WO2022247453A1 - 一种setd4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用 - Google Patents

一种setd4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用 Download PDF

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WO2022247453A1
WO2022247453A1 PCT/CN2022/083977 CN2022083977W WO2022247453A1 WO 2022247453 A1 WO2022247453 A1 WO 2022247453A1 CN 2022083977 W CN2022083977 W CN 2022083977W WO 2022247453 A1 WO2022247453 A1 WO 2022247453A1
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cancer
protein
dek
tumor
seq
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杨卫军
杨尧顺
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浙江大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors

Definitions

  • the present invention relates to the application of a SETD4 protein inhibitor in the preparation of drugs for activating dormant tumor cells, especially DEK protein combined with clinical tumor resection, radiotherapy, chemotherapy, targeting and immunotherapy to activate and eliminate dormant tumor cells, thereby realizing tumor cure , as well as the clinical application of curing various cancers.
  • Cancer is still one of the major diseases of human health.
  • the annual number of new cancer patients worldwide exceeds 10 million, and the death toll due to cancer exceeds 6.7 million.
  • the current treatment methods for cancer mainly include surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Although these treatments clear and kill most tumor cells, they fail to clear a small population of dormant tumor cells within the tumor.
  • dormant tumor cells in the tumor are small, they are highly resistant to various clinical treatments. They usually have a strong ability to form tumors in vivo and tumorspheres in vitro. Activated dormant tumor cells can promote tumor progression and metastasis, and are the main factor in the formation of malignant tumors.
  • Dormant tumor cells can survive clinical radiotherapy, chemotherapy, targeted and immunotherapy, and can quickly activate and produce a large number of tumor cells after treatment, thereby inducing tumor recurrence and metastasis in cancer patients after surgery. Therefore, how to kill and remove dormant tumor cells is a bottleneck problem that cannot be broken through in the clinical cure of tumors.
  • SET domain-containing family proteins have the activity of histone arginine methyltransferase, and play an important role in regulating chromatin structure and regulating gene transcription and expression in the process of cell proliferation.
  • SETD4 histone arginine methyltransferase
  • SETD4 epigenetically regulates the dormancy of breast tumor cells by the same mechanism, revealing the evolutionary conservation mechanism of stem cell dormancy regulated by SETD4, and found that SETD4 dormant tumor cells exist in various types of clinical tumor patients Such as liver cancer, lung cancer, pancreatic cancer, ovarian cancer, uterine cancer, etc., compared with early-stage tumor patients, the number of SETD4 dormant tumor cells increased significantly (Document 2). Therefore, the use of SETD4 protein can mark such dormant tumor cells, which provides an important target for eliminating dormant tumor cells. This is the first research report on the molecular marking of dormant tumor cells in various tumors, and the first research report on the discovery of the epigenetic mechanism of tumor cell dormancy through histone modification (H4K20me3).
  • DEK as a nuclear factor protein, can bind to chromatin and participate in the regulation of cell proliferation, differentiation, apoptosis, aging, DNA damage repair and maintenance of stem cell characteristics.
  • oncoprotein it is highly expressed in various tumor cells and is related to tumor recurrence and metastasis.
  • DEK protein can be synthesized and secreted by tumor cells and taken up by other cells, so it is a protein with intracellular and intercellular activity.
  • DEK proteins can be released from cells and enter their target cells in the form of free and contained exosomes.
  • DEK was highly expressed in the activation process of Artemia dormant embryos, and found that DEK played an important role in the activation of dormant cells by reducing the expression of SETD4 protein and H4K20me3 (Document 3 ).
  • DEK protein is highly expressed in activated dormant tumor stem cells and tumor cells, inhibiting the expression of DEK protein can significantly inhibit the activation of dormant tumor cells, and adding exogenous DEK protein can promote the activation of dormant tumor cells. It was found that the activated dormant tumor cells lost the ability to resist radiotherapy and chemotherapy, and the simultaneous treatment of radiotherapy and chemotherapy with DEK protein could eliminate the dormant tumor cells, thereby achieving a complete cure of the tumor.
  • the purpose of the present invention is to provide the application of a SETD4 protein inhibitor in the preparation of drugs for activating dormant tumor cells, especially the exogenous DEK protein can enter and activate the dormant tumor cells, making them lose the ability to resist radiotherapy and chemotherapy.
  • Clinical surgery, radiotherapy and chemotherapy, targeted and immunotherapy, and DEK protein treatment can eliminate dormant tumor cells, thereby achieving a complete cure of the tumor.
  • the present invention provides an application of a SETD4 protein inhibitor in the preparation of a drug for activating dormant tumor cells.
  • the SETD4 protein inhibitor involved in the present invention includes DEK protein, and the DEK protein has a conserved sequence shown in SEQ ID NO.25.
  • DEK protein has more than 95% similarity to the amino acid sequence of the NLS domain shown in SEQ ID NO.2.
  • DEK protein has more than 95% similarity to the amino acid sequence of the SAP domain shown in SEQ ID NO.3.
  • DEK protein has more than 95% similarity to the amino acid sequence of the pseudo-SAP domain shown in SEQ ID NO.4.
  • the DEK protein has one or both of the pseudo-SAP domain shown in SEQ ID NO.4 or the SAP domain shown in SEQ ID NO.3, and also has the NLS structure shown in SEQ ID NO.2 area.
  • the DEK protein has more than 95% similarity to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.22.
  • DEK protein is selected from human DEK protein, has the amino acid sequence shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.5.
  • the DEK protein is selected from murine DEK protein, has the amino acid sequence shown in SEQ ID NO.22, and the coding gene sequence is shown in SEQ ID NO.21.
  • the dormant tumor cells or tumors described in the present invention include head and neck tumors, chest tumors, digestive system tumors, genitourinary system tumors, bone and soft tissue tumors, lymphatic and blood system tumors, and other tumors; wherein head and neck tumors include brain cancer, Eye cancer, ear tumor, jaw tumor, neck tumor, nasal cavity cancer, sinus cancer, nasopharyngeal cancer, gum cancer, tongue cancer, soft and hard palate cancer, jaw cancer, mouth floor cancer, oropharyngeal cancer, lip cancer, Cancer of the maxillary sinus, cancer of the skin and mucous membranes of the face, laryngeal cancer, salivary gland tumors, thyroid cancer, meningioma, ependymoma, pituitary tumor, epithelial neuroblastoma, neuroectodermal and paraganglionic tumors; thoracic tumors Including lung cancer, esophageal cancer, breast cancer, mediastinal tumor and thymus cancer; digestive system
  • the present invention provides a SETD4 protein inhibitor delivery protein for activating dormant tumor cells
  • the delivery protein includes delivery of DEK protein
  • the delivery of DEK protein is a medically acceptable carrier containing DEK protein
  • said Carriers include exosomes, liposomes or nanomaterials.
  • the exosome refers to a vesicle secreted by a cell with a size of 30-150 nm and a double membrane structure.
  • the delivery of the DEK protein is carried out by a tumor cell line Isolate exosomes containing DEK protein from culture medium, or insert DEK protein coding genes into various gene expression vectors, overexpress DEK protein in various cell lines (recommended tumor cells) and produce exosomes containing DEK protein body.
  • the DEK protein-containing exosomes obtained by inserting the DEK protein coding gene into the gene expression vector are prepared according to one of the following methods: (1) Construct a plasmid that overexpresses the DEK protein and transfect it into various cell lines Preparation of exosomes in China: Insert the DEK gene into the EcoR I and Xba I sites of the pEGFP-C1 plasmid, and screen to obtain the recombinant plasmid pEGFP-C1-DEK; use the liposome transfer agent lipo8000 (Biyuntian, product number: C0533 ) Transfer the above-mentioned recombinant plasmid into a cell line, collect the cell culture medium after overexpressing the DEK protein, separate and purify the exosome solution A containing the DEK protein from the culture medium; (2) construct a lentivirus overexpressing the DEK protein and It infects various cell lines to construct cell lines expressing DEK protein to prepare exosomes: insert the DE
  • exosomes obtained from the culture medium of tumor cell lines be prepared by exosome extraction reagent method: from tumor Add exosome isolation reagent (brand: Thermo Fisher, product number: 4478359) to the cell line culture medium, invert up and down 3 times to mix well, and then incubate overnight at 4°C. Then resuspend the pellet at the bottom of the centrifuge tube with PBS, which is the crude exosomes containing DEK protein.
  • cell lines of method (1) and (2) are preferably 4T1, EMT6, MCF7.
  • both methods (1) and (2) are used to separate and purify the exosome solution containing DEK protein from the culture medium: the exosomes are separated from the cell culture medium using an exosome extraction reagent, and the exosomes are obtained by flow cytometry The sorter sorted to obtain GFP-positive exosomes containing DEK protein with a particle size range of 50-200 nm.
  • the lentiviral packaging plasmid mixture described in method (2) is derived from the lentiviral packaging kit (Lentiviral Packaging Kit, purchased from Shanghai Yisheng Biology, article number: 41102ES10), consisting of pMDL, VSVG and pRSV-Rev composition.
  • the liposome refers to a microcapsule formed by a phospholipid bilayer artificial membrane in an aqueous solution.
  • the liposome containing DEK protein is prepared by a film hydration method: take dipalpalm Acylphosphatidylcholine (DPPC), cholesterol, distearoylphosphatidylacetamide-methoxypolyethylene glycol (2000DSPE-mPEG2000) were dissolved in chloroform, reduced pressure and rotary evaporation to form a uniform film, and evaporated overnight in vacuum Remove residual chloroform, add DEK protein-containing PBS solution, ice-bath at 25KHz for 20 minutes to sonicate the liposome membrane, oscillate on an oscillator for 5 minutes to fully hydrate, and form a turbid solution, then transfer the turbid solution to an EP tube, and use Probe ultrasound at 135W for 30 minutes to obtain a transparent and uniform blue liposome suspension; use a 10kD ultrafiltration tube to ultra
  • Collect the retentate which is a liposome suspension containing DEK protein; the preferred concentration of the DEK protein in PBS solution is 2mg/ml; the mass ratio of dipalmitoylphosphatidylcholine to cholesterol is 1:0.1, and the two The mass ratio of palmitoylphosphatidylcholine to distearoylphosphatidylacetamide-methoxypolyethylene glycol is 1:0.1, and the volumetric dosage of said chloroform is 0.1mL/mg based on the mass of dipalmitoylphosphatidylcholine ; The mass ratio of DEK protein in the PBS solution of dipalmitoylphosphatidylcholine and DEK protein is 1:0.2.
  • the nanomaterial refers to a solid colloidal particle with a size between 10-1000 nm that can be used as a carrier for conduction or delivery of drugs, and is composed of natural or synthetic macromolecules.
  • the carrier is a nanomaterial, it contains DEK protein
  • the nanomaterials were prepared by the improved solvent evaporation method: the PBS solution of DEK protein was transferred to the dichloromethane solution of polylactic acid-glycolic acid copolymer (PLGA), and 25KHz ultrasonic wave was used for 1 minute to form colostrum, and the colostrum was transferred into In an aqueous solution of 1% polyvinyl alcohol (PVA) with a volume concentration, ultrasonication at 25KHz was performed for 5 minutes to form a double emulsion, and stirred for 4 hours.
  • PVA polyvinyl alcohol
  • the precipitate was collected by centrifugation at 18,000 r/min, and the precipitate was freeze-dried at -55°C for 24 hours to obtain DEK-containing PLGA nanoparticles of protein; preferably, the DEK protein and polylactic acid-glycolic acid copolymer mass ratio is 1:0.1, the PBS solution concentration of the DEK protein is 25mg/L; the dichloromethane solution concentration of the PLGA is 20 mg/mL, and the volume dosage of the PVA aqueous solution is 1 mL/mg based on the amount of DEK protein in the PBS solution of DEK protein.
  • the present invention provides an application of a SETD4 protein inhibitor in the preparation of a reagent for activating dormant tumor cells, and the reagent includes DEK protein.
  • the activation is to deliver the SETD4 protein inhibitor to the dormant tumor cells to achieve the purpose of activating the dormant tumor cells.
  • the reagent can be used in clinical treatment of tumors and basic scientific research.
  • the agent for activating dormant tumor cells of the present invention also includes SETD4 protein inhibitors and drugs for eliminating tumor cells.
  • the SETD4 protein inhibitor includes DEK protein; the drug for eliminating tumor cells recommends paclitaxel or 5-fluorouracil.
  • the method for removing tumor cells described in the present invention is to combine SETD4 protein inhibitors with conventional methods such as radiotherapy and chemotherapy to achieve the purpose of eliminating dormant tumor cells. Specifically, it refers to combining SETD4 protein inhibitors (especially It is the DEK protein, preferably delivered to tumor cells through exosomes, liposomes and nanomaterials) to activate and eliminate dormant tumor cells, so as to achieve clinical tumor cure without metastasis and recurrence.
  • the definition and scope of application of the DEK protein, dormant tumor cells or tumors in the third aspect are the same as those defined in the first aspect.
  • the SETD4 protein inhibitor is delivered to dormant tumor cells using the delivery protein described in the second aspect.
  • the present invention provides an application of a SETD4 protein inhibitor in the preparation of an antitumor drug, the antitumor drug includes the SETD4 protein inhibitor and a drug for removing tumor cells, and the SETD4 protein inhibitor includes DEK protein; paclitaxel or 5-fluorouracil is recommended as the drug for eliminating tumor cells.
  • the application is to inject SETD4 protein inhibitors (especially DEK protein in the form of exosomes, liposomes and nanocarriers) through intraperitoneal, intravenous or The tumor body is directly injected and delivered into the tumor, and at the same time, it is used in combination with drugs that eliminate tumor cells to achieve the purpose of tumor removal.
  • the definition and scope of application of the DEK protein, dormant tumor cells or tumors in the fourth aspect are the same as those defined in the first aspect.
  • the SETD4 protein inhibitor is delivered to dormant tumor cells using the delivery protein described in the second aspect.
  • the present invention provides a method for treating tumors with a SETD4 protein inhibitor, the method comprising: injecting SETD4 protein inhibitors through intravenous injection, intraperitoneal injection or intratumoral injection of SETD4 protein inhibitors in various stages of tumor patients.
  • the SETD4 protein inhibitor (especially the DEK protein) enters the nucleus, binds to the promoter of the SETD4 gene, closes the expression of SETD4, down-regulates the formation of heterochromatin, opens the expression of most genes, and activates dormant tumor cells, The activated dormant tumor cells are then killed by conventional methods such as radiotherapy and chemotherapy;
  • the SETD4 protein inhibitor includes DEK protein, and the DEK protein is injected in the form of delivery DEK protein, and the delivery DEK protein refers to the DEK protein-containing For exosomes, liposomes or nanomaterials, the delivery of DEK protein is the same as that described in the second aspect.
  • the present invention screens and obtains dormant tumor cells that can resist radiotherapy and chemotherapy in human and mouse breast cancer through radiotherapy and chemotherapy, and discovers a functional protein and its gene that can activate dormant tumor cells.
  • the full length of DNA of the gene is 1128bp (SEQ ID NO.5), which translates to encode a protein (SEQ ID NO.1) consisting of 375 amino acids, namely the DEK protein.
  • the exogenous DEK protein of the present invention (including exosomes containing DEK protein secreted by tumor cells) can enter and activate dormant tumor cells, making them lose the ability to resist radiotherapy and chemotherapy. Conventional methods remove dormant tumor cells.
  • the present invention can activate and eliminate dormant tumor cells in tumors in the tumor-bearing mice through simultaneous treatment with radiotherapy and chemotherapy and exosomes containing DEK protein, thereby realizing the complete cure of tumor-bearing mice, and clarifying the use of conventional methods such as radiotherapy and chemotherapy.
  • Methods Simultaneous treatment with DEK or exosomes containing DEK protein can eliminate dormant tumor cells (Figure 7). In this experiment, it was observed that the mice had no tumor recurrence or metastasis after treatment.
  • DEK protein activating dormant tumor cells in the present invention is as follows: deliver DEK protein to tumor tissue in vivo, and DEK protein can enter dormant tumor cells, bind to the promoters of genes such as SETD4, MYC and TP53, and down-regulate SETD4 And the expression of p53 protein, up-regulate the expression of MYC protein.
  • the beneficial effect of the present invention is mainly reflected in: because the dormant tumor cells in the tumor have the ability to resist various clinical tumor treatments including radiotherapy, chemotherapy, targeting and immunotherapy, etc., It is the main reason for tumor deterioration, metastasis and prognosis of tumor recurrence.
  • the invention provides a SETD4 protein inhibitor, especially the application of DEK protein in activating dormant tumor cells, clearing tumor cells and curing tumors.
  • the present invention delivers exogenous DEK protein to dormant tumor cells
  • exogenous DEK protein can combine with chromatin, reduce heterochromatin and increase euchromatin structure at the same time, thereby causing a series of gene transcription and expression, and then inducing Tumor cells switch from a dormant state to an activated state (see Figure 1 for the molecular mechanism), making them lose their resistance to various clinical treatments.
  • the dormant tumor cells in the body can be eliminated, and clinically realized Curative purpose of cancer without recurrence.
  • the present invention directs the transfer of DEK gene or delivery of DEK protein in resting tumor cells or tissues, which can activate the resting tumor cells and make them lose their resistance to tumor treatment, combined with standard radiotherapy and chemotherapy Thereby completely eradicating dormant tumor cells.
  • the SETD4 protein inhibitor of the present invention has a clearance rate of 100% for 4T1 dormant cells, EMT6 dormant cells and MCF7 dormant cells, and in vitro experiments have verified that the clearance rate for resting cells in clinical breast cancer patients is 100%.
  • the method of the present invention can be used for the treatment of very early tumors.
  • the combination of DEK exosomes and chemotherapy can be used to activate and completely eliminate dormant tumor cells, so as to prevent tumor recurrence. Realize the cure and treatment of various human cancers.
  • the method of the present invention can also be used for patients whose in situ tumors have been surgically resected, but there are still metastatic tumor cells in the whole body.
  • the combination of DEK exosomes and chemotherapy can activate and clear the dormant tumor cells transferred to other places.
  • the present invention is the first study and report on the activation of dormant tumor cells by SETD4 protein inhibitors, and it is also the first use of DEK protein combined with standard clinical treatment to cure cancer without recurrence and metastasis.
  • the present invention has significant clinical application value in the curative treatment of various human cancers.
  • Figure 1 is a schematic diagram of exosomes containing exogenous DEK protein activating dormant tumor cells and killing them in combination with radiotherapy and chemotherapy.
  • Figure 2 is the light micrographs of 4T1, EMT6 and MCF7 cells in adherent culture. Scale bar, 50 ⁇ m.
  • Figure 3 is the light micrographs of 4T1, EMT6 and MCF7 tumors. Ruler, 4mm.
  • Figure 4 is a light micrograph of cells digested from 4T1, EMT6 and MCF7 tumors. Scale bar, 50 ⁇ m.
  • Figure 5 is a light micrograph of cells resistant to radiotherapy and chemotherapy in 4T1, EMT6 and MCF7 tumors. Scale bar, 50 ⁇ m.
  • Figure 6 shows the immunoblotting results of SETD4, H3S10ph and PCNA in tumor cells resistant to radiotherapy and chemotherapy in 4T1, EMT6 and MCF7 tumors, and the internal references were GAPDH and H3, respectively.
  • Fig. 7 is the immunofluorescence results of ALDH1, CD44 and CD24 of dormant tumor cells in 4T1, EMT6 and MCF7 tumors. Scale bar, 50 ⁇ m. DAPI indicates staining of cell nuclei. Merge represents an overlay of four fluorescence channels.
  • Figure 8 is the particle size distribution curves of crude exosomes derived from 4T1, EMT6 and MCF7 cells.
  • Figure 9 is a transmission electron micrograph of crude exosomes derived from 4T1, EMT6 and MCF7 cells. Scale bar, 100nm. Arrows indicate exosomes.
  • Figure 10 is the immunoblotting results of DEK, CD9, CD81 and CD63 of crude exosomes derived from 4T1, EMT6 and MCF7 cells.
  • Figure 11 shows the western blot results and quantitative statistics of DEK, H3S10ph, PCNA and SETD4 after adding PBS and crude exosomes to 4T1, EMT6 and MCF7 dormant tumor cells, and the internal references were H3 and GAPDH, respectively.
  • Fig. 12 is the immunofluorescence results of ALDH1, CD44 and CD24 of activated dormant tumor cells in 4T1, EMT6 and MCF7. Scale bar, 50 ⁇ m. DAPI indicates staining of cell nuclei. Merge represents an overlay of four fluorescence channels.
  • Figure 13 is the light micrographs and statistical results of spheres formed in vitro by single cells of activated dormant tumor cells in 4T1, EMT6 and MCF7. Ruler, 2mm. Red arrows indicate tumorspheres.
  • Figure 14 shows the light microscope photos and statistical results of in situ tumor formation in vivo by 100, 10 or 1 activated dormant tumor cells in 4T1, EMT6 and MCF7, and 5 parallel controls were set. Ruler, 1cm.
  • Figure 15 is the immunofluorescence results of ALDH1, Ki67, SETD4 and DEK in solid tumor sections of 4T1, EMT6 and MCF7 dormant tumor cells.
  • a is the immunofluorescence results of detecting ALDH1, Ki67 and SETD4.
  • b is the immunofluorescence results of detecting ALDH1, Ki67 and DEK.
  • Scale bar 10 ⁇ m.
  • DAPI indicates staining of cell nuclei. Merge represents an overlay of four fluorescence channels. Arrows indicate ALDH1-positive and Ki67-negative tumor cells.
  • Figure 16 is the immunofluorescence results of ALDH1, Ki67, SETD4 and DEK in tumor cells activated by 4T1, EMT6 and MCF7 in solid tumor sections.
  • a is the immunofluorescence results of detecting ALDH1, Ki67 and SETD4.
  • b is the immunofluorescence results of detecting ALDH1, Ki67 and DEK.
  • Scale bar 10 ⁇ m.
  • DAPI indicates staining of cell nuclei. Merge represents an overlay of four fluorescence channels. Arrows indicate ALDH1-positive and Ki67-positive tumor cells.
  • Figure 17 is the western blot results of SETD4 and DEK before and after activation of 4T1, EMT6 and MCF7 dormant tumor cells (a) and their quantitative statistics (b, c), the internal references are GAPDH and H3 respectively.
  • Figure 18 is a map of the pFastBac TM HTA plasmid.
  • Figure 19 shows the human DEK protein (hDEK-GFP), mouse DEK protein (mDEK-GFP), human domain NLS mutant DEK protein (hDEK ⁇ NLS -GFP) and mouse domain NLS mutant DEK protein (mDEK ⁇ NLS -GFP ) stained with Coomassie brilliant blue. Arrows indicate the position of the protein.
  • M represents protein molecular weight standard.
  • Figure 20 is the Western blot results and quantitative statistics of DEK-GFP, DEK, H3S10ph, PCNA and SETD4 after adding PBS, DEK ⁇ NLS -GFP protein or DEK-GFP protein to 4T1, EMT6 and MCF7 dormant tumor cells, internal reference H3 and GAPDH, respectively.
  • Figure 21 is the immunofluorescence results and statistical charts of GFP and cCasp3 after adding PBS, DEK ⁇ NLS -GFP protein or DEK-GFP protein to 4T1, EMT6 and MCF7 dormant tumor cells, the scale bar is 50 ⁇ m.
  • DAPI indicates staining of cell nuclei.
  • Figure 22 is the trypan blue staining results and mortality statistics of 4T1, EMT6 and MCF7 dormant tumor cells after adding PBS, DEK ⁇ NLS -GFP protein or DEK-GFP protein. Scale bar, 50 ⁇ m.
  • Figure 23 is a map of the pLent-U6-RFP-Puro plasmid.
  • Figure 24 is the western blot and in vitro sphere formation test after DEK interference in activated 4T1, EMT6 and MCF7 dormant tumor cells.
  • a is the Western blot results of DEK, H3S10ph, PCNA and SETD4 and their quantitative statistics, and the internal references are H3 and GAPDH, respectively.
  • b is the light microscope photo of the in vitro spheroidization test and the statistical graph of spheroidization rate, the scale bar is 400 ⁇ m.
  • Figure 25 shows the results of immunoblotting of DEK-GFP, DEK, H3S10ph, PCNA and SETD4 in activated 4T1, EMT6 and MCF7 dormant tumor cells after interfering with DEK for one week and adding PBS, DEK ⁇ NLS -GFP protein or DEK-GFP protein And its quantitative statistical chart, the internal references are H3 and GAPDH respectively.
  • Figure 26 is a light microscope photo of the in vitro sphere formation test and a statistical graph of the sphere formation rate after interfering with DEK in activated 4T1, EMT6 and MCF7 dormant tumor cells for one week, adding PBS, DEK ⁇ NLS -GFP protein or DEK-GFP protein, Scale bar, 400 ⁇ m.
  • Figure 27 is the transmission electron micrograph of the cells and the relative level of heterochromatin in the nucleus after adding PBS, DEK ⁇ NLS -GFP protein or DEK-GFP protein to 4T1 (a), EMT6 (b) and MCF7 (c) dormant tumor cells summary graph. Scale bar, 1 ⁇ m.
  • Figure 28 is the Western blot results and quantification of H4K20me1, H4K20me2, H4K20me3, HP1- ⁇ , H3K9ac, H3K9me3 and H3K27me3 after adding PBS, DEK ⁇ NLS -GFP protein or DEK-GFP protein to 4T1, EMT6 and MCF7 dormant tumor cells
  • PBS PBS
  • DEK ⁇ NLS -GFP protein or DEK-GFP protein to 4T1
  • EMT6 and MCF7 dormant tumor cells
  • Figure 29 shows the distribution of peaks of DEK binding sites on chromosomes analyzed by binding site analysis (ChIP-Seq) in dormant MCF7 tumor cells after activation.
  • Figure 30 shows the distribution of peaks of DEK binding sites in gene regions in the activated MCF7 dormant tumor cells analyzed by ChIP-Seq.
  • Figure 31 is the Gene Ontology enrichment analysis of the gene where the peak of the DEK binding site in the activated MCF7 dormant tumor cells was analyzed by ChIP-Seq.
  • Figure 32 is the visualization result of ChIP-Seq analysis of DEK binding signals on SETD4, TP53 and MYC gene regions in activated MCF7 dormant tumor cells.
  • Figure 33 shows the overall signal of open genes in MCF7 dormant tumor cells before and after activation by ATAC-Seq analysis.
  • a is the distribution curve of reads relative to the TSS region
  • b is the heat map of reads relative to the TSS region.
  • Figure 34 shows the results of gene ontology enrichment analysis of genes with increased openness in MCF7 dormant tumor cells before and after activation by ATAC-Seq analysis.
  • Fig. 35 is the visualization result of ATAC-Seq signal on SETD4, TP53 and MYC gene regions of MCF7 dormant tumor cells before and after activation by ATAC-Seq analysis.
  • Figure 36 shows the results of RNA-Seq analysis of gene differences in MCF7 dormant tumor cells before and after activation.
  • a is the cluster heat map of differential genes
  • b is the volcano map of differential genes.
  • padj represents the corrected p-value.
  • Figure 37 is a gene ontology enrichment analysis of up-regulated genes in MCF7 dormant tumor cells before and after activation by RNA-Seq analysis.
  • Figure 38 is a gene ontology enrichment analysis of down-regulated genes in MCF7 dormant tumor cells before and after activation by RNA-Seq analysis.
  • Figure 39 is the GSEA analysis of differential genes in MCF7 dormant tumor cells before and after activation by RNA-Seq analysis.
  • a is the set of genes up-regulated in activated cells
  • b is the set of genes down-regulated in activated cells.
  • Figure 40 shows the results of GSEA analysis of the MYC-targeted gene set and the p53-targeted gene set.
  • a is the GSEA map
  • b is the heat map of the expression of significantly different genes in the gene set.
  • Figure 41 is the Western blot results and quantitative statistics of p53, p21, PUMA and MYC after adding PBS, DEK ⁇ NLS -GFP protein or DEK-GFP protein to 4T1, EMT6 and MCF7 dormant tumor cells, the internal reference is ⁇ -actin .
  • Figure 42 shows the immunofluorescence results and statistical results of cCasp3 after adding PBS and crude exosomes to 4T1, EMT6 and MCF7 dormant tumor cells, scale bar, 50 ⁇ m.
  • Figure 43 is the trypan blue staining results and mortality statistics of 4T1, EMT6 and MCF7 dormant tumor cells after adding PBS and crude exosomes. Scale bar, 50 ⁇ m.
  • Figure 44 is the Western blot results and quantitative statistics of H4K20me1, H4K20me2, H4K20me3, HP1- ⁇ , H3K9ac, H3K9me3 and H3K27me3 after adding PBS and crude exosomes to 4T1, EMT6 and MCF7 dormant tumor cells, and the internal references are respectively H4 and H3.
  • Figure 45 is the Western blot results and quantitative statistics of p53, p21, PUMA and MYC after adding PBS and crude exosomes to 4T1, EMT6 and MCF7 dormant tumor cells, and the internal reference is ⁇ -actin.
  • Figure 46 is a map of pEGFP-C1 plasmid.
  • Figure 47 is a map of the pLent-N-GFP plasmid.
  • Figure 48 is the particle size distribution curve of exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein derived from 4T1, EMT6 and MCF7 cells.
  • Figure 49 is a transmission electron micrograph of sorted exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein derived from 4T1, EMT6 and MCF7 cells. Scale bar, 100nm. Arrows indicate exosomes.
  • Figure 50 is the immunoblotting results of DEK-GFP, DEK, CD9, CD81 and CD63 of sorted exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein derived from 4T1, EMT6 and MCF7 cells.
  • Figure 51 is the immunoblotting results of DEK-GFP, DEK, H3S10ph, PCNA and SETD4 after adding PBS, sorted exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein to 4T1, EMT6 and MCF7 dormant tumor cells and Its quantitative statistical chart, the internal references are H3 and GAPDH respectively.
  • Figure 52 shows the immunofluorescence results and statistical results of cCasp3 after adding PBS, sorted exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein to 4T1, EMT6 and MCF7 dormant tumor cells, scale bar, 50 ⁇ m.
  • Figure 53 is the trypan blue staining results and mortality statistics of 4T1, EMT6 and MCF7 dormant tumor cells after adding PBS, sorted exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein. Scale bar, 50 ⁇ m.
  • Figure 54 shows the expression of H4K20me1, H4K20me2, H4K20me3, HP1- ⁇ , H3K9ac, H3K9me3 and H3K27me3 after adding PBS, sorted exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein in 4T1, EMT6 and MCF7 dormant tumor cells Western blot results and their quantitative statistical charts, the internal references are H4 and H3, respectively.
  • Figure 55 is the western blot results and quantitative statistics of p53, p21, PUMA and MYC after adding PBS, sorted exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein in 4T1, EMT6 and MCF7 dormant tumor cells , the internal reference is ⁇ -actin.
  • Figure 56 is the flow cytometric analysis results and statistical charts of the proportion of GFP-positive exosomes in mouse plasma after intraperitoneal injection of sorted exosomes containing exogenous DEK-GFP protein in mice.
  • Figure 57 is the immunofluorescence results of GFP in tumor slices after intraperitoneal injection of sorted exosomes containing DEK-GFP or DEK ⁇ NLS -GFP protein in mice. Scale bar, 50 ⁇ m. DAPI indicates nuclear staining.
  • Figure 58 shows the immunofluorescence results and proportion statistics of SETD4 in tumor slices after intraperitoneal injection of sorted exosomes containing DEK-GFP protein in mice for 24 hours. Scale bar, 50 ⁇ m. DAPI indicates nuclear staining.
  • Figure 59 shows the SETD4 immunofluorescence results of tumor sections of 4T1 tumor-bearing mice in the radiotherapy group, radiotherapy and injection of sorted exosomes containing DEK ⁇ NLS -GFP protein group, and radiotherapy and injection of exosomes containing DEK-GFP protein group and its proportion statistics. Scale bar, 50 ⁇ m. DAPI indicates nuclear staining.
  • Figure 60 shows the tumor sections of EMT6 tumor-bearing mice in the radiochemotherapy group, radiochemotherapy and injection of sorted exosomes containing DEK ⁇ NLS -GFP protein group, and radiotherapy and chemotherapy and injection of sorted exosomes containing DEK-GFP protein group SETD4 immunofluorescence results and their ratio statistics. Scale bar, 50 ⁇ m. DAPI indicates nuclear staining.
  • Figure 61 shows the radiotherapy and sorted exosome injection scheme and tumor diameter curve of 4T1 tumor-bearing mice.
  • Figure 62 shows the lung metastases of 4T1 tumor-bearing mice after combined treatment with radiotherapy and sorted exosomes.
  • a is the result of hematoxylin and eosin staining of lung tissue sections (scale bar, 1 mm)
  • b is the statistical map of the number of lung metastases; 1, 2, 3, and 4 in the figure indicate the enlarged areas.
  • Figure 63 is the survival curve of 4T1 tumor-bearing mice after combined treatment with radiotherapy and sorted exosomes.
  • Figure 64 shows the radiotherapy and chemotherapy of EMT6 tumor-bearing mice and the injection scheme of sorted exosomes and the tumor diameter curve.
  • Figure 65 shows the chemotherapy and sorted exosome injection scheme and tumor diameter curve of MCF7 tumor-bearing mice.
  • Fig. 66 is the corresponding relationship between the proportion of SETD4 cells and the TNM stage in breast cancer slices of clinical patients.
  • a is the sample information of clinical patients
  • b is the proportion of SETD4 cells in breast cancer slices of patients with clinical stages I, II and III.
  • Figure 67 is the data of dormant tumor cells obtained from clinical breast cancer patient samples and killed by MCF7 sorted exosomes containing DEK-GFP protein combined with chemotherapy.
  • a is the dormant tumor cells resistant to radiotherapy and chemotherapy obtained from clinical breast cancer patient samples, scale bar, 50 ⁇ m.
  • b is the activation of dormant tumor cells by adding MCF7 exosomes containing DEK-GFP protein, DAPI indicates nuclear staining, scale bar, 50 ⁇ m.
  • c is killing dormant tumor cells by adding MCF7 exosomes containing DEK-GFP protein, scale bar, 50 ⁇ m.
  • Figure 68 is the data of activation and elimination of dormant tumor cells by DEK combined with chemotherapy in various human tumor cells.
  • a is activation and removal of dormant tumor cells by DEK combined with chemotherapy in lung cancer H226 cells, scale bar, 50 ⁇ m.
  • b is activation and removal of dormant tumor cells by DEK combined with chemotherapy in gastric cancer MKN45 cells, scale bar, 50 ⁇ m.
  • c is activation and removal of dormant tumor cells by DEK combined with chemotherapy in prostate cancer PC-3 cells, scale bar, 50 ⁇ m.
  • d is activation and removal of dormant tumor cells by DEK combined with chemotherapy in cervical cancer HeLa cells, scale bar, 50 ⁇ m.
  • Phosphate buffer solution (PBS) used in the present invention formula: final concentration 137mM sodium chloride, final concentration 2.7mM potassium chloride, final concentration 10mM disodium hydrogen phosphate and final concentration 1.76mM potassium dihydrogen phosphate, solvent is water, the pH was adjusted to 7.4.
  • Embodiment 1 the acquisition of mouse xenograft tumor
  • the MCF7 cell line human breast cancer cells, molecularly characterized as luminal A type, was purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, the catalog number is TCHu74, and the cell culture medium is EMEM medium (Eagle's minimum Essential Medium, Corning, catalog number : 10-009-cv) with 10% fetal bovine serum and 1% penicillin-streptomycin.
  • 4T1 cell line a typical triple-negative breast cancer cell, with characteristics similar to clinical stage IV breast cancer, was purchased from the American Type Culture Collection (ATCC), catalog number is CRL-2539, and the cell culture medium is DMEM medium (Dulbecco's Modification of Eagle's Medium, Corning, catalog number: R10-013-cv) with 10% fetal bovine serum and 1% penicillin-streptomycin.
  • ATCC American Type Culture Collection
  • EMT6 cell line mouse breast cancer cells, purchased from the American Type Culture Collection (ATCC), the catalog number is CRL-2755, and the cell culture medium is Waymouth's medium (Gibco, catalog number: 31220072) with a volume concentration of 15% Fetal bovine serum and 1% penicillin-streptomycin.
  • Nod/Scid female mice aged 6-8 weeks were selected as inoculation objects for MCF7 cells.
  • PBS phosphate buffered solution
  • EDTA ethylenediaminetetraacetic acid
  • BALB/c female mice aged 6-8 weeks (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected as inoculation objects for 4T1 cells.
  • the 4T1 cell suspension was orthotopically injected into the mammary fat pad of the axilla of BALB/c female mice with a 1 mL micro-syringe, and each BALB/c female mouse was inoculated with 1 million 4T1 cells.
  • BALB/c female mice aged 6-8 weeks were selected as inoculation objects for EMT6 cells.
  • the EMT6 cell suspension was orthotopically injected into the mammary fat pad of BALB/c female mice with a 1 mL micro-syringe, and each BALB/c female mouse was inoculated with 200,000 EMT6 cells.
  • mice All mice were kept in a sterile environment, water and mouse food were continuously supplied, and the bedding was replaced in time. When the tumor volume reached about 500 mm 3 , the mice were euthanized for the next step to obtain solid tumors. All mouse experiments have passed the ethical review of animal experiments, and the experimenters abide by the ethical principles of experimental animal welfare when conducting experiments.
  • the three kinds of tumor-bearing mice in Example 1 were euthanized, and the solid tumors were surgically removed.
  • the light microscope photos are shown in FIG. 3 .
  • the digested cell solution was filtered through a 40 ⁇ m nylon membrane, and the filtrate was taken to be the cells digested from the solid tumor. See Figure 4 for the light microscope photo.
  • the digested cells from the above solid tumors were inoculated at a density of 800,000 cells/well in an ultra-low adsorption six-well plate (brand: Corning, item number: 3471).
  • DCCs dormant tumor cells
  • step 1 Separate the 3 kinds of dormant tumor cells (4T1-DCCs, EMT6-DCCs, MCF7-DCCs) obtained in step 1 from the culture medium, and add 0.2mL cell lysate (50mM Tris- HCl (pH 7.4), 150mM NaCl, 1% ethylphenyl polyethylene glycol (NP-40), 0.1% sodium dodecyl sulfate (SDS)), placed on ice for 10 minutes, centrifuged at 12000rpm 4°C for 10 minutes , transfer the supernatant to a new centrifuge tube, add 0.05mL protein loading buffer (250mM Tris hydrochloride, pH 6.8; 0.1g/ml sodium lauryl sulfate; 0.005g/ml ml bromophenol blue; 50% glycerol and 0.05g/ml ⁇ -mercaptoethanol, the solvent is water), boiled water bath for 10 minutes, centrifuged at 12000r
  • Mouse monoclonal anti-PCNA (abcam, catalog number: ab29), Rabbit monoclonal anti-H3S10ph (Cell signaling technology, catalog number: 53348) and Mouse monoclonal anti-SETD4Santa Cruz, catalog number: sc-134221) were respectively 2 ⁇ g Add to 2mL of blocking solution to configure the primary antibody dilution, then completely soak the PVDF membrane in the dilution, and incubate overnight at 4°C. The next day, the PVDF membrane was washed 4 times with TBS containing 0.1% Tween, 7 minutes each time.
  • the three kinds of dormant tumor cells (4T1-DCCs, EMT6-DCCs, MCF7-DCCs) obtained in step 1 were respectively fixed with 4% paraformaldehyde.
  • the fixed cell suspensions were dropped on the adhesive glass slides respectively. After the cells are stably attached, wash once with PBS for 5 minutes.
  • the slides were respectively immersed in PBS containing 0.25% Triton-100 and incubated at 25°C for 10 minutes, and washed once with PBS for 5 minutes.
  • the slides were respectively immersed in PBS (blocking solution) containing 1.5% donkey serum and incubated at 25° C. for 1 hour.
  • Rabbit monoclonal anti-ALDH1A1 (abcam, catalog number: ab52492), Rat monoclonal anti-CD44, FITC (eBiosciences, catalog number: 11-0441-82) and Alexa Add 2 ⁇ g of each 647 anti-CD24 (BioLegend, catalog number: 311109) antibody to 200 ⁇ L blocking solution (brand: Roche, catalog number: 11921681001) to configure the primary antibody dilution, and add the primary antibody dilution to the cells in the slide Just immerse on the sample and incubate overnight at 4°C. The next day, wash 3 times with PBS, 5 minutes each time.
  • MCF7 cell lines Five million MCF7 cell lines were inoculated into a 90 mm 3 cell culture dish, and the medium was 10 mL of EMEM medium (same as Example 1) supplemented with 1% penicillin-streptomycin. After culturing at 37°C for 24 hours, collect the cell culture medium, centrifuge at 1000rpm at 4°C for 10 minutes, take the supernatant, and centrifuge at 12000rpm at 4°C for 20 minutes, take the supernatant, which is the MCF7 cell culture medium.
  • DMEM medium (same as Example 1) supplemented with 1% penicillin-streptomycin. After culturing at 37°C for 24 hours, collect the cell culture medium, centrifuge at 1000rpm at 4°C for 10 minutes, take the supernatant, and centrifuge at 12000rpm at 4°C for 20 minutes, take the supernatant, which is the 4T1 cell culture medium.
  • EMT6 cell lines Five million EMT6 cell lines were inoculated into a 90 mm 3 cell culture dish, and the medium was 10 mL of Waymouth's medium (same as Example 1) supplemented with 1% penicillin-streptomycin. After culturing at 37°C for 24 hours, collect the cell culture medium, centrifuge at 1000rpm at 4°C for 10 minutes, take the supernatant, and centrifuge at 12000rpm at 4°C for 20 minutes, take the supernatant, which is the EMT6 cell culture medium.
  • exosome isolation reagent brand: Thermo Fisher, product number: 4478359
  • 20 mL of MCF7, 4T1 and MCF7 cell culture medium obtained in step 1 mix by inverting up and down 3 times, and incubate overnight at 4°C.
  • the above mixture was centrifuged at 10,000 rpm at 4°C for 60 minutes, the supernatant was removed, and the sediment at the bottom of the centrifuge tube was resuspended with 200 ⁇ L of PBS (the sediment was crude exosomes), and the BCA protein quantitative detection kit (brand: Sheng Worker, product number: C503021) to measure the total amount of protein in the crude exosome suspension, and prepare a crude exosome solution with a protein concentration of 20 ⁇ g/mL with PBS for subsequent use.
  • the BCA protein quantitative detection kit brand: Sheng Worker, product number: C503021
  • the particle size detection instrument (model: ZetaView, manufacturer: Particle Metrix) was used to detect the 20 ⁇ g/mL crude exosome solution obtained in step 2. The sizes are all around 150nm (Fig. 8).
  • the cells collected above were recorded as activated dormant tumor cells (A-DCCs), and activated dormant tumor cells 4T1A-DCCs, EMT6A-DCCs, and MCF7A-DCCs were obtained respectively.
  • the activated dormant tumor cells obtained above were subjected to immunofluorescence analysis of ALDH1, CD44 and CD24 (same as step 2 of Example 2), and high levels of ALDH1 and CD44 and low levels of CD24 were also found to be expressed ( FIG. 12 ).
  • Example 5 Activated dormant tumor cells have the ability to form spheres in vitro and tumors in vivo
  • the activated dormant tumor cells (4T1A-DCCs, EMT6A-DCCs, MCF7A-DCCs) obtained in Example 4 were inoculated into an ultra-low adsorption 96-well plate (brand : Corning, item number: 3469a). 200 ⁇ L of DMEM/F12 medium containing 10% serum replacement was added to each well, and cultured continuously at 37° C. for three weeks. It was found that the cells exhibited a very high ability to form tumor spheres in vitro ( FIG. 13 ). 2. Detection of tumorigenic ability in mice
  • mice 4 mice were injected, 2 mice were injected with 1 injection), the mice were raised in a sterile environment, water and mouse food were continuously supplied, and the litter was replaced in time.
  • the mice were euthanized when the diameter of the tumor reached the ethical limit within half a year, and the tumor was removed by surgery and photographed (Figure 14). It was found that the cells showed a strong ability to form tumors in mice.
  • One million 4T1-DCCs and one million EMT6-DCCs cells obtained by the method in Example 2 were respectively orthotopically inoculated into the fat pads of 8-week-old BALB/c female mice, and one million MCF7-DCCs cells were orthotopically inoculated into In the fat pad of 8-week-old Nod/Scid female mice.
  • the tumor volume reached about 500 mm 3 the mice were euthanized, and the 4T1-DCCs, EMT6-DCCs and MCF7-DCCs tumors were surgically removed.
  • Example 7 After activation, the DEK protein of dormant tumor cells significantly increased, and the SETD4 protein decreased significantly
  • step 2 of Example 2 using Rabbit polyclonal anti-DEK (Proteintech, catalog number: 16448-1-AP) antibody and Mouse monoclonal anti-SETD4 (Santa Cruz, catalog number: sc-134221) antibody, carried out dormancy Western blot analysis (same as Example 2) of tumor cells (acquired in step 1 of Example 2) and activated dormant tumor cells (acquired in Example 4), found that compared with dormant tumor cells, activated dormant tumor cells expressed DEK in a high amount protein, but the expression of SETD4 was significantly decreased (Fig. 17). The results showed that DEK protein plays an important role in the activation of dormant tumor cells.
  • primer F3 CCGGAATTCATGGTGAGCA
  • primer R3 CGCTCTAGATCAAGAAATTAG
  • primer F4 AGAATTCATGGTGAGCAAGGGCGA
  • primer R4 GCTCTAGATCAAGAAATTAGCTCTTTTACAGTTGT
  • Human source hEGFP-DEK gene (nucleotide sequence: SEQ ID NO.6, amino acid sequence: SEQ ID NO.7), human source hEGFP-DEK ⁇ NLS (nuclear localization sequence NLS deletion) gene (nucleotide sequence : SEQ ID NO.8, amino acid sequence: SEQ ID NO.9), mouse mEGFP-DEK gene (nucleotide sequence: SEQ ID NO.10, amino acid sequence: SEQ ID NO.11), mouse mEGFP-DEK ⁇ NLS (nuclear localization sequence NLS deletion) gene (nucleotide sequence: SEQ ID NO.12, amino acid sequence: SEQ ID NO.13) was connected to the EcoR of pFastBac TM HTA plasmid (purchased from Thermo Fisher, product number: 10712024) I and Xba I enzyme cutting sites ( Figure 18).
  • 100 ⁇ L of the resuspension was coated to contain a final concentration of 30 ⁇ L/mL kanamycin, a final concentration of 15 ⁇ L/mL gentamicin, a final concentration of 12 ⁇ L/mL tetracycline, 40 ⁇ L (20 mg/ml) X-gal (5- Bromo-4-chloro-3-indole- ⁇ -D-galactopyranoside, the solvent is dimethylformamide), 4 ⁇ L (200 mg/ml) IPTG (isopropyl- ⁇ -D-thiogalacto glucoside, the solvent is super-distilled water) in the LB solid medium (15g/L agar was added to the LB medium), and cultured in the dark at 37°C for 48 hours, and the white single colony was selected and inoculated into 2mL of the LB medium, 37 Shake at 220 rpm overnight.
  • X-gal Bromo-4-chloro-3-indole- ⁇ -
  • the plasmid was extracted with a kit (purchased from Invitrogen, product number: K210002) to obtain a recombinant bacmid.
  • the recombinant Bacmid was transfected into Sf9 cells (sourced from ATCC, catalog number: CRL-1711) using Cellfectin transfection reagent (Gibco, catalog number: 10362100).
  • the transfected cells were inoculated in Sf-900 medium (Gibco, catalog number: 10902179), cultured at 27°C for 72 hours, the cell culture medium was collected, centrifuged at 10,000xg for 20 minutes, and the supernatant was taken as the virus solution to obtain hDEK carrying human sources respectively.
  • -GFP gene murine mDEK-GFP gene, human hDEK ⁇ NLS -GFP gene, murine mDEK ⁇ NLS -GFP gene recombinant baculovirus.
  • the resuspended solution was sonicated (39 watts, 10 sec sonication, 50 sec pause, 3 min sonication duration).
  • the sonicated solution was centrifuged at 12000 rpm at 4°C for 20 minutes, and the supernatant was taken as the protein solution.
  • the above-mentioned protein solution was purified using a His-tagged protein purification kit (Beyontian, product number: P2226), and the purified protein was dissolved in PBS to prepare a 20 ⁇ g/mL protein solution, that is, a PBS solution of exogenous DEK protein ( mDEK-GFP, hDEK-GFP), NLS mutant DEK protein in PBS solution (hDEK ⁇ NLS -GFP, mDEK ⁇ NLS -GFP). Coomassie Brilliant Blue staining revealed that the purity of the protein was high and the protein size was correct ( FIG. 19 ).
  • Example 9 the addition of exogenous DEK protein activates dormant tumor cells
  • mDEK-GFP and mDEK ⁇ NLS-GFP were added to 4T1-DCCs and EMT6-DCCs, respectively, hDEK-GFP and hDEK ⁇ NLS -GFP were added to MCF7-DCCs, and cells were collected after culturing at 37°C for 20 hours.
  • exogenous DEK protein could activate dormant tumor cells, which were recorded as dormant tumor cells activated by exogenous DEK protein, and 4T1A-DCCs, EMT6A-DCCs, and MCF7A-DCCs activated by exogenous DEK protein were obtained respectively.
  • Example 10 In combination with chemotherapy, the addition of exogenous DEK protein can eliminate dormant tumor cells
  • Interference sequence 1 targeting human DEK GGATAGTTCAGATGATGAAC, SEQ ID NO.14, denoted as shDEK#H1;
  • Interference sequence 2 targeting human DEK GTGATGAAGATGAAAAGAAA, SEQ ID NO.15, denoted as shDEK#H2;
  • Interference sequence 1 targeting murine DEK GTGAAGAAATTACTGGCTGAT, SEQ ID NO.16, denoted as shDEK#M1;
  • Interference sequence 2 targeting murine DEK CGAACTCGTGAAGAGGATCTT, SEQ ID NO.17, denoted as shDEK#M2;
  • SW28 centrifuge tubes into the Beckman SW28 ultracentrifuge rotor in sequence, and centrifuge at 25000 rpm for 2 hours at 4°C. Carefully remove the SW28 tubing from the rotor. Pour off the supernatant, place the SW28 centrifuge tube upside down on a paper towel for 10 minutes to drain the remaining supernatant. Aspirate off remaining droplets. Add 1ml calcium- and magnesium-free PBS to each tube to resuspend the pellet at the bottom of the tube. Insert the SW28 centrifuge tube into a 50ml conical-bottom centrifuge tube, cover it, dissolve at 4°C for 2 hours, and shake gently every 20 minutes.
  • lentivirus shDEK#H1 ie, human DEK interference sequence 1
  • a kit (GeneCopoeia, product number: LT005) was used to measure the virus titer, and the virus titer was 10 8 pfu/mL.
  • shRNA lentiviruses targeting DEK, shDEK#H1, shDEK#H2 human DEK interference sequence 2, 6ml, virus titer 10 8 pfu/mL
  • shDEK#M1 mouse DEK interference sequence 2, 6ml , virus titer is 10 8 pfu/mL
  • shDEK#M2 murine DEK interference sequence 2, 6ml, virus titer is 10 8 pfu/mL
  • shCTRL interference control 6ml, virus titer is 10 8 pfu/mL mL.
  • the 3 kinds of activated dormant tumor cells (4T1A-DCCs, EMT6A-DCCs, MCF7A-DCCs) obtained in Example 4 were inoculated into low-adhesion 6-well plates at a density of 100,000 cells/well, and the medium was 3 mL of DMEM/Ham's F-12, after culturing at 37°C for 2 hours, add polybrene at a final concentration of 6 ⁇ g/mL to each well; add 10 ⁇ L of 10 8 to each well of 4T1A-DCCs and EMT6A-DCCs lentivirus shDEK#M1, shDEK#M2, shCTR prepared in step 1 in pfu/mL without adding lentivirus as untreated.
  • Collect cells after culturing at 37°C for 20 hours (denoted as mouse-derived interference sequence 1+mDEK-GFP, mouse-derived interference sequence 1+mDEK ⁇ NLS -GFP, mouse-derived interference sequence 1+PBS, shCTRL), and develop DEK-GFP, DEK , H3S10ph, PCNA and SETD4 antibody immunoblotting experiment (method is the same as step 2 of Example 2, primary antibody and secondary antibody are shown in Table 1).
  • EMT6A-DCCs cells mDEK-GFP and mDEK ⁇ NLS -GFP
  • EMT6A-DCCs cells after lentivirus shDEK#M2 and shCTRL interfered with DEK in step 2
  • MCF7A-DCCs cells after shDEK#H2 and shCTRL interfered with DEK (hDEK- GFP and hDEK ⁇ NLS -GFP) for immunoblotting.
  • Exogenous DEK protein causes the decrease of heterochromatin and the increase of euchromatin
  • the final concentrations of protein and NLS mutant DEK protein were both 50ng/mL, in which mDEK-GFP and mDEK ⁇ NLS-GFP were added to 4T1-DCCs and EMT6-DCCs respectively, and hDEK-GFP and hDEK ⁇ NLS -GFP were added to MCF7-DCCs respectively.
  • Spurr embedding agent ethylene dioxide cyclohexene, diglycidyl ether of polypropylene glycol, nonylsuccinic anhydride and dimethylethanolamine are polymerized from four substances, purchased from SPI-CHEM company
  • the sample was taken out and placed in a 10x10x5mm open cuboid plastic embedding mold (SAKURA, item number: 4566).
  • SAKURA 10x10x5mm open cuboid plastic embedding mold
  • the mold was filled with new Spurr embedding agent and placed in an oven at 70°C for standing overnight.
  • the embedded samples were obtained.
  • the samples were sliced in a LEICA EM UC7 ultramicrotome to obtain 70nm slices.
  • High-throughput sequencing (ATAC-seq) of the nuclear chromatin region accessible to transposase collect the MCF7-DCCs dormant tumor cells obtained in step 1 of Example 2 and the exogenous DEK protein activation obtained in Example 9 High-throughput sequencing (ATAC-Seq) of transposase-accessible nuclear chromatin regions in MCF7A-DCCs cells. The nuclei of the above two cell samples were extracted, and transposase mix was added, which contained transposase and two equimolar adapters Adapter 1 and Adapter 2, and incubated at 37°C for 30 minutes. The product was subjected to primer amplification, fragment length selection and purification, and after the library was obtained, it was sequenced on the machine. This operation was entrusted to Beijing Novogene Technology Co., Ltd. to complete.
  • the UCSC gene browser visualization analysis of ATAC-seq signals of dormant tumor cells and dormant tumor cells activated by exogenous DEK protein found that the ATAC signals of SETD4 and TP53 genes in activated dormant tumor cells were significantly lower than those of dormant tumor cells, and activated The ATAC signal of the MYC gene in dormant tumor cells was significantly higher than that in dormant tumor cells ( Figure 35), indicating that after activation of exogenous DEK protein in dormant tumor cells, the openness of SETD4 and TP53 genes decreased, and the openness of MYC gene increased.
  • RNA-seq High-throughput sequencing (RNA-seq) of the transcriptome: collect the MCF7-DCCs dormant tumor cells obtained in step 1 of Example 2 and the MCF7 A-DCCs cells activated by the exogenous DEK protein obtained in Example 9, Perform high-throughput sequencing (RNA-seq) of the transcriptome. use Stranded RNA-Seq Kit (Clontech, catalog number: 634836), 1 mL of TRIzol reagent (Thermo Fisher) was added to 1 g of the above cell sample, total RNA was extracted, and polyA-tailed mRNA was enriched by Oligo (dT) magnetic beads.
  • the UltraTM RNA Library Prep Kit was used to construct the transcriptome library and perform sequencing on the machine.
  • GSEA analysis Gene set enrichment analysis was carried out on the expression level of genes, and it was found that in the activated dormant tumor cells, the gene sets whose expression levels were upregulated mainly included DNA replication, G2M checkpoint, mitotic spindle, oxidative phosphorylation, Krebs cycle, E2F target genes, MYC target genes and fatty acid metabolism (Fig. 39), the down-regulated gene set mainly includes hypoxia, inflammatory response, complement, coagulation, epithelial-mesenchymal transition, p53 signaling pathway, TNFA signaling pathway, JAK-STAT3 signaling pathway and Kras signaling pathway. 25 genes in the MYC signaling pathway were significantly up-regulated, and 30 genes in the p53 signaling pathway were significantly down-regulated ( FIG. 40 ).
  • the final concentration of mutant DEK protein was 50ng/mL, and mDEK-GFP and mDEK ⁇ NLS-GFP were added to 4T1-DCCs and EMT6-DCCs respectively, and hDEK-GFP and hDEK ⁇ NLS - GFP were added to MCF7-DCCs respectively.
  • Example 13 Combined with chemotherapy, the addition of crude exosomes from tumor cell culture fluid can eliminate dormant tumor cells
  • Example 14 Regulatory mechanism of crude exosomes derived from tumor cell culture medium to activate dormant tumor cells
  • Example 15 Preparation of sorted exosomes containing exogenous DEK protein and domain NLS mutant DEK protein
  • Second use primer F1 (CCGGAATTCTATGTCCGCCT) and primer R1 (CGCTCTAGATCAAGAAATTAG) to perform PCR amplification on SEQ ID NO.5 and SEQ ID NO.19 to obtain the human DEK gene sequence covering the restriction site. Then the above sequence and the pEGFP-C1 plasmid were simultaneously subjected to double enzyme digestion treatment and ligation treatment with EcoR I and Xba I.
  • primer F2 AGAATTCTATGTCGGCGGCGGCGG
  • primer R2 GCTCTAGATCAAGAAATTAGCTCTTTTTACAGTTGT
  • the human source hDEK gene (nucleotide sequence: SEQ ID NO.5, amino acid sequence: SEQ ID NO.1), human source hDEK ⁇ NLS (nuclear localization sequence NLS deletion) (nucleotide sequence: SEQ ID NO.19 , amino acid sequence: SEQ ID NO.20), mouse mDEK gene (nucleotide sequence: SEQ ID NO.21, amino acid sequence: SEQ ID NO.22), mouse mDEK ⁇ NLS (nuclear localization sequence NLS deletion) gene (Nucleotide sequence: SEQ ID NO.23, amino acid sequence: SEQ ID NO.24) respectively inserted into the EcoR I and Xba I sites of the pEGFP-C1 plasmid (Youbao Biology, product number: VT1118) ( Figure 46) .
  • Recombinant plasmids pEGFP-C1-hDEK, pEGFP-C1-mDEK, pEGFP-C1-hDEK ⁇ NLS and pEGFP-C1-mDEK ⁇ NLS were respectively obtained, which were plasmids for overexpressing exogenous DEK protein and domain NLS mutant DEK protein.
  • Second use primer F1 (CCGGAATTCTATGTCCGCCT) and primer R1 (CGCTCTAGATCAAGAAATTAG) to perform PCR amplification on SEQ ID NO.5 and SEQ ID NO.19 to obtain the human DEK gene sequence covering the restriction site. Then the above sequence and the pLent-N-GFP plasmid were simultaneously subjected to double enzyme digestion treatment and ligation treatment with EcoR I and Xba I.
  • primer F2 AGAATTCTATGTCGGCGGCGGCGG
  • primer R2 GCTCTAGATCAAGAAATTAGCTCTTTTTACAGTTGT
  • the above-mentioned recombinant lentiviral expression vector and lentiviral packaging plasmid mixture were co-transfected into 293T cells, and the cells were collected after 72 hours of transfection
  • the culture supernatant is the virus liquid.
  • the virus titer is measured to obtain lentiviruses that overexpress exogenous DEK protein and domain NLS mutant DEK protein, namely lentiviruses hDEK, mDEK, hDEK ⁇ NLS and mDEK ⁇ NLS .
  • the 4T1-DCCs, EMT6-DCCs and MCF7-DCCs cells obtained in Example 2 were respectively inoculated according to about 3 million cells per 10 cm cell culture dish, and 10 mL of DMEM culture solution containing 10% serum and 1% antibiotic was added, and 37 Cultivate overnight. The next day, pour out the original culture medium and add new DMEM culture solution containing 10% serum and 1% antibiotics.
  • Exosomes were sorted by flow cytometry (Beckman moflo Astrios EQ) to obtain sorted exosomes and structural domains containing exogenous DEK proteins from different tumor cells that were GFP positive and had a particle size range of 50-150 nm.
  • the sorted exosomes of NLS mutant DEK protein were MCF7 exosomes containing hDEK-GFP protein, MCF7 exosomes containing hDEK ⁇ NLS -GFP protein, 4T1 exosomes containing mDEK-GFP protein, 4T1 Exosomes of mDEK ⁇ NLS -GFP protein, EMT6 exosomes containing mDEK-GFP protein, EMT6-exosomes containing mDEK ⁇ NLS -GFP protein.
  • polybrene polybrene
  • polybrene polybrene
  • a final concentration of 6 ⁇ g/mL and 10 ⁇ L of 10 8 pfu/mL lentivirus prepared in step 2 to each well (4T1-DCCs, EMT6-DCCs cells were added with mDEK and mDEK ⁇ NLS ; MCF7-DCCs cells added hDEK and hDEK ⁇ NLS ).
  • the exosomes were sorted by a flow sorter (Beckman moflo Astrios EQ) to obtain GFP-positive and sorted exosomes containing exogenous DEK proteins and domain NLS mutations from different sources with a particle size range of 50-150 nm
  • the sorted exosomes of DEK protein are MCF7 sorted exosomes containing hDEK-GFP protein, MCF7 sorted exosomes containing hDEK ⁇ NLS -GFP protein, and 4T1 sorted exosomes containing mDEK-GFP protein body solution, 4T1 sorted exosomes containing mDEK ⁇ NLS -GFP protein, EMT6 sorted exosomes containing mDEK-GFP protein, EMT6-sorted exosomes containing mDEK ⁇ NLS -GFP protein.
  • BCA protein quantitative detection kit brand: Sangon, product number: C503021
  • PBS PBS at a concentration of 200 ⁇ g/mL
  • sorting Exosome PBS solution for subsequent use.
  • Example 16 Addition of sorted exosomes containing exogenous DEK protein to activate dormant tumor cells
  • 4T1-DCCs cells were added with 2.5 ⁇ L PBS, 2.5 ⁇ L 200 ⁇ g/mL of 4T1 sorted exosomes containing mDEK-GFP protein prepared by transfection method of overexpressed lentivirus in Step 3 of Example 15, and 2.5 ⁇ L 200 ⁇ g/mL of Example 15
  • the final concentration of the sorted exosomes of GFP protein in the culture medium was 50 ng/mL.
  • EMT6-DCCs added PBS, EMT6 sorted exosomes containing mDEK-GFP protein PBS solution, EMT6-sorted exosomes containing mDEK ⁇ NLS -GFP protein PBS solution; MCF7-DCCs cells added PBS, MCF7 sorted exosomes PBS solution containing hDEK-GFP protein, MCF7 sorted exosomes PBS solution containing hDEK ⁇ NLS -GFP protein.
  • Example 17 Combined with chemotherapy, the addition of sorted exosomes containing exogenous DEK protein can clear dormant tumor cells 1. Effect of sorted exosomes combined with chemotherapy on cCasp3 signal of dormant tumor cells
  • Example 16 collect the cells after incubating at 37°C for 20 hours, and use the cleaved cysteine protease (cCasp3, abcam, product number: ab32042) antibody to carry out immunofluorescence experiment (the specific operation is the same as in Example 2 step 2, and the secondary antibody is replaced Fluorescently labeled donkey anti-rabbit for Alexa Fluor 594, Thermo Fisher, Cat. No. R37119). The samples were observed with a fluorescence microscope, and it was found that a large number of cCasp3 signals appeared, and a large number of cells underwent apoptosis ( FIG. 52 ).
  • cCasp3 cysteine protease
  • Example 18 Regulatory mechanism of sorted exosomes containing exogenous DEK protein to activate dormant tumor cells
  • Example 16 In the same manner as in Example 16, the cells were collected after culturing at 37° C. for 20 hours. Using antibodies against H4K20me1, H4K20me2, H4K20me3, HP1- ⁇ , H3K9ac, H3K9me3, and H3K27me3, Western blot analysis was carried out in the above cells (the method is the same as that in step 2 of Example 2, see Table 1 for the primary and secondary antibodies).
  • Example 16 In the same manner as in Example 16, the cells were collected after culturing at 37° C. for 20 hours. Using Mouse monoclonal anti-p53 (Santa Cruz, catalog number: sc-126), Rabbit monoclonal anti-p21 (Cell Signaling Technology, catalog number: 2947), Rabbit polyclonal anti-PUMA (abcam, catalog number: ab9643) and Mouse monoclonal The antibody of anti-c-Myc (Santa Cruz, catalog number: sc-40) was carried out in the above cells for western blot analysis (the method is the same as step 2 of Example 2, and the secondary antibody is shown in Table 1).
  • Example 19 Detection of injected sorted exosomes containing exogenous DEK protein in blood and tumor tissues of tumor-bearing mice
  • the exosomes were sorted in PBS solution, and 3 tumor-bearing mice were not injected intraperitoneally with substances as untreated controls.
  • Six tumor-bearing mice in the EMT6 group were injected intraperitoneally with 20 ⁇ g of total protein per 20 g of mouse body weight, and injected 200 ⁇ g/mL of EMT6 containing mDEK-GFP protein prepared by the overexpression lentiviral transfection method in Step 3 of Example 15.
  • the exosomes were sorted in PBS solution, and 3 tumor-bearing mice were not injected intraperitoneally with substances as untreated controls.
  • mice in the MCF7 group were injected intraperitoneally with 20 ⁇ g of total protein per 20 g of mouse body weight, and injected 200 ⁇ g/mL of MCF7 containing mDEK-GFP protein prepared by the overexpression lentiviral transfection method in Step 3 of Example 15.
  • the exosomes were sorted in PBS solution, and 3 tumor-bearing mice were not injected intraperitoneally with substances as untreated controls.
  • the mice were euthanized 24 hours and 7 days after the injection, and the plasma of the mice was collected.
  • Exosomes in the plasma were separated using an exosome extraction kit (Invitrogen, catalog number: 4484450), and 10 ⁇ L of exosomes were added to 100 ⁇ L of exosomes.
  • 9 ⁇ m sulfuric acid latex beads (dissolved in super-distilled water) with a mass concentration of 4%, incubated at 25°C for 10 minutes. Centrifuge at 1000 rpm at 25°C for 5 minutes, discard the supernatant, and resuspend with 100 ⁇ L PBS. Put the resuspension into flow cytometer to detect the ratio of GFP + beads.
  • the sorted exosomes were intraperitoneally injected into tumor-bearing mice and then entered the tumor tissue
  • mice were euthanized 24 hours after the injection, and the 4T1, EMT6, and MCF7 tumors were surgically removed.
  • the above tumors were placed in 4% paraformaldehyde at 4°C overnight, soaked in 30% sucrose aqueous solution for dehydration for 48 hours, and the tumors after soaking Take it out, put it into an open cuboid plastic mold of 10x10x5mm, fill it with OCT embedding agent (SAKURA, catalog number: 4583), put the mold on dry ice and let it stand for 5 minutes, take out the embedding block, Store at -80°C. Cut the embedding blocks into 10 ⁇ m tumor sections with a cryostat.
  • SAKURA OCT embedding agent
  • Example 20 Injecting sorted exosomes containing exogenous DEK protein to activate dormant tumor cells in tumor-bearing mice
  • 3 tumor-bearing mice in the 4T1 group were not intraperitoneally injected with substances as untreated controls, and 3 tumor-bearing mice were intraperitoneally injected with a total protein amount of 20 ⁇ g per 20 g of mouse body weight and injected with the overexpressed lentivirus transfected in step 3 of Example 15 Methods Prepare 200 ⁇ g/mL 4T1 PBS solution of sorted exosomes containing mDEK-GFP protein.
  • Three tumor-bearing mice in EMT6 group were not intraperitoneally injected with substances as untreated controls, and three tumor-bearing mice were intraperitoneally injected with a total protein amount of 20 ⁇ g per 20 g of mouse body weight and injected with overexpressed lentivirus transfection in Step 3 of Example 15 Methods Prepare 200 ⁇ g/mL EMT6 PBS solution of sorted exosomes containing mDEK-GFP protein.
  • Three tumor-bearing mice in the MCF7 group were not injected intraperitoneally with substances as untreated controls, and three tumor-bearing mice were injected intraperitoneally with a total protein amount of 20 ⁇ g per 20 g of mouse body weight for transfection of the overexpressed lentivirus in step 3 of Example 15 Methods Prepare 200 ⁇ g/mL MCF7-containing mDEK-GFP protein sorted exosome PBS solution. The mice were euthanized 24 hours after the injection, and the 4T1, EMT6, and MCF7 tumors were surgically removed.
  • the above tumors were placed in 4% paraformaldehyde at 4°C overnight, soaked in 30% sucrose aqueous solution for dehydration for 48 hours, and the tumors after soaking Take it out, put it into an open cuboid plastic mold of 10x10x5mm, fill it with OCT embedding agent (SAKURA, catalog number: 4583), put the mold on dry ice and let it stand for 5 minutes, take out the embedding block, Store at -80°C. Cut the embedding blocks into 10 ⁇ m tumor sections with a cryostat.
  • SAKURA OCT embedding agent
  • Example 21 Combined with radiotherapy and chemotherapy, injection of sorted exosomes containing exogenous DEK protein can eliminate dormant tumor cells in tumor-bearing mice
  • each tumor-bearing mouse in the radiotherapy + PBS group was injected intraperitoneally with PBS, and the injection volume of PBS was 100 ⁇ L; Inject the mice with a total protein amount of 20 ⁇ g and inject the 200 ⁇ g/mL 4T1 sorted exosome PBS solution containing mDEK-GFP protein prepared by the overexpression lentiviral transfection method in step 3 of Example 15; radiotherapy + 4T1 containing mDEK ⁇ Sorting of NLS -GFP protein In the exosome group, inject 200 ⁇ g/mL 4T1 containing mDEK prepared by the overexpression lentiviral transfection method in Step 3 of Example 15 at an amount of 20 ⁇ g of total protein per 20 g of mouse body weight ⁇ NLS -GFP protein sorting exosomes in PBS solution.
  • EMT6 cells were inoculated into the axillary mammary fat pad of BALB/c female mice at 6-8 weeks, and were divided into radiotherapy and chemotherapy + PBS group, radiotherapy and chemotherapy + EMT6 sorted exosomes group containing mDEK-GFP protein, and radiotherapy and chemotherapy + PBS group.
  • each tumor-bearing mouse in the radiochemotherapy+PBS group was intraperitoneally injected with PBS, and the injection volume of PBS was 100 ⁇ L;
  • the exosome group injected 200 ⁇ g/mL EMT6 sorted exosomes containing mDEK-GFP protein PBS solution prepared by the overexpression lentiviral transfection method in Step 3 of Example 15 at a total protein amount of 20 ⁇ g per 20 g of mouse body weight ;
  • Chemotherapy+EMT6-sorted exosome group containing mDEK ⁇ NLS -GFP protein was injected with a total protein amount of 20 ⁇ g per 20 g of mouse body weight, prepared by the overexpression lentiviral transfection method in Step 3 of Example 15 200 ⁇ g/mL EMT6-sorted exosomes PBS solution containing mDEK ⁇ NLS- GFP protein.
  • Example 22 Combined with radiotherapy and chemotherapy, injection of sorted exosomes containing exogenous DEK protein can cure breast cancer
  • mice On the 8th week after cell inoculation, all experimental mice were euthanized and surgically removed lung tissue samples, which were fixed and embedded and frozen for sectioning. Sections were washed once in PBS for 5 minutes; stained with hematoxylin staining solution for 10 minutes; rinsed off excess staining solution in tap water for 1 minute; washed once in distilled water for 2 minutes; stained with eosin staining solution for 2 minutes; Glycerin coverslips.
  • the stained slices were observed under a microscope, and it was found that compared with the radiotherapy + PBS group and the radiotherapy + 4T1 sorted exosome group containing mDEK ⁇ NLS -GFP protein, the sorted exosome group containing mDEK-GFP protein in radiotherapy + 4T1 Metastases of tumors were not found in the body group ( FIG. 62 ).
  • EMT6 cells Inoculate 200,000 EMT6 cells into the axillary mammary fat pad of BALB/c female mice at 6-8 weeks, and divide them into untreated group, radiochemotherapy+PBS group, radiochemotherapy+EMT6 sorted exocytosis containing mDEK-GFP protein Body group, radiotherapy and chemotherapy+EMT6-sorted exosomes group containing mDEK ⁇ NLS -GFP protein, 7 rats in each group.
  • the tumor diameter reached the ethical upper limit 6 weeks after the inoculation of cells in the chemoradiotherapy+PBS group and the chemoradiotherapy+EMT6-containing mDEK ⁇ NLS -GFP protein group, while in the chemoradiotherapy+EMT6 group containing mDEK-GFP protein
  • tumor growth was significantly inhibited (Figure 64).
  • the results showed that the use of sorted exosomes containing exogenous DEK protein and radiotherapy and chemotherapy could completely cure EMT6 tumors and inhibit tumor recurrence.
  • MCF7 cells were inoculated into the axillary mammary fat pad of Nod/Scid female mice at 6-8 weeks, and divided into untreated group, chemotherapy + PBS group, chemotherapy + MCF7 sorted exosomes group containing hDEK-GFP protein , Chemotherapy + MCF7 sorted exosomes group containing hDEK ⁇ NLS -GFP protein, 5 in each group.
  • the length and width of the tumor were measured every week after cell inoculation, and the tumor volume was calculated using the formula (length ⁇ width ⁇ width/2), and the growth curve of the tumor was drawn.
  • the results showed that the use of sorted exosomes containing exogenous DEK protein and chemotherapy could completely cure MCF7 tumors and eliminate tumor recurrence.
  • Example 23 the number of dormant tumor cells is closely related to the degree of deterioration of clinical breast cancer
  • Example 24 Acquisition, activation and killing of dormant tumor cells derived from clinical breast cancer samples
  • the tumor dissociation kit (Miltenyi, Cat. No.: 130-095-929) was used to process clinical breast cancer samples from two patients to obtain digested tumor cells, which were inoculated at a density of 800,000 cells/well in a super Low-adsorption six-hole plate (brand: Corning, item number: 3471).
  • DMEM/F12 medium (Corning, catalog number: 10-092-cv) containing 10% serum replacement (Thermo Fisher Scientific Cat#10828028), final concentration of 100nM paclitaxel and final concentration of 1mM 5-fluorouracil into each well, Cultivate at 37°C for 1 month, change the culture medium every 3 days, and irradiate 30Gy X-rays once a week in the first two weeks, and irradiate twice in total (7 days between the two times), each time irradiating for 10 minutes.
  • tumor cells resistant to radiotherapy and chemotherapy were screened out using a dead cell removal kit (Miltenyi Biotec, catalog number: 130-090-101), which were dormant tumor cells (a in FIG. 67 ).
  • the PBS solution of the sorted exosomes containing hDEK ⁇ NLS -GFP protein in MCF7 prepared by the overexpression lentiviral transfection method in step 3, so that the sorted exosomes containing hDEK-GFP protein in MCF7 and the sorted exosomes containing hDEK ⁇ NLS -GFP in MCF7
  • the final concentration of the sorted exosomes in the medium was 50 ng/mL.
  • the PBS solution of the sorted exosomes containing hDEK ⁇ NLS -GFP protein in MCF7 prepared by the overexpression lentiviral transfection method in step 3, so that the sorted exosomes containing hDEK-GFP protein in MCF7 and the sorted exosomes containing hDEK ⁇ NLS -GFP in MCF7
  • the final concentration of the sorted exosomes in the medium was 50 ng/mL.
  • Cell samples were collected after culturing at 37°C for 30 hours, and dead cells were detected by trypan blue staining.
  • Example 25 Activation and removal of dormant tumor cells by DEK combined with chemotherapy in various human tumor cells
  • the cell culture medium is RPMI-1640 medium (Gibco, catalog number: 31800022) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.
  • H226 cell lines were inoculated into 20 mL of culture medium. After culturing at 37°C for 24 hours, collect the cell culture medium, centrifuge at 1000rpm at 4°C for 10 minutes, take the supernatant, and centrifuge at 12000rpm at 4°C for 20 minutes, take the supernatant, which is the cell culture medium of H226. Add 1 mL of exosome isolation reagent (brand: Thermo Fisher, product number: 4478359) to the cell culture medium, invert up and down 3 times to mix well and incubate overnight at 4°C.
  • exosome isolation reagent brand: Thermo Fisher, product number: 4478359
  • the above mixture was centrifuged at 10,000 rpm at 4°C for 60 minutes, the supernatant was removed, and the sediment at the bottom of the centrifuge tube was resuspended with 200 ⁇ L of PBS (the sediment was crude exosomes), and the BCA protein quantitative detection kit (brand: Sheng Worker, product number: C503021) to measure the total amount of protein in the crude exosome suspension, and prepare a crude exosome solution with a protein concentration of 20 ⁇ g/mL with PBS for subsequent use.
  • the BCA protein quantitative detection kit brand: Sheng Worker, product number: C503021
  • PBS phosphate buffered solution
  • EDTA ethylenediaminetetraacetic acid
  • DMEM/F12 medium (Corning, catalog number: 10-092-cv) containing 10% serum replacement (Thermo Fisher Scientific Cat#10828028), final concentration of 100nM paclitaxel and final concentration of 1mM 5-fluorouracil into each well, Cultivate at 37°C for 1 month, change the culture medium every 3 days, and irradiate 30Gy X-rays once a week in the first two weeks, and irradiate twice in total (7 days between the two times), each time irradiating for 10 minutes.
  • dormant tumor cells resistant to radiochemotherapy were screened out using a dead cell removal kit (Miltenyi Biotec, catalog number: 130-090-101).
  • step 1 method cell line replacement is PC-3, medium replacement is: F-12 medium (Gibco, catalog number: 21700075) adds 10% fetal bovine serum and 1% penicillin-streptomycin ), and found that PC-3-derived crude exosomes combined with chemotherapy were 100% activated and cleared PC-3 dormant tumor cells.
  • step 1 of Example 25 the cell line is replaced by HeLa, and the medium is replaced by: adding 10% fetal bovine serum and 1% penicillin-streptomycin to the MEM medium (Gibco, catalog number: 41500034), it is found that HeLa-derived crude exosomes combined with chemotherapy 100% activated and eliminated HeLa dormant tumor cells.

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Abstract

本发明公开了一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用，所述SETD4蛋白抑制剂包括DEK蛋白，所述DEK蛋白具有SEQ ID NO.25所示保守区域。本发明提供一种SETD4蛋白抑制剂，特别是DEK蛋白在激活休眠肿瘤细胞、清除肿瘤细胞及治愈肿瘤中的应用，癌症患者的不同时期，定向在静息肿瘤细胞中转入DEK基因或投递DEK蛋白，可以激活静息肿瘤细胞，使其失去对肿瘤治疗的抵抗性。

Description

一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用 (一)技术领域
本发明涉及一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用，特别是DEK蛋白联合临床的肿瘤切除、放化疗、靶向及免疫治疗，激活并清除休眠肿瘤细胞，从而实现肿瘤治愈，以及在临床上治愈各种癌症的应用。
(二)背景技术
癌症目前依然是人类健康的主要疾病之一。全球年新发癌症患者人数超过一千万人，因癌症死亡人数超过670万人。目前癌症的治疗方法主要为外科手术、放疗、化疗、靶向治疗及免疫治疗为主。尽管这些治疗能够清除并杀死大部分的肿瘤细胞，然而却无法清除肿瘤内一小类群的休眠肿瘤细胞。肿瘤内的休眠肿瘤细胞数量虽少，但是具有极强的抵抗各种临床治疗的特征，它们通常具有极强的体内形成肿瘤和体外形成肿瘤球的能力。激活的休眠肿瘤细胞能够促进肿瘤的恶化和转移，是恶性肿瘤形成的主要因素。休眠肿瘤细胞能够在临床的放化疗、靶向及免疫治疗中存活，在治疗后能够快速激活并产生大量的肿瘤细胞，从而诱发肿瘤病人术后的肿瘤复发和转移。因此如何杀死并清除休眠肿瘤细胞是肿瘤临床治愈上无法突破的瓶颈问题。
研究报道，含有SET结构域的家族蛋白(SETD)具有组蛋白精氨酸甲基化转移酶的活性，在调控染色质结构及在细胞增殖过程中调控基因的转录表达上起重要的作用。我们经十余年的研究，建立了独有的卤虫胚胎干细胞休眠及激活的研究模型。使用该模型，我们筛选获得了细胞休眠调控的SETD4蛋白，阐明了SETD4通过催化组蛋白H4K20me3促进异染色质的形成，从而表观遗传调控卤虫休眠胚胎形成过程的分子机制(文献1)。在此基础上，我们发现SETD4以同样的机制表观遗传调控乳腺肿瘤细胞的休眠，揭示了由SETD4调控干细胞休眠的进化保守机制，同时发现SETD4休眠肿瘤细胞存在于各种不同类型临床肿瘤病人中如肝癌，肺癌，胰腺癌，卵巢癌，子宫癌等，与早期患者相比晚期肿瘤患者的SETD4休眠肿瘤细胞数量显著增加(文献2)。因此使用SETD4蛋白能够标记这类休眠的肿瘤细胞，为清除休眠肿瘤细胞提供了重要的靶点。这是首次在各种肿瘤中分子标记了休眠肿瘤细胞的研究报道，也是首次发现通过组蛋白修饰(H4K20me3)的肿瘤细胞休眠 的表观遗传机制的研究报道。
以前的研究表明，DEK作为细胞核因子蛋白能够与染色质相结合并参与调控细胞增殖、分化、凋亡、衰老，DNA损伤修复及干细胞特征维持。此外作为癌蛋白在各种肿瘤细胞中高量表达并与肿瘤的复发及转移相关。DEK蛋白能够被肿瘤细胞合成分泌并被另外的细胞所摄入，因此是具有细胞内及细胞间活性的蛋白。此外，DEK蛋白能够以自由及包含在外泌体中的形式从细胞中释放并进入到其目标细胞中。我们通过卤虫动物模型的研究发现了DEK，在卤虫休眠胚胎激活的过程中高量表达，并发现DEK通过降低SETD4蛋白的表达，降低H4K20me3从而在休眠细胞的激活起到了重要的作用(文献3)。本发明中，我们发现DEK蛋白在激活的休眠的肿瘤干细胞及肿瘤细胞中高量表达，抑制DEK蛋白的表达能够显著抑制休眠肿瘤细胞的激活，添加外源DEK蛋白能够促进休眠肿瘤细胞的激活。并发现激活后的休眠肿瘤细胞失去抵抗放化疗的能力，使用放化疗和DEK蛋白同时处理能够清除休眠肿瘤细胞，从而实现了肿瘤的完全治愈。
文献
1.Li Dai,Sen Ye,Hua-Wei Li,Dian-Fu Chen,Hong-Liang Wang,Sheng-Nan Jia,Cheng Lin,Jin-Shu Yang,Fan Yang,Hiromichi Nagasawa and Wei-Jun Yang*.SETD4 regulates cell quiescence and catalyzes the trimethylation of H4K20during diapause formation of Artemia.Molecular and Cellular Biology 37(7).pii,e00453-16(2017).
2.Sen Ye,Yan-Fu Ding,Wen-Huan Jia,Xiao-Li Liu,Jing-Yi Feng,Qian Zhu,Sun-Li Cai,Yao-Shun Yang,Qian-Yun Lu,Xue-Ting Huang,Jin-Shu Yang,Sheng-Nan Jia,Guo-Ping Ding,Yue-Hong Wang,Jiao-Jiao Zhou,Yi-Ding Chen and Wei-Jun Yang*.SET domain-containing protein 4epigenetically controls breast cancer stem cell quiescence.Cancer Research 79(18),4729–4743(2019).
3.Wen-Huan Jia,An-Qi Li,Jing-Yi Feng,Yan-Fu Ding,Sen Ye,Jin-Shu Yang and Wei-Jun Yang*.DEK terminates diapause by activation of quiescent cells in the crustacean Artemia.Biochemical Journal 476(12),1753–1769(2019).
(三)发明内容
本发明目的是提供一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用，特别是外源DEK蛋白能够进入并激活休眠的肿瘤细胞，使其失去抵抗放化疗的能力。临床手术、放化疗、靶向及免疫治疗和DEK蛋白同时处理能够清除休眠肿瘤细胞，从而实现了肿瘤的完全治愈。
本发明采用的技术方案是：
第一方面，本发明提供一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用。
本发明所涉及的SETD4蛋白抑制剂包括DEK蛋白，所述DEK蛋白具有SEQ ID NO.25所示保守序列。
进一步，所述DEK蛋白具有SEQ ID NO.2所示NLS结构域的氨基酸序列95％以上相似性。
进一步，所述DEK蛋白具有SEQ ID NO.3所示SAP结构域的氨基酸序列95％以上相似性。
进一步，所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域的氨基酸序列95％以上相似性。
进一步，所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域或SEQ ID NO.3所示SAP结构域中的一种或两种，且同时具有SEQ ID NO.2所示NLS结构域。
进一步，所述DEK蛋白具有SEQ ID NO.1或SEQ ID NO.22所示氨基酸序列95％以上相似性。
更进一步，所述DEK蛋白选自人源DEK蛋白，具有SEQ ID NO.1所示氨基酸序列，编码基因序列为SEQ ID NO.5所示。
更进一步，所述DEK蛋白选自鼠源DEK蛋白，具有SEQ ID NO.22所示氨基酸序列，编码基因序列为SEQ ID NO.21所示。
本发明所述休眠肿瘤细胞或肿瘤包括头颈部肿瘤，胸部肿瘤，消化系统肿瘤，泌尿生殖系统肿瘤，骨及软组织肿瘤，淋巴及血液系统肿瘤，其它肿瘤；其中头颈部肿瘤包括脑癌、眼癌、耳部肿瘤、颌骨肿瘤、颈部肿瘤、鼻腔癌、鼻窦癌、鼻咽癌、牙龈癌、舌癌、软硬腭癌、颌骨癌、口底癌、口咽癌、唇癌、上颌窦癌、颜面部皮肤黏膜的癌症、喉癌、涎腺肿瘤、甲状腺癌、脑膜瘤、室管膜瘤、垂体瘤、上皮神经母细胞瘤、神经外胚层肿瘤和副神经节肿瘤；胸部肿瘤包括肺癌、食管癌、乳腺癌、纵膈肿瘤和胸腺癌；消化系统肿瘤包括胃癌、大肠癌、肝癌、胰腺癌、胆管癌、小肠癌；泌尿生殖系统肿瘤包括肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、宫颈癌、子宫癌、卵巢癌、输卵管癌和阴道癌；骨及软组织肿瘤包括尤文氏肉瘤、脂肪肿瘤、卡波济肉瘤、平滑肌肿瘤、横纹肌肿瘤、血管肿瘤、滑膜肉瘤、纤维肉瘤和骨癌；淋巴及血液系统肿瘤包括恶性淋巴瘤、多发性骨髓瘤、白血病；其它肿瘤包括心脏肿瘤、 间皮肿瘤、纤维母细胞肿瘤、滋养细胞肿瘤和黑色素瘤。
第二方面，本发明提供一种用于激活休眠肿瘤细胞的SETD4蛋白抑制剂投递蛋白，所述投递蛋白包括投递DEK蛋白，所述投递DEK蛋白是含有DEK蛋白的医学可接受的载体，所述载体包括外泌体、脂质体或纳米材料。在第二方面所述的DEK蛋白、休眠肿瘤细胞或肿瘤的定义及适用范围与第一方面限定相同。
进一步，所述外泌体是指一种由细胞分泌的大小为30-150nm的具有双层膜结构的囊泡，当所述载体为外泌体时，所述投递DEK蛋白是由肿瘤细胞系培养液中分离获得含有DEK蛋白的外泌体，或将DEK蛋白编码基因接入各种基因表达载体，在各种细胞系(推荐肿瘤细胞)中过量表达DEK蛋白并产生含有DEK蛋白的外泌体。
进一步，所述将DEK蛋白编码基因接入基因表达载体获得的含DEK蛋白的外泌体按如下方法之一制备：(1)构建过表达DEK蛋白的质粒并将其转染到各种细胞系中制备外泌体：将DEK基因插入到pEGFP-C1质粒的EcoR Ⅰ和Xba Ⅰ位点上，筛选获得重组质粒pEGFP-C1-DEK；利用脂质体助转剂lipo8000(碧云天，货号：C0533)将上述重组质粒转入细胞系中，过量表达DEK蛋白后收集细胞培养液，从培养液中分离纯化含有DEK蛋白的外泌体溶液A；(2)构建过表达DEK蛋白的慢病毒并将其感染各种细胞系构建表达DEK蛋白的细胞株制备外泌体：将DEK基因分别插入到pLent-N-GFP的慢病毒表达载体的EcoR Ⅰ和Xba Ⅰ位点上，筛选获得重组慢病毒表达载体pLent-N-GFP-DEK；将重组慢病毒表达载体pLent-N-GFP-DEK和慢病毒包装质粒混合物共同转染293T细胞，转染72小时后收集细胞培养上清即为病毒液，浓缩纯化，获得过表达DEK蛋白的慢病毒；用过表达DEK蛋白的慢病毒感染细胞系并构建过表达DEK蛋白的细胞株；收集过表达DEK蛋白的细胞株的细胞培养液，从培养液中分离纯化含有DEK蛋白的外泌体溶液B。所述含有DEK蛋白的外泌体溶液A或含有DEK蛋白的外泌体溶液B实际均为含有DEK蛋白的外泌体溶液，为了区分不同方法获得的外泌体而命名，字母本身没有含义。
进一步，从肿瘤细胞系培养液中分离外泌体的方法有多种，都可以使用，本发明推荐由肿瘤细胞系培养液中分离获得外泌体采用外泌体抽提试剂法制备：从肿瘤细胞系培养液中加入外泌体分离试剂(品牌：Thermo Fisher，货号：4478359)，上下颠倒3次混匀后4℃孵育过夜，第二天，上述混合液10000rpm 4℃离心60分钟，去掉上清，用PBS重悬离心管底部的沉淀，即为含DEK蛋白的粗外泌体。
进一步，方法(1)和(2)细胞系均优选4T1、EMT6、MCF7。
进一步，方法(1)和(2)从培养液中分离纯化含有DEK蛋白的外泌体溶液的方法均为：利用外泌体抽提试剂从细胞培养液中分离获得外泌体，通过流式分选仪分选得到GFP阳性和粒径范围在50-200nm的含有DEK蛋白的外泌体。
进一步，方法(2)所述慢病毒包装质粒混合物源自慢病毒包装使用试剂盒(Lentiviral Packaging Kit，购自上海翊圣生物，货号：41102ES10)，由质量比5:3:2的pMDL，VSVG和pRSV-Rev组成。
进一步，所述脂质体是指由磷脂双分子层人工膜在水溶液中形成的微囊，所述载体为脂质体时，含DEK蛋白的脂质体采用薄膜水化法制备：取二棕榈酰磷脂酰胆碱(DPPC)、胆固醇、二硬脂酰磷脂酰乙酰胺-甲氧基聚乙二醇(2000DSPE-mPEG2000)溶于氯仿中，减压旋转蒸发成均匀的薄膜，真空过夜彻底挥发掉残留的氯仿，加入含DEK蛋白的PBS溶液，冰浴25KHz超声20min使脂质体膜脱落，在振荡器上震荡5min充分水化，形成浊液，然后将浊液转移到EP管中，用探头超声在功率135W下超声30min，得到透明均一的蓝色脂质体悬液；用10kD的超滤管以12000g的转速4℃超滤，每五分钟取出吹打一次并补充PBS洗去游离蛋白，收集截留液，即为含DEK蛋白的脂质体悬液；所述DEK蛋白的PBS溶液浓度优选2mg/ml；所述二棕榈酰磷脂酰胆碱与胆固醇质量比为1:0.1，所述二棕榈酰磷脂酰胆碱与二硬脂酰磷脂酰乙酰胺-甲氧基聚乙二醇质量比为1:0.1，所述氯仿体积用量以二棕榈酰磷脂酰胆碱质量计为0.1mL/mg；所述二棕榈酰磷脂酰胆碱与DEK蛋白的PBS溶液中DEK蛋白质量比为1:0.2。
进一步，所述纳米材料是指可作为传导或输送药物的载体，由天然或人工合成的高分子构成的大小在10-1000nm之间的固态胶体颗粒，所述载体为纳米材料时，含DEK蛋白的纳米材料按采用改良溶剂挥发法制备：将DEK蛋白的PBS溶液，转入聚乳酸-羟基乙酸共聚物(PLGA)的二氯甲烷溶液中，25KHz超声1分钟形成初乳，将初乳转入体积浓度1％聚乙烯醇(PVA)水溶液中，再次25KHz超声5分钟形成复乳，搅拌4h，待有机溶剂挥发后，18000r/min离心收集沉淀，将沉淀-55℃冷冻干燥24h，获得含DEK蛋白的PLGA纳米粒；优选的，所述DEK蛋白与聚乳酸-羟基乙酸共聚物质量比为1：0.1，所述DEK蛋白的PBS溶液浓度为25mg/L；所述PLGA的二氯甲烷溶液浓度为20mg/mL，所述PVA水溶液体积用量以DEK蛋白的PBS溶液中DEK蛋白质量计为1mL/mg。
第三方面，本发明提供一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞试剂中 的应用，所述试剂包括DEK蛋白。所述激活是将SETD4蛋白抑制剂投递到休眠肿瘤细胞中，达到激活休眠肿瘤细胞的目的。所述试剂能够用于肿瘤的临床治疗和基础科学研究。
本发明所述激活休眠肿瘤细胞试剂还包括SETD4蛋白抑制剂及清除肿瘤细胞的药物。所述SETD4蛋白抑制剂包括DEK蛋白；所述清除肿瘤细胞的药物推荐紫杉醇或5-氟尿嘧啶。本发明所述清除肿瘤细胞的方法是将SETD4蛋白抑制剂与放化疗等常规方法结合达到清除休眠肿瘤细胞的目的，具体是指在进行放化疗等常规治疗的同时，将SETD4蛋白抑制剂(特别是DEK蛋白，优选通过外泌体，脂质体及纳米材料)投递到肿瘤细胞中，达到激活并清除休眠肿瘤细胞的目的，以实现无转移，无复发的临床肿瘤治愈。在第三方面所述的DEK蛋白、休眠肿瘤细胞或肿瘤的定义及适用范围与第一方面限定相同。所述SETD4蛋白抑制剂投递到休眠肿瘤细胞采用第二方面所述投递蛋白。
第四方面，本发明提供一种SETD4蛋白抑制剂在制备抗肿瘤药物中的应用，所述抗肿瘤药物包括所述的SETD4蛋白抑制剂及清除肿瘤细胞的药物，所述SETD4蛋白抑制剂包括DEK蛋白；所述清除肿瘤细胞的药物推荐紫杉醇或5-氟尿嘧啶。所述的应用是在进行临床手术、放化疗、靶向或免疫治疗的同时将SETD4蛋白抑制剂(特别是DEK蛋白以外泌体，脂质体及纳米载体的形式)，通过腹腔、静脉注射或瘤体直接注射投递到肿瘤中，同时与清除肿瘤细胞的药物组合使用达到清除肿瘤的目的。在第四方面所述的DEK蛋白、休眠肿瘤细胞或肿瘤的定义及适用范围与第一方面限定相同。所述SETD4蛋白抑制剂投递到休眠肿瘤细胞采用第二方面所述投递蛋白。
第五方面，本发明提供一种应用SETD4蛋白抑制剂治疗肿瘤的方法，所述方法为：在各种时期的肿瘤病人中，通过静脉注射、腹腔注射或瘤体内注射SETD4蛋白抑制剂，将SETD4蛋白抑制剂投递到肿瘤中，从而激活休眠肿瘤细胞，并在临床手术、放化疗、靶向或免疫治疗的作用下杀死并彻底清除休眠的肿瘤细胞，实现无转移，无复发的临床肿瘤治愈；所述SETD4蛋白抑制剂(特别是DEK蛋白)进入细胞核，结合在SETD4基因的启动子上，关闭SETD4的表达，下调异染色质的形成，开放大部分基因的表达，激活休眠的肿瘤细胞，再通过放化疗等常规方法，杀死激活后的休眠肿瘤细胞；所述SETD4蛋白抑制剂包括DEK蛋白，所述DEK蛋白以投递DEK蛋白的形式注射，所述投递DEK蛋白是指含有DEK蛋白的外泌体、脂质体或纳米材料，所述投递DEK蛋白同第二方面所述。在第五方面所述的DEK蛋白、休眠肿瘤细胞或 肿瘤的定义及适用范围与第一方面限定相同。
本发明通过放化疗在人及小鼠乳腺癌中筛选获得了能够抵抗放化疗治疗的休眠肿瘤细胞，同时发现了一个可以激活休眠肿瘤细胞的功能蛋白及其基因，该基因的DNA全长为1128bp(SEQ ID NO.5)，翻译编码一个由375个氨基酸组成的蛋白质(SEQ ID NO.1)，即DEK蛋白。本发明外源DEK蛋白(包括肿瘤细胞分泌的含有DEK蛋白的外泌体)能够进入并激活休眠的肿瘤细胞，使其失去抵抗放化疗的能力，激活后的休眠肿瘤细胞，再通过放化疗等常规方法清除休眠肿瘤细胞。本发明在荷瘤小鼠中通过放化疗和含DEK蛋白外泌体同时处理能够激活并清除体内肿瘤中的肿瘤休眠细胞，从而实现了荷瘤小鼠的完全治愈，明确了使用放化疗等常规方法和DEK或含DEK蛋白的外泌体同时处理能够清除休眠肿瘤细胞(图7)，本实验中观察到小鼠在治疗后肿瘤未见复发、未见转移。
本发明DEK蛋白激活休眠肿瘤细胞的机理为：在体内将DEK蛋白投递到肿瘤组织中，DEK蛋白能够进入到休眠的肿瘤细胞中，结合在SETD4，MYC及TP53等基因的启动子上，下调SETD4和p53蛋白的表达，上调MYC蛋白的表达。通过降低H4K20me3水平，降低异染色质结构水平并上升常染色质结构的水平，进而上调细胞增殖分裂相关的胞内信号转导、基因表达、细胞周期和细胞代谢、细胞的转录翻译、细胞呼吸、细胞代谢、DNA修复等途径，下调细胞滞育、染色体沉默、基因沉默、低氧代谢、p53信号通路、上皮间充质转化等途径，从而激活休眠的肿瘤细胞，在放化疗的条件下杀死并清除休眠的肿瘤细胞，以实现无转移，无复发的临床肿瘤治愈。
与现有的肿瘤临床治疗的方法相比，本发明有益效果主要体现在：由于在肿瘤中存在的休眠肿瘤细胞具有抵抗包括放疗，化疗，靶向及免疫治疗等各种临床肿瘤治疗的能力，是肿瘤恶化、转移及预后肿瘤复发的主要原因，本发明提供一种SETD4蛋白抑制剂，特别是DEK蛋白在激活休眠肿瘤细胞、清除肿瘤细胞及治愈肿瘤中的应用。
本发明将外源DEK蛋白投递到休眠肿瘤细胞，外源DEK蛋白能够与染色质相结合，降低异染色质的同时升高常染色质的结构，从而引起一系列的基因转录及表达，进而诱导肿瘤细胞从休眠状态转换到激活状态(分子机制见图1)，使其失去对各种临床治疗抵抗能力，同时结合放化疗等目前已有的治疗方法达到清除体内休眠肿瘤细胞，在临床上实现无复发癌症治愈目的。本发明在癌症患者的不同时期，定向在静息肿瘤细胞或组织中转入DEK基因或投递DEK蛋白，可以激活静息肿瘤细胞，使其失 去对肿瘤治疗的抵抗性，结合标准的放化疗治疗从而彻底清除休眠肿瘤细胞。本发明SETD4蛋白抑制剂对于4T1休眠细胞、EMT6休眠细胞和MCF7休眠细胞清除率为100％，体外实验验证临床乳腺癌病人静息细胞清除率为100％。本发明方法可用于很早期的肿瘤治疗，当肿瘤的尺寸小到无法用手术进行治疗时，可以通过DEK外泌体和化疗的联合使用，激活并彻底清除休眠肿瘤细胞，杜绝肿瘤的复发，进而实现人类各种癌症的治愈治疗。本发明方法还可用于原位的肿瘤已经被手术切除，但是全身还存在转移肿瘤细胞的病人，通过DEK外泌体和化疗的联合使用，激活并清除转移到其它地方的休眠肿瘤细胞。
本发明是首例关于SETD4蛋白抑制剂激活休眠肿瘤细胞的研究和报道，也是首次使用DEK蛋白与标准临床治疗联合处理治愈癌症，并且无复发与转移。本发明在各种人类癌治愈治疗的临床上有着重大的应用价值。
(四)附图说明
图1为含外源DEK蛋白的外泌体激活休眠肿瘤细胞，结合放化疗将其杀死的模式图。
图2为4T1、EMT6和MCF7细胞在贴壁培养时的光镜照片。标尺，50μm。
图3为4T1、EMT6和MCF7肿瘤的光镜照片。标尺，4mm。
图4为从4T1、EMT6和MCF7肿瘤中消化下来的细胞光镜照片。标尺，50μm。
图5为4T1、EMT6和MCF7肿瘤中抵抗放化疗的细胞的光镜照片。标尺，50μm。
图6为4T1、EMT6和MCF7肿瘤中抵抗放化疗的肿瘤细胞的SETD4、H3S10ph和PCNA的免疫印迹结果，内参分别为GAPDH和H3。
图7为4T1、EMT6和MCF7肿瘤中休眠肿瘤细胞的ALDH1、CD44和CD24的免疫荧光结果。标尺，50μm。DAPI表示细胞核的染色。Merge表示四个荧光通道的叠加图。
图8为4T1、EMT6和MCF7细胞来源的粗外泌体的粒径分布曲线。
图9为4T1、EMT6和MCF7细胞来源的粗外泌体的透射电镜照片。标尺，100nm。箭头指示外泌体。
图10为4T1、EMT6和MCF7细胞来源的粗外泌体的DEK、CD9、CD81和CD63的免疫印迹结果。
图11为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图，内参分别为H3和 GAPDH。
图12为4T1、EMT6和MCF7中激活的休眠肿瘤细胞的ALDH1、CD44和CD24的免疫荧光结果。标尺，50μm。DAPI表示细胞核的染色。Merge表示四个荧光通道的叠加图。
图13为4T1、EMT6和MCF7中激活的休眠肿瘤细胞的单颗细胞体外成球的光镜照片和统计结果。标尺，2mm。红色箭头指示肿瘤球。
图14为4T1、EMT6和MCF7中100颗、10颗或者1颗激活的休眠肿瘤细胞在体内原位成瘤的光镜照片和统计结果，设置5个平行对照。标尺，1cm。
图15为4T1、EMT6和MCF7休眠肿瘤细胞实体瘤切片中的ALDH1、Ki67、SETD4和DEK的免疫荧光结果。a为检测ALDH1、Ki67和SETD4的免疫荧光结果。b为检测ALDH1、Ki67和DEK的免疫荧光结果。标尺，10μm。DAPI表示细胞核的染色。Merge表示四个荧光通道的叠加图。箭头指示ALDH1阳性且Ki67阴性的肿瘤细胞。
图16为4T1、EMT6和MCF7激活肿瘤细胞实体瘤切片中的ALDH1、Ki67、SETD4和DEK的免疫荧光结果。a为检测ALDH1、Ki67和SETD4的免疫荧光结果。b为检测ALDH1、Ki67和DEK的免疫荧光结果。标尺，10μm。DAPI表示细胞核的染色。Merge表示四个荧光通道的叠加图。箭头指示ALDH1阳性且Ki67阳性的肿瘤细胞。
图17为4T1、EMT6和MCF7休眠肿瘤细胞激活前后的SETD4和DEK的免疫印迹结果(a)及其定量统计图(b、c)，内参分别为GAPDH和H3。
图18为pFastBac TMHT A质粒的图谱。
图19为人源DEK蛋白(hDEK-GFP)、鼠源DEK蛋白(mDEK-GFP)、人源结构域NLS突变DEK蛋白(hDEK ΔNLS-GFP)和鼠源结构域NLS突变DEK蛋白(mDEK ΔNLS-GFP)的考马斯亮蓝染色。箭头指示蛋白的位置。M表示蛋白分子量标准。
图20为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEK ΔNLS-GFP蛋白或DEK-GFP蛋白后的DEK-GFP、DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图，内参分别为H3和GAPDH。
图21为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEK ΔNLS-GFP蛋白或DEK-GFP蛋白后细胞的GFP和cCasp3的免疫荧光结果及其统计图，标尺，50μm。 DAPI表示细胞核的染色。
图22为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEK ΔNLS-GFP蛋白或DEK-GFP蛋白后细胞的台盼蓝染色结果及其死亡率统计图。标尺，50μm。
图23为pLent-U6-RFP-Puro质粒的图谱。
图24为在激活的4T1、EMT6和MCF7休眠肿瘤细胞中进行DEK干涉后的免疫印迹和体外成球试验。a为DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图，内参分别为H3和GAPDH。b为体外成球试验的光镜照片和成球率的统计图，标尺，400μm。
图25为在激活的4T1、EMT6和MCF7休眠肿瘤细胞中干涉DEK一周后加入PBS、DEK ΔNLS-GFP蛋白或DEK-GFP蛋白后细胞的DEK-GFP、DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图，内参分别为H3和GAPDH。
图26为在激活的4T1、EMT6和MCF7休眠肿瘤细胞中干涉DEK一周后加入PBS、DEK ΔNLS-GFP蛋白或DEK-GFP蛋白后的体外成球试验的光镜照片和成球率的统计图，标尺，400μm。
图27为在4T1(a)、EMT6(b)和MCF7(c)休眠肿瘤细胞中加入PBS、DEK ΔNLS-GFP蛋白或DEK-GFP蛋白后细胞的透射电镜照片及细胞核中异染色质的相对水平统计图。标尺，1μm。
图28为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEK ΔNLS-GFP蛋白或DEK-GFP蛋白后的H4K20me1、H4K20me2、H4K20me3、HP1-α、H3K9ac、H3K9me3和H3K27me3的免疫印迹结果及其定量统计图，内参分别为H4和H3。
图29为结合位点分析法(ChIP-Seq)分析在激活后的MCF7休眠肿瘤细胞中DEK结合位点的峰在染色体上的分布情况。
图30为ChIP-Seq分析在激活后的MCF7休眠肿瘤细胞中DEK结合位点的峰在基因区域中的分布情况。
图31为ChIP-Seq分析在激活后的MCF7休眠肿瘤细胞中DEK结合位点的峰所在基因的基因本体富集分析。
图32为ChIP-Seq分析在激活后的MCF7休眠肿瘤细胞中DEK在SETD4、TP53和MYC基因区域上结合信号的可视化结果。
图33为ATAC-Seq分析激活前后的MCF7休眠肿瘤细胞中开放基因的整体信号情况。a为reads相对于TSS区域的分布曲线，b为reads相对于TSS区域的热图。
图34为ATAC-Seq分析在激活前后的MCF7休眠肿瘤细胞中开放程度上升的基因进行基因本体富集分析的结果。
图35为ATAC-Seq分析激活前后的MCF7休眠肿瘤细胞在SETD4、TP53和MYC基因区域上ATAC-Seq信号的可视化结果。
图36为RNA-Seq分析激活前后的MCF7休眠肿瘤细胞基因差异的结果。a为差异基因的聚类热图，b为差异基因的火山图。padj表示校正后的p值。
图37为RNA-Seq分析激活前后的MCF7休眠肿瘤细胞中上调基因的基因本体富集分析。
图38为RNA-Seq分析激活前后的MCF7休眠肿瘤细胞中下调基因的基因本体富集分析。
图39为RNA-Seq分析激活前后的MCF7休眠肿瘤细胞中差异基因的GSEA分析。a为在激活后的细胞中上调的基因集，b为在激活后的细胞中下调的基因集。
图40为MYC靶向基因集和p53靶向基因集的GSEA分析结果。a为GSEA图，b为基因集中显著差异基因的表达量热图。
图41为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEK ΔNLS-GFP蛋白或DEK-GFP蛋白后的p53、p21、PUMA和MYC的免疫印迹结果及其定量统计图，内参为β-actin。
图42为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的cCasp3的免疫荧光结果及其统计结果，标尺，50μm。
图43为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的台盼蓝染色结果及其死亡率统计图。标尺，50μm。
图44为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的H4K20me1、H4K20me2、H4K20me3、HP1-α、H3K9ac、H3K9me3和H3K27me3的免疫印迹结果及其定量统计图，内参分别为H4和H3。
图45为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的p53、p21、PUMA和MYC的免疫印迹结果及其定量统计图，内参为β-actin。
图46为pEGFP-C1质粒的图谱。
图47为pLent-N-GFP质粒的图谱。
图48为4T1、EMT6和MCF7细胞来源的含有DEK-GFP或者DEK ΔNLS-GFP蛋白的外泌体的粒径分布曲线。
图49为4T1、EMT6和MCF7细胞来源的含有DEK-GFP或者DEK ΔNLS-GFP蛋白的分选外泌体的透射电镜照片。标尺，100nm。箭头指示外泌体。
图50为4T1、EMT6和MCF7细胞来源的含有DEK-GFP或者DEK ΔNLS-GFP蛋白的分选外泌体的DEK-GFP、DEK、CD9、CD81和CD63的免疫印迹结果。
图51为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEK ΔNLS-GFP蛋白的分选外泌体后的DEK-GFP、DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图，内参分别为H3和GAPDH。
图52为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEK ΔNLS-GFP蛋白的分选外泌体后的cCasp3的免疫荧光结果及其统计结果，标尺，50μm。
图53为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEK ΔNLS-GFP蛋白的分选外泌体后的台盼蓝染色结果及其死亡率统计图。标尺，50μm。
图54为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEK ΔNLS-GFP蛋白的分选外泌体后的H4K20me1、H4K20me2、H4K20me3、HP1-α、H3K9ac、H3K9me3和H3K27me3的免疫印迹结果及其定量统计图，内参分别为H4和H3。
图55为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEK ΔNLS-GFP蛋白的分选外泌体后的p53、p21、PUMA和MYC的免疫印迹结果及其定量统计图，内参为β-actin。
图56为在小鼠中腹腔注射含外源DEK-GFP蛋白的分选外泌体后小鼠血浆中含有GFP阳性外泌体的比例检测的流式分析结果和统计图。
图57为在小鼠中腹腔注射含有DEK-GFP或者DEK ΔNLS-GFP蛋白的分选外泌体后的肿瘤切片中GFP的免疫荧光结果。标尺，50μm。DAPI表示细胞核染色。
图58为在小鼠中腹腔注射含有DEK-GFP蛋白的分选外泌体24小时后的肿瘤切片中SETD4的免疫荧光结果及其比例统计。标尺，50μm。DAPI表示细胞核染色。
图59为4T1荷瘤小鼠在放疗组、放疗且注射含有DEK ΔNLS-GFP蛋白的分选外泌体组和放疗且注射含有DEK-GFP蛋白的外泌体组中肿瘤切片的SETD4免疫荧光结果及其比例统计。标尺，50μm。DAPI表示细胞核染色。
图60为EMT6荷瘤小鼠在放化疗组、放化疗且注射含有DEK ΔNLS-GFP蛋白的分 选外泌体组和放化疗且注射含有DEK-GFP蛋白的分选外泌体组中肿瘤切片的SETD4免疫荧光结果及其比例统计。标尺，50μm。DAPI表示细胞核染色。
图61为4T1荷瘤小鼠的放疗及分选外泌体注射方案和瘤径曲线。
图62为4T1荷瘤小鼠在接受放疗和分选外泌体的联合治疗后的肺转移灶情况。a为肺组织切片的苏木素伊红染色结果(标尺，1mm)，b为肺转移灶数目的统计图；图中1、2、3、4表示放大区域。
图63为4T1荷瘤小鼠在接受放疗和分选外泌体的联合治疗后的生存曲线。
图64为EMT6荷瘤小鼠的放化疗及分选外泌体注射方案和瘤径曲线。
图65为MCF7荷瘤小鼠的化疗及分选外泌体注射方案和瘤径曲线。
图66为临床病人的乳腺癌切片中SETD4细胞比例与TNM分期的对应关系。a为临床病人的样本信息，b为临床分期Ⅰ、Ⅱ和Ⅲ的病人乳腺癌切片中的SETD4细胞比例。
图67为从临床乳腺癌病人样品中获得休眠肿瘤细胞，并且利用MCF7含DEK-GFP蛋白的分选外泌体结合化疗将其杀死的数据。a为从临床乳腺癌病人样品中获得抵抗放化疗的休眠肿瘤细胞，标尺，50μm。b为在休眠肿瘤细胞中加入MCF7含DEK-GFP蛋白的外泌体使其激活，DAPI表示细胞核染色，标尺，50μm。c为在休眠肿瘤细胞中加入MCF7含DEK-GFP蛋白的外泌体将其杀死，标尺，50μm。
图68为在各种人肿瘤细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞的数据。a为在肺癌H226细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞，标尺，50μm。b为在胃癌MKN45细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞，标尺，50μm。c为在前列腺癌PC-3细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞，标尺，50μm。d为在宫颈癌HeLa细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞，标尺，50μm。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
本发明实施例中百分浓度除特别说明外均为体积浓度。
本发明所用磷酸缓冲液(PBS)，配方：终浓度137mM氯化钠、终浓度2.7mM氯化钾、终浓度10mM的十二水合磷酸氢二钠和终浓度1.76mM磷酸二氢钾，溶剂为水，pH调至7.4。
本发明实施例中免疫印迹试验所用一抗及相应二抗：
表1、蛋白对应的一抗和二抗
Figure PCTCN2022083977-appb-000001
实施例1、小鼠移植瘤的获取
1、细胞系
MCF7细胞系，人乳腺癌细胞，分子表征为luminal A型，购自中国科学院典型培养物保藏委员会细胞库，目录号为TCHu74，细胞培养基是EMEM培养基(Eagle's  minimum Essential Medium，Corning，目录号:10-009-cv)中添加10％的胎牛血清和1％的青霉素-链霉素。
4T1细胞系，典型的三阴性乳腺癌细胞，特征类似于临床Ⅳ期的乳腺癌，购自美国菌种保藏中心(ATCC)，目录号为CRL-2539，细胞培养基是DMEM培养基(Dulbecco’s Modification of Eagle's Medium，Corning，目录号:R10-013-cv)中添加10％的胎牛血清和1％的青霉素-链霉素。
EMT6细胞系，小鼠乳腺癌细胞，购自美国菌种保藏中心(ATCC)，目录号为CRL-2755，细胞培养基是Waymouth’s培养基(Gibco，目录号:31220072)中添加体积浓度15％的胎牛血清和1％的青霉素-链霉素。
从液氮罐中取出第五代4T1、EMT6和MCF7细胞的冻存管各1支，37℃水浴3分钟后，1000rpm 25℃离心5分钟，去掉上清，分别加入1mL上述的培养基重悬，重悬液分别接种到含9mL培养基的直径10cm的细胞培养皿中。将细胞培养皿培养在37℃，5％CO 2的湿润环境中，贴壁细胞的光镜照片见图2。
2、小鼠移植瘤的获取
MCF7细胞选取6-8周龄的Nod/Scid雌鼠(购自上海斯莱克实验动物有限责任公司)作为接种对象。1000万MCF7细胞中加入1mL含2500U/mL胰蛋白酶和0.02％乙二胺四乙酸(EDTA)的磷酸缓冲液(PBS)，25℃静置孵育40秒，加入0.5mL胎牛血清中和，将壁上的细胞吹打下来，1000rpm 25℃离心5分钟，去上清。取底部的0.5g细胞沉淀用1mL体积比1:1的EMEM培养基(Corning，目录号:10-009-cv)与基质胶(Corning BioCoat，目录号:354234)的混合液重悬，获得1mL含1000万MCF7细胞的悬液。采用1mL的微量注射器将MCF7细胞悬液原位注射到Nod/Scid雌鼠腋下的乳腺脂肪垫中，每只Nod/Scid鼠接种100万颗MCF7细胞。
4T1细胞选取6-8周龄的BALB/c雌鼠(北京维通利华实验动物技术有限公司)作为接种对象。1000万4T1细胞中加入1mL含2500U/mL胰蛋白酶和0.02％EDTA的PBS，25℃静置孵育40秒，加入0.5mL胎牛血清中和，将壁上的细胞吹打下来，1000rpm 25℃离心5分钟，去上清。取底部的0.5g细胞沉淀用1mL的DMEM培养基重悬，获得1mL含1000万4T1细胞的悬液。采用1mL的微量注射器将4T1细胞悬液原位注射到BALB/c雌鼠腋下的乳腺脂肪垫中，每只BALB/c雌鼠接种100万颗4T1细胞。
EMT6细胞选取6-8周龄的BALB/c雌鼠(北京维通利华实验动物技术有限公司) 作为接种对象。400万EMT6细胞中加入0.5mL含2500U/mL胰蛋白酶和0.02％EDTA的PBS，25℃静置孵育40秒，加入0.25mL胎牛血清中和，将壁上的细胞吹打下来，1000rpm 25℃离心5分钟，去上清。取底部的0.2g细胞沉淀，用2mL的Waymouth’s培养基重悬，获得2mL含400万EMT6细胞的悬液。采用1mL的微量注射器将EMT6细胞悬液原位注射到BALB/c雌鼠腋下的乳腺脂肪垫中，每只BALB/c雌鼠接种20万颗EMT6细胞。
所有的小鼠都饲养在无菌的环境中，水和鼠粮持续供应，垫料及时更换，饲养至肿瘤体积达到500mm 3左右时，安乐死小鼠，用于下一步的获取实体瘤。小鼠实验均通过动物实验的伦理审查，实验者在进行实验时遵守实验动物的福利伦理原则。
实施例2、抵抗放化疗的休眠肿瘤细胞的获取和鉴定
1、实体瘤中获取抵抗放化疗的休眠肿瘤细胞
将实施例1中三种荷瘤小鼠安乐死，手术取出实体瘤，光镜照片见图3。先用剪刀将瘤子物理切割成小块，再进一步剪成碎末，将1.5g瘤子碎末放入30mL消化液(DMEM培养基中含有1250U/mL的胰蛋白酶，0.01％EDTA，2000U/mL胶原酶Ⅳ)中，37℃水浴20分钟，每隔5分钟手动摇匀。消化后的细胞液通过40μm的尼龙膜过滤，取滤液，即为从实体瘤中消化下来的细胞，光镜照片见图4。
上述实体瘤中消化下来的细胞按80万颗/孔的密度接种在超低吸附的六孔盘(品牌：Corning，货号:3471)中。每孔加入3mL含有10％血清替代物(Thermo Fisher Scientific Cat#10828028)，终浓度100nM紫杉醇和终浓度1mM的5-氟尿嘧啶的DMEM/F12培养基(Corning，目录号：10-092-cv)，在37℃培养1个月，每隔3天换一次培养液，前两周中每周照射30Gy X射线一次，总共照射2次(两次间隔7天)，每次照射10分钟。一个月后，使用死细胞去除试剂盒(Miltenyi Biotec，目录号：130-090-101)筛选出抵抗放化疗的肿瘤细胞，即休眠肿瘤细胞(DCCs)，分别获得休眠肿瘤细胞4T1-DCCs、EMT6-DCCs、MCF7-DCCs，光镜照片见图5。
2、休眠肿瘤细胞的特征鉴定
(1)免疫印迹实验检测PCNA、H3S10ph和SETD4
分别将步骤1中得到3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)从培养基中分离出来，在每种细胞各400万颗中加入0.2mL细胞裂解液(50mM Tris-HCl(pH 7.4)，150mM NaCl，1％乙基苯基聚乙二醇(NP-40)，0.1％十 二烷基硫酸钠(SDS))，冰上放置10分钟，12000rpm 4℃离心10分钟，将上清转移到新的离心管中，加入0.05mL的蛋白上样缓冲液(250mM三羟甲基氨基甲烷盐酸盐，pH 6.8；0.1g/ml十二烷基硫酸钠；0.005g/ml溴酚蓝；50％甘油和0.05g/mlβ-巯基乙醇，溶剂为水)，沸水浴10分钟，12000rpm 25℃离心10分钟，取上清，即为蛋白样品，转至新的离心管中用于后续的上样。取3μL蛋白样品进行聚丙烯酰胺凝胶电泳，将凝胶中的蛋白转移到PVDF膜(聚偏二氟乙烯膜)上(转膜液：25mM三羟甲基氨基甲烷，pH 8.3，192mM甘氨酸和10％甲醇，溶剂为水)。TBS缓冲液(三羟甲基氨基甲烷12.1g和氯化钠17.5g溶到1500mL蒸馏水中，滴加盐酸将pH调至7.4，蒸馏水定容至2000mL)洗1遍，5分钟；封闭液(品牌：Roche,货号:11921681001)封闭1小时。分别将Mouse monoclonal anti-PCNA(abcam，目录号:ab29)，Rabbit monoclonal anti-H3S10ph(Cell signaling technology，目录号:53348)和Mouse monoclonal anti-SETD4Santa Cruz，目录号:sc-134221)的抗体各2μg加入到2mL的封闭液中配置成一抗稀释液，再将PVDF膜完全浸泡在所述稀释液中，4℃孵育过夜。第二天，PVDF膜用含0.1％吐温的TBS洗4遍，每遍7分钟。将16.7μL的表1对应二抗加入到50mL的封闭液中配置成二抗稀释液，将PVDF膜完全浸泡在二抗稀释液中，25℃孵育40分钟。PVDF膜用含0.1％吐温的TBS洗4遍，每遍7分钟。在PVDF膜中加入辣根过氧化氢酶底物Clarity Max Western ECL Substrate(BIO-RAD，货号：1705062)，浸没处理，用显影仪检测。免疫印迹实验发现，3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)均表达极低水平的细胞分裂指标蛋白PCNA和H3S10ph，但是高量表达细胞静息蛋白SETD4，免疫印迹图见图6。
(2)免疫荧光检测ALDH1、CD44和CD24
分别将步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)用4％的多聚甲醛固定。固定后的细胞悬液分别滴在粘附性的载玻片上。待细胞稳定附着后，用PBS洗1遍，5分钟。将载玻片分别浸入含0.25％Triton-100的PBS中25℃孵育10分钟，PBS洗1遍，5分钟。再将载玻片分别浸入含1.5％驴血清的PBS(封闭液)中25℃孵育1小时。将Rabbit monoclonal anti-ALDH1A1(abcam，目录号:ab52492)、Rat monoclonal anti-CD44,FITC(eBiosciences，目录号：11-0441-82)和Alexa
Figure PCTCN2022083977-appb-000002
647 anti-CD24(BioLegend，目录号：311109)抗体各2μg加入到200μL封闭液(品牌：Roche,货号:11921681001)中配置成一抗稀释液， 将一抗稀释液滴加到载玻片中的细胞样品上，浸没即可，4℃孵育过夜。第二天，PBS洗3遍，每次5分钟。将1μg Alexa Fluor 594荧光标记驴抗兔(Thermo Fisher scientific，货号R37119)的抗体加入到200μL的封闭液(品牌：Roche,货号:11921681001)中配置成二抗稀释液，将二抗稀释液滴加到ALDH1的载玻片中的细胞样品上，浸没即可，25℃孵育2小时。将200μL细胞核染色DAPI(碧云天，目录号:C1005)滴加到载玻片中的细胞样品上，浸没即可，25℃孵育10分钟。用50％甘油封片。样品用荧光显微镜进行观察，发现上述细胞均表达高水平的ALDH1和CD44及低水平的CD24(图7)，表明分离获得的休眠肿瘤细胞为肿瘤干细胞。
实施例3、肿瘤细胞培养液来源粗外泌体的制备和鉴定
1、肿瘤细胞培养液的获取
500万颗的MCF7细胞系接种到90mm 3的细胞培养皿中，培养基是10mL添加1％的青霉素-链霉素的EMEM培养基(同实施例1)。37℃培养24小时后收集细胞的培养液，1000rpm 4℃离心10分钟，取上清，12000rpm 4℃离心20分钟，取上清，即为MCF7细胞培养液。
500万颗的4T1细胞系接种到90mm 3的细胞培养皿中，培养基是10mL添加1％的青霉素-链霉素的DMEM培养基(同实施例1)。37℃培养24小时后收集细胞的培养液，1000rpm 4℃离心10分钟，取上清，12000rpm 4℃离心20分钟，取上清，即为4T1细胞培养液。
500万颗的EMT6细胞系接种到90mm 3的细胞培养皿中，培养基是10mL添加1％的青霉素-链霉素的Waymouth’s培养基(同实施例1)。37℃培养24小时后收集细胞的培养液，1000rpm 4℃离心10分钟，取上清，12000rpm 4℃离心20分钟，取上清，即为EMT6细胞培养液。
2、粗外泌体的制备
在20mL步骤1中获得的MCF7、4T1和MCF7细胞培养液中分别加入1mL的外泌体分离试剂(品牌：Thermo Fisher，货号：4478359)，上下颠倒3次混匀后4℃孵育过夜。第二天，上述混合液10000rpm 4℃离心60分钟，去掉上清，用200μL的PBS重悬离心管底部的沉淀(沉淀即为粗外泌体)，利用BCA蛋白质定量检测试剂盒(品牌：生工，货号:C503021)测定粗外泌体悬液中的蛋白总量，用PBS配制蛋白浓度为20μg/mL的粗外泌体溶液，用于后续的使用。
3、粗外泌体的鉴定
(1)粗外泌体粒径分布
使用粒径检测仪器(型号:ZetaView,厂家:Particle Metrix)对步骤2中获得的20μg/mL粗外泌体溶液进行检测，发现粗外泌体的粒径范围均为50-300nm，主体粒径大小均在150nm附近(图8)。
(2)粗外泌体形态特征
将2μL的步骤2中获得的20μg/mL粗外泌体溶液滴加到铜网上并覆盖铜网表面(直径3.05mm，孔径90μm，厚度18μm，200目)，25℃孵育10秒钟，滤纸吸除多余的粗外泌体。将2μL含2％醋酸双氧铀的水溶液滴加到铜网上，25℃孵育10秒钟，滤纸吸除多余的醋酸双氧铀。将铜网放在40℃中烘干5分钟后，放入透射电子显微镜(型号:JEM-1200EX,厂家：NEC)中，电压调节到120kV，观察到双层膜结构的茶托状囊泡，符合外泌体的形态特征(图9)。
(3)免疫印迹检测粗外泌体中DEK、CD9、CD81和CD63
使用Rabbit polyclonal anti-DEK(Proteintech,目录号:16448-1-AP)、Rabbit monoclonal anti-CD9(Abcam，目录号：ab92726)、Rabbit monoclonal anti-CD81(Abcam，目录号：ab109201)和Mouse monoclonal anti-CD63(Abcam，目录号：ab59479)的抗体，对步骤2中获得的20μg/mL粗外泌体溶液开展了DEK和外泌体通用的指标分子CD9、CD81和CD63的免疫印迹分析(方法同实施例2步骤2)，发现粗外泌体表达高水平的DEK蛋白和CD9、CD81和CD63蛋白(图10)。以上的结果逐一验证了粗外泌体的大小、形态和分子特征，并且说明粗外泌体中含有DEK蛋白。
实施例4、肿瘤细胞培养液来源粗外泌体激活休眠肿瘤细胞
1、粗外泌体激活休眠肿瘤细胞
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL PBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体溶液使其在培养基中的终浓度50ng/mL；37℃培养20小时后收集细胞，使用Rabbit polyclonal anti-DEK(Proteintech,目录号:16448-1-AP)、Mouse monoclonal anti-PCNA(abcam，目录号:ab29)，Rabbit monoclonal anti-H3S10ph(Cell signaling technology，目录号:53348)和Mouse monoclonal anti-SETD4(Santa Cruz，目录号:sc-134221)的抗体，以H3和GAPDH为内参，开展了免疫印迹分析(方法同实施 例2步骤2)，发现DEK，PCNA和H3S10ph的水平上升，SETD4的水平下降(图11)。研究结果表明外源添加粗外泌体能够激活休眠肿瘤细胞。将上述收集的细胞记为激活的休眠肿瘤细胞(A-DCCs)，分别获得激活的休眠肿瘤细胞4T1A-DCCs、EMT6A-DCCs、MCF7A-DCCs。
2、免疫荧光检测激活的休眠肿瘤细胞ALDH1、CD44和CD24
将上述获得的激活的休眠肿瘤细胞开展ALDH1、CD44和CD24的免疫荧光分析(同实施例2步骤2)，同样发现表达高水平的ALDH1和CD44及低水平的CD24(图12)。
实施例5、激活后的休眠肿瘤细胞具有体外成球和体内成瘤的能力
1、体外肿瘤球形成的能力检测
将实施例4中获取的激活的休眠肿瘤细胞(4T1A-DCCs、EMT6A-DCCs、MCF7A-DCCs)通过流式细胞分选仪，按1颗/孔的密度接种到超低吸附96孔板(品牌:Corning,货号:3469a)中。每个孔中加入200μL含10％血清替代物的DMEM/F12培养基，37℃连续培养三周，发现该细胞表现出极高的体外肿瘤球形成能力(图13)。2、小鼠体内成瘤的能力检测
分别将100颗、10颗和1颗实施例4中获取的激活的休眠肿瘤细胞(4T1A-DCCs、EMT6A-DCCs、MCF7A-DCCs)稀释在DMEM培养基和基质胶(Corning BioCoat，目录号:354234)以体积比1:1的混合液100μL中，即为不同细胞含量的细胞液。将100μL的各细胞液原位注射到6-8周龄的Nod/Scid雌鼠腋下的乳腺脂肪垫中(EMT6和4T1各个细胞含量分别注射5只，MCF7中100颗注射5只，10颗注射4只，1颗注射2只)，将小鼠饲养在无菌的环境中，水和鼠粮持续供应，垫料及时更换。饲养时间在半年以内，瘤子的直径到达伦理上限时安乐死小鼠，手术取出肿瘤部位进行拍照(图14)，发现该细胞表现出极强的小鼠体内成瘤的能力。
实施例6、实体瘤内休眠及激活的休眠肿瘤细胞的鉴定
1、休眠肿瘤细胞
将实施例2方法获得的100万颗4T1-DCCs和100万颗EMT6-DCCs细胞分别原位接种至8周BALB/c雌鼠的脂肪垫中，将100万颗MCF7-DCCs细胞原位接种至8周Nod/Scid雌鼠脂肪垫中。待瘤子体积达到500mm 3左右时，分别安乐死小鼠，手术取出4T1-DCCs、EMT6-DCCs和MCF7-DCCs肿瘤。将上述肿瘤分别放入4％多聚甲醛中4℃过夜，4℃浸泡到质量浓度30％蔗糖水溶液中脱水48小时，浸泡后 的肿瘤捞出，放到10x10x5mm的敞开长方体型塑料材质的模具中，往其中加满OCT包埋剂(SAKURA，目录号：4583)，将模具放在干冰上静置5分钟，将包埋块取出，-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切片。肿瘤切片用PBS洗1遍，5分钟。放入含0.25％Triton-100的PBS中，25℃孵育10分钟。放入含1.5％驴血清的PBS中25℃孵育1小时。按实施例2步骤2方法分别开展Mouse monoclonal anti-ALDH1A1(BD Pharmingen,目录号:611195)抗体、Rat monoclonal anti-Ki67(eBioscience,目录号:14-5698-82)抗体和Rabbit polyclonal anti-SETD4(Sigma-Aldrich,目录号:HPA024073)抗体以及Mouse monoclonal anti-ALDH1A1(BD Pharmingen,目录号:611195)抗体、Rat monoclonal anti-Ki67(eBioscience,目录号:14-5698-82)抗体和Rabbit polyclonal anti-DEK(Proteintech,目录号:16448-1-AP)抗体的免疫荧光分析，Alexa Fluor 488荧光标记驴抗鼠免疫球蛋白(Thermo Fisher scientific，货号A21202)对应ALDH1，Alexa Fluor 594荧光标记羊抗大鼠免疫球蛋白(Thermo Fisher scientific，货号A11007)对应Ki67，Alexa Fluor 647荧光标记驴抗兔免疫球蛋白(Thermo Fisher scientific，货号A31573)对应SETD4和DEK样品，用荧光显微镜进行观察。结果发现ALDH1阳性且Ki67阴性的肿瘤细胞中，SETD4高表达且DEK低表达(图15)，说明DEK在这一类静息的肿瘤细胞中低表达。
2、激活肿瘤细胞
将步骤1中4T1-DCCs、EMT6-DCCs、MCF7-DCCs分别替换为实施例4方法获得的4T1A-DCCs、EMT6A-DCCs、MCF7A-DCCs，其他操作相同。
ALDH1阳性且Ki67阳性的肿瘤细胞中，SETD4低表达且DEK高表达(图16)，说明DEK在这一类激活的肿瘤细胞中高表达。
实施例7、激活后的休眠肿瘤细胞DEK蛋白显著上升，SETD4蛋白显著下降
采用实施例2步骤2的方法，使用Rabbit polyclonal anti-DEK(Proteintech，目录号：16448-1-AP)抗体和Mouse monoclonal anti-SETD4(Santa Cruz,目录号:sc-134221)抗体，开展了休眠肿瘤细胞(实施例2步骤1获取)和激活的休眠肿瘤细胞(实施例4获取)的免疫印迹分析(同实施例2)，发现与休眠肿瘤细胞相比，激活的休眠肿瘤细胞高量表达DEK蛋白，但是SETD4表达显著下降(图17)。研究结果表明DEK蛋白对休眠肿瘤细胞的激活有着重要的作用。
实施例8、外源DEK蛋白及结构域NLS突变DEK蛋白的制备
1、制备表达外源DEK蛋白及结构域突变DEK蛋白的杆状病毒
先用引物F3(CCGGAATTCATGGTGAGCA)和引物R3(CGCTCTAGATCAAGAAATTAG)对SEQ ID NO.6和SEQ ID NO.8进行PCR扩增，得到含有酶切位点的人源hEGFP-DEK序列和人源hEGFP-DEK △NLS序列。再对上述的序列和pFastBac TMHT A质粒同时进行EcoR Ⅰ和Xba Ⅰ的双酶切处理和连接处理。
同样地，用引物F4(AGAATTCATGGTGAGCAAGGGCGA)和引物R4(GCTCTAGATCAAGAAATTAGCTCTTTTACAGTTGT)对SEQ ID NO.10和SEQ ID NO.12进行PCR扩增，得到含有酶切位点的鼠源hEGFP-DEK序列和鼠源hEGFP-DEK △NLS序列。再对上述的序列和pFastBac TMHT A质粒同时进行EcoR Ⅰ和Xba Ⅰ的双酶切处理和连接处理。
分别将人源hEGFP-DEK基因(核苷酸序列：SEQ ID NO.6，氨基酸序列：SEQ ID NO.7)、人源hEGFP-DEK △NLS(核定位序列NLS缺失)基因(核苷酸序列：SEQ ID NO.8、氨基酸序列：SEQ ID NO.9)、鼠源mEGFP-DEK基因(核苷酸序列：SEQ ID NO.10，氨基酸序列：SEQ ID NO.11)、鼠源mEGFP-DEK △NLS(核定位序列NLS缺失)基因(核苷酸序列：SEQ ID NO.12，氨基酸序列：SEQ ID NO.13)连入pFastBac TMHT A质粒(购自Thermo Fisher，货号:10712024)的EcoR Ⅰ和Xba Ⅰ的酶切位点(图18)中。
将连入上述基因后的pFastBac TMHT A质粒转化大肠杆菌DH10Bac感受态细胞(购自Gibco,货号：10361012)，42℃水浴45秒。在转化后的大肠杆菌中加入1mL LB培养基(胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，溶剂为水，pH＝7.4)，37℃220rpm摇4小时。1000rpm离心5分钟，去掉上清，加入100μL LB培养基重悬。100μL重悬液涂布至含有终浓度30μL/mL卡那霉素、终浓度15μL/mL庆大霉素、终浓度12μL/mL四环霉素、40μL(20mg/ml)X-gal(5-溴-4-氯-3-吲哚-β-D-吡喃半乳糖苷，溶剂为二甲基甲酰胺)，4μL(200mg/ml)IPTG(异丙基-β-D-硫代半乳糖苷，溶剂为超蒸水)的LB固体培养基(LB培养基中加入15g/L琼脂)中，37℃避光培养48小时，挑选其中的白色单菌落接种到2mL的LB培养基中，37℃220rpm摇过夜。第二天用试剂盒(购自Invitrogen,货号：K210002)进行质粒抽提，得到重组杆状病毒质粒(Bacmid)。将重组Bacmid利用Cellfectin转染试剂(Gibco,货号:10362100)转染至Sf9细胞(ATCC来源，货号：CRL-1711)中。转染后的细胞接种在Sf-900培养基(Gibco,货号:10902179)中，27℃培养72小时，收集细胞培养液， 10000xg离心20分钟，取上清为病毒液，分别获得携带人源hDEK-GFP基因、鼠源mDEK-GFP基因、人源hDEK △NLS-GFP基因、鼠源mDEK △NLS-GFP基因的重组杆状病毒。
2、外源DEK蛋白及结构域突变DEK蛋白的表达和纯化
Sf9细胞按照50万/mL的密度接种到100ml的Sf-900培养基(Gibco,货号:10902179)中，27℃培养72小时，加入10mL滴度为10 8pfu/mL的上述重组杆状病毒(携带人源DEK-GFP、鼠源DEK-GFP或人源DEK ΔNLS-GFP、鼠源DEK ΔNLS-GFP基因，共4个基因序列)，27℃培养5天，1200rpm离心10分钟，去掉上清，细胞沉淀用裂解液(50mM磷酸二氢钠和300mM氯化钠溶于双蒸水中，pH＝8.0)重悬。重悬后的溶液进行超声处理(39瓦特，10秒超声，50秒停顿，超声时长3分钟)。超声后的溶液12000rpm 4℃离心20分钟，取上清即为蛋白溶液。利用His标签蛋白纯化试剂盒(碧云天，货号：P2226)对上述的蛋白溶液进行纯化，纯化后的蛋白溶于PBS中，制备成20μg/mL的蛋白溶液，即外源DEK蛋白的PBS溶液(mDEK-GFP、hDEK-GFP)、NLS突变DEK蛋白的PBS溶液(hDEK ΔNLS-GFP、mDEK ΔNLS-GFP)。考马斯亮蓝染色发现蛋白的纯度较高，蛋白大小正确(图19)。
实施例9、外源DEK蛋白的添加激活休眠肿瘤细胞
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL，使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL。其中4T1-DCCs和EMT6-DCCs中各自分别加入mDEK-GFP和mDEK ΔNLS-GFP，MCF7-DCCs中分别加入hDEK-GFP和hDEK ΔNLS-GFP，37℃培养20小时后收集细胞。取部分细胞，使用Rabbit polyclonal anti-DEK(Proteintech,目录号:16448-1-AP)，Mouse monoclonal anti-PCNA(abcam，目录号:ab29)，Rabbit monoclonal anti-H3S10ph(Cell signaling technology，目录号:53348)和Mouse monoclonal anti-SETD4(Santa Cruz，目录号:sc-134221)的抗体，开展了免疫印迹分析(方法同实施例2步骤2，二抗见表1)，发现DEK，PCNA和H3S10ph的水平上升，SETD4的水平下降(图20)。研究结果表明外源添加DEK蛋白能够激活休眠肿瘤细胞，将其记为外源DEK蛋白激活的休眠肿瘤细胞，分别获得外源DEK蛋白激活的4T1A-DCCs、EMT6A-DCCs、MCF7A-DCCs。
实施例10、结合化疗，外源DEK蛋白添加能够清除休眠肿瘤细胞
1、DEK蛋白结合化疗对休眠肿瘤细胞cCasp3信号的影响
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL，使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL，其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEK ΔNLS-GFP，MCF7-DCCs中分别添加hDEK-GFP和hDEK ΔNLS-GFP。37℃培养20小时后收集细胞，使用切割的Rabbit monoclonal anti-cleaved Caspase-3(abcam,货号：ab32042)抗体开展免疫荧光实验(具体操作同实施例2步骤2，二抗更替为Alexa Fluor 594荧光标记驴抗兔，Thermo Fisher，货号：R37119)。样品用荧光显微镜进行观察，发现cCasp3的信号大量出现，细胞出现大量的凋亡(图21)。
2、DEK蛋白结合化疗处理不同时间对休眠肿瘤细胞的影响
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL，使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL，其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEK ΔNLS-GFP，MCF7-DCCs中分别添加hDEK-GFP和hDEK ΔNLS-GFP。37℃培养0小时、5小时、20小时、25小时、30小时后收集细胞样品，台盼蓝染色检测死亡的细胞，发现原本抵抗放化疗的休眠肿瘤细胞逐渐全部死亡(图22)。
以上的结果表明结合化疗，外源DEK蛋白添加能够清除休眠肿瘤细胞。
实施例11、在激活的休眠肿瘤细胞中干涉DEK使细胞重新进入静息状态
1、制备表达靶向DEK的小发卡RNA(shRNA)慢病毒
(1)序列
靶向人源DEK的干涉序列1：GGATAGTTCAGATGATGAAC，SEQ ID NO.14，记为shDEK#H1；
靶向人源DEK的干涉序列2：GTGATGAAGATGAAAAGAAA，SEQ ID NO.15， 记为shDEK#H2；
靶向鼠源DEK的干涉序列1：GTGAAGAAATTACTGGCTGAT，SEQ ID NO.16，记为shDEK#M1；
靶向鼠源DEK的干涉序列2：CGAACTCGTGAAGAGGATCTT，SEQ ID NO.17，记为shDEK#M2；
杂乱的干涉序列：ATGTTAACAGCTGTACTGGTG，SEQ ID NO.18，记为shCTRL。
(2)重组慢病毒表达载体
将5段靶向DEK的shRNA序列分别插入到pLent-U6-RFP-Puro慢病毒表达载体(维真生物，货号：LT88024，图23)的BamH Ⅰ和Mlu Ⅰ位点上，分别获得含人源DEK1(SEQ ID NO.14)重组慢病毒表达载体，含人源DEK2(SEQ ID NO.15)重组慢病毒表达载体，含鼠源DEK1(SEQ ID NO.16)重组慢病毒表达载体，含鼠源DEK2(SEQ ID NO.17)重组慢病毒表达载体和含杂乱序列(SEQ ID NO.18)重组慢病毒表达载体。
(3)靶向DEK的shRNA慢病毒
将上述含人源DEK1(SEQ ID NO.14)重组慢病毒表达载体和慢病毒包装质粒混合物(pMDL，VSVG，pRSV-Rev，质量比例为5:3:2，购自上海翊圣生物，货号：41102ES10)共同转染293T细胞(ATCC，货号：ACS-4500)，具体为：取一个洁净无菌离心管，加入750μL不含抗生素和血清的DMEM培养液，分别加入4.5μg慢病毒包装质粒混合物和1.5μg含人源DEK1(SEQ ID NO.14)重组慢病毒表达载体，并用枪轻轻吹打混匀，再加入24μl脂质体转染试剂Lipo8000(Beyotime，目录号：C0533)，用枪轻轻吹打混匀，获得转染试剂和质粒的混合物。将转染试剂和质粒的混合物750μL均匀滴加到293T细胞中，37℃培养72小时。转染72小时后收集细胞培养上清，即为病毒液。取6个SW28离心管，每个SW28离心管中加入约32ml的病毒液。取6支10ml的移液管，分别吸取4ml质量浓度20％的蔗糖水溶液，将移液管一直插入到每支SW28离心管的底部，缓慢将蔗糖水溶液打出4ml。用PBS调整各管的重量，使对应的SW28离心管之间的重量相差不超过0.1g。按次序将所有6个SW28离心管放入Beckman SW28超速离心转头中，4℃，25000rpm离心2小时。小心将SW28管子从转头中取出。倒掉上清，将SW28离心管倒扣在纸巾上放置10分钟使剩余的上清流干。吸掉剩余的液滴。每管中加入1ml不含钙和镁的PBS重悬管 底的沉淀。将SW28离心管插入到50ml锥底离心管中，盖上盖子，4℃溶解2小时，每隔20分钟轻轻震荡。4℃，500rpm离心1分钟，使溶液集中于管底。用200μl移液器轻柔吹打使沉淀重悬，避免产生泡沫。将所有管中的液体集中到一个新的离心管中，即为浓缩纯化后的病毒液6ml，记为慢病毒shDEK#H1(即人源DEK干涉序列1)。使用试剂盒(GeneCopoeia，货号：LT005)进行病毒滴度的测定，病毒滴度为10 8pfu/mL。
将含人源DEK1(SEQ ID NO.14)重组慢病毒表达载体分别替换为含人源DEK2(SEQ ID NO.15)重组慢病毒表达载体，含鼠源DEK1(SEQ ID NO.16)重组慢病毒表达载体，含鼠源DEK2(SEQ ID NO.17)重组慢病毒表达载体和含杂乱序列(SEQ ID NO.18)重组慢病毒表达载体，其他操作相同。最终分别获得靶向DEK的shRNA慢病毒shDEK#H1，shDEK#H2(人源DEK干涉序列2，6ml，病毒滴度为10 8pfu/mL)，shDEK#M1(鼠源DEK干涉序列2，6ml，病毒滴度为10 8pfu/mL)，shDEK#M2(鼠源DEK干涉序列2，6ml，病毒滴度为10 8pfu/mL)，shCTRL(干涉对照6ml，病毒滴度为10 8pfu/mL)。
2、在激活的休眠肿瘤细胞中干涉DEK使细胞重新进入静息状态
(1)干涉DEK使休眠肿瘤细胞重新进入静息状态
分别将实施例4获取的3种激活的休眠肿瘤细胞(4T1A-DCCs、EMT6A-DCCs、MCF7A-DCCs)以10万颗细胞/孔的密度接种到低贴6孔板中，培养基为3mL的DMEM/Ham's F-12，37℃培养2小时后，在每个孔中加入终浓度6μg/mL的聚凝胺(polybrene)；在4T1A-DCCs和EMT6A-DCCs培养孔中各自分别加入10μL 10 8pfu/mL的步骤1制备的慢病毒shDEK#M1、shDEK#M2、shCTR，同时不添加慢病毒作为未处理。在MCF7A-DCCs培养孔中分别加入10μL 10 8pfu/mL的步骤1制备的慢病毒shDEK#H1、shDEK#H2、shCTRL，同时不添加慢病毒作为未处理。37℃培养24小时后更换成新的培养基，继续培养72小时，收集细胞(记为干涉DEK后的细胞)，开展Rabbit polyclonal anti-DEK、Rabbit monoclonal anti-H3S10ph、Mouse monoclonal anti-PCNA和Mouse monoclonal anti-SETD4抗体的免疫印迹实验(方法同实施例2步骤2，二抗见表1)，发现Ki67，PCNA和H3S10ph的水平下降，SETD4的水平上升(图24中a)。
(2)干涉DEK后的休眠肿瘤细胞成瘤能力受到抑制
将上述步骤(1)干涉DEK后的细胞1万颗接种到3mL含10％血清替代物的 DMEM/Ham's F-12培养基中，37℃培养一周，发现形成肿瘤球的能力受到抑制(图24中b)。
以上结果表明在激活的休眠肿瘤细胞中干涉DEK使细胞重新进入静息状态。
3、DEK干涉导致的休眠肿瘤细胞中添加外源DEK可重新激活休眠肿瘤细胞
(1)分别将步骤2中慢病毒shDEK#M1和shCTRL干涉DEK后的4T1A-DCCs细胞按4000颗细胞/孔的密度接种到5mL含10％血清替代物的DMEM/Ham's F-12培养基中。shDEK#M1干涉DEK后的4T1A-DCCs分别添加12.5μL的PBS、12.5μL 20μg/mL实施例8步骤2中制备的mDEK-GFP的PBS溶液和12.5μL 20μg/mL实施例8步骤2中制备的mDEK ΔNLS-GFP的PBS溶液，使mDEK-GFP和mDEK ΔNLS-GFP在培养基中的终浓度均为50ng/mL；shCTRL干涉DEK后的4T1A-DCCs细胞不添加PBS或蛋白。37℃培养20小时后收集细胞(记为鼠源干涉序列1+mDEK-GFP、鼠源干涉序列1+mDEK △NLS-GFP、鼠源干涉序列1+PBS、shCTRL)，开展DEK-GFP、DEK、H3S10ph、PCNA和SETD4抗体的免疫印迹实验(方法同实施例2步骤2，一抗和二抗见表1)。同样条件下，对步骤2中慢病毒shDEK#M2和shCTRL干涉DEK后的EMT6A-DCCs细胞(mDEK-GFP和mDEK ΔNLS-GFP)，shDEK#H2和shCTRL干涉DEK后的MCF7A-DCCs细胞(hDEK-GFP和hDEK ΔNLS-GFP)进行免疫印迹实验。
发现PCNA和H3S10ph的水平回升，SETD4的水平回降(图25)。
(2)将上述步骤(1)重新加入DEK蛋白的shDEK干涉细胞(即鼠源干涉序列1+mDEK-GFP的4T1A-DCCs和EMT6A-DCCs细胞、人源干涉序列1+mDEK-GFP的MCF7A-DCCs细胞)1万颗接种到3mL含10％血清替代物的DMEM/Ham's F-12培养基中，37℃培养一周，体外成球试验的光镜照片和成球率的统计图发现形成肿瘤球的能力得到恢复(图26)。
以上结果表明DEK干涉导致的休眠肿瘤细胞中添加外源DEK可重新激活休眠肿瘤细胞。
实施例12、外源DEK蛋白激活休眠肿瘤细胞的调控机制
1、外源DEK蛋白引起异染色质的下降，常染色质的上升
(1)将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL的PBS、实施例8步骤2中制备的20μg/mL的外源 DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL，使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL，其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEK ΔNLS-GFP，MCF7-DCCs中分别添加hDEK-GFP和hDEK ΔNLS-GFP。37℃培养20小时后收集细胞。将细胞加入2.5％的戊二醛水溶液中4℃固定过夜。第二天，倒掉固定液，用PBS漂洗样品三次，每次15分钟。用1％的锇酸水溶液25℃孵育样品1-2h。取出锇酸废液，用PBS漂洗样品三次，每次15分钟。用梯度浓度(30％，50％，70％，80％，90％和95％)的乙醇水溶液25℃孵育样品各15分钟。100％乙醇25℃孵育样品20分钟。纯丙酮25℃孵育样品20分钟。Spurr包埋剂(二氧化乙烯环己烯、聚丙烯乙二醇的二缩水甘油醚、壬基琥珀酸酐和二甲基乙醇胺四种物质聚合而成，购自SPI-CHEM公司)与纯丙酮的混合液(V/V＝1/1)25℃孵育样品1小时。Spurr包埋剂与丙酮的混合液(V/V＝3/1)25℃孵育样品3小时。纯Spurr包埋剂25℃孵育样品过夜。第二天，将样品捞出，放在10x10x5mm的敞开长方体型塑料材质的包埋模具(SAKURA，货号：4566)里，模具中倒满新的Spurr包埋剂，放到70℃烘箱中静置过夜。第二天，得到包埋好的样品。样品在LEICA EM UC7型超薄切片机中切片，获得70nm的切片。在切片中滴加柠檬酸铅溶液(21.33g的硝酸铅和1.76g的柠檬酸钠加入到30mL的双蒸水中，用力振荡30分钟，至溶液呈乳白色浑浊状后，加入1mol/L的氢氧化钠水溶液8mL，使溶液变得清亮透明，再加双蒸水定容到50mL)，浸没即可，25℃染色10分钟，滤纸吸除多余的染色液。在切片中滴加醋酸双氧铀50％乙醇饱和溶液(2g醋酸双氧铀加到100mL的50％乙醇中，充分搅拌10分钟，静置1天后取上清液使用)，浸没即可，25℃染色10分钟，滤纸吸除多余的染色液。用透射电镜观察完成染色的切片。发现在休眠肿瘤细胞中添加了PBS或外源NLS突变DEK蛋白后，细胞核内异染色质水平没有显著性的改变，而在休眠肿瘤细胞中添加了外源DEK蛋白后，细胞核内异染色质水平显著下降(图27)。
(2)将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL，使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL，其中 4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEK ΔNLS-GFP，MCF7-DCCs中分别添加hDEK-GFP和hDEK ΔNLS-GFP。37℃培养20小时后收集细胞。使用Mouse monoclonal anti-H4K20me1(Santa Cruz，目录号：sc-134221)、Rabbit polyclonal anti-H4K20me2(abcam，目录号：ab9052)、Rabbit polyclonal anti-H4K20me3(abcam，目录号：ab9053)、Rabbit polyclonal anti-HP1α(Cell Signaling Technology，目录号：2616)、Rabbit polyclonal anti-H3K9ac(abcam，目录号：ab10812)、Rabbit monoclonal anti-H3K9me3(abcam，目录号：ab176916)和Mouse monoclonal anti-H3K27me3(abcam，目录号：ab6002)，在上述细胞中开展免疫印迹分析(方法同实施例2步骤2，H4K20me1和H3K27me3对应偶联辣根过氧化酶驴抗鼠二抗，H4K20me2、H4K20me3、H3K9ac、HP1α和H3K9me3对应偶联辣根过氧化酶驴抗兔二抗)。发现与在休眠肿瘤细胞中添加了PBS或外源NLS突变DEK蛋白相比，在休眠肿瘤细胞中添加了外源DEK蛋白后，组成型异染色质的分子指标H4K20me3和异染色质形成关键蛋白HP1-α的水平显著下降，常染色质的分子指标H3K9ac的水平显著上升，兼性异染色质的分子指标H3K9me3和H3K27me3的水平没有显著性的变化(图28)。
2、DEK蛋白在染色质上结合位点的基因检测
(1)染色质免疫沉淀试验的高通量测序(ChIP-seq)：收集实施例9中的外源DEK蛋白激活的MCF7A-DCCs细胞，使用试剂盒EZ ChIP Kit(Millipore，目录号：17-371)和DEK(Proteintech，目录号：16448-1-AP)抗体开展DEK蛋白的染色质免疫沉淀试验(ChIP-Seq)。1g细胞中加入1mL 4％多聚甲醛(溶于PBS)25℃孵育10分钟。加入1mL 1M甘氨酸(溶于PBS)25℃孵育10分钟。1000rpm离心5分钟，去上清，在细胞沉淀中加入1.5mL裂解液(50mM Tris，pH8.1，1％SDS和0.1μmol/L蛋白酶抑制剂混合物(Sigma-Aldrich，货号：P8340))，吹打均匀，冰上放置10分钟。悬液进行超声(39瓦特，10秒超声，50秒停顿，总超声时长2分钟)。4℃12000rpm离心10分钟，取上清，加入60μL的ChIP Blocked Protein G Agarose(Sigma-Aldrich，货号：16-201D)和3μg Rabbit polyclonal anti-DEK抗体4℃孵育过夜。3000rpm离心2分钟，去掉上清，取沉淀。TE缓冲液(10mM Tris-HCI和1mM EDTA，pH＝8.0)洗3遍，每遍5分钟。洗涤后的沉淀中加入200μL洗脱液(10μL 20％SDS水溶液；20μL 1M碳酸氢钠水溶液和170μL双蒸水)25℃孵育15分钟，3000rpm离心2分钟，将上清转移至新的离心管中。在上清中加入8μL 5M氯化钠水溶液，65℃ 水浴孵育5小时。加入1μL RNase A 37℃水浴孵育30分钟。加入4μL 0.5M EDTA水溶液；8μL 1M Tris-HCl和1μL Proteinase K(Sigma-Aldrich，货号：20-298)，45℃水浴孵育1小时。利用DNA纯化试剂盒(Solarbio，货号：D1300)提取DNA。将获得的DNA构建文库(包括末端修复、加A、加接头、长度筛选、PCR扩增)并进行高通量测序(该操作委托北京诺禾致源科技股份有限公司完成)。
(2)测序结果的分析：DEK结合位点的峰分布在所有23条染色体上(图29)，DEK结合位点的峰主要集中在启动子区域，占64.35％(其中内含子区域占10.5％，基因间区域占11.81％)(图30)。对DEK结合位点的峰对应的基因进行基因本体富集分析(GO富集分析)发现，DEK结合的基因主要与胞内信号转导、个体发育和蛋白结合相关(图31)。对DEK的结合信号进行可视化分析发现，DEK结合在SETD4、TP53和MYC基因的启动子区域上(图32)。
3、外源DEK蛋白引起染色质的开放水平上升
(1)易接近转座酶核染色质区域的高通量测序(ATAC-seq)：收集实施例2步骤1中获得的MCF7-DCCs休眠肿瘤细胞和实施例9中获得的外源DEK蛋白激活的MCF7A-DCCs细胞，开展易接近转座酶核染色质区域的高通量测序(ATAC-Seq)。提取上述两种细胞样品的细胞核，加入转座酶mix，mix中包含转座酶和两种等摩尔的接头Adapter 1和Adapter 2，37℃下孵育30分钟。将产物进行引物扩增、片段长度选择和纯化，获得文库后上机测序，该操作委托北京诺禾致源科技股份有限公司完成。
(2)测序结果的分析：激活后的休眠肿瘤细胞中的整体信号强度显著高于休眠的肿瘤细胞，表明休眠肿瘤细胞在激活后开放的基因区域大幅增加(图33)。将在激活的休眠肿瘤细胞中信号上调的基因进行基因本体富集分析(GO富集分析)，发现上调的基因主要与胞内信号转导、基因表达、细胞周期、代谢过程、生物合成过程、催化活性和激酶活性等生物过程相关(图34)。对休眠肿瘤细胞和外源DEK蛋白激活的休眠肿瘤细胞的ATAC-seq信号进行UCSC基因浏览器可视化分析发现，激活的休眠肿瘤细胞中的SETD4和TP53基因的ATAC信号显著低于休眠肿瘤细胞，激活的休眠肿瘤细胞中的MYC基因的ATAC信号显著高于休眠肿瘤细胞(图35)，说明休眠肿瘤细胞在外源DEK蛋白激活之后，SETD4和TP53基因的开放程度下降，MYC基因的开放程度上升。
4、外源DEK蛋白引起基因表达的变化
(1)转录组的高通量测序(RNA-seq)：收集实施例2步骤1中获得的MCF7-DCCs休眠肿瘤细胞和实施例9中获得的外源DEK蛋白激活的MCF7 A-DCCs细胞，开展转录组的高通量测序(RNA-seq)。采用
Figure PCTCN2022083977-appb-000003
Stranded RNA-Seq Kit(Clontech，货号：634836)，在上述的细胞1g样品中加入1mL TRIzol试剂(Thermo Fisher)，抽提总RNA，通过Oligo(dT)磁珠富集带polyA尾的mRNA。使用Illumina的
Figure PCTCN2022083977-appb-000004
UltraTM RNA Library Prep Kit进行转录组文库的构建，上机测序。使用HISAT2软件将测序片段的序列比对到参考基因组UCSC人类参考基因组hg38上，根据基因比对在参考基因组上的位置信息，统计每个基因从起始到终止范围内覆盖的信号数量，进行基因表达水平的定量分析。进一步对基因表达水平的数据进行统计学分析，筛选不同样本间表达水平显著差异的基因。
(2)测序结果的分析：发现在激活后的休眠肿瘤细胞中，有2119个上调的基因和3338个下调的基因(图36)。对得到的差异基因进行基因本体分析(GO分析)，发现在激活后的休眠肿瘤细胞中，上调的基因主要与细胞激活过程相关(GO期转换成G1期、细胞增殖、细胞转录、细胞呼吸和代谢过程)(图37)，下调的基因主要与细胞静息相关(细胞周期的负调控、甲基化依赖的染色质沉默、p53通路、翻译终止、细胞死亡和蛋白泛素化)(图38)。在基因的表达水平上开展基因集富集分析(GSEA分析)，发现在激活后的休眠肿瘤细胞中，表达水平上调的基因集主要包括DNA复制、G2M检查点、有丝分裂纺锤体、氧化磷酸化、三羧酸循环、E2F靶向基因、MYC靶向基因和脂肪酸代谢(图39)，表达水平下调的基因集主要包括低氧、炎症反应、补体、凝血、上皮间充质转化、p53信号通路、TNFA信号通路、JAK-STAT3信号通路和Kras信号通路。MYC信号通路中有25个基因显著上调，p53信号通路中有30个基因显著下调(图40)。
5、外源DEK蛋白添加引起P53水平的下调和MYC水平的上调
将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL，使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL，其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEK ΔNLS-GFP，MCF7-DCCs中分别添加hDEK-GFP和hDEK ΔNLS-GFP。37℃培养20小时后收集细胞。使用Mouse  monoclonal anti-p53(Santa Cruz，目录号：sc-126)、Rabbit monoclonal anti-p21(Cell Signaling Technology，目录号：2947)、Rabbit polyclonal anti-PUMA(abcam，目录号：ab9643)和Mouse monoclonal anti-c-Myc(Santa Cruz，目录号：sc-40)的抗体，在上述细胞中开展免疫印迹分析(方法同实施例2步骤2，二抗见表1)。发现与休眠肿瘤细胞和休眠肿瘤细胞中添加DEK △NLS-GFP蛋白20小时相比，在休眠肿瘤细胞中添加DEK-GFP蛋白20小时的细胞中，p53、p21和PUMA的水平显著下降，MYC的水平显著上升(图41)。
实施例13、结合化疗，肿瘤细胞培养液来源粗外泌体添加能够清除休眠肿瘤细胞
1、粗外泌体结合化疗检测细胞cCasp3信号
将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL PBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体溶液使其在培养基中的终浓度50ng/mL，37℃培养20小时后收集细胞，使用Rabbit monoclonal anti-cleaved Caspase-3(abcam,货号：ab32042)抗体开展免疫荧光实验(具体操作同实施例2步骤2，二抗更替为Alexa Fluor 594荧光标记驴抗兔，Thermo Fisher，货号：R37119)。样品用荧光显微镜进行观察，发现cCasp3的信号大量出现，细胞出现大量的凋亡(图42)。
2、粗外泌体结合化疗处理时间对休眠肿瘤细胞活力影响
将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL PBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体溶液使其在培养基中的终浓度50ng/mL，37℃培养0小时、5小时、20小时、25小时、30小时后收集细胞样品，台盼蓝染色检测死亡的细胞，发现原本抵抗放化疗的休眠肿瘤细胞逐渐全部死亡(图43)。
以上的结果表明结合化疗，肿瘤细胞培养液来源粗外泌体添加能够清除休眠肿瘤细胞。
实施例14、肿瘤细胞培养液来源粗外泌体激活休眠肿瘤细胞的调控机制
1、粗外泌体的添加引起异染色质水平的下调和常染色质水平的上调
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基 中，每种细胞分别添加25μL PBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体的PBS溶液使其在培养基中的终浓度50ng/mL，37℃培养20小时后收集细胞。使用Mouse monoclonal anti-H4K20me1(Santa Cruz，目录号：sc-134221)、Rabbit polyclonal anti-H4K20me2(abcam，目录号：ab9052)、Rabbit polyclonal anti-H4K20me3(abcam，目录号：ab9053)、Rabbit polyclonal anti-HP1α(Cell Signaling Technology，目录号：2616)、Rabbit polyclonal anti-H3K9ac(abcam，目录号：ab10812)、Rabbit monoclonal anti-H3K9me3(abcam，目录号：ab176916)和Mouse monoclonal anti-H3K27me3(abcam，目录号：ab6002)的抗体，在上述细胞中开展免疫印迹分析(方法同实施例2步骤2，二抗见表1)。发现与在休眠肿瘤细胞中添加PBS相比，在休眠肿瘤细胞中添加了粗外泌体后，组成型异染色质的分子指标H4K20me3和异染色质形成关键蛋白HP1-α的水平显著下降，常染色质的分子指标H3K9ac的水平显著上升，兼性异染色质的分子指标H3K9me3和H3K27me3的水平没有显著性的变化(图44)。
2、粗外泌体的添加引起P53信号通路的下调和MYC信号通路的上调
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物的DMEM/F12培养基中，每种细胞分别添加25μL PBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体PBS溶液使其在培养基中的终浓度50ng/mL，37℃培养20小时后收集细胞。使用Mouse monoclonal anti-p53(Santa Cruz，目录号：sc-126)、Rabbit monoclonal anti-p21(Cell Signaling Technology，目录号：2947)、Rabbit polyclonal anti-PUMA(abcam，目录号：ab9643)和Mouse monoclonal anti-c-Myc(Santa Cruz，目录号：sc-40)的抗体，在上述细胞中开展免疫印迹分析(方法同实施例2步骤2，二抗见表1)。发现与在休眠肿瘤细胞中添加PBS相比，在休眠肿瘤细胞中添加了粗外泌体后，p53、p21和PUMA的水平显著下降，MYC的水平显著上升(图45)。
实施例15、含有外源DEK蛋白及结构域NLS突变DEK蛋白的分选外泌体的制备
1、构建过表达外源DEK蛋白及结构域NLS突变DEK蛋白的质粒
先用引物F1(CCGGAATTCTATGTCCGCCT)和引物R1(CGCTCTAGATCAAGAAATTAG)对SEQ ID NO.5和SEQ ID NO.19进行PCR扩增，得到涵盖酶切位点的人源DEK基因序列。再对上述的序列和pEGFP-C1质粒同时进行EcoR Ⅰ和Xba Ⅰ的双酶切处理和连接处理。
同样地，用引物F2(AGAATTCTATGTCGGCGGCGGCGG)和引物R2(GCTCTAGATCAAGAAATTAGCTCTTTTACAGTTGT)对SEQ ID NO.21和SEQ ID NO.23进行PCR扩增，得到涵盖酶切位点的鼠源DEK基因序列。再对上述的序列和pEGFP-C1质粒同时进行EcoR Ⅰ和Xba Ⅰ的双酶切处理和连接处理。
将人源hDEK基因(核苷酸序列：SEQ ID NO.5，氨基酸序列：SEQ ID NO.1)、人源hDEK △NLS(核定位序列NLS缺失)(核苷酸序列：SEQ ID NO.19，氨基酸序列：SEQ ID NO.20)、鼠源mDEK基因(核苷酸序列：SEQ ID NO.21，氨基酸序列：SEQ ID NO.22)、鼠源mDEK △NLS(核定位序列NLS缺失)基因(核苷酸序列：SEQ ID NO.23，氨基酸序列：SEQ ID NO.24)分别插入到pEGFP-C1质粒(优宝生物，货号：VT1118)(图46)的EcoR Ⅰ和Xba Ⅰ位点上。分别获得重组质粒pEGFP-C1-hDEK，pEGFP-C1-mDEK，pEGFP-C1-hDEK △NLS和pEGFP-C1-mDEK △NLS，即为过表达外源DEK蛋白及结构域NLS突变DEK蛋白的质粒。
2、构建过表达外源DEK蛋白及结构域NLS突变DEK蛋白的慢病毒
先用引物F1(CCGGAATTCTATGTCCGCCT)和引物R1(CGCTCTAGATCAAGAAATTAG)对SEQ ID NO.5和SEQ ID NO.19进行PCR扩增，得到涵盖酶切位点的人源DEK基因序列。再对上述的序列和pLent-N-GFP质粒同时进行EcoR Ⅰ和Xba Ⅰ的双酶切处理和连接处理。
同样地，用引物F2(AGAATTCTATGTCGGCGGCGGCGG)和引物R2(GCTCTAGATCAAGAAATTAGCTCTTTTACAGTTGT)对SEQ ID NO.21和SEQ ID NO.23进行PCR扩增，得到涵盖酶切位点的鼠源DEK基因序列。再对上述的序列和pLent-N-GFP质粒同时进行EcoR Ⅰ和Xba Ⅰ的双酶切处理和连接处理。
将步骤1的hDEK基因、hDEK △NLS基因、mDEK基因、mDEK △NLS基因分别插入到pLent-N-GFP的慢病毒表达载体(维真生物，货号：LT88008)(图47)的EcoRⅠ和Xba Ⅰ位点上。分别获得重组慢病毒表达载体pLent-N-GFP-hDEK，pLent-N-GFP-mDEK，pLent-N-GFP-hDEK △NLS和pLent-N-GFP-mDEK △NLS。采用实施例11方法，将上述重组慢病毒表达载体和慢病毒包装质粒混合物(pMDL，VSVG，pRSV-Rev，质量比例为5:3:2)共同转染293T细胞，转染72小时后收集细胞培养上清即为病毒液，浓缩纯化后进行病毒滴度的测定，获得过表达外源DEK蛋白及结构域NLS突变DEK蛋白的慢病毒，即慢病毒hDEK，mDEK，hDEK △NLS和mDEK △NLS。
3、分选外泌体的获取与鉴定
(1)分选外泌体的制备
1)过表达质粒转染方法：
①分别将实施例2获得的4T1-DCCs、EMT6-DCCs和MCF7-DCCs细胞按照每个10cm细胞培养盘约300万细胞进行接种，加入10mL含10％血清和1％抗生素的DMEM培养液，37℃培养过夜。第二天，倒去原来的培养基，加入新的含10％血清和1％抗生素的DMEM培养液。
②取一个洁净无菌离心管，加入750μL不含抗生素和血清的DMEM培养液，分别加入步骤1制备的15μg重组质粒(pEGFP-C1-hDEK，pEGFP-C1-mDEK，pEGFP-C1-hDEK △NLS或pEGFP-C1-mDEK △NLS)，并用枪轻轻吹打混匀，再加入24μl脂质体转染试剂Lipo8000(Beyotime，目录号：C0533)，用枪轻轻吹打混匀，获得转染试剂和质粒的混合物。
③将转染试剂和质粒的混合物750μL均匀滴加到上述①的细胞培养盘(4T1-DCCs、EMT6-DCCs细胞分别加入pEGFP-C1-mDEK和pEGFP-C1-mDEK △ NLS；MCF7-DCCs细胞加入pEGFP-C1-hDEK和pEGFP-C1-hDEK △NLS)中，37℃培养72小时。收集细胞培养液，利用外泌体抽提试剂(Invitrogen，目录号:4478359)从细胞培养液中分离获得外泌体(同实施例3步骤2中的方法)。将外泌体通过流式分选仪(Beckman moflo Astrios EQ)，分选得到GFP阳性和粒径范围在50-150nm的来自不同肿瘤细胞的含有外源DEK蛋白的分选外泌体及结构域NLS突变DEK蛋白的分选外泌体，分别为MCF7含hDEK-GFP蛋白的外泌体、MCF7含hDEK △ NLS-GFP蛋白的外泌体、4T1含mDEK-GFP蛋白的外泌体、4T1含mDEK △NLS-GFP蛋白的外泌体、EMT6含mDEK-GFP蛋白的外泌体、EMT6-含mDEK △NLS-GFP蛋白的外泌体。
2)过表达慢病毒转染方法：分别将实施例2获得的4T1-DCCs、EMT6-DCCs和MCF7-DCCs细胞按照每个10cm细胞培养盘约300万细胞进行接种，加入10mL含10％血清和1％抗生素的DMEM培养液，37℃培养过夜。第二天，在每个孔中加入终浓度6μg/mL的聚凝胺(polybrene)和10μL的步骤2制备的10 8pfu/mL慢病毒(4T1-DCCs、EMT6-DCCs细胞分别加入mDEK和mDEK △NLS；MCF7-DCCs细胞加入hDEK和hDEK △NLS)。37℃培养24小时后更换成新的培养基，继续培养72小时，收集细胞培养液，利用外泌体抽提试剂(Invitrogen，目录号:4478359)从细胞培养液中分离获得外泌体(同实施例3步骤2中的方法)。将外泌体通过流式分 选仪(Beckman moflo Astrios EQ)，分选得到GFP阳性和粒径范围在50-150nm的不同来源的含有外源DEK蛋白的分选外泌体和结构域NLS突变DEK蛋白的分选外泌体，分别为MCF7含hDEK-GFP蛋白的分选外泌体、MCF7含hDEK △NLS-GFP蛋白的分选外泌体、4T1含mDEK-GFP蛋白的分选外泌体溶液、4T1含mDEK △ NLS-GFP蛋白的分选外泌体、EMT6含mDEK-GFP蛋白的分选外泌体、EMT6-含mDEK △NLS-GFP蛋白的分选外泌体。
利用BCA蛋白质定量检测试剂盒(品牌：生工，货号:C503021)测定分选外泌体中的蛋白总量，将分选外泌体以200μg/mL的浓度溶解到PBS中，制成分选外泌体PBS溶液，用于后续的使用。
(2)分选外泌体的鉴定：使用粒径检测仪器(型号:ZetaView,厂家:Particle Metrix)对步骤(1)过表达慢病毒转染方法中获得的200μg/mL分选外泌体PBS溶液进行检测，发现分选外泌体的粒径范围均为30-150nm，主体粒径大小均在70nm附近(图48)。对步骤(1)中获得的200μg/mL分选外泌体PBS溶液进行透射电子显微镜的检测(方法同实施例3步骤3)，观察到双层膜结构的茶托状囊泡，符合外泌体的形态特征(图49)。使用Rabbit polyclonal anti-DEK(Proteintech,目录号:16448-1-AP)、Rabbit monoclonal anti-CD9(Abcam，目录号：ab92726)、Rabbit monoclonal anti-CD81(Abcam，目录号：ab109201)和Mouse monoclonal anti-CD63(Abcam，目录号：ab59479)的抗体，对步骤(1)中获得的200μg/mL分选外泌体溶液开展了DEK、CD9、CD81和CD63的免疫印迹分析(方法同实施例2步骤2，一抗、二抗见表1)，发现分选外泌体表达高水平的CD9、CD81和CD63蛋白(图50)。以上的结果逐一验证了分选外泌体的大小、形态和分子特征。
实施例16、含有外源DEK蛋白的分选外泌体添加激活休眠肿瘤细胞
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10％血清替代物、终浓度100nM紫杉醇和终浓度1mM 5-氟尿嘧啶的DMEM/F12培养基中。4T1-DCCs细胞分别添加2.5μL PBS、2.5μL200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的4T1含mDEK-GFP蛋白的分选外泌体PBS溶液、2.5μL200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的4T1含mDEK △NLS-GFP蛋白的分选外泌体PBS溶液，使4T1含mDEK-GFP蛋白的分选外泌体和4T1含mDEK △NLS-GFP蛋白的分选外泌体在培养基中的终浓度均为50ng/mL。同样条件下，EMT6-DCCs添加PBS、EMT6含mDEK-GFP 蛋白的分选外泌体PBS溶液、EMT6-含mDEK △NLS-GFP蛋白的分选外泌体PBS溶液；MCF7-DCCs细胞添加PBS、MCF7含hDEK-GFP蛋白的分选外泌体PBS溶液、MCF7含hDEK △NLS-GFP蛋白的分选外泌体PBS溶液。37℃培养20小时后收集细胞，使用Rabbit polyclonal anti-DEK、Rabbit monoclonal anti-H3S10ph、Mouse monoclonal anti-PCNA和Mouse monoclonal anti-SETD4的抗体，开展了免疫印迹分析(方法同实施例2步骤2，二抗见表1)，发现DEK，PCNA和H3S10ph的水平上升，SETD4的水平下降(图51)。研究结果表明含有外源DEK蛋白的分选外泌体添加能够激活休眠肿瘤细胞。
实施例17、结合化疗，含有外源DEK蛋白的分选外泌体添加能够清除休眠肿瘤细胞1、分选外泌体结合化疗对休眠肿瘤细胞cCasp3信号的影响
同实施例16方法，37℃培养20小时后收集细胞，使用切割的半胱氨酸蛋白酶(cCasp3，abcam,货号：ab32042)抗体开展免疫荧光实验(具体操作同实施例2步骤2，二抗更替为Alexa Fluor 594荧光标记驴抗兔，Thermo Fisher，货号：R37119)。样品用荧光显微镜进行观察，发现cCasp3的信号大量出现，细胞出现大量的凋亡(图52)。
2、分选外泌体结合化疗处理时间对休眠肿瘤细胞的影响
同实施例16方法，37℃培养20小时改为在37℃培养0小时、5小时、20小时、25小时、30小时后收集细胞样品，台盼蓝染色检测死亡的细胞，发现原本抵抗放化疗的休眠肿瘤细胞逐渐全部死亡(图53)。
以上的结果表明结合化疗，含有外源DEK蛋白的分选外泌体添加能够清除休眠肿瘤细胞。
实施例18、含有外源DEK蛋白的分选外泌体激活休眠肿瘤细胞的调控机制
1、分选外泌体的添加引起异染色质水平的下调和常染色质水平的上调
同实施例16方法，37℃培养20小时后收集细胞。使用H4K20me1、H4K20me2、H4K20me3、HP1-α、H3K9ac、H3K9me3和H3K27me3的抗体，在上述细胞中开展免疫印迹分析(方法同实施例2步骤2，一抗、二抗见表1)。发现与在休眠肿瘤细胞中添加PBS和含外源结构域突变DEK蛋白的分选外泌体相比，在休眠肿瘤细胞中添加了含外源DEK蛋白的分选外泌体后，组成型异染色质的分子指标H4K20me3和异染色质形成关键蛋白HP1-α的水平显著下降，常染色质的分子指标H3K9ac的水平显著上升，兼性异染色质的分子指标H3K9me3和H3K27me3的水平没有显著性的变化 (图54)。
2、分选外泌体的添加引起P53信号通路的下调和MYC信号通路的上调
同实施例16方法，37℃培养20小时后收集细胞。使用Mouse monoclonal anti-p53(Santa Cruz，目录号：sc-126)、Rabbit monoclonal anti-p21(Cell Signaling Technology，目录号：2947)、Rabbit polyclonal anti-PUMA(abcam，目录号：ab9643)和Mouse monoclonal anti-c-Myc(Santa Cruz，目录号：sc-40)的抗体，在上述细胞中开展免疫印迹分析(方法同实施例2步骤2，二抗见表1)。发现与在休眠肿瘤细胞中添加PBS和含外源结构域突变DEK蛋白的分选外泌体相比，在休眠肿瘤细胞中添加了含外源DEK蛋白的分选外泌体后，p53、p21和PUMA的水平显著下降，MYC的水平显著上升(图55)。
实施例19、在荷瘤小鼠的血液和肿瘤组织中检测注射的含有外源DEK蛋白的分选外泌体
1、分选外泌体腹腔注射到荷瘤小鼠体内后在血液中滞留
将100万颗4T1和100万颗EMT6细胞分别原位接种至8周BALB/c雌鼠的脂肪垫中，将100万颗MCF7细胞原位接种至8周Nod/Scid雌鼠脂肪垫中，待瘤子体积达到500mm 3左右时，分为4T1组、EMT6组和MCF7组，每组9只小鼠。4T1组6只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL的4T1含mDEK-GFP蛋白的分选外泌体PBS溶液，3只荷瘤小鼠腹腔不注射物质作为未处理对照。EMT6组6只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL的EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液，3只荷瘤小鼠腹腔不注射物质作为未处理对照。MCF7组6只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL的MCF7含mDEK-GFP蛋白的分选外泌体PBS溶液，3只荷瘤小鼠腹腔不注射物质作为未处理对照。分别在注射24小时后和7天后安乐死小鼠，收集小鼠的血浆，利用外泌体抽提试剂盒(Invitrogen,货号：4484450)分离血浆内的外泌体，在100μL外泌体中加入10μL的质量浓度4％的9μm硫酸乳胶珠子(溶于超蒸水中)，25℃孵育10分钟。1000rpm 25℃离心5分钟，去掉上清，用100μL PBS重悬。将重悬液放到流式分析仪中检测GFP +的珠子比例。发现在荷瘤小鼠的血液中检测到注射的含有外源DEK蛋白的分选外泌体(图56)，说明分选外泌体腹腔 注射到荷瘤小鼠体内后能在血液中滞留。
2、分选外泌体腹腔注射到荷瘤小鼠体内后进入肿瘤组织
将100万颗4T1和100万颗EMT6细胞分别原位接种至8周BALB/c雌鼠的脂肪垫中，将100万颗MCF7细胞原位接种至8周Nod/Scid雌鼠脂肪垫中，待瘤子体积达到500mm 3左右时，分为4T1组、EMT6组和MCF7组，每组6只小鼠。4T1组3只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK-GFP蛋白的分选外泌体PBS溶液，3只小鼠按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK △NLS-GFP蛋白的分选外泌体PBS溶液。EMT6组3只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液，3只小鼠按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK △NLS-GFP蛋白的分选外泌体PBS溶液。MCF7组3只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL MCF7含mDEK-GFP蛋白的分选外泌体PBS溶液，3只小鼠按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL MCF7含mDEK △NLS-GFP蛋白的分选外泌体PBS溶液。
在注射24小时后安乐死小鼠，手术取出4T1、EMT6和MCF7肿瘤，将上述肿瘤放入4％多聚甲醛中4℃过夜，浸泡到质量浓度30％蔗糖水溶液中脱水48小时，浸泡后的肿瘤捞出，放到10x10x5mm的敞开长方体型塑料材质的模具中，往其中加满OCT包埋剂(SAKURA，目录号：4583)，将模具放在干冰上静置5分钟，将包埋块取出，-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切片。对肿瘤切片开展Rabbit polyclonal anti-SETD4(Sigma-Aldrich,目录号:HPA024073)抗体的免疫荧光实验(方法同实施例2)。发现在肿瘤组织中有大量的GFP信号(图57)，表明含有外源蛋白DEK或结构域突变DEK蛋白的分选外泌体注射到荷瘤小鼠体内后进入肿瘤组织。
实施例20、注射含有外源DEK蛋白的分选外泌体激活荷瘤小鼠体内休眠肿瘤细胞
将100万颗4T1和100万颗EMT6细胞分别原位接种至8周BALB/c雌鼠的脂肪 垫中，将100万颗MCF7细胞原位接种至8周Nod/Scid雌鼠脂肪垫中，待瘤子体积达到500mm 3左右时，分为4T1组、EMT6组、MCF7组，每组6只小鼠。4T1组3只荷瘤小鼠腹腔不注射物质作为未处理对照，3只荷瘤小鼠腹腔按20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK-GFP蛋白的分选外泌体PBS溶液。EMT6组3只荷瘤小鼠腹腔不注射物质作为未处理对照，3只荷瘤小鼠腹腔按20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液。MCF7组3只荷瘤小鼠腹腔不注射物质作为未处理对照，3只荷瘤小鼠腹腔按20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL MCF7含mDEK-GFP蛋白的分选外泌体PBS溶液。在注射24小时后安乐死小鼠，手术取出4T1、EMT6和MCF7肿瘤，将上述肿瘤放入4％多聚甲醛中4℃过夜，浸泡到质量浓度30％蔗糖水溶液中脱水48小时，浸泡后的肿瘤捞出，放到10x10x5mm的敞开长方体型塑料材质的模具中，往其中加满OCT包埋剂(SAKURA，目录号：4583)，将模具放在干冰上静置5分钟，将包埋块取出，-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切片。对肿瘤切片开展Rabbit polyclonal anti-SETD4(Sigma-Aldrich,目录号:HPA024073)抗体的免疫荧光实验(方法同实施例2)。发现相比于未处理组，腹腔注射含外源DEK蛋白的分选外泌体后，SETD4细胞占总细胞的比例显著降低(图58)，表明注射含外源DEK蛋白的分选外泌体能够100％激活荷瘤小鼠体内休眠肿瘤细胞。
实施例21、结合放化疗，注射含有外源DEK蛋白的分选外泌体能够清除荷瘤小鼠体内休眠肿瘤细胞
1、外泌体结合放疗对休眠肿瘤细胞的影响
在6-8周BALB/c雌鼠的腋下乳腺脂肪垫中接种100万颗4T1细胞，分为放疗+PBS组、放疗+4T1含mDEK-GFP蛋白的分选外泌体组、放疗+4T1含mDEK △NLS-GFP蛋白的分选外泌体组，每组3只。在接种后的第6天和第9天，放疗+PBS组每只荷瘤小鼠腹腔注射PBS，PBS注射量为100μL；放疗+4T1含mDEK-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK-GFP蛋白的分选外泌体PBS溶液；放疗+4T1含mDEK △NLS-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20 μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK △NLS-GFP蛋白的分选外泌体PBS溶液。在接种后的第7天、第10天和第13天分别接受一次腋下范围内的3分钟45秒的20Gy X射线辐照。在接种后第21天，安乐死实验小鼠，手术取出瘤子，将肿瘤放入4％多聚甲醛中4℃过夜，浸泡到质量浓度30％蔗糖水溶液中脱水48小时，浸泡后的肿瘤捞出，放到10x10x5mm的敞开长方体型塑料材质的模具中，往其中加满OCT包埋剂(SAKURA，目录号：4583)，将模具放在干冰上静置5分钟，将包埋块取出，-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切片。在肿瘤切片中开展Rabbit polyclonal anti-SETD4(Sigma-Aldrich,目录号:HPA024073)抗体的免疫荧光分析(方法同实施例2)，发现相比于放疗+PBS组和放疗+4T1含mDEK △NLS-GFP蛋白的外泌体组，放疗+4T1含mDEK-GFP蛋白的外泌体组中，只发现极少量SETD4阳性细胞的存在(图59)。结果表明结合放疗，注射含有外源DEK蛋白的分选外泌体能够100％清除4T1荷瘤小鼠体内休眠肿瘤细胞。
2、外泌体结合放化疗对休眠肿瘤细胞的影响
在6-8周BALB/c雌鼠的腋下乳腺脂肪垫中接种20万颗EMT6细胞，分为放化疗+PBS组、放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组、放化疗+EMT6-含mDEK △NLS-GFP蛋白的分选外泌体组，每组3只。在接种后的第6天、第9天和第12天，放化疗+PBS组每只荷瘤小鼠腹腔注射PBS，PBS注射量为100μL；放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液；放化疗+EMT6-含mDEK △NLS-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6-含mDEK △NLS-GFP蛋白的分选外泌体PBS溶液。在接种后的第7天、第10天和第13天分别接受一次腋下范围内的3分钟45秒的20Gy X射线辐照，在接种后的第6天、第12天和第18天分别腹腔注射1次剂量为3mg/kg小鼠体重的紫杉醇。在接种后第21天，安乐死实验小鼠，手术取出瘤子，将肿瘤放入4％多聚甲醛中4℃过夜，浸泡到质量浓度30％蔗糖水溶液中脱水48小时，浸泡后的肿瘤捞出，放到10x10x5mm的敞开长方体型塑料材质的模具中，往其中加满OCT包埋剂(SAKURA，目录号：4583)，将模具放在干冰上静置5分钟，将包埋块取出，-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切 片。在肿瘤切片中开展SETD4抗体(Sigma-Aldrich,目录号:HPA024073)的免疫荧光分析(方法同实施例2)，发现相比于放化疗+PBS组和放化疗+EMT6-含mDEK △ NLS-GFP蛋白的外泌体组，放化疗+EMT6含mDEK-GFP蛋白的外泌体组中，只发现极少量SETD4阳性细胞的存在(图60)。结果表明结合放化疗，注射含有外源DEK蛋白的分选外泌体能够100％清除EMT6荷瘤小鼠体内休眠肿瘤细胞。
实施例22、结合放化疗，注射含有外源DEK蛋白的分选外泌体能够治愈乳腺癌
1、放疗结合分选外泌体治愈4T1移植瘤小鼠
在6-8周BALB/c雌鼠的腋下乳腺脂肪垫中接种100万颗4T1细胞，分为未处理组、放疗+PBS组、放疗+4T1含mDEK-GFP蛋白的分选外泌体组、放疗+4T1含mDEK △NLS-GFP蛋白的分选外泌体组，每组11只。在接种后的第6天和第9天，未处理组不注射物质；放疗+PBS组每只荷瘤小鼠腹腔注射PBS，PBS注射量为100μL；放疗+4T1含mDEK-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL4T1含mDEK-GFP蛋白的分选外泌体PBS溶液；放疗+4T1含mDEK △NLS-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK △NLS-GFP蛋白的分选外泌体PBS溶液。在接种后的第7天、第10天和第13天分别接受一次腋下范围内的3分钟45秒的20Gy X射线辐照。在接种细胞后每周测量肿瘤的长度和宽度，利用公式(长度×宽度×宽度/2)计算出肿瘤体积，绘制肿瘤的生长曲线，发现无处理组在接种细胞4周后瘤体直径达到伦理上限，单纯放疗组和放疗且注射含外源结构域突变DEK蛋白的分选外泌体组在接种细胞8周后流体直径达到约400mm 3，而在放疗且注射含外源DEK蛋白的分选外泌体组中，没有发现肿瘤(图61)。
在接种细胞后的第8周，对所有实验小鼠安乐死后手术取出肺组织的样品，固定包埋后冷冻切片。切片在PBS中洗1遍，5分钟；苏木素染色液染色10分钟；自来水中冲洗掉多余的染色液，1分钟；蒸馏水洗1遍，2分钟；伊红染色液染色2分钟；用50％的甘油封片。染色后的片子在显微镜下观察，发现相比于放疗+PBS组、放疗+4T1含mDEK △NLS-GFP蛋白的分选外泌体组，在放疗+4T1含mDEK-GFP蛋白的分选外泌体组中找不到肿瘤的转移灶(图62)。
对所有的实验鼠进行生存曲线分析，发现相比于未处理组的35天、放疗+PBS组的56天和放疗+4T1含mDEK △NLS-GFP蛋白的外泌体组的57天，在放疗+4T1 含mDEK-GFP蛋白的外泌体组中，小鼠的中位生存期延长到了100天(图63)。结果表明使用含外源DEK蛋白的分选外泌体和放疗能够完全治愈4T1肿瘤，消除肿瘤的复发和转移，改善小鼠的生存，治疗后1年内小鼠的肿瘤未见复发且未见转移。
2、放化疗结合分选外泌体治愈EMT6移植瘤小鼠
在6-8周BALB/c雌鼠的腋下乳腺脂肪垫中接种20万颗EMT6细胞，分为未处理组、放化疗+PBS组、放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组、放化疗+EMT6-含mDEK △NLS-GFP蛋白的分选外泌体组，每组7只。在接种后的第6天、第9天和第12天，未处理组不注射物质；放化疗+PBS组每只荷瘤小鼠腹腔注射100μL的PBS；放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液；放化疗+EMT6-含mDEK △NLS-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6-含mDEK △ NLS-GFP蛋白的分选外泌体PBS溶液。在接种后的第7天、第10天和第13天分别接受一次腋下范围内的3分钟45秒的20Gy X射线辐照，在接种后的第6天、第12天和第18天分别腹腔注射1次剂量为3mg/kg小鼠体重的紫杉醇。在接种细胞后每周测量肿瘤的长度和宽度，利用公式(长度×宽度×宽度/2)计算出肿瘤体积，绘制肿瘤的生长曲线，发现未处理组在接种细胞3周后瘤体直径达到伦理上限，放化疗+PBS组和放化疗+EMT6-含mDEK △NLS-GFP蛋白的外泌体组在接种细胞6周后瘤体直径达到伦理上限，而在放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组中，肿瘤的生长受到显著抑制(图64)。结果表明使用含外源DEK蛋白的分选外泌体和放化疗能够完全治愈EMT6肿瘤，抑制肿瘤的复发。
3、化疗结合分选外泌体治愈MCF7移植瘤小鼠
在6-8周Nod/Scid雌鼠的腋下乳腺脂肪垫中接种100万颗MCF7细胞，分为未处理组、化疗+PBS组、化疗+MCF7含hDEK-GFP蛋白的分选外泌体组、化疗+MCF7含hDEK △NLS-GFP蛋白的分选外泌体组，每组5只。在接种后的第3-8周，未处理组不注射物质；化疗+PBS组每只小鼠每周腹腔注射1次PBS和1次剂量为3mg/kg小鼠体重的紫杉醇，PBS注射量为100μL；化疗+MCF7含hDEK-GFP蛋白的分选外泌体组每周腹腔注射1次剂量为3mg/kg小鼠体重的紫杉醇和1次剂量为1μg/g小鼠体重的实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL MCF7含 hDEK-GFP蛋白的分选外泌体PBS溶液；化疗+MCF7含hDEK △NLS-GFP蛋白的分选外泌体组每周腹腔注射1次剂量为3mg/kg小鼠体重的紫杉醇和1次剂量为1μg/g小鼠体重的实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL MCF7含hDEK △NLS-GFP蛋白的分选外泌体PBS溶液。在接种细胞后每周测量肿瘤的长度和宽度，利用公式(长度×宽度×宽度/2)计算出肿瘤体积，绘制肿瘤的生长曲线，发现未处理组在接种细胞5周后瘤体直径达到伦理上限，化疗+PBS组和化疗+MCF7含hDEK △NLS-GFP蛋白的外泌体组在接种细胞12周后瘤体直径到伦理上限，而在化疗+MCF7含hDEK-GFP蛋白的外泌体组中，没有发现肿瘤(图65)。结果表明使用含外源DEK蛋白的分选外泌体和化疗能够完全治愈MCF7肿瘤，消除肿瘤的复发。
实施例23、休眠肿瘤细胞的数量与临床乳腺癌的恶化程度密切相关
我们收集了8例Ⅰ期、12例Ⅱ期和3例Ⅲ期的乳腺癌病人石蜡包埋样品，切片后进行SETD4的免疫荧光实验(方法同实施例2)，发现Ⅲ期样品中的SETD4细胞比例远远高于Ⅰ期和Ⅱ期样品(图66)。结果表明静息肿瘤细胞的数量越多，临床乳腺癌的肿瘤恶化程度越高。
实施例24、临床乳腺癌样品来源休眠肿瘤细胞的获取，激活和杀死
1、病人样品中获取抵抗放化疗的肿瘤细胞
利用肿瘤解离试剂盒(美天旎,货号：130-095-929)处理2个病人的临床乳腺癌样品，得到消化下来的肿瘤细胞，并将其按80万颗/孔的密度接种在超低吸附的六孔盘(品牌：Corning，货号:3471)中。每孔加入3mL含有10％血清替代物(Thermo Fisher Scientific Cat#10828028)，终浓度100nM紫杉醇和终浓度1mM的5-氟尿嘧啶的DMEM/F12培养基(Corning，目录号：10-092-cv)，在37℃培养1个月，每隔3天换一次培养液，前两周中每周照射30Gy X射线一次，总共照射2次(两次间隔7天)，每次照射10分钟。一个月后，使用死细胞去除试剂盒(Miltenyi Biotec，目录号：130-090-101)筛选出抵抗放化疗的肿瘤细胞，即为休眠肿瘤细胞(图67中a)。
2、DEK蛋白结合化疗对休眠肿瘤细胞SETD4和Ki67信号的影响
分别将上述2个病人来源的休眠肿瘤细胞各20万颗接种至10mL含有10％血清替代物、终浓度100nM紫杉醇和终浓度1mM 5-氟尿嘧啶的DMEM/F12培养基中。在上述细胞中分别添加25μL PBS、25μL 200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的MCF7含hDEK-GFP蛋白的分选外泌体PBS溶液、25μL 200μg/mL实施 例15步骤3中过表达慢病毒转染方法制备的MCF7含hDEK △NLS-GFP蛋白的分选外泌体PBS溶液，使MCF7含hDEK-GFP蛋白的分选外泌体和MCF7含hDEK △NLS-GFP蛋白的分选外泌体在培养基中的终浓度均为50ng/mL。37℃培养20小时后收集细胞，使用Rabbit polyclonal anti-DEK(Proteintech,货号：16448-1-AP)、Rabbit polyclonal anti-SETD4(Sigma-Aldrich,货号：HPA024073)和Rabbit monoclonal anti-Ki67(abcam,货号：ab16667)抗体开展免疫荧光实验(具体操作同实施例2步骤2，使用的二抗均是Alexa Fluor 594荧光标记驴抗兔，Thermo Fisher，货号：R37119)。样品用荧光显微镜进行观察，发现相较于添加PBS的对照组和添加MCF7含hDEK △NLS-GFP蛋白的分选外泌体PBS溶液的对照组，在休眠肿瘤细胞中添加MCF7含hDEK-GFP蛋白的分选外泌体PBS溶液后，细胞中SETD4的水平下降，Ki67的水平上升(图67中b)。研究结果表明含有外源DEK蛋白的分选外泌体添加能够100％激活临床乳腺癌样本来源的休眠肿瘤细胞。
3、DEK蛋白结合化疗对休眠肿瘤细胞死亡的影响
分别将上述2个病人来源的休眠肿瘤细胞各20万颗接种至10mL含有10％血清替代物、终浓度100nM紫杉醇和终浓度1mM 5-氟尿嘧啶的DMEM/F12培养基中。在上述细胞中分别添加25μL PBS、25μL 200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的MCF7含hDEK-GFP蛋白的分选外泌体PBS溶液、25μL 200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的MCF7含hDEK △NLS-GFP蛋白的分选外泌体PBS溶液，使MCF7含hDEK-GFP蛋白的分选外泌体和MCF7含hDEK △NLS-GFP蛋白的分选外泌体在培养基中的终浓度均为50ng/mL。37℃培养30小时后收集细胞样品，台盼蓝染色检测死亡的细胞，发现原本抵抗放化疗的休眠肿瘤细胞在添加MCF7含hDEK-GFP蛋白的分选外泌体后全部死亡(图67中c)。研究结果表明添加含有外源DEK蛋白的分选外泌体结合化疗能够100％杀死临床乳腺癌样本来源的休眠肿瘤细胞。
实施例25、在各种人肿瘤细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞
1.肺癌细胞系H226来源粗外泌体结合化疗100％激活并清除H226休眠肿瘤细胞
H226细胞系(来源见表2)，细胞培养基是RPMI-1640培养基(Gibco，目录号:31800022)中添加10％的胎牛血清和1％的青霉素-链霉素。
1000万颗H226细胞系接种到20mL的培养基中。37℃培养24小时后收集细胞的培养液，1000rpm 4℃离心10分钟，取上清，12000rpm 4℃离心20分钟，取上清， 即为H226的细胞培养液。在细胞培养液中加入1mL的外泌体分离试剂(品牌：Thermo Fisher，货号：4478359)，上下颠倒3次混匀后4℃孵育过夜。第二天，上述混合液10000rpm 4℃离心60分钟，去掉上清，用200μL的PBS重悬离心管底部的沉淀(沉淀即为粗外泌体)，利用BCA蛋白质定量检测试剂盒(品牌：生工，货号:C503021)测定粗外泌体悬液中的蛋白总量，用PBS配制蛋白浓度为20μg/mL的粗外泌体溶液，用于后续的使用。
100万颗H226细胞中加入0.1mL含2500U/mL胰蛋白酶和0.02％乙二胺四乙酸(EDTA)的磷酸缓冲液(PBS)，25℃静置孵育40秒，加入0.2mL胎牛血清中和，将壁上的细胞吹打下来，1000rpm 25℃离心5分钟，去上清，沉淀用1mL的细胞培养基重悬。将上述的重悬液按80万颗/孔的密度接种在超低吸附的六孔盘(品牌：Corning，货号:3471)中。每孔加入3mL含有10％血清替代物(Thermo Fisher Scientific Cat#10828028)，终浓度100nM紫杉醇和终浓度1mM的5-氟尿嘧啶的DMEM/F12培养基(Corning，目录号：10-092-cv)，在37℃培养1个月，每隔3天换一次培养液，前两周中每周照射30Gy X射线一次，总共照射2次(两次间隔7天)，每次照射10分钟。一个月后，使用死细胞去除试剂盒(Miltenyi Biotec，目录号：130-090-101)筛选出抵抗放化疗的休眠肿瘤细胞。
将1000颗上述的休眠肿瘤细胞接种至2mL含有10％血清替代物、终浓度100nM紫杉醇和终浓度1mM 5-氟尿嘧啶的DMEM/F12培养基中，并加入5μL上述制备的20μg/mL粗外泌体溶液使其在培养基中的终浓度为50ng/mL。37℃培养30小时后，发现细胞的死亡率为100％，H226来源粗外泌体结合化疗100％激活并清除H226休眠肿瘤细胞。
2.胃癌细胞系MKN45来源粗外泌体结合化疗100％激活并清除MKN45休眠肿瘤细胞
同实施例25步骤1方法(细胞系更替为MKN45)，发现MKN45来源粗外泌体结合化疗100％激活并清除MKN45休眠肿瘤细胞。
3.前列腺癌细胞系PC-3来源粗外泌体结合化疗100％激活并清除PC-3休眠肿瘤细胞
同实施例25步骤1方法(细胞系更替为PC-3，培养基更替为：F-12培养基(Gibco，目录号:21700075)中添加10％的胎牛血清和1％的青霉素-链霉素)，发现PC-3来源粗外泌体结合化疗100％激活并清除PC-3休眠肿瘤细胞。
4.宫颈癌细胞系HeLa来源粗外泌体结合化疗100％激活并清除HeLa休眠肿瘤细胞
同实施例25步骤1方法(细胞系更替为HeLa，培养基更替为：MEM培养基(Gibco，目录号:41500034)中添加10％的胎牛血清和1％的青霉素-链霉素)，发现HeLa来源粗外泌体结合化疗100％激活并清除HeLa休眠肿瘤细胞。
研究结果表明使用DEK激活并清除休眠肿瘤细胞可以适用于各种不同类型的人癌症的治疗。
表2、DEK蛋白对于肿瘤细胞激活并清除效果
Figure PCTCN2022083977-appb-000005

Claims (71)

  1. 一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用。
  2. 如权利要求1所述的应用，其特征在于所述SETD4蛋白抑制剂包括DEK蛋白。
  3. 如权利要求2所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.25所示保守序列。
  4. 如权利要求3所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.2所示NLS结构域的氨基酸序列95％以上相似性。
  5. 如权利要求3所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.3所示SAP结构域的氨基酸序列95％以上相似性。
  6. 如权利要求3所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域的氨基酸序列95％以上相似性。
  7. 如权利要求3所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域或SEQ ID NO.3所示SAP结构域中的一种或两种，且同时具有SEQ ID NO.2所示NLS结构域。
  8. 如权利要求3-7之一所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.1或SEQ ID NO.22所示氨基酸序列95％以上相似性。
  9. 如权利要求8所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.1所示氨基酸序列。
  10. 如权利要求8所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.22所示氨基酸序列。
  11. 如权利要求1所述的应用，其特征在于所述休眠肿瘤细胞包括源自下列肿瘤：头颈部肿瘤、胸部肿瘤、消化系统肿瘤、泌尿生殖系统肿瘤、骨及软组织肿瘤、淋巴及血液系统肿瘤。
  12. 如权利要求11所述的应用，其特征在于所述休眠肿瘤细胞选自脑癌、眼癌、耳部肿瘤、颌骨肿瘤、颈部肿瘤、鼻腔癌、鼻窦癌、鼻咽癌、牙龈癌、舌癌、软硬腭癌、颌骨癌、口底癌、口咽癌、唇癌、上颌窦癌、颜面部皮肤黏膜的癌症、喉癌、涎腺肿瘤、甲状腺癌、脑膜瘤、室管膜瘤、垂体瘤、上皮神经母细胞瘤、神经外胚层肿瘤、副神经节肿瘤、肺癌、食管癌、乳腺癌、纵膈肿瘤、胸腺癌、胃癌、大肠癌、肝癌、胰腺癌、胆管癌、小肠癌、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、宫颈癌、子宫癌、卵巢癌、输卵管癌、阴道癌、尤文氏肉瘤、脂肪肿瘤、卡波济肉瘤、平滑肌肿瘤、横纹肌肿瘤、血管肿瘤、滑膜肉瘤、纤维肉瘤、骨癌、恶性淋巴瘤、多发性骨髓瘤、白血病、心脏肿瘤、 间皮肿瘤、纤维母细胞肿瘤、滋养细胞肿瘤、黑色素瘤。
  13. 一种用于激活休眠肿瘤细胞的SETD4蛋白抑制剂的投递蛋白，其特征在于所述投递蛋白包括投递DEK蛋白，所述投递DEK蛋白为含有DEK蛋白的医学可接受的载体。
  14. 如权利要求13所述的投递蛋白，其特征在于所述载体包括外泌体、脂质体或纳米材料。
  15. 如权利要求13所述的投递蛋白，其特征在于所述DEK蛋白具有SEQ ID NO.25所示保守序列。
  16. 如权利要求15所述的投递蛋白，其特征在于所述DEK蛋白具有SEQ ID NO.2所示NLS结构域的氨基酸序列95％以上相似性。
  17. 如权利要求15所述的投递蛋白，其特征在于所述DEK蛋白具有SEQ ID NO.3所示SAP结构域的氨基酸序列95％以上相似性。
  18. 如权利要求15所述的投递蛋白，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域的氨基酸序列95％以上相似性。
  19. 如权利要求15所述的投递蛋白，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域或SEQ ID NO.3所示SAP结构域中的一种或两种，且同时具有SEQ ID NO.2所示NLS结构域。
  20. 如权利要求15-19之一所述的投递蛋白，其特征在于所述DEK蛋白具有SEQ ID NO.1或SEQ ID NO.22所示氨基酸序列95％以上相似性。
  21. 如权利要求20所述的投递蛋白，其特征在于所述DEK蛋白具有SEQ ID NO.1所示氨基酸序列。
  22. 如权利要求20所述的投递蛋白，其特征在于所述DEK蛋白具有SEQ ID NO.22所示氨基酸序列。
  23. 如权利要求14所述的投递蛋白，其特征在于所述载体为外泌体时，所述投递DEK蛋白是由肿瘤细胞系培养液中分离获得含有DEK蛋白的外泌体，或将DEK蛋白编码基因接入基因表达载体，在细胞系中过量表达DEK蛋白并产生含有DEK蛋白的外泌体。
  24. 如权利要求23所述的投递蛋白，其特征在于所述将DEK蛋白编码基因接入基因表达载体获得含DEK蛋白的外泌体按如下方法之一制备：(1)构建过表达DEK蛋白的质粒并将其转染到细胞系中制备外泌体：将DEK基因插入到pEGFP-C1质粒的EcoRⅠ和XbaⅠ位点上，筛选获得重组质粒pEGFP-C1-DEK；利用脂质体助转剂lipo8000将上述重组质粒转入细胞系中，过量表达DEK蛋白后收集细胞培养液，从培养液中分离纯化含有 DEK蛋白的外泌体溶液A；(2)构建过表达DEK蛋白的慢病毒并将其感染细胞系构建表达DEK蛋白的细胞株制备外泌体：将DEK基因分别插入到pLent-N-GFP的慢病毒表达载体的EcoRⅠ和XbaⅠ位点上，筛选获得重组慢病毒表达载体pLent-N-GFP-DEK；将上述重组慢病毒表达载体和慢病毒包装质粒混合物共同转染293T细胞，转染72小时后收集细胞培养上清即为病毒液，浓缩纯化，获得过表达DEK蛋白的慢病毒；用过表达DEK蛋白的慢病毒感染细胞系并构建过表达DEK蛋白的细胞株；收集过表达DEK蛋白的细胞株中的细胞培养液，从培养液中分离纯化含有DEK蛋白的外泌体溶液B；所述慢病毒包装质粒混合物为质量比5:3:2的pMDL、VSVG和pRSV-Rev。
  25. 如权利要求23所述的投递蛋白，其特征在于所述细胞系包括下列肿瘤：头颈部肿瘤，胸部肿瘤，消化系统肿瘤，泌尿生殖系统肿瘤，骨及软组织肿瘤，淋巴及血液系统肿瘤。
  26. 如权利要求25所述的投递蛋白，其特征在于所述细胞系选自脑癌、眼癌、耳部肿瘤、颌骨肿瘤、颈部肿瘤、鼻腔癌、鼻窦癌、鼻咽癌、牙龈癌、舌癌、软硬腭癌、颌骨癌、口底癌、口咽癌、唇癌、上颌窦癌、颜面部皮肤黏膜的癌症喉癌、涎腺肿瘤、甲状腺癌、脑膜瘤、室管膜瘤、垂体瘤、上皮神经母细胞瘤、神经外胚层肿瘤、副神经节肿瘤、肺癌、食管癌、乳腺癌、纵膈肿瘤、胸腺癌、胃癌、大肠癌、肝癌、胰腺癌、胆管癌、小肠癌、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、宫颈癌、子宫癌、卵巢癌、输卵管癌、阴道癌、尤文氏肉瘤、脂肪肿瘤、卡波济肉瘤、平滑肌肿瘤、横纹肌肿瘤、血管肿瘤、滑膜肉瘤、纤维肉瘤、骨癌、恶性淋巴瘤、多发性骨髓瘤、白血病、心脏肿瘤、间皮肿瘤、纤维母细胞肿瘤、滋养细胞肿瘤、黑色素瘤。
  27. 如权利要求14所述的投递蛋白，其特征在于所述载体为脂质体时，含DEK蛋白的脂质体采用薄膜水化法制备：取二棕榈酰磷脂酰胆碱、胆固醇、二硬脂酰磷脂酰乙酰胺-甲氧基聚乙二醇溶于氯仿中，减压旋转蒸发成均匀的薄膜，真空过夜彻底挥发掉残留的氯仿，加入含DEK蛋白的PBS溶液，冰浴25KHz超声20min使脂质体膜脱落，在振荡器上震荡充分水化，形成浊液；然后将浊液在功率135W下超声30min，得到脂质体悬液；用10kD的超滤管以12000g的转速4℃超滤，每五分钟取出吹打一次并补充PBS洗去游离蛋白，收集截留液，即为含DEK蛋白的脂质体悬液。
  28. 如权利要求27所述的投递蛋白，其特征在于所述二棕榈酰磷脂酰胆碱与胆固醇质量比为1:0.1，所述二棕榈酰磷脂酰胆碱与二硬脂酰磷脂酰乙酰胺-甲氧基聚乙二醇质量比为1:0.1，所述氯仿体积用量以二棕榈酰磷脂酰胆碱质量计为0.1mL/mg；所述二棕榈酰 磷脂酰胆碱与DEK蛋白的PBS溶液中DEK蛋白质量比为1:0.2。
  29. 如权利要求14所述的投递蛋白，其特征在于所述载体为纳米材料时，含DEK蛋白的纳米材料采用改良溶剂挥发法制备：将DEK蛋白的PBS溶液中，转入聚乳酸-羟基乙酸共聚物的二氯甲烷溶液中，25KHz超声1分钟形成初乳，将初乳转入体积浓度1％聚乙烯醇水溶液中，再次25KHz超声5分钟形成复乳，搅拌4h，待有机溶剂挥发后，18000r/min离心收集沉淀，将沉淀-55℃冷冻干燥24h，获得含DEK蛋白的PLGA纳米材料。
  30. 如权利要求29所述的投递蛋白，其特征在于所述DEK蛋白的PBS溶液中DEK蛋白与聚乳酸-羟基乙酸共聚物质量比为1：0.1，所述聚乙烯醇水溶液体积用量以DEK蛋白的PBS溶液中DEK蛋白质量计为1mL/mg。
  31. 一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞试剂中的应用。
  32. 如权利要求31所述的应用，其特征在于所述试剂包括DEK蛋白。
  33. 如权利要求32所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.25所示保守序列。
  34. 如权利要求33所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.2所示NLS结构域的氨基酸序列95％以上相似性。
  35. 如权利要求33所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.3所示SAP结构域的氨基酸序列95％以上相似性。
  36. 如权利要求33所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域的氨基酸序列95％以上相似性。
  37. 如权利要求33所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域或SEQ ID NO.3所示SAP结构域中的一种或两种，且同时具有SEQ ID NO.2所示NLS结构域。
  38. 如权利要求33-37之一所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.1或SEQ ID NO.22所示氨基酸序列95％以上相似性。
  39. 如权利要求38所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.1所示氨基酸序列。
  40. 如权利要求38所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.22所示氨基酸序列。
  41. 如权利要求31所述的应用，其特征在于所述的应用是将SETD4蛋白抑制剂投递到休眠肿瘤细胞中，达到激活休眠肿瘤细胞的目的。
  42. 如权利要求31所述的应用，其特征在于所述肿瘤细胞包括源自下列肿瘤：头颈部肿瘤，胸部肿瘤，消化系统肿瘤，泌尿生殖系统肿瘤，骨及软组织肿瘤，淋巴及血液系统肿瘤。
  43. 如权利要求42所述的应用，其特征在于所述肿瘤细胞选自脑癌、眼癌、耳部肿瘤、颌骨肿瘤、颈部肿瘤、鼻腔癌、鼻窦癌、鼻咽癌、牙龈癌、舌癌、软硬腭癌、颌骨癌、口底癌、口咽癌、唇癌、上颌窦癌、颜面部皮肤黏膜的癌症喉癌、涎腺肿瘤、甲状腺癌、脑膜瘤、室管膜瘤、垂体瘤、上皮神经母细胞瘤、神经外胚层肿瘤、副神经节肿瘤、肺癌、食管癌、乳腺癌、纵膈肿瘤、胸腺癌、胃癌、大肠癌、肝癌、胰腺癌、胆管癌、小肠癌、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、宫颈癌、子宫癌、卵巢癌、输卵管癌、阴道癌、尤文氏肉瘤、脂肪肿瘤、卡波济肉瘤、平滑肌肿瘤、横纹肌肿瘤、血管肿瘤、滑膜肉瘤、纤维肉瘤、骨癌、恶性淋巴瘤、多发性骨髓瘤、白血病、心脏肿瘤、间皮肿瘤、纤维母细胞肿瘤、滋养细胞肿瘤、黑色素瘤。
  44. 如权利要求32～37之一或39～43之一所述的应用，其特征在于所述试剂包括SETD4蛋白抑制剂及清除肿瘤细胞的药物。
  45. 如权利要求44所述的应用，其特征在于所述清除肿瘤细胞的药物包括紫杉醇或5-氟尿嘧啶。
  46. 一种SETD4蛋白抑制剂在制备抗肿瘤药物中的应用，其特征在于所述抗肿瘤药物包括所述的SETD4蛋白抑制剂及清除肿瘤细胞的药物。
  47. 如权利要求46所述的应用，其特征在于所述SETD4蛋白抑制剂包括DEK蛋白。
  48. 如权利要求47所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.25所示保守序列。
  49. 如权利要求48所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.2所示NLS结构域的氨基酸序列95％以上相似性。
  50. 如权利要求48所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.3所示SAP结构域的氨基酸序列95％以上相似性。
  51. 如权利要求48所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域的氨基酸序列95％以上相似性。
  52. 如权利要求48所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域或SEQ ID NO.3所示SAP结构域中的一种或两种，且同时具有SEQ ID NO.2所示NLS结构域。
  53. 如权利要求47-52之一所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.1或SEQ ID NO.22所示氨基酸序列95％以上相似性。
  54. 如权利要求53所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.1所示氨基酸序列。
  55. 如权利要求53所述的应用，其特征在于所述DEK蛋白具有SEQ ID NO.22所示氨基酸序列。
  56. 如权利要求46所述的应用，其特征在于所述清除肿瘤细胞的药物包括紫杉醇或5-氟尿嘧啶。
  57. 如权利要求46所述的应用，其特征在于所述肿瘤包括头颈部肿瘤，胸部肿瘤，消化系统肿瘤，泌尿生殖系统肿瘤，骨及软组织肿瘤，淋巴及血液系统肿瘤。
  58. 如权利要求57所述的应用，其特征在于所述肿瘤包括脑癌、眼癌、耳部肿瘤、颌骨肿瘤、颈部肿瘤、鼻腔癌、鼻窦癌、鼻咽癌、牙龈癌、舌癌、软硬腭癌、颌骨癌、口底癌、口咽癌、唇癌、上颌窦癌、颜面部皮肤黏膜的癌症喉癌、涎腺肿瘤、甲状腺癌、脑膜瘤、室管膜瘤、垂体瘤、上皮神经母细胞瘤、神经外胚层肿瘤、副神经节肿瘤、肺癌、食管癌、乳腺癌、纵膈肿瘤、胸腺癌、胃癌、大肠癌、肝癌、胰腺癌、胆管癌、小肠癌、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、宫颈癌、子宫癌、卵巢癌、输卵管癌、阴道癌、尤文氏肉瘤、脂肪肿瘤、卡波济肉瘤、平滑肌肿瘤、横纹肌肿瘤、血管肿瘤、滑膜肉瘤、纤维肉瘤、骨癌、恶性淋巴瘤、多发性骨髓瘤、白血病、心脏肿瘤、间皮肿瘤、纤维母细胞肿瘤、滋养细胞肿瘤、黑色素瘤。
  59. 一种应用SETD4蛋白抑制剂治疗肿瘤的方法，其特征在于所述方法为：在各种时期的肿瘤病人中，通过静脉注射、腹腔注射或瘤体内注射SETD4蛋白抑制剂，将SETD4蛋白抑制剂投递到肿瘤中，从而激活休眠肿瘤细胞，并在临床手术、放疗、化疗、靶向或免疫的作用下杀死并彻底清除休眠的肿瘤细胞，实现无转移，无复发的临床肿瘤治愈。
  60. 如权利要求59所述的方法，其特征在于所述SETD4蛋白抑制剂包括DEK蛋白。
  61. 如权利要求60所述的方法，其特征在于所述DEK蛋白具有SEQ ID NO.25所示保守序列。
  62. 如权利要求61所述的方法，其特征在于所述DEK蛋白具有SEQ ID NO.2所示NLS结构域的氨基酸序列95％以上相似性。
  63. 如权利要求61所述的方法，其特征在于所述DEK蛋白具有SEQ ID NO.3所示SAP结构域的氨基酸序列95％以上相似性。
  64. 如权利要求61所述的方法，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域的氨基酸序列95％以上相似性。
  65. 如权利要求61所述的方法，其特征在于所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域或SEQ ID NO.3所示SAP结构域中的一种或两种，且同时具有SEQ ID NO.2所示NLS结构域。
  66. 如权利要求61～65之一所述的方法，其特征在于所述DEK蛋白具有SEQ ID NO.1或SEQ ID NO.22所示氨基酸序列95％以上相似性。
  67. 如权利要求66所述的方法，其特征在于所述DEK蛋白具有SEQ ID NO.1所示氨基酸序列。
  68. 如权利要求66所述的方法，其特征在于所述DEK蛋白具有SEQ ID NO.22所示氨基酸序列。
  69. 如权利要求59所述的方法，其特征在于所述DEK蛋白以投递DEK蛋白的形式注射，所述投递DEK蛋白是指含有DEK蛋白的外泌体、脂质体或纳米材料。
  70. 如权利要求59所述的方法，其特征在于所述肿瘤包括头颈部肿瘤，胸部肿瘤，消化系统肿瘤，泌尿生殖系统肿瘤，骨及软组织肿瘤，淋巴及血液系统肿瘤。
  71. 如权利要求70所述的方法，其特征在于所述肿瘤包括脑癌、眼癌、耳部肿瘤、颌骨肿瘤、颈部肿瘤、鼻腔癌、鼻窦癌、鼻咽癌、牙龈癌、舌癌、软硬腭癌、颌骨癌、口底癌、口咽癌、唇癌、上颌窦癌、颜面部皮肤黏膜的癌症喉癌、涎腺肿瘤、甲状腺癌、脑膜瘤、室管膜瘤、垂体瘤、上皮神经母细胞瘤、神经外胚层肿瘤、副神经节肿瘤、肺癌、食管癌、乳腺癌、纵膈肿瘤、胸腺癌、胃癌、大肠癌、肝癌、胰腺癌、胆管癌、小肠癌、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、宫颈癌、子宫癌、卵巢癌、输卵管癌、阴道癌、尤文氏肉瘤、脂肪肿瘤、卡波济肉瘤、平滑肌肿瘤、横纹肌肿瘤、血管肿瘤、滑膜肉瘤、纤维肉瘤、骨癌、恶性淋巴瘤、多发性骨髓瘤、白血病、心脏肿瘤、间皮肿瘤、纤维母细胞肿瘤、滋养细胞肿瘤、黑色素瘤。
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