CN115970006A - A method for detecting the autophagy level of living tumor cells - Google Patents

Abstract

The present disclosure provides a method for detecting autophagy levels in a living tumor cell, the method comprising: screening tumor cell strains stably expressing GFP-mRFP-LC3b and mCErulean; inoculating a tumor cell strain stably expressing GFP-mRFP-LC3b and mCErulean into a donor, and then carrying out living body imaging on a tumor area of the donor; injecting a heat restriction simulation drug into a donor at intervals of a plurality of days to induce autophagy of tumor cells, continuously imaging a tumor metastasis site of a living body for a plurality of days, and collecting fluorescent signals of at least GFP, mRFP and mCErulean after determining a tumor metastasis; the autophagy level of the tumor cells in vivo was determined from the optical imaging of the living body at different time points and the fluorescence signals collected. Compared with the in vitro section and the cell level, the living body imaging can reflect the autophagy level change of the tumor cells in the living body more truly; in addition, the invention can track the dynamic change of autophagy level of tumor cells in the growth and metastasis processes through long-term living body imaging.

Description

A method for detecting the autophagy level of living tumor cells

technical field

The present disclosure relates to the field of biotechnology, in particular to a method for detecting the autophagy level of living tumor cells.

Background technique

Autophagy is an evolutionarily conserved process involving the cell's own structure through the lysosome mechanism, which is responsible for transporting damaged organelles, misfolded proteins, and other macromolecules to the lysosome for degradation and reuse. The occurrence of autophagy depends on whether the process of autophagic flow is completed, and autophagic flow means the complete dynamic process of autophagy, including the formation of autophagosomes, the fusion of autophagosomes and lysosomes, and the subsequent degradation and degradation of contents. Recycle.

Autophagy has a multi-level relationship with tumors. Before normal cells become cancerous, intracellular autophagy maintains cell homeostasis and prevents cell transformation: for example, removing dysfunctional mitochondria, redox-active protein aggregates, pathogens, and gene damage Substances that inhibit the accumulation of genotoxic substances; help DNA repair, ensure the accuracy of mitosis; ensure the metabolic homeostasis of cells, etc. For cancerous cells, autophagy can help cancer cells maintain metabolism and redox homeostasis in the tumor microenvironment, preserve cancer cell stemness, and support tumor growth. Extracellularly, autophagy can limit the chronic inflammation of precancerous cells by degrading endogenous pro-inflammatory substances on the one hand; on the other hand, autophagy can promote immunogenic death, release ATP, and promote the recruitment of antigen-presenting cells with activation.

The current study found that the level of autophagy was higher in tumor cells in the early stage of metastasis. High levels of autophagy can promote the growth of tumor metastasis and implantation, and inhibit anoikis, but the research on autophagy for metastatic tumors is still in a blank stage. Therefore, in order to reveal the changes in the level of tumor autophagy during the process of tumor metastasis, it is necessary to detect the level of autophagy in tumor cells. At present, the detection of autophagy levels in cells is mainly divided into three categories. The first category is the observation of autophagosomes by electron microscopy, which can clearly observe the different forms of autophagy at each stage, but cannot dynamically detect changes in autophagy. The second category is to detect the expression of autophagy-related proteins by Western Blot. The third type is the method of fluorescent labeling, in which LC3b is the main labeling target. However, the method of characterizing tumor autophagy level by LC3 fluorescent labeling is currently limited to the cell level in vitro or the tissue section level at a single time point. The current methods for detecting the level of autophagy are mainly applied to cells cultured in vitro, and the level of autophagy in living cells is rarely detected. In particular, there is currently a lack of methods for observing changes in the level of autophagy in vivo.

Contents of the invention

The present disclosure provides a method for detecting the autophagy level of living tumor cells, so as to at least solve the above technical problems existing in the prior art.

According to the first aspect of the present disclosure, a method for detecting the autophagy level of living tumor cells is provided, the method comprising:

Screen tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean;

A tumor cell line stably expressing GFP-mRFP-LC3b and mCerulean was inoculated into the donor, and then the tumor area of the donor was imaged in vivo;

Inject caloric restriction mimic drugs into the donor after a few days interval to induce tumor cell autophagy, perform in vivo imaging of the donor's tumor metastases for several consecutive days, and collect at least the fluorescent signals of GFP, mRFP, and mCerulean after the tumor metastases are determined ;

The level of autophagy in living tumor cells was determined based on in vivo optical imaging and collected fluorescence signals at different time points.

In one possible embodiment, the screening of tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean includes:

Construction of the PB vector plasmid comprising the mRFP-GFP-LC3b fragment;

Transfect the PB vector plasmid into tumor cells expressing mCerulean;

The transfected tumor cells were cultured, and multiple tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean were screened.

In one possible embodiment, monoclonal cells whose growth rate is consistent with that of normal B16 cells and whose autophagosome fluorescence is clearly visible are screened from multiple tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean.

In one possible embodiment, the transfected tumor cells are placed in RPMI1640 medium containing 10% fetal bovine serum.

In one possible embodiment, the culture medium is cultured at 37°C, containing 5% CO ₂ .

In one possible embodiment, the fluorescence signals of GFP, mRFP, and mCerulean are collected. The collection parameters are: GFP is excited at 488nm and received at 510nm-560nm; mRFP is excited at 561nm and received at 580-650nm; mCerulean is excited at 440nm and received at 450-500nm .

In a possible embodiment, the method further includes: taking high-definition photographs of tumor metastasis sites with a 60-fold objective lens, so as to determine autophagy spots in tumor cells.

In a possible embodiment, the calorie restriction mimic drug is used to induce autophagy, and the calorie restriction mimic drug includes one of hydroxycitrate (Hydroxycitrate, HC), spermidine, rapamycin, and metformin. kind.

The invention provides a method for detecting the autophagy level of living tumor cells. First, the tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean are screened; then the tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean are inoculated into donors In vivo imaging of the donor's tumor area is then performed; several days later, the donor is injected with caloric restriction mimic drugs to induce tumor cell autophagy, and the donor's tumor metastases are continuously imaged for multiple days in vivo, and GFP is collected. , mRFP, and mCerulean fluorescence signals; and then determine the autophagy level of donor tumor cells according to in vivo optical imaging and collected fluorescence signals at different time points. Compared with in vitro section and cell level detection methods, the detection method of the present invention can more truly reflect the changes in the autophagy level of tumor cells in vivo; in addition, the present invention can track the growth and metastasis of tumor cells through long-term in vivo imaging. Dynamic changes in autophagy levels during the process.

It should be understood that what is described in this section is not intended to identify key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will be readily understood through the following description.

Description of drawings

The above and other objects, features and advantages of exemplary embodiments of the present disclosure will become readily understood by reading the following detailed description with reference to the accompanying drawings. In the drawings, several embodiments of the present disclosure are shown by way of illustration and not limitation, in which:

In the drawings, the same or corresponding reference numerals denote the same or corresponding parts.

Fig. 1 shows a schematic flow chart of a method for detecting the autophagy level of living tumor cells according to an embodiment of the present disclosure;

Figure 2 shows the construction and characterization of LC3b ^mRFP-GFP -mCerulean-B16 cells in the embodiment of the present disclosure: wherein (A) schematic diagram of mRFP-GFP-LC3b plasmid fragment; (B) 4 stable strains transfected with PB plasmid system Confocal images of subcutaneous tumor sections of mCerulean-B16 (LC3b ^mRFP-GFP- mCerulean-B16) cell lines expressing mRFP-GFP-LC3b, scale bar: 30 μm; (C) four LC3b ^mRFP-GFP- mCerulean-B16 stable cells Subcutaneous tumor growth curve of strain; (D) In vivo confocal images of 1#LC3b ^mRFP-GFP- mCerulean-B16 cells shown in B and C in CXCR6 ^GFP/+ mice formed liver metastases at day 5, scale bar : 20μm;

Figure 3 shows the long-term imaging of the liver window in the embodiment of the present disclosure to visualize the autophagy level of tumor cells during the development of liver metastasis: (A) Schematic diagram of long-term imaging of liver metastasis in vivo, and transsplenic liver metastasis at the same time on day 0 Modeling and liver window surgery; (B) In vivo confocal imaging images of CXCR6 ^GFP/+ mice transferred LC3b ^mRFP-GFP -mCerulean-B16 cells via spleen and liver from day 1 to day 8, the upper row is untreated Group, the lower row is the HC-treated group with intraperitoneal injection of 2 mg hydroxycitrate (HC) on the third day, the objective lens is 20 times, the green is CXCR6 cells, LC3b ^GFP in tumor cells, and the red is in tumor cells LC3b ^mRFP of LC3b, scale bar: 50 μm; (C) Corresponding to in vivo confocal images taken with a 60x objective lens on day 1, day 3 and day 4 in A, scale bar: 20 μm.

Detailed ways

In order to make the purpose, features, and advantages of the present disclosure more obvious and understandable, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present disclosure. Obviously, the described The embodiments are only some of the embodiments of the present disclosure, but not all of them. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without making creative efforts belong to the protection scope of the present disclosure.

At present, the detection of autophagy levels in cells is mainly divided into three categories. The first category is the observation of autophagosomes by electron microscopy, which can clearly observe the different forms of autophagy at each stage, but cannot dynamically detect changes in autophagy. The second category is to detect the expression of autophagy-related proteins by Western Blot. The third type is the method of fluorescent labeling, in which LC3b is the main labeling target. However, the method of characterizing tumor autophagy level by LC3 fluorescent labeling is currently limited to the cell level in vitro or the tissue section level at a single time point. The current methods for detecting the level of autophagy are mainly applied to cells cultured in vitro, and rarely detect the level of autophagy in living cells. In particular, there is currently a lack of long-term in vivo imaging research methods for the level of autophagy in tumor cells in vivo.

Based on the above problems, the present invention provides a method for detecting the autophagy level of living tumor cells, as shown in Figure 1, which is a schematic flow chart of the method, and the method includes:

S1. Screening tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean;

Among them, GFP is green fluorescent protein, mRFP is red fluorescent protein, and LC3b is an autophagy marker.

Specifically, screen tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean, including:

S11, constructing the PB vector plasmid comprising the mRFP-GFP-LC3b fragment; as shown in Figure 2 (A) is a schematic diagram of the constructed PB vector plasmid comprising the mRFP-GFP-LC3b fragment;

S12. Transfecting the PB vector plasmid into tumor cells expressing mCerulean;

S13, culturing the transfected tumor cells, and screening multiple tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean.

The transfected tumor cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. The medium was placed in an incubator with a temperature of 37° C. and 5% CO ₂ in the incubator.

From the transfected tumor cells, several tumor cell lines with 100% stable expression of GFP-mRFP-LC3b and mCerulean were screened. For example, in this example, four mCerulean-B16 cell lines with stable expression of mRFP-GFP-LC3b were screened. Mark them as 1#, 2#, 3#, 4# respectively, as shown in Figure 2(B). In order to verify the tumorigenicity of tumor cells, the above four cell lines were inoculated subcutaneously in mice, and the results are shown in Figure 2(C), showing that all of them can form tumors.

From the above four tumor cell lines stably expressing GFP-mRFP-LC3b, the monoclonal cells whose growth rate was consistent with that of normal B16 cells and whose autophagosome fluorescence was clearly visible were screened. According to Figure 2(B)-(C), the autophagy level of 1# cells is normal, and the fluorescence brightness is moderate, and the tumor formation speed is the fastest, so 1# cells are selected for subsequent experiments.

S2. Inoculating a tumor cell line stably expressing GFP-mRFP-LC3b and mCerulean into the donor, and then performing live imaging on the tumor area of the donor;

As shown in Figure 3(A), in this example, mice were used as living research objects, and the 1# cells determined in step S1, that is, tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean, were injected into the CXCR6 ^{GFP/ +} In mice, it can be operated according to the liver metastasis model of cancer cells. The specific method is: install the liver window model in the mouse body and at the same time make the fluorescent protein-labeled tumor cell suspension (1# cells), 2×10 ⁵ 1# The cells were injected into the mice through the spleen to establish a tumor liver metastasis model, and the day of the operation was taken as day 0 for recording, and the tumor metastasis was further detected by an in vivo confocal imaging system. As shown in Figure 2(D), after injection of 1# cells, LC3b autophagy dots in tumor cells can be seen according to 60x magnification in vivo imaging. Because the acidic environment of late autophagosomes can cause quenching of GFP signal, but has no effect on mRFP signal, LC3b-GFP signal in 1# cells represents early autophagy, and LC3b-mRFP signal represents early autophagy and late autophagy. Because tumor cells themselves express mCerulean, GFP cells can be distinguished by fluorescence overlay, so it can be used in optical in vivo imaging of GFP fluorescent reporter mice.

S3. Inject caloric restriction analog drugs into the donor after a few days interval to induce tumor cell autophagy, perform in vivo imaging on the tumor metastases in the living body for several consecutive days, and collect the fluorescent signals of GFP, mRFP, and mCerulean after determining the tumor metastases ;

After the injection of 1# cells, live imaging of liver tumor metastases can be performed continuously every day, or at intervals of 1 or more days, which is not limited in the present invention. For example, in this embodiment, after the injection of 1# cells, in vivo imaging of liver metastasis sites was performed on the first day, the third day, the fourth day, the fifth day, the sixth day and the eighth day respectively. Intraperitoneal injection of a caloric restriction mimic in a group of mice at the end of day 3 restricted cellular energy metabolism, thereby inducing autophagy. Caloric restriction simulating drugs include one of hydroxycitrate, spermidine, rapamycin, and metformin. In this example, the caloric restriction simulant uses hydroxycitrate as an example. At the end of the third day, a group of mice 2 mg of hydroxycitrate (Hydroxycitrate, HC) were injected intraperitoneally, and they were named as HC-treated group. After the tumor metastases were found, the fluorescent signals of GFP, mRFP, and mCerulean were collected.

Specific acquisition parameters are as follows: GFP is excited at 488nm and received at 510nm-560nm; mRFP is excited at 561nm and received at 580-650nm; mCerulean is excited at 440nm and received at 450-500nm.

S4. Determine the autophagy level of living tumor cells according to in vivo optical imaging and collected fluorescence signals at different time points.

Through continuous multi-day in vivo optical imaging and fluorescent signals, the process of tumor liver metastasis and changes in the autophagy level of tumor cells can be further analyzed and observed. As shown in Figure 3(B), a small number of cells colonized the hepatic sinusoids on the first day after the tumor was injected through the spleen, and began to gradually proliferate and grow. As autophagy progressed, fluorescence quenched due to GFP entering late autophagosomes , while mRFP remains fluorescent in late autophagosomes, so cells that appear reddish during imaging have higher levels of autophagy. It can be seen that in the process of tumor metastasis, the level of autophagy is very high in the early stage (the first day of metastasis), and with the development of tumor metastasis, the metastases gradually turn green on the 4th to 6th day, indicating that the level of autophagy begins to decline, while In the advanced stage, tumor metastases gradually turn red, indicating that the level of autophagy has increased (Fig. 3(B) upper row). After intraperitoneal injection of hydroxycitrate HC on the third day of liver metastasis, the level of autophagic flux in metastatic foci of HC-treated mice significantly increased on day 4 and day 5, and the level of autophagy increased on day 6. Gradually decreased (Figure 3(B) lower row), indicating that the caloric restriction mimic HC can effectively increase the autophagy level of tumor cells in metastatic foci in the short term. In order to see autophagosomes more clearly, this example uses a 60x objective lens (numerical aperture 1.42) to take high-definition shots of the transfer sites on the 1st, 3rd and 4th days after liver transfer, and the results are shown in the figure 3(C) showed that the tumor cells had a large number of red late autophagosomes on the first day after transfer, and then the red autophagosomes gradually disappeared on the third and fourth days, indicating that the autophagy level of tumor cells gradually decreased and returned. However, 12 hours after the injection of the caloric restriction mimic HC on the third day, that is, the LC3b-GFP signal level in the metastases of the HC-treated group on the fourth day decreased, and the overall metastases appeared reddish, indicating that the level of autophagy increased, accompanied by Infiltration of CXCR6-GFP cells.

The above results show that, through the combination of the liver window imaging model and the tumor cell line that stably characterizes the autophagy level, the present invention visualizes the change law of the autophagy level in the process of liver metastasis: that is, the tumor at the initial stage of metastasis maintains a high level of autophagy in order to adapt to the new environment ; With the development of metastasis, the level of autophagy gradually decreases. In the late stage of metastasis, the level of autophagy in metastases will increase again due to the inability to meet the energy required by a large amount of tumor growth; Drugs can induce the level of autophagy in tumor cells.

In summary, the present invention provides a method for detecting the autophagy level of living tumor cells. First, the tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean are screened; then the tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean are inoculated into the donor, and then perform in vivo imaging of the donor's tumor area; inject caloric restriction mimic drugs into the donor after several days to induce tumor cell autophagy, and perform in vivo imaging of the tumor metastases in the living body for several consecutive days to determine After the tumor metastases, the fluorescence signals of GFP, mRFP, and mCerulean were collected; the autophagy level of tumor cells in vivo was determined according to the in vivo optical imaging and the collected fluorescence signals at different time points. Compared with in vitro slices and cell levels, the detection method of the present invention can more truly reflect the changes in the autophagy level of tumor cells in vivo; in addition, the present invention can track the growth and metastasis of tumor cells through long-term in vivo imaging Dynamic changes in autophagy levels.

It should be understood that steps may be reordered, added or deleted using the various forms of flow shown above. For example, each step described in the disclosure of the present disclosure may be executed in parallel, sequentially, or in a different order, as long as the desired result of the technical solution disclosed in the present disclosure can be achieved, no limitation is imposed herein.

In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present disclosure, "plurality" means two or more, unless otherwise specifically defined.

The above is only a specific implementation of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope of the present disclosure. should fall within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be determined by the protection scope of the claims.

Claims (8)

1. A method for detecting autophagy level of living tumor cells, characterized in that, the method comprises: Screen tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean; A tumor cell line stably expressing GFP-mRFP-LC3b and mCerulean was inoculated into the donor, and then the tumor area of the donor was imaged in vivo; Inject caloric restriction mimic drugs into the donor after a few days interval to induce tumor cell autophagy, perform in vivo optical imaging of the donor's tumor metastases for several consecutive days, and collect at least the fluorescence of GFP, mRFP, and mCerulean after confirming the tumor metastases Signal; The level of autophagy in living tumor cells was determined based on in vivo optical imaging and collected fluorescence signals at different time points. 2. The method according to claim 1, wherein the screening of tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean comprises: Construction of the PB vector plasmid comprising the mRFP-GFP-LC3b fragment; Transfer the PB vector plasmid into the tumor cells expressing mCerulean to transfect the tumor cells; The transfected tumor cells were cultured, and multiple tumor cell lines stably expressing GFP-mRFP-LC3b and mCerulean were screened. 3. The method according to claim 2, characterized in that the growth rate is consistent with that of normal B16 cells and the autophagosome fluorescence is clearly visible from the screening of multiple tumor cell lines stably expressing GFP-mRFP-LC3b cell. 4. The method according to claim 2, wherein the transfected tumor cells are placed in RPMI1640 medium containing 10% fetal bovine serum. 5. The method according to claim 4, characterized in that, the culture medium is cultivated at 37°C, containing 5% CO ₂ . 6. The method according to claim 1, wherein the fluorescent signals of GFP, mRFP and mCerulean are collected, and the collection parameters are: GFP is excited at 488nm and received at 510nm to 560nm; mRFP is excited at 561nm and received at 580 to 650nm; mCerulean Excitation at 440nm, reception at 450-500nm. 7. according to the method described in claim 1, it is characterized in that, the method also comprises: A 60x objective lens was used to take high-definition shots of the tumor metastases to determine the autophagosomes in the tumor cells. 8. The method according to claim 1, wherein the calorie restriction mimic drug is used to induce autophagy, and the calorie restriction mimic drug includes hydroxycitrate, spermidine, rapamycin, metformin One of.

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