CN110669111B

CN110669111B - Cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparation of anti-cancer drugs

Info

Publication number: CN110669111B
Application number: CN201910967207.9A
Authority: CN
Inventors: 王立新; 黄芳
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2021-07-09
Anticipated expiration: 2039-10-12
Also published as: CN110669111A

Abstract

The invention discloses a cancer stem cell-like drug-resistant cell-derived ubiquitination protein and application thereof in preparation of anti-cancer drugs, and the cancer stem cell-like drug-resistant cell-derived ubiquitination protein is mainly prepared by the following method: (1) inducing cancer cell drug resistance by using chemotherapeutic drugs to obtain a cancer stem cell-like drug-resistant cell strain enriched with cancer stem cells; (2) coupling the ubiquitinated protein recruiting protein to an adjuvant to obtain a coupled adjuvant; (3) culturing the cancer stem cell-like drug-resistant cell strain, inhibiting degradation of ubiquitinated protein, and then recruiting the ubiquitinated protein by using the coupling adjuvant obtained in the step (2) to obtain the ubiquitinated protein. The invention changes the source of ubiquitinated protein on the original basis, prepares and obtains ubiquitinated protein with the specificity of cancer stem cell-like drug-resistant tumor cell antigen, induces the immune response with the specificity of cancer stem cell-like drug-resistant tumor cell, and greatly improves the tumor treatment effect of the vaccine by combining with STING agonist.

Description

Cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparation of anti-cancer drugs

Technical Field

The invention relates to a cancer stem cell-like drug-resistant cell-derived ubiquitination protein and application thereof in preparation of anti-cancer drugs, belonging to the technical field of tumor vaccines.

Background

With the continuous development of oncology, immunology, molecular biology and other disciplines, tumor immunotherapy is becoming an emerging treatment method beyond traditional treatments such as surgery, chemotherapy, radiotherapy and the like. Tumor immunotherapy kills tumors, inhibits tumor growth and metastasis by stimulating and enhancing the body's own anti-tumor immune response. In clinical studies, immunotherapy has achieved dramatic results in melanoma, lung cancer, ovarian cancer, breast cancer, etc., and some immunotherapies have received FDA approval in the united states. Taking breast cancer as an example, breast cancer still is a common cancer of women at present, and is the second leading cause of death of women, and the incidence rate is also increased year by year. The current conventional treatment for breast cancer includes conventional cancer treatment methods using surgery, radiation therapy and chemotherapy, and also includes targeted therapy and hormone therapy. Clinical data show that breast cancer has high heterogeneity, high metastasis, easy recurrence and poor prognosis, most tumor patients are not sensitive to chemotherapy, radiotherapy and the like, long-term chemotherapy and targeted therapy can also cause drug resistance of breast cancer patients, and particularly for patients who appear or develop into stage IV diseases, the 5-year survival rate is still poor. Therefore, new means and treatment strategies are urgently needed for clinical treatment of breast cancer, and the emergence of immunotherapy in recent years is expected to become a new opportunity for treating breast cancer. Immunotherapy including tumor vaccines has the advantages of strong targeting property, small toxic and side effects and the like, and clinically tests with a plurality of achievements are achieved, and especially therapeutic tumor vaccines are more and more concerned by people.

The important factor for the tumor vaccine to play a role is the tumor-associated antigens (TAAs), the tumor vaccine is mainly formed by cross presenting the TAAs by professional antigen presenting cells (pAPC), inducing initial T cell response, recognizing the TAAs by activated T cells and specifically killing target cells. The applicant finds that autophagosome DRibble rich in ubiquitination protein is an effective antigen carrier, tumor antigen carried by the DRibble can be taken up by DC, and specific CD8 is induced by a cross-presentation mode⁺T cell response. Further research shows that ubiquitinated protein Ub is a main component of DRibble playing an anti-tumor role, and applicant can directly enrich ubiquitinated protein in tumor cell lysate rich in ubiquitinated protein through Vx3 fusion protein (His-Vx3-eGFP) with three ubiquitin molecule binding motifs (UIM) in earlier stages to play an anti-tumor role on various tumor models. In order to further optimize the ubiquitinated protein vaccine, the applicant covalently couples His-Vx3-eGFP protein to nano aluminum, and by utilizing the physical characteristics of the nano aluminum, the ubiquitinated protein can be quickly, simply and efficiently recruited by a one-step centrifugation method, and meanwhile, the nano aluminum is used as an immune adjuvant, so that the immune effect of the ubiquitinated protein is further improved, the ubiquitinated protein-nano aluminum adjuvant composite vaccine is successfully prepared, and the anti-tumor effect can be effectively exerted on a tumor-bearing mouse model.

The breast cancer patients are easy to have metastasis and generate tumor drug resistance, cancer stem-like breast cancer tumor cells are enriched in the drug-resistant tumor cells, and the cancer stem cells are a small part of cell groups which exist in tumor tissues and have self-renewal, proliferation and differentiation functions and are considered as key factors and even sources of tumor recurrence, metastasis and drug resistance. The development of a new treatment strategy taking cancer stem cells as targets, the elimination of the cancer stem cells which are mainly targeted while paying attention to the overall reduction of the tumor volume is expected to overcome the occurrence of tumor recurrence, metastasis and drug tolerance, and the new concept in the field of tumor treatment is formed.

The existing conventional therapeutic tumor vaccine is usually aimed at primary drug-intolerant non-metastatic breast cancer, and can not play an effective role in some breast cancer patients who are in late stage or have metastasis or drug resistance.

Disclosure of Invention

The purpose of the invention is as follows: in order to overcome the defects of the prior art, the invention aims to provide a cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparing an anti-cancer drug, in particular application of the ubiquitinated protein in combination with Sting agonist DMXAA in treating metastatic cancer.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

a cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is mainly prepared by the following steps:

(1) establishment of cancer-resistant cells: inducing cancer cell drug resistance by using chemotherapeutic drugs to obtain a cancer stem cell-like drug-resistant cell strain enriched with cancer stem cells;

(2) modification of the adjuvant: coupling the ubiquitinated protein recruiting protein to an adjuvant to obtain a coupled adjuvant;

(3) recruiting ubiquitinated proteins: culturing the cancer stem cell-like drug-resistant cell strain, inhibiting degradation of ubiquitinated protein, and then collecting ubiquitinated protein by using the coupling adjuvant obtained in the step (2), so as to obtain the ubiquitinated protein derived from the cancer stem cell-like drug-resistant cell.

In step (1), the cancer cells are preferably breast cancer cells, and the chemotherapeutic drug may be selected from clinical commonly used drugs for breast cancer patients, including alkylating agents (such as cyclophosphamide, melphalan, etc.), antimetabolic drugs (such as fluorouracil, methotrexate, etc.), antibiotics (such as adriamycin, epirubicin, mitomycin C, etc.), alkaloids (such as vincristine, colchicine, etc.), and taxols (such as docetaxel, etc.), preferably the antibiotics epirubicin. Other similar cancer cells are also suitable, for example, the cancer cells can also be selected from lung cancer cells, and the chemotherapy drug cisplatin is used for inducing the drug resistance of the lung cancer cells, and the like.

In the step (1), the drug resistance of the cancer cells is induced by combining a low concentration gradient increment method with a high-dose intermittent impact method, and the method is preferably as follows:

culturing cancer cells, changing into a culture medium containing chemotherapeutic drugs when the cells grow to 55-85%, gradually increasing the drug-containing concentration of the culture medium from (0.01-5) mu g/ml to (100) -500 mu g/ml, culturing, changing the culture medium once every 1-3 days, and carrying out passage.

In step (2), the ubiquitinated protein recruiting protein may be selected from a Ubiquitin Antibody (e.g., Ubiquitin Antibody, K11-link Specific Polyubiquitin Antibody, K48-link Specific Polyubiquitin Antibody, K63-link Specific Polyubiquitin Antibody, etc.) or a Ubiquitin binding protein, preferably a Ubiquitin binding protein such as Vx3 protein, etc., and more preferably Vx3 protein.

For preparation and purification of Vx3 protein, tag protein such as His protein, MyC protein, GST protein or HA protein can be selected for labeling, preferably His protein.

The adjuvant is selected from a nano calcium phosphate adjuvant, a nano ferroferric oxide adjuvant, a manganese adjuvant or nano aluminum, and the nano aluminum is preferred.

Vx3 protein can be adsorbed or covalently coupled to nano-aluminum adjuvant, preferably by covalent coupling method with coupling agent. More preferably, the coupling agent is selected from triethoxysilane, which is linked to the nano-aluminum adjuvant through an aldehyde group and then reacts with His-Vx3 protein to generate a stable carbon-nitrogen double bond through an amino group and a carboxyl group.

The His-Vx3 protein can be prepared by the following method:

and (3) recovering the Escherichia coli transformed with the His-Vx3 expression plasmid, inducing the expression protein of the Escherichia coli, and extracting the His-Vx3 protein by adopting a nickel ion chromatography.

The His-Vx3 protein is a Vx3(A7) fusion protein with a ubiquitin binding domain, the Vx3(A7) fusion protein is an artificial construct which is connected in series with three UIMs (ubiquitin-interacting moieties) derived from Vps27(a yeast homology of HRS, Hepatocyte growth factor-regulated tyrosine kinase substrate), and the protein is a protein which is reported in recent years and is artificially constructed and connected in series with the ubiquitin binding domain and has high affinity to ubiquitinated protein.

In the step (3), the method for inhibiting the degradation of ubiquitinated protein is preferably: the cultured cancer stem cell-like drug-resistant cells grow to 75-85%, a proteasome inhibitor (such as PSI, lactacystin, MG132 or bortezomib, preferably bortezomib) and ammonium chloride are added, the addition amount is respectively (200-.

And (3) after inhibiting degradation of ubiquitinated protein in the drug-resistant cells, treating the cells by using a cell lysate, centrifuging to obtain a supernatant, incubating the supernatant with the coupled nano aluminum particles to collect ubiquitinated protein, and centrifuging at a low temperature (2-15 ℃) to obtain a precipitate, namely the ubiquitinated protein derived from the cancer stem cell-like drug-resistant cells. The co-incubation is preferably performed overnight at 4 ℃.

The invention also provides application of the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein in preparation of anti-cancer drugs. Preferably, when the ubiquitinated protein source is a breast cancer cell, it can be used to treat breast cancer.

Preferably, the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is used for preparing a drug for treating drug-resistant metastatic cancer, and further preferably, when the ubiquitinated protein is derived from breast cancer cells, the ubiquitinated protein can be used for treating drug-resistant metastatic breast cancer.

The invention finally provides application of the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein combined Sting agonist in preparation of anti-cancer drugs. Preferably, when the ubiquitinated protein source is breast cancer cells, the combination of the ubiquitinated protein and the Sting agonist can be used for treating breast cancer, and the effect is more remarkable.

Preferably, the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is combined with Sting agonist to be applied to preparation of drugs for treating drug-resistant metastatic cancer. Preferably, when the ubiquitinated protein source is a breast cancer cell, the combination of the ubiquitinated protein and a Sting agonist can be used for treating drug-resistant metastatic breast cancer, and the effect is more remarkable.

The Sting agonist is selected from DMXAA or 2'3' -c-di-Amp, etc., or 2'3' -cGAMP, 3'3' -cGAMP, c-di-AMP, etc., which have similar structure to 2'3' -c-di-Amp, etc., and preferably DMXAA or 2'3' -c-di-Amp.

Aiming at drug-resistant and multiple metastatic cancer patients generated by clinical chemotherapy, particularly breast cancer patients, the invention prepares a novel vaccine of drug-resistant cell-derived ubiquitinated protein in a cancer stem cell shape by utilizing a highly metastatic mouse 4T1 model simulating human IV-stage breast cancer, solves the problems of drug resistance and high metastasis of breast cancer tumor, and explores the anti-tumor effect and mechanism of the novel vaccine. The method comprises the steps of inducing cancer stem cell-like epirubicin-resistant cells by an in vitro low-concentration gradient increment combined large-dose intermittent impact method, further identifying drug resistance and cancer stem cell characteristics in vivo and in vitro, successfully inducing the cancer stem cell-like epirubicin-resistant cells 4T1/EPB, and establishing a breast cancer drug-resistant metastatic tumor-bearing mouse model through the 4T1/EPB cells.

In addition, the invention connects the ubiquitinated protein of the drug-resistant tumor cell source of the cancer stem cell sample with the nano-aluminum to prepare a novel vaccine on the basis of the original vaccine, combines with Sting agonist DMXAA and the like, explores a new immunotherapy strategy, researches and observes the treatment effect on a drug-resistant high-metastatic breast cancer 4T1/EPB mouse model, and provides an experimental basis for the clinical research of the treatment of multiple chemotherapy drug-resistant and metastatic cancer patients.

Compared with the existing research, the research of the invention has the following advantages:

1. the vaccine alpha-Al prepared by the invention₂O₃The alpha-Al of the previous generation nano aluminum vaccine is greatly improved by the-His-Vx 3-Ub (4T1/EPB)₂O₃The anti-tumor effect of His-Vx3-Ub (4T1/WT) on 4T1/WT breast cancer tumor-bearing mice is obviously prolongedSurvival time of the mouse;

2. the vaccine of the invention alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) in 4T1/EPB drug-resistant highly metastatic breast cancer tumor-bearing mice can effectively inhibit tumor growth and tumor lung metastasis and remarkably prolong the survival time of the mice;

3. the vaccine of the invention alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) combined with Sting agonist DMXAA or 2'3' -c-di-Amp can cause the regression of most of mouse tumors in 4T1/EPB drug-resistant highly metastatic breast cancer tumor-bearing mice, and tumor-regressed tumor-bearing mice have immunological memory and can resist the re-attack of 4T1/EPB tumors.

The technical effects are as follows: compared with the prior art, the method changes the source of the ubiquitinated protein on the basis of the prior art, prepares the ubiquitinated protein with the specificity of the cancer stem cell-like drug-resistant tumor cell antigen, induces the immune response with the specificity of the cancer stem cell-like drug-resistant tumor cell, and combines the STING agonist, thereby greatly improving the treatment effect of the vaccine, and particularly having more remarkable treatment effect on the drug-resistant metastatic cancer.

Drawings

FIG. 1: induction and drug resistance identification of mouse breast cancer epirubicin resistant cells 4T 1/EPB; A. detecting a drug resistance index; B. detecting the gene expression level of the drug-resistance related gene; C. detecting the protein expression level of the drug resistance related gene; D. detecting the sensitivity of the chemotherapy drugs in the mice; E. detection of metastasis in two tumor-established mouse models.

FIG. 2: identification of mouse breast cancer epirubicin resistant cell 4T1/EPB cancer stem cell: A.4T1/EPB cancer stem cell surface marker CD44⁺CD24^-/lowDetection of the subpopulation; detecting a 4T1/EPB cancer stem cell marker acetaldehyde dehydrogenase 1(ALDH 1); C.4T1/EPB migration ability detection; D.4T1/EPB invasion capacity detection; E.4T1/EPB tumorigenicity detection.

FIG. 3: alpha-Al₂O₃His-Vx 3-recruitment of ubiquitinated proteins from 4T1/WT and 4T1/EPB cells, respectively; A. detecting the loading amount of the sample protein; detection of the expression of ub.

FIG. 4: alpha-Al₂O₃Detection of the anti-tumor effect of the His-Vx3-Ub (4T1/EPB) vaccine; A. alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) vaccine treated the tumor growth curve of mice in a 4T1/WT tumor-bearing mouse model; B. alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃-the survival time of His-Vx3-Ub (4T1/EPB) vaccine treated mice in a 4T1/WT tumor-bearing mouse model; C. alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) vaccine for treatment of metastasis in mice in a 4T1/WT tumor-bearing mouse model; D. alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) vaccine treated the tumor growth curve of mice in a 4T1/EPB tumor-bearing mouse model; E. alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃-the survival time of His-Vx3-Ub (4T1/EPB) vaccine treated mice in a 4T1/WT tumor-bearing mouse model; F. alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) vaccine treated mice for metastasis in the 4T1/WT tumor-bearing mouse model.

FIG. 5: observation of alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) and alpha-Al₂O₃Detection of the immune response induced by His-Vx3-Ub (4T1/WT) and its specificity: A. CD3 in spleen cells of mice after different immunizations⁺CD8⁺IFN-γ⁺The proportion of T cells of (a); B. CD3 in spleen cells of mice after different immunizations⁺CD8⁺IFN-γ⁺(iii) statistics of T cell ratios of (a); C. and (3) detecting the IFN-gamma level in spleen cells of mice after different immunizations.

FIG. 6: alpha-Al₂O₃Detection of the Effect of His-Vx3-Ub (4T1/EPB) vaccine Mixed Sting agonists on BMDC: A. alpha-Al₂O₃Detection of BMDC secreting type I interferon IFN- β by His-Vx3-Ub (4T1/EPB) mixed Sting agonist 2'3' -c-di-Amp; BETA, ALPHA-Al₂O₃Detection of BMDC secreting type I interferon IFN- β by His-Vx3-Ub (4T1/EPB) mixed Sting agonist DMXAA.

FIG. 7: alpha-Al₂O₃Detection of the anti-tumor effect of His-Vx3-Ub (4T1/EPB) vaccine in combination with Sting agonist DMXAA; A. alpha-Al₂O₃-tumor growth curve of His-Vx3-Ub (4T1/WT) vaccine in combination with Sting agonist DMXAA for treatment of 4T1/EPB tumor-bearing mice; B. alpha-Al₂O₃-the survival time of 4T1/EPB tumor-bearing mice treated with His-Vx3-Ub (4T1/WT) vaccine in combination with Sting agonist DMXAA; C. alpha-Al₂O₃-transfer profile of His-Vx3-Ub (4T1/WT) vaccine in combination with Sting agonist DMXAA for treatment of 4T1/EPB tumor-bearing mice; D. alpha-Al₂O₃Detection of immunological memory induced by His-Vx3-Ub (4T1/EPB) vaccine in combination with Sting agonist DMXAA.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples.

Example 1 preparation of cancer Stem cell-like drug-resistant cell-derived ubiquitinated protein

1. Induction of mouse cancer stem cell-like breast cancer epirubicin resistant cells 4T 1/EPB:

(1) induction of epirubicin-resistant cells 4T 1/EPB:

selecting Epirubicin (EPB) to induce the drug resistance of 4T1 breast cancer cells (4T1/WT) of mice by a low-concentration gradient increasing and large-dose intermittent impact method in vitro, purchasing 4T1/WT in an ATCC cell bank, recovering and freezing the 4T1/WT cells, culturing the cells by using RPMI-1640 culture medium containing 10% (V/V) fetal calf serum, 100U/mL penicillin and 100U/mL streptomycin till the cells grow to about 60%, changing the culture medium into a drug-added culture medium, gradually increasing the drug-added concentration of the culture medium of 2.0 mu g/mL from the beginning to 250 mu g/mL, culturing the cells in a constant-temperature culture box of 37 ℃ and 5% (V/V) CO2, changing the liquid once every 3 days, and conventionally digesting the cells by using 0.25% (g/mL) of trypsin.

(2) IC50 detection of induced cells:

the CCK8 method detects the IC50 of the induced cells and 4T1/WT cells to the EPB, and the drug resistance index reaches 35.0 calculated as shown in figure 1A, which indicates that the induced cells reach high drug resistance and are named epirubicin resistant cell strains (4T 1/EPB).

2. Drug resistance identification of mouse breast cancer epirubicin resistant cell 4T1/EPB

(1) Detection of drug resistance gene of 4T 1/EPB:

a) when 4T1/WT cells and 4T1/EPB cells were cultured to 80% in the conventional tumor cell culture method, 1X 10 of the digestion count was obtained⁶Extracting RNA from individual cells with TRIzol, preparing sample, detecting gene expression levels of multidrug resistance gene (MDR1), glutathione S-transferase-pi (GST-pi), Breast Cancer Resistance Protein (BCRP), and matrix metalloproteinase-7 (MMP7), and collecting 1 × 10⁶Adding RIPA cell lysate to ice for 40 times, centrifuging, collecting supernatant, taking a proper amount of supernatant, adding SDS-PAGE loading buffer, transferring protein to a PVDF membrane after electrophoresis, and adding an anti-MDR 1 antibody and an anti-BCRP antibody. And (3) incubating overnight at 4 ℃, washing the membrane by PBST 5 times in the next day, adding corresponding secondary HRP-labeled antibody for 5min each time, incubating for 1h at room temperature, adding a color reagent for PBST 5 times for 5min each time, and photographing to detect the protein expression levels of the MDR1 and the BCRP drug-resistant gene. As shown in fig. 1B and 1C, the expression level of the drug-resistant gene of 4T1/EPB cells was significantly increased compared to 4T 1/WT.

b) The sensitivity of 4T1/EPB to the chemotherapeutic drug EPB was tested in mice: 12 BALB/c female 6-8 week old mice were inoculated with 1X 10 of the vaccine at the left and right breast pads, respectively⁴4T1/WT and 4T1/EPB cells, this is day 0, on day 3 of palpation of the tumor, were randomly divided into 2 groups, Normal Saline (NS) control and EPB groups, and mice received treatment with 500. mu.g/500. mu.l of EPB or saline tail vein on days 7 and 14, and both tumor growth was observed. As shown in FIG. 1D, 4T1/WT was found to be sensitive to EPB with significant inhibition of tumor size, whereas 4T1/EPB was not sensitive to EPB with no inhibition of tumor growth.

c) Detection of metastasis in two mouse models of tumor establishment: 24 BALB/c female 6-8 week mice were randomly divided into 2 groups, and 1X 10 mice were inoculated on day 0 to the breast pads of the two groups of mice⁴Two mouse tumor models were established with 4T1/WT and 4T1/EPB cells, and on day 3 of tumor palpation, the 4T1/WT and 4T1/EPB groups were randomized into 4 groups, 4T1/WT + NS and 4T1/EPB + NS groups treated with saline, 4T1/WT + EPB and 4T1/EPB + EPB groups treated with EPB, and 7 th and 1 st EPB groups, respectivelyAfter 4 days of 500. mu.g/500. mu.l EPB or saline tail vein treatment, mice were euthanized at day 30 and observed for metastasis in both models, as shown in FIG. 1E, with 67% mice in the 4T1/WT mouse model and 30% mice receiving EPB treatment, while 100% mice in the 4T1/EPB mouse model and 100% mice receiving EPB treatment, indicating that 4T1/EPB cells had enhanced metastatic potential over 4T1/WT and little effect of EPB treatment on metastasis inhibition, indicating that the 4T 1/EPB-induced mouse model is a mouse model of multiple metastatic breast cancer.

3. Identification of cancer stem cell of mouse breast cancer epirubicin resistant cell 4T1/EPB

(1) Detection of 4T1/EPB cancer stem cell surface marker:

a) when 4T1/WT cells and 4T1/EPB cells were cultured to 80% in the conventional tumor cell culture method, 1X 10 of the digestion count was obtained⁶Staining individual cells according to the instruction, detecting the subgroup CD44 according with the surface mark of breast cancer stem cells by using a flow cytometer⁺CD24^-/lowSubgroup, CD44 of 4T1/WT, as shown in FIG. 2A⁺CD24^-/lowThe subpopulation ratio was 28% and the 4T1/EPB subpopulation ratio was 81%, indicating an enrichment of stem cells in 4T 1/EPB.

(2) Detection of 4T1/EPB cancer stem cell marker acetaldehyde dehydrogenase 1(ALDH 1):

when 4T1/WT cells and 4T1/EPB cells were cultured to 80% in the conventional tumor cell culture method, 1X 10 of the digestion count was obtained⁶The cells were stained according to the instructions and tested by flow cytometry to meet the breast cancer stem cell marker ALDH1, as shown in FIG. 2B, 4T1/WT showed positive expression of ALDH1 of 0.8% and 4T1/EPBALDH1 of 11.6%, indicating that 4T1/EPB is enriched in stem cells.

(3)4T1/EPB migration and invasion capacity detection:

a)4T1/EPB migration capability test: respectively culturing 4T1/WT cells and 4T1/EPB cells to 80% according to conventional tumor cell culture method, digesting the cells, washing with PBS and serum-free medium for 1 time, suspending the cells with serum-free medium,counting, adjusting the concentration to 7 × 10³Ml, insert was removed and placed in a 24-well plate, 500. mu.l of lower chamber medium (containing 5% serum) was added first, and 200. mu.l of cell suspension was added to the insert. The insert was put into a 24-well plate, at 37 ℃ for 36h, and the statistics of photographs after staining were obtained, as shown in FIG. 2C, the migration ability of 4T1/EPB was significantly improved compared with that of 4T 1/WT;

b)4T1/EPB invasion ability test: the upper face of the bottom membrane of the Transwell cell was coated with 50mg/L matrigel 1:8 dilution and air dried at 4 ℃. Respectively culturing 4T1/WT cells and 4T1/EPB cells to 80% according to conventional tumor cell culture method, digesting cells, washing with PBS and serum-free medium for 1 time, suspending cells with serum-free medium, counting, and adjusting concentration to 1 × 10³Ml, insert was removed and placed in a 24-well plate, 500. mu.l of lower chamber medium (containing 5% serum) was added first, and 200. mu.l of cell suspension was added to the insert. Putting insert into a 24-well plate, keeping the temperature at 37 ℃ for 24h, and taking a picture for counting after dyeing, wherein the invasion capacity of 4T1/EPB is obviously improved compared with that of 4T1/WT as shown in figure 2D;

(4) detection of 4T1/EPB tumorigenic experiment:

when 4T1/WT cells and 4T1/EPB cells were cultured to 80% in the conventional tumor cell culture method, the number of digestions was counted, and 5 gradients (2X 10) were set, respectively²、2×10³、2×10⁴、2×10⁵、2×10⁶)5 BALB/c mice at 6-8 weeks per gradient, one mouse was inoculated with 4T1/WT tumor on the left breast pad and 4T1/EPB tumor on the right breast pad, and the tumor formation of each gradient mouse was observed. As shown in FIG. 2E, a minimum of 2X 10 is required⁵The mouse can be tumorigenic only by 4T1/WT tumor cells, and the mouse can be tumorigenic only by 200 4T1/EPB tumor cells, which shows that the tumorigenic capacity of the 4T1/EPB tumor is obviously improved, and the cancer stem cell property of the 4T1/EPB is enhanced.

In conclusion, the cancer stem cell-like mouse breast cancer epirubicin resistant cell line 4T1/EPB has been successfully prepared, and the mouse model induced by 4T1/EPB is a mouse model of multiple metastatic breast cancer.

4、α-Al₂O₃His-Vx3 recruit ubiquitinated proteins from 4T1/WT and 4T1/EPB cells, respectivelyPreparation of alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) vaccine

(1) Treatment of tumor cells: respectively culturing 4T1/WT and 4T1/EPB cells, adding 400nmol/L bortezomib and 80mmol/L ammonium chloride when the cells grow to 80%, intervening the tumor cells for 166 hours, digesting the tumor cells, washing for three times by PBS, centrifuging to take out cell precipitates, adding protease inhibitor, phosphatase inhibitor and RP-619 RIPA lysate to lyse the cells at low temperature, inhibiting the degradation of ubiquitinated protein, standing on ice for 40 minutes, centrifuging at high speed and low temperature for 40 minutes, and taking supernatant to obtain the tumor cell lysate rich in ubiquitinated protein.

(2)α-Al₂O₃-preparation of Vx3 nanoparticles:

a) preparing nano aluminum particles: weighing dried nano aluminum (alpha-Al)₂O₃)500mg, placing in a 150mL flat-bottom test tube, adding 1629.39mg of triethoxysilane and 37.58mL of absolute ethanol, stirring and reacting for 12h at room temperature, placing the reaction product at 15000g, centrifuging for 10min to remove the supernatant, washing the precipitate with a large amount of absolute ethanol three times, taking the precipitate obtained in the previous step, adding 3.2784mL of glutaraldehyde (25%) and 20.605mL of ultrapure water, stirring and reacting for 2h at room temperature, placing the reaction product at 15000g, centrifuging for 10min to remove the supernatant, washing the precipitate with a large amount of ultrapure water three times, taking the precipitate to obtain alpha-Al₂O₃；

b) His-Vx3 protein was covalently coupled to the nano-aluminum particles: resuscitating escherichia coli transformed with His-Vx3 expression plasmid, coating the escherichia coli on an LB plate containing 50 mu g/ml ampicillin, after 14h, selecting a monoclonal antibody to an LB culture solution containing ampicillin, carrying out shake culture at 37 ℃ for-8 h, sucking 1ml, inoculating the solution to 100ml of LB culture solution, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 100mM to induce the solution at low temperature for 16h when the solution grows to logarithmic phase, carrying out low-temperature high-speed centrifugation on the obtained solution to obtain bacteria, Washing the bacteria for three times by PBS, adding 100 mu g/ml lysozyme after buffer resuspension to act on ice for 40min, then acting the bacteria on an ultrasonic crusher for 50min, carrying out high-speed low-temperature centrifugation to obtain a supernatant, adding the supernatant into a nickel ion affinity chromatographic column, carrying out shake reaction at 4 ℃ overnight, Washing the protein by Washing buffer on the next day, eluting the His-Vx3 protein by an Elution buffer, and finally obtaining the Escherichia colialpha-Al of (2)₂O₃Stirring and reacting with His-Vx3 protein at 4 ℃ overnight, and centrifuging at low temperature and high speed the next day to collect the covalent coupling product (alpha-Al) of nano aluminum and His-Vx3 protein₂O₃-His-Vx3)；

(3) With alpha-Al₂O₃-His-Vx3 nanoparticles recruit ubiquitinated proteins to prepare alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) vaccine:

the 4T1/WT and 4T1/EPB cell lysates collected in (1) were mixed with the alpha-Al cell lysate obtained in (2)₂O₃Incubating at-His-Vx 34 deg.C overnight, and centrifuging at low temperature and high speed the next day to obtain alpha-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB), and unbound cell lysate of the supernatant was also collected after centrifugation.

(4)α-Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃Identification of His-Vx3-Ub (4T1/EPB) vaccine:

alpha-Al obtained in (3)₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃Re-suspending His-Vx3-Ub (4T1/EPB) with a proper amount of PBS, sucking 100 mu l of the re-suspended solution, centrifuging at a high speed, removing the supernatant, adding 100 mu l of SDS-PAGE loading buffer into the obtained tumor cell lysate (1) and the unbound tumor cell lysate (3) respectively, carrying out boiling water bath for 5min, centrifuging, taking the supernatant, carrying out SDS-PAGE, and adding an anti-Ub antibody to detect the Ub protein; the results are shown in FIG. 3A, with

bands

2 and 5 representing total cell lysates of 4T1/WT and 4T1/EPB, respectively,

bands

3 and 6 representing unbound cell lysates of 4T1/WT and 4T1/EPB, respectively, and

bands

4 and 7 representing prepared α -Al, respectively₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB), 12.5% SDS-PAGE electrophoretic staining showed consistent sample loading for each group. As shown in FIG. 3B, the total cell lysates of 4T1/WT and 4T1/EPB represented by

bands

1 and 4 had a certain amount of Ub protein, the unbound cell lysates of 4T1/WT and 4T1/EPB represented by

bands

2 and 5 had a very low amount of Ub protein, and the prepared α -Al represented by bands 3 and 6₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃-His-Vx3-Ub(4T1/EPB) has the largest Ub protein content, which is obviously more than that of total cell lysate and unbound cell lysate, indicating that alpha-Al₂O₃His-Vx3 can be recruited to a large number of ubiquitinated proteins in 4T1/WT and 4T1/EPB, and alpha-Al is successfully prepared₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃-His-Vx3-Ub(4T1/EPB)。

Example 2: alpha-Al observations in 4T1/WT and 4T1/EPB tumor-bearing mouse models, respectively₂O₃His-Vx3-Ub (4T1/WT) (abbreviated as UPs (4T1/WT) in the drawing) and alpha-Al₂O₃Therapeutic Effect of His-Vx3-Ub (4T1/EPB) (abbreviated as UPs (4T1/EPB) in the drawing)

1. Observation of alpha-Al in the 4T1/WT mouse model₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃Therapeutic Effect of His-Vx3-Ub (4T1/EPB) vaccine

Day 0 female BALB/c mice were inoculated with 1X 10 inoculations at the right breast pad of day 0³4T1/WT tumor cells, mice were randomly divided into saline control group (NS), alpha-Al on day 5₂O₃His-Vx3-Ub (4T1/WT) treatment group and alpha-Al₂O₃Three groups of-His-Vx 3-Ub (4T1/EPB) treatment groups were treated three times on

days

5, 7, and 9, and the treatment effect and the survival time of the mice were observed. As shown in FIG. 4A, α -Al₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) has inhibitory effect on 4T1/WT tumor, but alpha-Al₂O₃The tumor growth inhibition effect of-His-Vx 3-Ub (4T1/EPB) is more obvious, the survival time of tumor-bearing mice is remarkably prolonged as shown in figure 4B, the tumor metastasis is inhibited as shown in figure 4C, and in conclusion, the alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) showed more significant antitumor effect in the 4T1/WT tumor model.

2. Observation of alpha-Al in a 4T1/EPB mouse model₂O₃His-Vx3-Ub (4T1/WT) and alpha-Al₂O₃Therapeutic Effect of His-Vx3-Ub (4T1/EPB) vaccine:

day 0 female BALB/c mice were inoculated with 1X 10 inoculations at the right breast pad of day 0³4T1/WT tumor cells, mice were randomly assigned to saline control group (NS) on day 5,α-Al₂O₃His-Vx3-Ub (4T1/WT) treatment group and alpha-Al₂O₃Three groups of-His-Vx 3-Ub (4T1/EPB) treatment groups were treated three times on

days

5, 7, and 9, and the treatment effect and the survival time of the mice were observed. As shown in FIG. 4D, α -Al₂O₃His-Vx3-Ub (4T1/WT) showed no significant inhibition of 4T1/EPB tumor growth, whereas alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) significantly inhibited the growth of 4T1/EPB tumors, and both vaccines significantly prolonged the survival time of 4T1/EPB tumor-bearing mice, but α -Al₂O₃The elongation effect of His-Vx3-Ub (4T1/EPB) was better, as shown in FIG. 4F, alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) significantly inhibited tumor metastasis in 4T1/EPB tumor-bearing mice, while alpha-Al₂O₃Inhibition of 4T1/EPB tumor metastasis by-His-Vx 3-Ub (4T1/WT) was not evident, and in conclusion, α -Al₂O₃His-Vx3-Ub (4T1/EPB) showed more significant antitumor effect in the 4T1/EPB tumor model.

3. Observation of alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) and alpha-Al₂O₃Detection of the immune response induced by His-Vx3-Ub (4T1/WT) and its specificity:

day 0 9 female BALB/c mice at 6-8 weeks were randomly divided into NS groups, alpha-Al₂O₃His-Vx3-Ub (4T1/WT) group and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) group, which were immunized three times on

days

1, 3 and 5, respectively, and mouse spleen was taken after euthanizing mice on day 12 to prepare cell suspension, 5X 10³Each well/48 wells, cultured in vitro, and combined with 5X 10³Inactivated 4T1/WT and 4T1/EPB tumor cells were co-cultured for 48h, and levels of IFN- γ secreted from spleen cells were measured. As shown in FIGS. 5A and 5B, alpha-Al₂O₃Mouse splenocytes obtained after immunization with His-Vx3-Ub (4T1/EPB) vaccine, CD3 in coculture with two inactivated tumor cells⁺CD8⁺IFN-γ⁺Has the highest proportion of T cells and has obvious statistical difference compared with other groups, as shown in figure 5C, alpha-Al₂O₃The level of IFN-gamma secreted by splenocytes of mice immunized by His-Vx3-Ub (4T1/EPB) vaccine is highest, and has obvious statistical difference compared with other groups, which indicates that the alpha-Al vaccine is high in the content of IFN-gamma₂O₃His-Vx3-Ub (4T1/EPB) vaccine induced stronger tumor drug resistant cell specific immune response.

Example 3 Observation of alpha-Al in a tumor-bearing mouse model of 4T1/EPB₂O₃Therapeutic Effect of His-Vx3-Ub (4T1/EPB) in combination with Sting agonist DMXAA.

1. Observation of alpha-Al₂O₃Detection of the Effect of His-Vx3-Ub (4T1/EPB) vaccine Mixed Sting agonists on BMDC:

according to the conventional method for culturing mouse BMDC, the leg bone marrow of BALB/c mice of 6-8 weeks is cultured in RPMI-1640 medium containing GM-CSF20ng/ml, IL-420 ng/ml, 10% (V/V) fetal calf serum, 100U/ml penicillin and 100U/ml streptomycin, and changed every 2 days, and 1X 10 is taken on the sixth day⁶Each hole/48 holes are plated with CM group, LPS positive control group and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) group and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) Mixed Sting agonist 2'3' -c-di-Amp (purchased from Invivogen) group or DMXAA (purchased from MCE) group, co-cultured with BMDC for 48h, and IFN- β levels secreted by BMDC were measured by ELISA, as shown in FIGS. 6A and 6B, and α -Al₂O₃His-Vx3-Ub (4T1/EPB) hybrid Sting agonists, whether mixed with 2'3' -c-di-Amp or mixed with DMXAA group, BMDC secreted IFN- β levels significantly higher than α -Al alone₂O₃His-Vx3-Ub (4T1/EPB) group and Sting agonist alone, indicating alpha-Al₂O₃The ability of BMDC to secrete type I interferon IFN-beta is remarkably improved by combining His-Vx3-Ub (4T1/EPB) with Sting agonist, and the IFN-beta can further activate local 8 alpha⁺DC, activated CD8 alpha⁺DCs were directed to CD8 in lymph nodes⁺The T cells cross present tumor antigens, activate specific T cells and further play an anti-tumor role.

2. Observation of alpha-Al on a tumor-bearing mouse model of 4T1/EPB₂O₃Therapeutic Effect of His-Vx3-Ub (4T1/EPB) in combination with Sting agonist DMXAA, and with α -Al₂O₃His-Vx3-Ub (4T1/WT) comparison:

day 0 female BALB/c mice were inoculated with 1X 10 inoculations at the right breast pad of day 0³4T1EPB tumor cells, mice were randomly divided into NS group, DMXAA-treated group, alpha-Al on day 5₂O₃His-Vx3-Ub (4T1/WT) treatment group, alpha-Al₂O₃His-Vx3-Ub (4T1/WT) in combination with DMXAA treatment group, alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) treatment group and alpha-Al₂O₃Six groups of-His-Vx 3-Ub (4T1/EPB) in combination with DMXAA treatment group, all treated with 500. mu.g of chemotherapeutic agent EPB, three times on

days

8, 10 and 12, respectively, and observed for treatment effect and survival time of mice, as shown in FIG. 7A, using DMXAA alone, α -Al alone₂O₃His-Vx3-Ub (4T1/WT) group and alpha-Al₂O₃The growth of the-His-Vx 3-Ub (4T1/WT) and DMXAA team 4T1/EPB tumor has certain inhibition effect, but the inhibition effect is not obvious, and the alpha-Al is singly used₂O₃the-His-Vx 3-Ub (4T1/EPB) treatment group has obvious inhibition effect on the growth of 4T1/EPB tumor, and alpha-Al₂O₃The anti-tumor effect of the combination of His-Vx3-Ub (4T1/EPB) and DMXAA group was most pronounced, with regression of 5 tumors in 6 tumor-bearing mice in this group. As shown in FIG. 7B, alpha-Al alone is used₂O₃His-Vx3-Ub (4T1/EPB) treatment group and alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) in combination with DMXAA significantly prolonged the survival time of tumor-bearing mice compared to the other groups. As shown in FIG. 7C, alpha-Al alone was used₂O₃His-Vx3-Ub (4T1/EPB) treatment group and alpha-Al₂O₃The combination of His-Vx3-Ub (4T1/EPB) and DMXAA group remarkably inhibits the metastasis of tumor-bearing mice, alpha-Al₂O₃The best effect of the combination of His-Vx3-Ub (4T1/EPB) and DMXAA group inhibiting metastasis was obtained, as described above, with alpha-Al₂O₃The combination of-His-Vx 3-Ub (4T1/EPB) and DMXAA group further improves alpha-Al₂O₃Therapeutic efficacy of the His-Vx3-Ub (4T1/EPB) vaccine resulted in regression of 4T1/EPB tumor-bearing mice.

3. Observation of alpha-Al₂O₃His-Vx3-Ub (4T1/EPB) in combination with DMXAA induced immunological memory in mice:

selecting tumor-regressed mice from step 2 as vaccine group, and the same number of untreated BALB/c female mice as control group, and inoculating 1 × 10 mice into the two groups⁴4T1/EPB tumorTumor cells, and observing the growth condition of the tumor. As shown in FIG. 7D, 4T1/EPB tumor growth was observed in the control group of mice, whereas no tumor growth was observed in the vaccine group of mice that resisted secondary challenge with the 4T1/EPB tumor.

Claims

1. A cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is characterized in that the drug-resistant cell-derived ubiquitinated protein is mainly prepared by the following method:

(3) recruiting ubiquitinated proteins: culturing the cancer stem cell-like drug-resistant cell strain, inhibiting degradation of ubiquitinated protein, performing cell lysis treatment, centrifuging cell lysate to obtain supernatant, and then recruiting ubiquitinated protein by using the coupling adjuvant obtained in the step (2) to obtain the ubiquitinated protein derived from the cancer stem cell-like drug-resistant cells.

2. The cancer stem cell-like drug-resistant cell-derived ubiquitinated protein of claim 1, wherein in step (1), the cancer cell is a breast cancer cell and the chemotherapeutic is selected from the group consisting of alkylating agents, antimetabolite drugs, antibiotics, alkaloids, and taxanes.

3. The cancer stem cell-like resistant cell-derived ubiquitinated protein of claim 2, wherein the chemotherapeutic is epirubicin.

4. The cancer stem cell-like drug-resistant cell-derived ubiquitinated protein of claim 1, wherein in step (1), the cancer cell drug resistance is induced by a combination of low-concentration gradient increment and a high-dose intermittent impact method, as follows:

5. The cancer stem cell-like resistant cell-derived ubiquitinated protein of claim 1, wherein in step (2), the ubiquitinated protein recruiting protein is selected from a ubiquitin antibody or a ubiquitin binding protein;

the adjuvant is selected from a nano calcium phosphate adjuvant, a nano ferroferric oxide adjuvant, a manganese adjuvant or nano aluminum.

6. The cancer stem cell-like resistant cell-derived ubiquitinated protein of claim 5, wherein the ubiquitinated protein recruiting protein is Vx3 protein.

7. The cancer stem cell-like resistant cell-derived ubiquitinated protein of claim 1, wherein in the step (3), the method for inhibiting degradation of ubiquitinated protein comprises: culturing the cells to grow to 75-85%, adding a proteasome inhibitor and ammonium chloride in the amount of (200-500) nmol/L and (50-100) mmol/L respectively, and interfering the cells for 100-100 hours to inhibit the degradation of ubiquitinated protein.

8. Use of the cancer stem cell-like resistant cell-derived ubiquitinated protein of any one of claims 1 to 7 for the preparation of an anticancer drug.

9. The use according to claim 8, wherein the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is used for preparing a drug for treating drug-resistant metastatic cancer.

10. Use of the cancer stem cell-like resistant cell-derived ubiquitinated protein of any one of claims 1 to 7 in combination with Sting agonists for the preparation of an anti-cancer medicament.

11. The use of claim 10, wherein the cancer stem cell-like resistant tumor cell-derived ubiquitinated protein is used in combination with Sting agonists for the preparation of a medicament for the treatment of drug-resistant metastatic cancer.

12. The use according to claim 10, wherein the Sting agonist is selected from DMXAA or 2'3' -c-di-Amp.