table 1 and fig. 4).
Table 1Pearson coefficient and Mander coefficient profile
The distribution of these intracellular red autofluorophores is consistent with the metabolic pathways of lipofuscin. It was also observed that when MDA-MB-231 cells (F-H in fig. 3) and L929 fibroblasts (fig. 6) were induced to apoptosis by cisplatin, red autofluorescence increased and exhibited similar excitation spectra (fig. 5) and emission spectra (D in fig. 3). In addition to the Annexin-V/PI assay, apoptosis was further confirmed by targeting Caspase 3 in these red fluorescent cells (FIG. 3I). These spectral and imaging evidence demonstrate that stress-induced lipofuscin-like red fluorescence is closely related to the apoptotic process and may originate from the view that lysosomal lipofuscin accumulates acutely.
Long-term monitoring of apoptosis pharmacodynamic characteristics.
The time and dose-dependent relationship of red autofluorescence and apoptosis of cisplatin-treated MDA-MB-231 cells was analyzed. From the Annexin V/PI assay, apoptosis was time and dose dependent after 30. Mu.M cisplatin was added to stimulate cells, and the apoptosis rate was 45.5% after 72h treatment (FIG. 7A). A small number of red fluorescent cells appeared in the field of view of the control group (0. Mu.M, 0-72 h). Consistent with the Annexin V/PI experiment, in addition, the density of red fluorescent cells increased with increasing drug incubation time and concentration (a-B in fig. 8, a-D in fig. 7).
To further examine the extent of early apoptosis, the drugs were washed off at 12 and 24h after treatment, and further cultured in fresh medium for 24h, with cell apoptosis rates rising from 5.9% (12 h w/drug) and 12.0% (24 h w/drug) to 13.1% (12 h w/+24h w/o drug) and 27.4% (24 h w/+24h w/o drug), respectively (E in fig. 7). They follow the trend of increasing apoptosis with increasing drug incubation time (C, D in fig. 7). This confirms that apoptosis has begun at 12h of treatment, but conventional Annexin/PI assays have difficulty showing a tendency to apoptosis at this early time point. In contrast, the red fluorescence of the 10 μm and 30 μm dose groups increased 12h after treatment (B in fig. 8), 12h earlier than the 5 μm dose group (fig. 9), and the 30 μm dose group reached the threshold of lipofuscin-like fluorescence 48h after treatment (average intensity 8.968), 12h earlier than the 5 μm and 10 μm dose groups (fig. 9). In addition, the mean fluorescence lifetime also reported early treatment responses, from 171ps before treatment to 287.7ps at 12h after treatment. This lifetime increase eventually reaches 507ps after 48-72h of treatment, which means that the chemical environment of the lipofuscin-like fluorophores remains unchanged after 48h of treatment (C in fig. 8). Thus, if a threshold is set for the average lifetime of the photon counting and lipofuscin-like fluorescence in the cell, the therapeutic response can be measured 24h earlier than in the conventional method, and even the apoptotic response can be observed 12h earlier than possible with the Annexin-V/PI staining method.
Fluorescence Lifetime Imaging Microscopy (FLIM) distinguishes between apoptosis and necrosis.
High concentration of H 2 O 2 And shikonin, which is a traditional Chinese medicine anticancer agent, can induce cell necrosis. With 1mM H 2 O 2 And 5. Mu.g/mL shikonin treated MDA-MB-21 cells for 6 hours, the necrotic cells were 46.9% and 59.5%, respectively. Under these conditions, it was also found that there was also a significant increase in red fluorescence in necrotic cellsAdd (FIGS. 10 and 11). Their spectral shape is similar to apoptotic cells. This means that the intensity of lipofuscin-like red autofluorescence may indicate cell senescence but not the type of cell death.
To distinguish between necrosis and apoptosis, this example introduces an additional parameter, the two-photon lifetime of lipofuscin-like fluorescence. In contrast to intensity measurements, fluorescence lifetime measurements can avoid imaging due to photobleaching and do not require calibration under excitation conditions. In addition, fluorescence lifetime may reflect the effect of environmental factors on fluorophore charge relaxation, including solution pH, presence of quenchers, self-aggregation, and binding to macromolecules. In the previous data, it was found that life span measurement of lipofuscin-like fluorescence could indicate the transition of cells from normal state to apoptosis. If the increase in apoptotic life is due to the lysosomal environment of lipofuscin, it is reasonable to infer that disruption of lysosomes in necrosis may greatly alter the fluorescence life of lipofuscin. This can provide a marker-free indicator to distinguish between common necrosis and apoptosis.
According to the lipofuscin FLIM analysis (a-D in fig. 12), there were two major lifetime components τ1=400-700 ps and τ2=2200 ps in apoptotic cells, with the ratios 72% (α1) and 28% (α2), respectively (C, D in fig. 12). Whereas the fluorescence lifetime of the control group was significantly shortened, τ1=100 to 300ps, τ2=1500 ps, α1=88%. From the α2- τ1 two-dimensional scatter plot, it can be seen that the data points for apoptotic cells are better separated from the data points for living cells (B in fig. 12). In addition, similar measurements were made 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 (a-D in fig. 13). The apoptotic status of L929 cells was also confirmed by Annexin V/PI staining flow cytometry (E in FIG. 13).
Knowing FLIM can distinguish between apoptotic and normal cells, further validating its role in distinguishing between necrosis and apoptosis. Although 1mM H 2 O 2 Both violaxadiin induced an increase in red two-photon fluorescence in necrotic cells, but in combination withThe increased fluorescence decay times observed during apoptosis compared to those they shortened (E-G in fig. 12). As expected, no lysosomes were found in necrotic cells after LysoTracker labeling (H in fig. 12). Therefore, lysosomes were presumed to be severely damaged during necrosis, resulting in release of accumulated lipofuscin into the cytoplasm (I in fig. 12), altering the fluorescence lifetime of lipofuscin-like fluorophores. Furthermore, it is desirable to demonstrate the ability of FLIM to distinguish between necrosis and apoptosis at cellular resolution. Apoptosis was induced using the endoplasmic membrane stressor TG and cisplatin. Necrosis was induced with 1mM H2O2 and 5. Mu.g/mL shikonin. Cell status was confirmed by Annexin V/PI staining flow cytometry (fig. 14). All treated cells showed strong red lipofuscin-like TPF at the end of the experiment, the type of death was clearly shown by color attachment FLIM data for the mean lifetime τm (a in fig. 15). Cells after TG treatment showed little necrosis (even yellow), H 2 O 2 The treated cells showed a small amount of apoptosis (in cyan particles). Necrotic cells τ1=200 ps and τ2=1300 ps were much shorter than apoptotic cells (B in fig. 15). The short-lived component contributes more (α1=80%), and the long-lived component α2 contributes less (C in fig. 15). These results demonstrate that FLIM of lipofuscin-like TPF can support label-free differentiation of dead and apoptotic cells through its lifetime and subcellular structure. The method of the invention provides key information for assessing the type of cell death that occurs after exposure to different doses of drug.
To further elucidate the value of fluorescence lifetime in determining cell death types, ovarian cancer cell line OVCA429 was treated with 5uM, 15uM and 30 uM cisplatin. As shown in fig. 15D, time and dose dependent studies showed that red autofluorescence of OVCA429 cells increased with increasing cisplatin dose and incubation time. Interestingly, FLIM data showed that treatment at different doses resulted in significantly different life characteristics (E in fig. 15; a-B in fig. 16) corresponding to different types of cell death. Cisplatin (5. Mu.M) at low doses induced apoptosis, and cells necrosis at 15. Mu.M and 30. Mu.M. In the two-dimensional scatter plots of α2 and τ1, separation of cells in different states can be easily observed (F in FIG. 15). We demonstrate this by specific Annexin V/PI analysis. Apoptosis was observed when cells were treated with 5. Mu.M cisplatin for 2 days. However, at doses of 15 μm and 30 μm, the necrosis rate increased to 16.2% and 29.2%, respectively (C in fig. 16). In summary, the combination of fluorescence intensity and fluorescence lifetime pattern of lipofuscin-like fluorophores not only reveals drug sensitivity, but also determines the effect of dose on cell death type.
A label-free optical probe for use in a 3D tumor model therapeutic response.
The dynamics of OCVC429 cell sphere treatment was observed based on molecular imaging of stress-induced lipofuscin autofluorescence in OVCA429 cells (red in a in fig. 17). After treatment with 5, 15 and 30. Mu.M cisplatin, lipofuscin fluorescence intensity in the OVCA429 sphere model increased significantly within 2 days (A, B in FIG. 17). Red fluorescence in the cells on day 2 was longer in lifetime than that of the control group (C in fig. 17). This stress-induced red fluorescence and increased lifetime was closely related to the extent of cellular senescence as demonstrated by Annexin-V/PI staining (C in fig. 18).
However, while cell sphere models have been widely recognized as a common research tool for drug screening, they fail to recreate the microenvironment of the tumor. Today, organoids are defined as a 3D cell aggregate derived from primary tissue or stem cells and can be divided into stented and non-stented types. They can self-update and reveal organ function. The tumor organoids can maintain the organ cell type and genetic characteristics of the original tumor, which ensures the reproduction of the tumor microenvironment. To identify early markers that can determine apoptosis, such label-free optical methods are used to enable rapid and long-term monitoring of organoid models derived from human breast tumors. Based on imaging of lipofuscin fluorescence in 3D tumor organoids, pharmacokinetics dependent on cisplatin dose (1, 5 and 30 μm) and incubation time was observed (fig. 19). After the treatment, an increase in fluorescence intensity and lifetime of lipofuscin in the organoids was observed within four days (D-F in FIG. 17). Annexin-V/PI staining demonstrated that this cisplatin-induced red fluorescence in human tumor organoids correlated closely with the extent of cellular senescence (FIG. 19). In contrast, organoids composed of cisplatin-resistant MDA-MB-231 cells did not show significant increases in intensity (FIG. 20). This result suggests 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.
Application in 3D tumor section culture model.
In addition, the efficacy of anticancer drugs was evaluated in real-time in a three-dimensional tumor slice culture (3D-TSC) model using a label-free metabolic imaging method.
The 3D-TSC can even preserve tumor microenvironment and immune cells, which is critical for assessing immune checkpoint blocking therapies. The 3D-TSC treatment kinetics over 4 days were studied by imaging drug-induced lipofuscin in cells (red channel of a in fig. 21) and collagen fibers (green color of a in fig. 21). After treatment with 25. Mu.M cisplatin, 2.5. Mu.g/mL. Alpha. PD-1 and 2.5. Mu.g/mL. Alpha. PD-L1, an increase in fluorescence intensity of lipofuscin in the tumor sections was observed, and the fluorescence lifetime increased from 573.4ps to a longer 950ps under treatment with. Alpha. PD-and. Alpha. PD-L1 compared to the control group (FIG. 21B). However, for cisplatin treatment, lipofuscin fluorescence was slightly lower in intensity and lifetime than the immunotherapy group (B in fig. 21). Meanwhile, stress-induced red fluorescence intensity and lifetime changes are closely related to cell viability (fig. 22). This phenomenon may mean that immunotherapy eliminates more tumor cells than cisplatin treatment. The technique is applied to tumor samples (PDTS) from patients, colon cancer and nasopharyngeal cancer samples from two patients. After 7 days of alpha PD-L1 treatment, the fluorescence intensity of lipofuscin of colon cancer was significantly increased. Lipofuscin fluorescence remained almost stable after immunotherapy for nasopharyngeal carcinoma (a-B in fig. 23).
To further confirm that this approach can support label-free detection of cell death in 3D-TSCs, live tumor cells were identified by GFP labeling in mouse gfp+ tumor sections. During the treatment time, it was confirmed that the enhancement of lipofuscin fluorescence intensity was accompanied by the disappearance of gfp+ cells (green in C in fig. 23). Thus, the results indicate that lipofuscin-like fluorescence can be used as a label-free and valuable optical probe for pharmacodynamics in 3D-TSC and long-term monitoring. This practical approach can be translated into clinical practice.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims (10)

1. A method for identifying apoptosis and/or necrosis, comprising: detecting fluorescence life of lipofuscin and/or lipofuscin adsorption material in the cell to be detected in real time, and identifying apoptosis and/or necrosis according to the length of fluorescence life;
the method is not directed to the diagnosis or treatment of a disease.
2. The method of identifying apoptosis and/or necrosis as defined in claim 1, further comprising setting an apoptosis threshold and/or necrosis threshold for fluorescence lifetime;
if the fluorescence lifetime of the cells to be detected is more than or equal to the apoptosis threshold value, judging the cells to be detected as apoptotic cells;
and if the fluorescence lifetime of the cell to be detected is less than or equal to the necrosis threshold value, judging the cell to be detected as necrotic cell.
3. The method of claim 2, wherein the fluorescence lifetime is the decay time of fluorescence emission after lipofuscin transmission through single photon or two photon excitation;
preferably, the fluorescence lifetime refers to the decay time of fluorescence emission after lipofuscin is excited by two photons.
4. The method for identifying apoptosis and/or necrosis as defined in claim 3, wherein said detection conditions of fluorescence lifetime of lipofuscin are: the excitation wavelength of single photon is 500-560 nm, the excitation wavelength of two photons is 970-1140 nm, and the emission wavelength of lipofuscin is 550-800 nm.
5. A method of identifying apoptosis and/or necrosis as claimed in claim 3, wherein said parameters related to fluorescence lifetime of lipofuscin comprise: at least one of the fluorescence lifetime τ1, the fluorescence lifetime τ2, the relative proportion α1 of the τ1 onset intensities and the relative proportion α2 of the τ2 onset intensities is determined based on relevant parameters when identifying the apoptotic and/or necrotic state of the cell.
6. The method of identifying apoptosis and/or necrosis as set forth in any of claims 1-5, wherein said test cells are selected from any of the group consisting of cell spheroid model, organoid, 3D tumor section model, primary tissue culture and embryonic cells.
7. The method of claim 6, wherein the test cell is any one of a 3D tumor slice model, primary tissue culture, and embryonic cells.
8. The method of claim 6, wherein the thickness of the 3D tumor section and primary tissue culture is 200-300 μm.
9. The method of claim 8, wherein the 3D tumor section model and the primary tissue culture acquisition method are: the tumor tissue or normal tissue is wrapped by gel and then is sectioned.
10. Use of a method for identifying apoptosis and/or necrosis according to any of claims 1-9 in the screening of drugs or the evaluation of drug efficacy.
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