Marker tracing system for tumor cell dormancy and reactivation, and use thereof

Abstract

Provided in the present invention is a marker tracing system (DormTracer) for tumor cell dormancy and reactivation. Specifically, the system integrates a gene switch and a site-specific recombination technology, and drives the expression of a reporter gene by means of a recombinase fused with a dormancy marker protein, so that the reporter gene is affected by the dormancy history of a cell without affecting the circulation of the dormancy and proliferation of the cell itself. Therefore, the system can trace the dormancy history of a tumor cell rather than merely monitor the instantaneous state of the cell. The system can distinguish tumor cells in the three states of proliferation, dormancy and reactivation after dormancy.

Description

A labeling and tracing system for tumor cell dormancy and awakening and its application

[0001] This application claims priority to Chinese application No. 2025100853483, filed on January 17, 2025, the contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of tumor biology, and more specifically, this invention relates to a labeling and tracing system for tumor cell dormancy and awakening and its application. Background Technology

[0003] Even after successful surgical resection or adjuvant therapy, metastasis and recurrence frequently occur in cancer patients. Tumor cells that have spread to distant organs can enter a dormant state of cell cycle arrest lasting from months to decades, evading treatments such as radiotherapy and chemotherapy that target proliferating cells. Research suggests that when the microenvironment of dormant tumor cells becomes suitable for growth, these cells re-enter a proliferative state, leading to distant metastasis and recurrence. However, current research on dormant tumor cells is primarily based on the transient expression levels of dormancy markers p27, proliferation markers Ki67, or related reporter systems, as well as the transient incorporation levels of thymine analogs such as EdU. This data only reflects the current proliferative state of tumor cells and cannot distinguish whether currently proliferating tumor cells have previously been dormant. Therefore, it cannot prove that recurrent metastatic lesions originate from dormant tumor cells that have awakened from dormancy.

[0004] When tumor cells enter a dormant state, they are in the G0 cell cycle. At this time, the p27 protein enters the nucleus and binds to the cyclin-dependent kinase (CDK) complex, preventing the G0-G1 cell cycle transition. When tumor cells are in a proliferative state, the p27 protein is degraded or arrested in the cytoplasm, allowing the G0-G1 cell cycle transition to proceed. Therefore, the current proliferative state can be indicated by detecting the p27 protein level in the cell nucleus. Oki et al. constructed the p27-deficient mutant p27K. - p27K - The degradation and cytoplasmic arrest of p27K are influenced by the cell proliferation state, but it cannot bind to the Cyclin-CDK complex. Therefore, it can be used to indicate the cell's own dormancy state without affecting the cell cycle process, and is widely used in tumor dormancy research. However, based on p27K... - The reporting system can only indicate the current proliferation status of cells and cannot track whether they have ever entered a dormant state.

[0005] Therefore, there is an urgent need to develop a tracking system that can simultaneously display whether tumor cells have ever entered a dormant phase and whether they are currently proliferating or dormant. Summary of the Invention

[0006] The purpose of this invention is to provide a tracking system that can simultaneously display whether tumor cells have entered a dormant phase and whether they are currently proliferating or dormant.

[0007] In a first aspect of the invention, a dormTracer system for tracing and identifying dormant and awakened tumor cells is provided, the system comprising:

[0008] A. Doxycycline-Tet-on system: System A is used to activate Cre-p27K. - Gene expression Cre-p27K - Fusion proteins;

[0009] B.(Cre-p27K - )-loxP system: The B system is used to enable reporter gene expression, and the reporter gene is used to indicate whether tumor cells are undergoing a dormant process;

[0010] Among them, when tumor cells are dormant, Cre-p27K - The fusion protein enters the nucleus and induces reporter gene expression; during tumor cell proliferation, Cre-p27K... - The fusion protein is degraded or retained outside the nucleus, and the reporter gene is not expressed.

[0011] In another preferred embodiment, the DormTracer system further includes a proliferation marker detection system.

[0012] In another preferred embodiment, the proliferation marker detection system is used to indicate whether tumor cells are currently in a proliferative state.

[0013] In another preferred embodiment, the proliferation marker is selected from the group consisting of Ki67, PCNA, and / or EdU.

[0014] In another preferred embodiment, in system A, in the presence of doxycycline, the Tet transcription activator rtTA binds to the Tet response element TRE, inducing the downstream target gene (Cre-p27K) of the TRE. - )Express.

[0015] In another preferred embodiment, the Cre-p27K - The fusion protein is a fusion protein of Cre recombinase and p27 mutant.

[0016] In another preferred embodiment, p27 is a marker protein of cell dormancy or aging.

[0017] In another preferred embodiment, the p27 mutant does not produce the p27 protein and does not affect the cell's own dormant proliferation cycle.

[0018] In another preferred embodiment, p27 is replaced by p21, p16, or any other dormant or aging marker protein.

[0019] In another preferred embodiment, system A is replaced by a doxycycline-Tet-off system, or a tamoxifen-Cre-ERT system, or a gibberellin-induced expression system, or any other system for activating Cre-p27K. - Gene expression Cre-p27K - A system of fusion proteins.

[0020] In another preferred embodiment, in system B, Cre-p27K is activated during cell dormancy. - The fusion protein enters the cell nucleus, and the Cre recombinase cleaves the Stop sequence in the middle of the loxP site, enabling the reporter gene mCherry to be expressed.

[0021] In another preferred embodiment, system B is replaced by the Dre-Rox system, the Flp / Flpo-FRT system, the Vika-vox system, or any other system used to induce reporter gene expression during cell dormancy.

[0022] In another preferred embodiment, the reporter gene in system B is mCherry.

[0023] In another preferred embodiment, the reporter gene in system B is replaced with GFP, or RFP, or any other fluorescent protein expression gene.

[0024] In another preferred embodiment, the reporter gene in system B is replaced with Luciferase, or other bioluminescent protein expression genes, or reporter genes that can be used to detect chemical reactions, or TAG or HA marker proteins, or any other reporter gene that can be used to mark gene recombination events.

[0025] In another preferred embodiment, the DormTracer system is used to distinguish tumor cells in three states: proliferating, dormant, and dormant-awakened.

[0026] In another preferred embodiment, when the reporter gene is expressed but the proliferation marker is not expressed, the tumor cells are in a dormant state; when the reporter gene is not expressed but the proliferation marker is expressed, the tumor cells are in a proliferating state; when both the reporter gene and the proliferation marker are expressed, the tumor cells are in a state of being previously dormant but now awakened and proliferating.

[0027] In another preferred embodiment, the DormTracer system is also used to label the proliferation, dormancy, dormancy awakening, aging, or other non-proliferative states of stem cells, immune cells, or any other cells with dormant-proliferative cycle characteristics.

[0028] In another preferred embodiment, the DormTracer system is also used to label tumor cells in three states in mice: proliferating, dormant, and dormant-awakened.

[0029] In another preferred embodiment, the DormTracer system is also used to mark the proliferation, dormancy, dormancy awakening, or aging states of stem cells, immune cells, or other cells in mice.

[0030] In another preferred embodiment, the DormTracer system is also used to track the origin of metastatic lesions in mice.

[0031] In a second aspect of the invention, a method for screening a gene library is provided, wherein the gene is a gene that regulates dormancy or proliferation, the method comprising:

[0032] a. Without modifying the candidate gene, the proportions of the three states of proliferation, dormancy, and dormancy awakening of target cells are detected using the DormTracer system as described in the first aspect of the present invention;

[0033] b. In the case where the candidate gene has been modified, the proportion of the three states of target cells—proliferation, dormancy, and dormancy awakening—is detected using the DormTracer system as described in the first aspect of the present invention.

[0034] c. Compare the results in step a and step b.

[0035] In another preferred embodiment, the modification includes gene knockout, gene mutation, or increase in gene expression.

[0036] In another preferred embodiment, the detection is performed by immunofluorescence microscopy.

[0037] In another preferred embodiment, the proportions of the three states of proliferation, dormancy, and dormancy awakening are shown by fluorescence staining results.

[0038] In a third aspect of the invention, a drug screening method is provided, wherein the drug is a drug that regulates dormancy or proliferation, the method comprising:

[0039] a. In the case of co-culturing candidate drugs with cells, the proportion of target cells in the three states of proliferation, dormancy and dormancy awakening is detected using the DormTracer system as described in the first aspect of the present invention;

[0040] b. In the absence of a candidate drug, the proportions of the target cells in the three states of proliferation, dormancy, and dormancy awakening are detected using the DormTracer system as described in the first aspect of the present invention.

[0041] c. Compare the results in step a and step b.

[0042] In another preferred embodiment, the candidate drug is a drug that eliminates dormant tumor cells or inhibits the awakening of dormant tumor cells.

[0043] In another preferred embodiment, if the proportion of dormant / awakened / proliferating target cells increases after using the candidate drug, it suggests that the drug has the effect of promoting cell proliferation.

[0044] In another preferred embodiment, if the proportion of dormant target cells increases after using the candidate drug, it suggests that the drug has the effect of promoting cell dormancy.

[0045] In a fourth aspect of the invention, a method for investigating the effects of an exogenous environment on tumor dormancy / awakening is provided, the method comprising:

[0046] a. Without altering the external environment, the proportions of tumor cells in the three states of proliferation, dormancy, and dormancy awakening are detected using the DormTracer system as described in the first aspect of the present invention;

[0047] b. Under conditions of external environmental change, the proportion of tumor cells in the three states of proliferation, dormancy, and dormancy awakening is detected using the DormTracer system as described in the first aspect of the present invention;

[0048] c. Compare the results in step a and step b.

[0049] In another preferred embodiment, the external environmental change includes changes in living environment and / or lifestyle.

[0050] In a fifth aspect of the invention, a method for in vitro monitoring of tumor cell dormancy is provided, the method comprising:

[0051] a. Introducing the tracing system described in the first aspect of the present invention into tumor cells;

[0052] b. After a period of time, observe the dormant / proliferative markers in the tumor cells.

[0053] In a sixth aspect of the invention, a method for monitoring tumor cell dormancy in vivo is provided, the method comprising:

[0054] a. Introducing the tracing system described in the first aspect of the present invention into tumor cells;

[0055] b. Inoculate the tumor cells into the body;

[0056] c. After a period of time, observe the dormant / proliferative markers in the tumor cells in vivo.

[0057] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0058] Figure 1 shows a schematic diagram of the embodiments of the present invention: (A) In the presence of doxycycline (Dox), rtTA induces the expression of Cre-p27K- fusion protein. Cre-p27K- fusion protein enters the nucleus in dormant tumor cells. Cre recombinase cleaves the Stop sequence in the middle of the loxP site, resulting in the expression of the reporter gene mCherry. At the same time, the expression of Ki67 is detected to track whether dormant tumor cells have awakened and entered the proliferative state; (B) Cartoon schematic diagram of the detection of three states of tumor cells in the present invention. Under doxycycline treatment, proliferating tumor cells are mCherry negative and Ki67 positive, dormant tumor cells are mCherry positive and Ki67 negative, and dormant awakened tumor cells are mCherry positive and Ki67 positive; (C) The traditional dormant reporter system can only detect the transient level of p27 protein in tumor cells, so it can only distinguish between the two states of proliferation and dormancy. The present invention can track whether tumor cells have entered the dormant state by detecting whether mCherry is expressed. Combined with the expression of Ki67, it can distinguish between tumor cells in the three states of proliferation, dormancy, and dormant awakening.

[0059] Figure 2 illustrates the in vitro labeling, tracking, and reactivation verification of dormant tumor cells: (A) A schematic diagram of P2 cells expressing the DormTracer system cultured under three conditions: sufficient serum, starvation, and post-starvation serum supplementation, with the addition of doxycycline; (B) Under sufficient serum or starvation conditions, 24 hours before detection, the immunofluorescence staining results of mCherry and EdU showed that mCherry was mainly expressed in EdU-negative dormant tumor cells; (C) Under the three conditions of sufficient serum, starvation, and post-starvation serum supplementation, the immunofluorescence staining results of mCherry and the proliferation marker Ki67 showed that DormTracer can track whether P2 cells have entered a dormant state and can label three cell states: proliferation, dormancy, and dormancy reactivation. Scale bar represents 50 μm.

[0060] Figure 3 shows the labeling, tracking, and awakening verification of dormant tumor cells in mice: (A) Immunofluorescence staining was performed after inoculating GFP-labeled P1 or P2 tumor cells via the tail vein. The EdU staining results and statistics showed that P1-inoculated mice had a large number of EdU-positive proliferating tumor cells in the lungs, which could form metastatic foci. P2-inoculated mice mainly had EdU-negative dormant tumor cells in the lungs, indicating that P1 was a lung metastatic proliferating tumor cell and P2 was a lung metastatic dormant tumor cell; (B) Schematic diagram of the experimental strategy for tracing dormant disseminated tumor cells and awakening dormant tumor cells in the mouse lungs according to the present invention; (C) Five days after inoculating P1 and P2 cells via the tail vein, the immunofluorescence staining results and statistics of mCherry and Ki67 showed that more mCherry-positive and Ki67-negative dormant cells were detected in the lungs of mice inoculated with lung metastatic dormant P2 cells, and more mCherry-negative and Ki67-positive proliferating cells were detected in the lungs of mice inoculated with metastatic proliferating P1 cells. Scale bar represents 50 μm.

[0061] Figure 4 shows the use of the tracing system of the present invention to track the source of metastatic lesions: H / E staining, immunofluorescence staining, and statistical analysis of mouse lung tissue 50 days after tail vein inoculation with P1 and P2 cells. The H / E staining scale bar represents 500 μm, and the immunofluorescence staining scale bar represents 50 μm. Detailed Implementation

[0062] Through extensive and in-depth research, the inventors have developed a labeling and tracing system (DormTracer) for tumor cell dormancy and awakening. Specifically, this system combines the doxycycline-Tet-on system and (Cre-p27K) - The 10xP system, reporter gene expression system, and proliferation marker detection can distinguish tumor cells in three states: proliferating, dormant, and dormant-awakened. This invention was developed based on these principles.

[0063] the term

[0064] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0065] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0066] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0067] In this invention, "dormant marker" and "dormant marker protein" can be used interchangeably.

[0068] In this invention, "proliferation marker" and "proliferation marker protein" can be used interchangeably.

[0069] Doxycycline-Tet-on system

[0070] The Tet-on system regulates the expression of target proteins by inducing drugs (such as doxycycline, tetracycline, and doxycycline) to alter the conformation of regulatory proteins.

[0071] The Tet-on system consists of a regulatory expression vector and a reactive expression vector. The regulatory expression vector contains the human cytomegalovirus early promoter (PhCMV) and the antisense Tet transcriptional activator (rtTA). The reactive expression vector consists of a Tet-responsive element (TRE), a CMV promoter (PminCMV), and the target gene.

[0072] In this invention, in the presence of doxycycline, the antisense Tet transcription activator rtTA binds to the Tet response element TRE, inducing the expression of the downstream target gene (Cre-p27K-).

[0073] rtTA DNA sequence (SEQ ID NO.1):

[0074] rtTA amino acid sequence (SEQ ID NO.2):

[0075] Inducing downstream target gene (Cre-p27K) -Other systems for expression include: doxycycline-Tet-off system, tamoxifen-Cre-ERT system, or gibberellin-induced expression system.

[0076] Fusion protein (Cre-p27K) - )-loxP system

[0077] Site-specific recombination involves the cleavage, exchange, or rearrangement of DNA sequences between specific DNA sites catalyzed by recombinases. Cre-loxP and Dre-roxP recombination systems are widely used for genetic pedigree tracing to investigate the origin, development, and function of specific cell populations. These recombination systems contain recombinases such as Cre or Dre and two recognition sites such as loxP or roxP. If the recognition sites are located on the same DNA strand and oriented in the same direction, the recombinase deletes the sequence in the middle of the recognition site. If the recognition sites are located on the same DNA strand and oriented in opposite directions, the recombinase induces the sequence in the middle of the recognition site to flip. If the recognition sites are located on two different DNA strands and oriented in the same direction, the recombinase induces the exchange of DNA between the two strands or a chromosomal translocation. Site-specific recombination can control the expression of reporter genes in specific cell types and stably transmit them to offspring. Therefore, combining an inducible expression system with a site-specific recombination system allows for the control of reporter gene expression in specific cell types under specific conditions or at specific times, enabling genetic tracing.

[0078] In this invention, p27 serves as a cell dormancy marker, being degraded during cell proliferation and expressed during cell dormancy. Cre-p27K - This is a fusion protein of Cre recombinase and the p27 mutant. p27K - It does not produce p27 protein, thus not affecting the cell's own dormancy-proliferation cycle. During cell proliferation, Cre-p27K... - The fusion protein is degraded or arrested in the cytoplasm, and the reporter gene mCherry is not expressed; when the cell is dormant, Cre-p27K... - The fusion protein is not degraded and thus enters the cell nucleus. Its coupled Cre protein can delete the transcription termination element at the loxP site in the cell nucleus, leading to the expression of the reporter gene mCherry. + This indicates that the cell is currently in a dormant phase or has previously experienced a dormant phase.

[0079] Cre-p27K - DNA sequence (SEQ ID NO.3):

[0080] Cre-p27K - Amino acid sequence (SEQ ID NO.4):

[0081] Induced reporter gene expression systems also include: the Dre-Rox system, the Flp / Flpo-FRT system, and the Vika-vox system.

[0082] Dormant marker proteins also include p21 and p16.

[0083] Reporter gene expression system

[0084] The cell dormancy marker p27 degrades after the cell exits dormancy and is re-expressed when the cell re-enters dormancy, thus it can only detect whether the cell is currently in a dormant state.

[0085] In this invention, when cells enter a dormant phase, Cre-p27K... - The fusion protein is not degraded, thereby inducing the expression of the reporter gene mCherry. The reporter gene is not degraded as the cell exits dormancy, thus enabling the cell to show its dormancy history, even if the cell is not currently in dormancy.

[0086] mCherry DNA sequence (SEQ ID NO.5):

[0087] mCherry amino acid sequence (SEQ ID NO.6):

[0088] The reporter genes also include: GFP, YFP, Luciferase, HA, TAG, etc.

[0089] Proliferation marker detection

[0090] Ki-67 is a nuclear protein involved in the cell cycle. Ki-67 is expressed in the G1, S, G2, and M phases of cell division. After the M phase, Ki-67 is rapidly degraded, so cells in the G0 phase do not express Ki-67.

[0091] In this invention, Ki-67 + Used to indicate that a cell is currently in a proliferating state.

[0092] The proliferation markers also include CFSE, BrdU, EdU, and PCNA.

[0093] DormTracer system

[0094] The DormTracer system of this invention combines the doxycycline-Tet-on system and (Cre-p27K) -The 100xP system and proliferation marker detection can distinguish tumor cells in three states: proliferating, dormant, and dormant-awakened. When the reporter gene is expressed but the proliferation marker is not, the tumor cells are in a dormant state; when the reporter gene is not expressed but the proliferation marker is expressed, the tumor cells are in a proliferating state; when both the reporter gene and the proliferation marker are expressed, the tumor cells are in a state of formerly dormant but now awakened and proliferating.

[0095] Compared with the prior art, the advantages of the present invention are as follows:

[0096] 1. Compared with systems that directly detect cell dormancy or proliferation: The DormTracer system of this invention can distinguish between three states of tumor cells: proliferation, dormancy, and dormancy awakening. It can not only detect the current state of the cell, but also trace the historical state of the cell, whether it has entered a dormant period and then revived and proliferated, which can be used to reflect the stability of tumor cells.

[0097] 2. Compared with dormant tracing systems using transcriptomics, the DormTracer system of this invention is more intuitive and easier to operate.

[0098] 3. The DormTracer system of the present invention can also be used for gene library screening to explore the molecular mechanisms of tumor cell regulation of dormancy and dormancy awakening.

[0099] 4. The DormTracer system of the present invention can also be used for compound library screening to screen compounds that affect the entry of tumor cells into a dormant state and the awakening of dormant cells, and further screen targeted drugs that eliminate dormant tumor cells or inhibit the awakening of dormant tumor cells.

[0100] 5. The DormTracer system of the present invention can also be used to investigate the effects of exogenous factors such as living environment and lifestyle on tumor dormancy and awakening, or to construct mouse models.

[0101] 6. The DormTracer system of the present invention can also be used to explore the dormancy and awakening regulation mechanisms of non-tumor cells such as stem cells and immune cells.

[0102] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0103] Example 1. The DormTracer system is used to distinguish between tumor cells in three states: proliferating, dormant, and dormant-awakening.

[0104] Cre-p27K was controlled using the Tet-on inducible expression system. - Expression of the fusion protein: In the presence of doxycycline, the rtTA protein binds to the TRE promoter, resulting in Cre-p27K... - Protein expression. In proliferating tumor cells, p27K - The protein is degraded or arrested in the cytoplasm; its coupled Cre protein cannot enter the nucleus; the S transcription termination element between loxP sites is present; the reporter gene mCherry is not expressed; and the proliferation marker Ki67 is expressed. In dormant tumor cells, p27K... - The protein is not degraded and enters the cell nucleus. Its coupled Cre protein can delete the transcription termination element in the middle of the loxP site in the cell nucleus. The reporter gene mCherry is expressed, but the proliferation marker Ki67 is not expressed. After the dormant tumor cells are awakened, the recombined DNA sequence is inherited by the offspring. The reporter gene mCherry is expressed, and the proliferation marker Ki67 is expressed (Figure 1A).

[0105] Therefore, by using tumor cells that express this tracking system and GFP, three cell states can be identified: proliferating tumor cells that express GFP and Ki67, dormant tumor cells that express GFP and mCherry, and dormant-awakened tumor cells that simultaneously express GFP, mCherry, and Ki67 (Figure 1B).

[0106] Traditional reporting systems can only distinguish between tumor cells that are currently in a proliferating or dormant state. This system can record the dormancy history, thus it can mark tumor cells in three states: proliferating, dormant, and dormant-awakened. It can also trace whether recurrent metastatic lesions originated from proliferating tumor cells or dormant-awakened tumor cells (Figure 1C).

[0107] Example 2. DormTracer system for tracking tumor cell dormancy history

[0108] 2,000 MCF10CA1h-P2 tumor cells expressing this tracking system and GFP were cultured in normal medium for 12 hours to adhere to the culture vessel. Then, 1 μg / mL doxycycline was added and the cells were cultured in serum-supplemented medium for 72 hours, serum-free medium for 72 hours, or serum-free medium for 48 hours followed by serum-supplemented medium for 48 hours. 3 μg / mL EdU was added 24 hours before detection. Immunofluorescence detection technology was used to verify that this system can label tumor cells in three states: proliferating, dormant, and dormant-awakened (Figure 2A).

[0109] In tumor cells cultured in serum-sufficient or serum-free medium, 87.9% of EdU-positive proliferating cells did not express mCherry, while 87.3% of EdU-negative dormant cells expressed mCherry, indicating that this system can label dormant tumor cells and has high detection efficiency (Figure 2B).

[0110] Under three culture conditions, the results of mCherry and Ki67 detection showed that under sufficient serum conditions, 92.0% of the cells were in a proliferative state of mCherry-negative and Ki67-positive; under serum starvation conditions, 69.8% of the cells were in a dormant state of mCherry-positive and Ki67-negative; and under serum starvation followed by serum supplementation conditions, 81.1% of the cells entered a dormant awakening state of mCherry-positive and Ki67-positive (Figure 2C).

[0111] The above results indicate that this system can distinguish between proliferating and dormant tumor cells, and can track dormant history and label dormant and awakened cells.

[0112] Example 3. The DormTracer system was used to label three cell states—proliferation, dormancy, and dormancy-awakening—in mice.

[0113] 1×10 6 Cell subsets P1 and P2, expressing GFP and derived from MCF10CA1h, were injected intravenously into nude mice. Twenty-four hours before testing, each mouse was intraperitoneally injected with 100 μg of EdU. Immunofluorescence staining was used to detect EdU incorporation in P1 and P2 cells disseminated in the lungs at different time points. The results showed that the proportion of EdU-positive proliferating cells in the P1 cell population was higher than that in P2, and these cells formed metastatic lesions in the lungs, while the P2 cell population remained in a dormant state and was EdU-negative for a long period (Figure 3A). P1 and P2 cells expressing GFP and DormTracer were injected intravenously into nude mice. Seventy-two hours later, the mice were treated with 0.2 g / L doxycycline in their drinking water for three days. The expression of mCherry and Ki67 in disseminated tumor cells in the lungs was then detected (Figure 3B). Five days after tail vein injection, the proportion of proliferating tumor cells (mCherry-negative, Ki67-positive) in cell population P1 was significantly higher than that in P2, while the proportion of dormant tumor cells (mCherry-positive, Ki67-negative) was significantly lower in P1 than in P2. There was no significant difference in the proportion of dormant-awakened cells (mCherry-positive, Ki67-positive) (Figure 3C). These results indicate that the DormTracer system can label three cell states—proliferation, dormancy, and dormancy-awakened—in mice.

[0114] Example 4. DormTracer system for tracing the source of metastatic lesions

[0115] Fifty days after tail vein injection, all five mice injected with P1 cells developed lung metastases and recurrence, with an average of 30 lung metastases per mouse. In contrast, only one of the five mice injected with P1 cells developed lung metastases and recurrence, with a total of seven lung metastases. This indicates that the DormTracer system does not alter the metastatic, proliferative, or dormant characteristics of tumor cells. Immunofluorescence staining revealed the expression of mCherry and Ki67 in P1 and P2 metastases. Approximately 60.5% of P1 metastases originated from dormant to awakened tumor cells, and 39.5% originated from proliferating tumor cells. Among the P2 metastases, six originated from dormant to awakened tumor cells, and only one originated from proliferating tumor cells (Figure 4).

[0116] These results demonstrate that the DormTracer system can record the dormancy history of tumor cells and trace the origin of metastatic lesions.

[0117] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A marker tracer system (DormTracer) for tumor cell dormancy and awakening, characterized in that, The system comprises: A. Doxycycline-Tet-on system: The A system is used to turn on Cre-p27K - gene expression Cre-p27K - fusion protein, the p27 is a cell dormancy marker protein; B. (Cre-p27K - ) -loxP system: the B system is used to turn on the expression of a reporter gene, which is used to indicate whether tumor cells have undergone the dormancy process; Wherein, when the tumor cell is in dormancy, the Cre-p27K - fusion protein enters the nucleus and induces the expression of the reporter gene; when the tumor cell is proliferating, the Cre-p27K - fusion protein is degraded or retained outside the nucleus, and the reporter gene is not expressed.

2. The tracking system of claim 1, wherein, The DormTracer system further comprises a proliferation marker detection system for indicating whether the tumor cells are currently in a proliferative state.

3. The tracking system of claim 1, wherein, The A system is replaced by a doxycycline-Tet-off system, or a tamoxifen-Cre-ERT system, or a gibberellin inducible expression system, or any other system for turning on Cre-p27K - gene expression Cre-p27K - fusion protein system.

4. The tracking system of claim 1, wherein, The Cre-p27K - The fusion protein is a fusion protein of Cre recombinase and a p27 mutant which does not produce a p27 protein and does not affect the cell's own dormant proliferation cycle.

5. The tracking system of claim 1, wherein, The p27 is replaced by p21, p16 or any other dormancy or aging marker protein.

6. The tracking system of claim 1, wherein, The B system is replaced by Dre-Rox system, Flp / Flpo-FRT system, Vika-vox system, or any other system for inducing reporter gene expression when cells are dormant.

7. The tracking system of claim 1, wherein, The reporter gene is selected from mCherry, GFP, RFP.

8. The tracking system of claim 1, wherein, The DormTracer system is used to distinguish tumor cells in three states: proliferation, dormancy and dormancy awakening.

9. The tracking system of claim 1, wherein, The DormTracer system is also used for dormancy tracking, aging state tracking, or other non-proliferative state tracking of tumor cells, stem cells, immune cells or any other cells with dormancy-proliferation cycle.

10. The tracking system of claim 1, wherein, The DormTracer system is also used to track the source of metastases in vivo.

11. A method for screening a gene library, the gene being a gene that regulates dormancy or proliferation, characterized in that, The method comprises: a. detecting the proportions of the three states of proliferation, dormancy and dormancy awakening of target cells using the DormTracer system of claim 1 without knocking out or modifying the candidate gene; b. detecting the proportions of the three states of proliferation, dormancy and dormancy awakening of target cell using the DormTracer system of claim 1 with modifying the candidate gene; c. comparing the results in step a and step b.

12. A method of screening for a drug, the drug being a drug that modulates dormancy or proliferation, characterized in that, The method comprises: a. detecting the proportions of the three states of proliferation, dormancy, and dormancy awakening of target cells using the DormTracer system of claim 1 with the candidate drug co-cultured with the cells; b. detecting the proportions of the three states of proliferation, dormancy, and dormancy awakening of the target cells using the DormTracer system of claim 1 without the candidate drug; c. comparing the results in step a and step b.

13. A method of investigating the effect of an exogenous environment on tumor dormancy / awakening, comprising: a) providing a tumor cell; b) exposing the tumor cell to an exogenous environment; and c) determining whether the tumor cell is dormant or awake. 13 The method comprises: a. Detecting the proportions of the three states of proliferation, dormancy, and dormancy awakening of tumor cells using the DormTracer system of claim 1 without changing the exogenous environment; b. Detecting the proportions of the three states of proliferation, dormancy, and dormancy awakening in tumor cells using the DormTracer system of claim 1 with changing the exogenous environment; c. comparing the results in step a and step b.

14. A method of monitoring tumor cell dormancy in vitro, characterized in that, The method comprises: a. introducing the tracking system of claim 1 into tumor cells; b. after a period of time, observing the dormancy / proliferation markers in the tumor cells.

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