WO2010124498A1

WO2010124498A1 - A resistance-screened tumor stem cell, its antigen composition, an anti-tumor dendritic cell loading with said antigens, their preparation methods, uses and kits thereof as well as a dendritic cell vaccine

Info

Publication number: WO2010124498A1
Application number: PCT/CN2009/075307
Authority: WO
Inventors: Li Shen; Limin Hao; Yajun Wang; Yanling Ma; Qiming Huang
Original assignee: Beijing Cellonis Biotechnology Co., Ltd
Priority date: 2009-04-30
Filing date: 2009-12-04
Publication date: 2010-11-04

Abstract

Resistance-screened tumor stem cells, their preparation method, use of such tumor stem cells for manufacturing anti-tumor medicaments and a kit for preparing said tumor stem cells. An antigen composition of said tumor stem cells, its preparation method, use of such antigen composition for manufacturing anti-tumor medicaments and a kit for preparing said antigen composition. Dendritic cells loading with said antigens obtained by pulsing normal dendritic cells with said antigen composition of said tumor stem cells, its preparation method, use of such dendritic cells for manufacturing anti-tumor medicaments, a kit for preparing such dendritic cells, and a vaccine of such dendritic cells. The medicaments prepared from the resistance-screened tumor stem cells, the antigen composition of such tumor stem cells and the dendritic cells loading with said antigens provided by the present invention, can induce immune response in vitro and in vivo to kill tumor stem cells which cause tumor recurrence, which make it possible to overcome tumor resistance and to radically cure tumor recurrence and metastasis.

Description

A RESISTANCE-SCREENED TUMOR STEM CELL, ITS ANTIGEN COMPOSITION, AN ANTI-TUMOR DENDRITIC CELL LOADING WITH SAID ANTIGENS, THEIR PREPARATION METHODS, USES AND KITS THEREOF AS WELL AS A DENDRITIC CELL VACCINE

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a tumor stem cell (TSC), its preparation method, use of said tumor stem cells for the manufacturing of an anti-tumor medicament, and a kit for preparing said tumor stem cells. The invention also relates to an antigen composition derived from said tumor stem cells, its preparation method, use of such antigen composition for the manufacturing of an anti-tumor medicament and a kit for preparing the antigen composition derived from tumor stem cells.

More particularly, this invention relates to a resistance-screened tumor stem cell, its preparation method, use of said resistance-screened tumor stem cells for the manufacturing of an anti-tumor medicament, and a kit for preparing said resistance-screened tumor stem cells. The invention also relates to an antigen composition derived from said resistance-screened tumor stem cells, its preparation method, use of such antigen composition for the manufacturing of an anti-tumor medicament and a kit for preparing the antigen composition derived from resistance-screened tumor stem cells.

The present invention still relates to a dendritic cell, its preparation method, use of such dendritic cells for the manufacturing of an anti-tumor medicament, a kit for preparing said dendritic cells, and a dendritic cell vaccine.

More particularly, the present invention relates to a dendritic cell with said antigens obtained by pulsing normal dendritic cells with said antigen compositions of said resistance-screened tumor stem cells, its preparation method, use of such dendritic cells for the manufacturing of an anti-tumor medicament, a kit for preparing such dendritic cells, and a vaccine of such dendritic cells. BACKGROUND OF THE INVENTION

At present, there are a number of methods and techniques for the treatment of cancer, among which may be included: radiotherapy, chemotherapy, and surgery. The common characteristic for all of these techniques as well as any other presently known technique is that they can not completely affect a radical cure. The main reason for it is that: among normal tumor cells, there are tumor stem cells, which possess biological characteristics of stem cells, such as self-renewal, multipotent differentiation, high resistant (e.g. high drug-resistant to chemotherapy, high radiation resistance to radiotherapy). Moreover, a few of stem tumor cells even can drive the formation of tumor. Therefore, tumor stem cells are the essential reason for tumor formation and development, including tumor development, treatment-resistant, recurrence and metastasis.

The methods for isolating tumor stem cells from patients' tumor tissues or screening tumor stem cells from tumor cell lines in vitro have been disclosed. For example, Irving L. Weissman et al. isolated and screened AML stem cells expressing CD 96 (mark of a stem cell) from a patent of acute myeloid leukaemia (See Naoki Hosen, Christopher Y. Park, Naoya Tatsumi, Yusuke Oji, Haruo Sugiyama, Martin Gramatzki, Alan M. Krensky, and Irving L. Weissman, CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia, PNAS, Jun. 26, 2007, vol.104, no.26:l 1008-11013); Xiu-wu Bian et al. isolated tumor stem cells from a human glioblastoma cell line U87 (See Shi-cang Yu, Yi-fang Ping, Liang Yi, Zhi-hua Zhou, Jian-hong Chen, Xiao-hong Yao, Lei Gao, Ji Ming Wang, Xiu-wu Bian, Isolation and characterization of tumor stem cells from a human glioblastoma cell line U87, Cancer Letters, Feb.3, 2008, vol.265: 124-134).

Moreover, the related art has attempted to use immunotherapy for cancer treatment, i.e., obtaining antigens by lysing tumor stem cells, making dendritic cells from a patient load with said antigens. The resultant dendritic cells targeting tumor stem cells may use to treat cancer. For example, WO2008/039874 discloses the method for isolating stem-like cells from the brain tumors, and loading dendritic cells with the antigens prepared from said stem-like cells, and using the resultant dendritic cells for treatment of cancers.

The related art only discloses how to get the related tumor stem cells, but it does not teach any extra operation of the obtained tumor stem cells. Moreover, the above therapies of the related art target tumor stem cells, but they still exist the problems of highly frequent emergences of treatment-resistant, recurrence and metastasis after radiotherapy and chemotherapy.

DESCRIPTION OF THE INVENTION

In order to overcome the problems of a cancer immunotherapy using ordinary tumor stem cells as targets, which are highly frequent emergences of treatment-resistant, recurrence and metastasis after radiotherapy and chemotherapy, the object of the present invention is to provide a resistance-screened tumor stem cell, its preparation method, use of such resistance-screened tumor stem cells for the manufacturing of an anti-tumor medicament and a kit for preparing the tumor stem cells as well as an antigen composition of said tumor stem cells, its preparation method, use of such antigen composition for the manufacturing of an anti-tumor medicament and a kit for preparing the antigen composition of tumor stem cells.

The further object of the present invention is to provide a dendritic cell loading with said antigens obtained by pulsing normal dendritic cells with said antigen compositions of said tumor stem cells, its preparation method, use of such dendritic cells for the manufacturing of an anti-tumor medicament, a kit for preparing such dendritic cells, and a vaccine of such dendritic cells.

The related art believes that all of tumor stem cells will be killed after being exposed to radiation and/or being contacted with a high-dose chemotherapeutic agent(s). However, the inventors of the present invention surprisingly found that after being exposed to a certain dose of radiation and/or being contacted with certain concentration of a chemotherapeutic agent(s), not only some of tumor stem cells do survive, but also they become the tumor stem cells with strong resistance. So it is very hard to kill said resistance-screened tumor stem cells by the ordinary dose of radiation exposure and/or the ordinary concentration of a chemotherapeutic agent(s). In an aspect, this application provides a method for preparing a resistance-screened tumor stem cell, characterized in that the method comprises: a step of performing radiation exposure screening and/or chemotherapeutic agent screening to tumor stem cells; wherein said radiation exposure screening is performed by exposing tumor stem cells to a radiation source of ray, wherein said ray is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent screening is performed by making tumor stem cells contact with a chemotherapeutic agent(s); wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide.

In a further aspect, this application also provides a tumor stem cell obtained by the above method.

In a further aspect, this application also provides a method for preparing an antigen composition of tumor stem cells, which comprises the steps of washing and re-suspending tumor stem cells with saline and then lysing the obtained tumor stem cells, characterized in that said tumor stem cells are the resistance-screened tumor stem cells of the present invention.

In a further aspect, this application also provides an antigen composition of tumor stem cells obtained by the above method.

In a further aspect, this application also provides use of the tumor stem cells of the present invention and use of the antigen composition of tumor stem cells of the present invention for the manufacturing of an anti-tumor medicament.

In a further aspect, this application also provides a kit for preparing the tumor stem cells of the present invention, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(b) a radiotherapeutic radioisotope source(s) and/or a chemotherapeutic agent(s);

(c) an enzyme(s) for digesting cells; (d) a cytokine(s); and

(e) operating instruction; wherein said radiotherapeutic radioisotope source is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide; wherein said operating instruction includes a method for preparing a resistance-screened tumor stem cell of the present invention.

In a further aspect, this application also provides a kit for preparing the antigen composition of tumor stem cells of the present invention, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(c) an enzyme(s) for digesting cells;

(d) a cytokine(s); and

(e) operating instruction; wherein said radiotherapeutic radioisotope source is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide; wherein said operating instruction includes a method for preparing an antigen composition of tumor stem cells of the present invention.

In a further aspect, this application still provides a method for preparing an anti-tumor dendritic cell, which comprises obtaining an antigen composition of tumor stem cells by lysing said tumor stem cells, making normal dendritic cells contact with said antigen composition of tumor stem cells, and then obtaining an anti-tumor dendritic cell loading with the antigens of said tumor stem cells, characterized in that said tumor stem cells are the tumor stem cells of the present invention.

In a further aspect, this application also provides an anti-tumor dendritic cells obtained by the above method.

In a further aspect, this application also provides a dendritic cell vaccine, which comprises dendritic cells, characterized in that said dendritic cells are the anti-tumor dendritic cells of the present invention.

In a further aspect, this application also provides use of the anti-tumor dendritic cells of the present invention for the manufacturing of an anti-tumor medicament.

In a further aspect, this application also provides a kit for preparing anti-tumor dendritic cells of the present invention, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(c) an enzyme(s) for digesting cells;

(d) at least one of B27, N2, epidermal growth factor, fibroblast growth factor and insulin;

(e) basic medium for dendritic cells;

(f) granulocyte macrophage colony stimulating factors;

(g) at least one of interleukin-4, interferon α and tumor necrosis factor; and

(h) operating instruction; wherein said radiotherapeutic radioisotope source is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide; wherein said operating instruction includes a method a method for preparing an anti-tumor dendritic cell of the present invention.

Since the tumor stem cells of the present invention are screened by radiation exposure and/or a chemotherapeutic agent(s), the obtained resistance-screened tumor stem cells get some characteristics different from the normal tumor stem cells(e.g., they express many new specific proteins), which are more closed to the tumor stem cells in tumor tissue in vivo after radiotherapy and/or chemotherapy. Therefore, the medicaments prepared from the resistance-screened tumor stem cells, the antigen composition of tumor stem cells and the dendritic cells loading with the antigens of tumor stem cells provided by the present invention, can induce immune response in vitro and in vivo to kill tumor stem cells which cause tumor recurrence. They make it possible to overcome tumor resistant and to radically cure tumor recurrence and metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a cell survival rate curve of cerebral glioma cancer stem cells of preparation example 1 screened by γ-ray radiation.

Fig.2 is a cell survival rate curve of lung cancer stem cells of preparation example 5 screened by γ-ray radiation.

Fig.3 is a cell survival rate curve of cerebral glioma cancer stem cells of preparation example 1 repeatedly screened by γ-ray radiation of 9 Gys.

Fig.4 shows a photograph of western blotting of resistant proteins' expression for glioma cerebral glioma stem cells screened by radiation exposure and a chemotherapeutic agent.

Fig.5 shows a photograph depicting electrophoresis results of the resistant proteins' expression of glioma cerebral glioma stem cells of preparation example 1, breast cancer stem cells of preparation example 3, and liver cancer stem cells of preparation example 6, after being screened by radiation exposure and a chemotherapeutic agent.

Fig.6 shows the results of phenotype of DCs loaded with antigens determined by flow cytometry, wherein the DCs are loaded with antigens of resistance-screened breast cancer stem cells. Fig.7 shows the results of immune response of cytotoxic T cells (CTL) to the related tumor stem cells, wherein the CTL cells are obtained from the DCs loaded with antigen compositions. Said DCs are obtained from preparation example 1 screened by radiation exposure and a chemotherapeutic agent in table 8, preparation example 5 by radiation exposure (total dose 9Gy, dose rate 200cGy/min and linear distance 50cm), and preparation example 6 screened by the chemotherapeutic agent in table 4.

Fig.8 shows comparative graph illustrating results of immune response of cytotoxic T cells (CTL) to breast cancer stem cells with or without screening by radiation exposure and a chemotherapeutic agent, wherein said CTL cells are obtained by DCs loaded with antigen compositions which are prepared with preparation example 2 with screening by radiation exposure and a chemotherapeutic agent in table 8.

DETAILED DESCRIPTION

In an aspect, this application provides a method for preparing a resistance-screened tumor stem cell, characterized in that the method comprises: a step of performing radiation exposure screening and/or chemotherapeutic agent screening to tumor stem cells; wherein said radiation exposure screening is performed by exposing tumor stem cells to a radiation source of ray, wherein said ray is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent screening is performed by making tumor stem cells contact with a chemotherapeutic agent(s); wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide.

As used herein, Gy refers to the total dose unit of radiation exposure, lGy=100rad. And cGy refers to the dose unit of radiation absorbed by per gram of specified body tissue; lcGy=lrad, namely lGy=100rad=100cGy. Said dose rate of radiation exposure represents the radiation dose (dosage) being absorbed per unit of time, which is often expressed in cGy/min. Based on the data identified above, time of radiation exposure = total dose of radiation exposure/ dose rate.

Preferably, the linear distance between said radiation source of ray and tumor stem cells ranges from about 10cm to about 5m; the total dose of said radiation exposure ranges from about 2Gy to about 1 OGy; the dose rate of said radiation exposure ranges from about 0.4cGy/min to about 1200cGy/min. More preferably, the linear distance between said radiation source of ray and tumor stem cells ranges from about 20cm to about 2m; the total dose of said radiation exposure ranges from about 3Gy to about 9Gy; the dose rate of said radiation exposure ranges from about 2cGy/min to about 600cGy/min.

Preferably, the ray of the present invention may be x-ray and/or γ-ray. The ray of the present invention may be provided by any conventional radioactive isotope for radiotherapy, such as Co60, Irl92, Cs-137, Am-241 and Se75 emitting γ-ray.

As used herein, the final concentration of said chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.1 mg/ml to about 100mg/ml; wherein the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about Ih to about 72h. Preferably, the final concentration of said chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 50mg/ml; wherein the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12h to about 36h. If the contacting time is excessively long or the final concentration of a chemotherapeutic agent(s) is excessively high, it will kill all tumor stem cells, and no resistance-screened tumor stem cells can be obtained; whereas the "screening" can not take its effect, and only tumor stem cells without resistance can be obtained. The contact may be performed one time or several times, e.g. 1-10 times, preferably 4-6 times. When the contact is performed several times, the chemotherapeutic agent(s) and dose of each time may be identical, such as the greatest concentration that tumor stem cells can be tolerated, i.e. interval inducement of high concentration; and the chemotherapeutic agent(s) and dose of each time may be different, such as gradual concentration changes of a same agent or different agents from small to great or from great to small, i.e. gradual increase or decrease of the concentrations.

In a further aspect, this application may use any chemotherapeutic agent(s) to perform the resistant screening, such as, but not limited to: an alkylating agent(s) selected from the group consisting of mechlorethamine, cyclophosphamide, isofosfamide, formylmerphalan, sarcoclorin, chlorambucil and thiotepa; nitrosoureas selected from the group consisting of carmustine, lomustine, semustine, nimustine, busulfan and estramustine; an anti-metabolite(s) selected from the group consisting of methotrexate, fluorouracil, tegafur, tegafur-uracil, carmofur, fluorodeoxyuridine, floxuridine, ancitabine, cytosine arabinoside and gemcitabine; anti-tumor antibiotics selected from the group consisting of actinomycin D, mitomycin, bleomycin, pingyangmycin, adriamycin, perarubicin, epirubicin, idamycin and mitoxantrone; herb-chemotherapeutics selected from the group consisting of vincristine, vinblastine, vindesine, vinorelbine, etoposide, teniposide, hydroxycamptothecine, irinotecan, topotecan, paclitaxel, docetaxel, harringtonine and hydroxyl-paclitaxel; hormone and incretion chemotherapeutics selected from the group consisting of prednisone, dexamethasone, testosterone propionate, diethylstilbestrol, flutamide, progestin, megestrol, tamoxifen, estrogen receptor antagonist, toremifene, aminoglutethimide, formestane, letrozole and gonadotropin releasing hormone antagonist; an antibody repressor(s) selected from the group consisting of epidermal growth factor inhibitor-gefϊtinib, tarceva, cetuximab and nimotuzumab. Further preferably, said chemotherapeutic agent(s) is selected from the group consisting of 4-hydroxyl-cyclophosphamide, gefitinib, tarceva, 5-fluorouracil, doxorubicin, taxol and cisplatin.

In some embodiment, a preparation method of resistance-screened tumor stem cells provided by the present invention may include only the steps of radiation exposure screening, but exclude the steps of a chemotherapeutic agent(s) screening. In this situation, the linear distance between said radiation source of ray and tumor stem cells ranges from about 20cm to about 2m; the total dose of said radiation exposure ranges from about 3Gy to about 9Gy; the dose rate of said radiation exposure ranges from about 2cGy/min to about 600cGy/min. Preferably, the linear distance between the radiation source and tumor stem cells ranges from about 50cm to about Im; the total dose of said radiation exposure ranges from about 6Gy to about 9Gy, and the dose rate of said radiation exposure ranges from about 2cGy/min to about 200cGy/min.

In some embodiment, a preparation method of resistance-screened tumor stem cells provided by the present invention may include only the steps of a chemotherapeutic agent(s) screening, but exclude the steps of radiation exposure screening. In this situation, the final concentration of the chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 50mg/ml; the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12h to about 36h. Preferably, the final concentration of the chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 20mg/ml, the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12 to about 24h.

More preferably, a preparation method of resistance-screened tumor stem cells provided by the present invention includes both the steps of a chemotherapeutic agent(s) screening and radiation exposure screening. In this situation, the linear distance between said radiation source of ray and tumor stem cells ranges from about 20cm to about 2m; the total dose of said radiation exposure ranges from about 3Gy to about 9Gy; the dose rate of said radiation exposure ranges from about 2cGy/min to about 600cGy/min; the final concentration of the chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 50mg/ml; the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12h to about 36h. Further preferably, the linear distance between the radiation source and tumor stem cells ranges from about 50cm to about Im; the total dose of said radiation exposure ranges from about 6Gy to about 9Gy, and the dose rate of said radiation exposure ranges from about 2cGy/min to about 200cGy/min; the final concentration of the chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 20mg/ml, the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12 to about 24h. Moreover, the radiation source(s) of radiation exposure and the chemotherapeutic agent(s) used during the course of screening tumor stem cells are as same as those used in the radiotherapy and chemotherapy to the patient, from who tumor stem cells are isolated.

The present invention may be applicable to all kinds of tumors which may be obtained tumor stem cells from, such as, but not limited to: breast cancer, glioma, lung cancer, brain tumor, nasopharyngeal carcinoma, hepatocellular carcinoma, carcinoma ventriculi, colon cancer, melanoma, osteosarcoma, renal cell carcinoma, prostate cancer, ovarian carcinoma, acute myelogenous leukemia, multiple myeloma, pancreatic cancer and metastatic cancer. Wherein said metastatic cancer may be selected from metastatic brain cancer, metastatic lung cancer, metastatic liver cancer and metastatic neck cancer.

In another aspect of the present invention, a method for preparing a resistance-screened tumor stem cell further comprises isolating tumor stem cells obtained from the tumor ex vivo and/or tumor cell lines. Generally, tumor tissues ex vivo isolated from the patient by operation belong to abandoned tumor tissues. The invention may use any conventional method to obtain tumor cells, and isolate tumor stem cells from said tumor cells (see WO 2008/039874). Moreover, tumor stem cells may also be isolated from culture of corresponding commercially- available cell lines. For example, the cell lines from American Type Culture Collection (ATCC) may be used to isolate the related tumor stem cells shown in Table 1.

Table 1

In a further aspect, this application also provides a resistance-screened tumor stem cell(s) obtained by any of the above methods. The proteins expressed by tumor stem cells after being screened by any of the methods of this invention are different from the proteins expressed without screening. For example, said screened tumor stem cells specifically express the proteins selected from the group consisting of epithelial growth factor receptor, P-glucoprotein, multidrug-associated protein, protein coded by ataxia-telangiectasia mutation gene (ATM), ataxia-telangiectasia Rad3 related protein and hypoxia inducible factor. On the other hand, the step of the screening may be monitored by determining the specially expressed proteins.

Tumor stem cells share the biological properties of normal stem cells of the same type (such as the same phenotypes and the markers), for example, CD34+/CD38- is an antigen present in tumor stem cells obtained from leukemia, CD44+/CD24- is an antigen present in tumor stem cells obtained from breast cancer, CD133+ is an antigen present in tumor stem cells obtained from neuroglioma and melanoma.

In some embodiment, the present invention uses a method of Side population (SP) to enrich tumor stem-like cells from tumor cell lines. "Side population" cells are a small subset of cells which have the capability to strongly efflux the DNA-binding fluorescent dye entering their nuclei, displaying low stain or no stain in flow cytometric analysis or under a fluorescent microscope. SP cells may express the molecular markers associating with tumor stem cells, such as nestin, CD90, CD44, Musashi-l(Msi-l), maternal embryomic leucine zipper kinase(MELK), glioma-associated antigen- 1 (GIi-I), stromal cell derived factor, B-cell-specifϊc Moloney leukemia virus insertion site-l(Bmi-l), patched homolog (PTCH), phosphoserine phosphatase (PSP), breast cancer-resistant protein 1 (BCRPl), 6-methylguanine-DNA methyltransferase(MGMT), stromal cell-derived factor receptor CXCR4, integrin, epithelial growth factor receptor(EGFR), survivin, nucleostemin(NS),and so on. Molecular markers of normal stem cells and/or those of tumor cells are used to identify tumor stem cells. For example, NS is nucleoprotein, as an important marker for stem cells, which may be used to determine the extent of cell differentiation. Furthermore, the genome of a tumor stem cell usually behaves instability to environment. After a treatment of radiotherapy and/or chemotherapy, most of tumor cells were killed; tumors became smaller or even seemed to be disappeared. But a few of tumor cells may be endued with the properties of stem cells by the treatment of radiotherapy and/or chemotherapy; malignancy mutations may occur, and the tumor may show high drug-resistant to chemotherapy, high radiation resistance to radiotherapy.

In some embodiment, a method for preparing an antigen composition of tumor stem cells, which comprises the steps of washing and re-suspending tumor stem cells with saline and then lysing the obtained tumor stem cells, wherein said tumor stem cells are the resistance-screened tumor stem cells of the present invention. As used herein, the number of times for washing tumor stem cells with saline may be decided by determining residual components of medium in washing solution. The amount of residual components of medium is deemed to be suitable to the extent that: when an antigen composition(s) of tumor stem cells of the present invention is administered (injected), residual components of the medium and a chemotherapeutic agent(s) shall not result in any indisposition for patient.

It may use any conventional method for lysing cells in the art to lyse tumor stem cells of the present invention. In view of not bring the new materials to the lysate, said step of lysing tumor stem cells preferably comprises heat shock and/or repeated freeze-thaw to tumor stem cells; wherein the temperature of said heat shock ranges from about 37°C to about 45°C, more preferably, from about 38°C to about 43°C; wherein the persistence time of said heat shock ranges from about Ih to about 6h, more preferably, from about 2h to about 4h; wherein the number of times of said repeated freeze-thaw ranges from 3 times to 5 times; wherein the time interval between two times ranges from about lOmin to about 2h, more preferably, from about 30min to about Ih; wherein the temperature of said freezing ranges from about -120⁰C to about -196°C (Liquid nitrogen, drikold, and so on may be used.), more preferably, from about -15O⁰C to about -196⁰C; wherein the temperature of said thawing ranges from about 1O⁰C to about 37°C, more preferably, from about 20⁰C to about 37°C. After being performed heat shock, tumor stem cells may produce heat shock proteins, which will exist in the lysate of said tumor stem cells. The existence of heat shock proteins may enhance the ability of immune response of dendritic cells loaded with antigens of tumor stem cells. Therefore, said step of lysing tumor stem cells more preferably comprises heat shock and repeated freeze-thaw to tumor stem cells; wherein the temperature of said heat shock ranges from about 37°C to about 45°C; wherein the persistence time of said heat shock ranges from about Ih to about 6h; wherein the number of times of said repeated freeze-thaw ranges from 3 times to 5 times; wherein the time interval between two times ranges from about lOmin to about 2h; wherein the temperature of said freezing ranges from about -120⁰C to about -196°C; wherein the temperature of said thawing ranges from about 20⁰C to about 37°C .

After lysing, cell debris in the lysate is usually removed by centrifugation and filtration. For example, after centrifugation (600rpm for 1 min), the resultant supernatant may be filtered through a 0.45 μm filter membrane, and the resultant filtrate is collected. Then one of the base components of an antigen composition of the present invention is saline. An antigen composition(s) of the tumor stem cells provided by the present invention may be stored at -80⁰C.

In a further aspect, this application provides use of the tumor stem cells of the present invention and use of the antigen composition of tumor stem cells of the present invention for the manufacturing of an anti-tumor medicament.

In a further aspect, this application provides a kit for preparing the tumor stem cells of the present invention, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(c) an enzyme(s) for digesting cells;

(d) a cytokine(s); and

The basic medium of the tumor stem cells of present invention may be any conventional basic medium capable of culturing tumor stem cells in the art, for example, DMEM/F-12, and so on. The culture system for tumor stem cells comprises tumor stem cells and about 3ml to about 50ml of the basic medium for tumor stem cells, which is incubated at 37 ⁰C, in a 5% CO₂ incubator. Radiation source for radiotherapy may provide that: the linear distance between said radiation source of ray and tumor stem cells ranges from about 10cm to about 5m; the total dose of said radiation exposure ranges from about 2Gy to about 10Gy; the dose rate of said radiation exposure ranges from about 0.4cGy/min to about 1200cGy/min. Said chemotherapeutic agent(s) may provide the relative conditions for chemotherapy defined by the above methods of the present invention such as that: the final concentration of said chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 20mg/ml; wherein the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12h to about 24h.

Said enzyme(s) for digesting cells may be any conventional enzyme(s) capable of digesting the cells in the art, preferably pancreatin and/or accutase. As used herein, when the volume of the basic medium for tumor stem cells reaches about 3ml to about 50ml, pancreatin and/or accutase preferably used as the agent for isolating tumor stem cells from tumor tissue, it may be used at conventional dose in the art, for example, its volume is 1/5-1/6 fold comparing to basic medium, in this situation, the volume of the pancreatin and/or accutase are about 0.5ml to about 10ml.

Said cytokine(s) is selected from the group consisting of B27, N2, epidermal growth Factor, basic fibroblast growth factor and insulin. As used herein, the final concentration of B27 reaches about 2ng/ml to about 50ng/ml, when B27 is added to the basic medium for tumor stem cells (for example DMEM/F12). The final concentration of N2 reaches about 2ng/ml to about 50ng/ml when N2 is added to the basic medium for tumor stem cells (for example DMEM/F12). B27 and N2 are cell culture additives which are available commercially, such as American Biotech and Gibco Company. As used herein, the final concentration of epidermal growth factor reaches about 5ng/ml to about 100ng/ml when it is added to the basic medium for tumor stem cells (for example DMEM/F12). As used herein, the final concentration of basic fibroblast growth factor reaches about 5ng/ml to about 100ng/ml when it is added to the basic medium for tumor stem cells(for example DMEM/F12). As used herein, the final concentration of insulin reaches about lmg/ml to about 50mg/ml when it is added to the basic medium for tumor stem cells (for example DMEM/F12).

The kit mentioned above may further comprise unnecessary animal serum albumin. As used herein, an animal serum (or sera) may be human serum albumin and/or bovine serum albumin, which may also be used as a terminator for digest enzyme. It may be used at conventional dose in the art, for example, its volume is relative to the volume of said digestive enzyme, and namely it is about 0.1ml to about 10ml. The ratio between the volume of the animal serum and that of digestive enzyme is about 1 :1 to about 1 :5.

In a further aspect, this application further provides a kit for preparing the antigen composition of tumor stem cells of the present invention, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(c) an enzyme(s) for digesting cells;

(d) a cytokine(s); and

The basic medium of the tumor stem cells of present invention may be any conventional basic medium capable of culturing tumor stem cells in the art, for example, DMEM/F-12, and so on. The culture system for tumor stem cells comprises tumor stem cells and about 3ml to about 50ml of the basic medium for tumor stem cells, which is incubated at 37 ⁰C, in a 5% CO2 incubator. Radiation source for radiotherapy may provide that: the linear distance between said radiation source of ray and tumor stem cells ranges from about 10cm to about 5m; the total dose of said radiation exposure ranges from about 2Gy to about 10Gy; the dose rate of said radiation exposure ranges from about 0.4cGy/min to about 1200cGy/min. Said chemotherapeutic agent(s) may provide the relative conditions for chemotherapy defined by the above methods of the present invention such as that: the final concentration of said chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 20mg/ml; wherein the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12h to about 24h.

Said antigen composition(s) of the tumor stem cells is obtained from tumor stem cells of the present invention, whose concentration ranges from about Ix IO⁵ cell/ml to about l xlθ⁸cell/ml, preferably from about l>< 10⁵ to about 5>< 10⁷ cell/ml. If the cell concentration is excessively high, it may cause a patient immune system's over-reaction to the compositions. On the contrary, if the cell concentration is excessively low, it cannot induce any immune response in a patient. Said antigen composition(s) of the tumor stem cells may further comprise human serum albumin, wherein based on the total volume of said composition, the content of human serum albumin ranges from about 1 percent weight in volume to about 5 percent weight in volume, preferably, from about 2 percent weight in volume to about 3 percent weight in volume.

In a further aspect, this application further provides a method for preparing an anti-tumor dendritic cell, which comprises obtaining an antigen composition of tumor stem cells by lysing said tumor stem cells, making normal dendritic cells contact with said antigen composition of tumor stem cells, and then obtaining an anti-tumor dendritic cell loading with the antigens of said tumor stem cells, wherein said tumor stem cells are the tumor stem cells of the present invention. As used herein, the antigen composition(s) of the tumor stem cells is the antigen composition(s) of the tumor stem cells provided by the present invention. Preferably, said normal dendritic cells and the tumor stem cells for preparing an antigen composition are derived from a same individual, which may relieve side effects of immunologic rejection.

Said tumor stem cells are washed in the medium for dendritic cells for 3 times before lysing them to prepare the antigen compositions of the tumor stem cells. The persistence time for making normal dendritic cells contact with said antigen composition of tumor stem cells may be the conventional persistence time in the art. Preferably, the persistence time ranges from about Ih to about 24h, more preferably, from about Ih to about 12h.

In another aspect, this application further provides the dendritic cells with anti-tumor activities by any method mentioned above.

In another aspect, this application further provides a dendritic cell vaccine, which comprises dendritic cells, characterized in that said dendritic cells are the anti-tumor dendritic cells of the present invention.

Before the preparation for a dendritic cell vaccine, said anti-tumor dendritic cells may be washed and re-suspended in saline. As used herein, the number of times for washing said anti-tumor dendritic cells with saline may be decided by determining residual components of medium in washing solution. The amount of residual components of medium is deemed to be suitable to the extent that: when a dendritic cell vaccine of the present invention is administered (injected), residual components of the medium and a chemotherapeutic agent(s) shall not result in any indisposition for patient. Said vaccine comprises the anti-tumor dendritic cells provided by this invention, whose concentration ranges from about I X lO⁵ to about 5 X 10⁷ cell/ml, preferably, the concentration ranges from about 5 X 10⁵ to about 1 X 10⁷cell/ml. The vaccine further comprises may further comprise human serum albumin, wherein based on the total volume of said vaccine, the content of human serum albumin ranges from about 1 percent weight in volume to about 5 percent weight in volume, preferably, from about 2 percent weight in volume to about 3 percent weight in volume.

In a further aspect, this application provides a kit for preparing anti-tumor dendritic cells of the present invention, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(c) an enzyme(s) for digesting cells;

(e) basic medium for dendritic cells;

(f) granulocyte macrophage colony stimulating factors;

(g) at least one of interleukin-4, interferon α and tumor necrosis factor; and (h) operating instruction; wherein said radiotherapeutic radioisotope source is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide; wherein said operating instruction includes a method of any above methods for preparing an anti-tumor dendritic cell.

As used herein, the final concentration of B27 reaches about 2ng/ml to about 50ng/ml, when B27 is added to the basic medium for tumor stem cells (for example DMEM/F12). The final concentration of N2 reaches about 2ng/ml to about 50ng/ml when N2 is added to the basic medium for tumor stem cells (for example DMEM/F12). B27 and N2 are cell culture additives which are available commercially, such as American Biotech and Gibco Company. As used herein, the final concentration of epidermal growth factor reaches about 5ng/ml to about lOOng/ml when it is added to the basic medium for tumor stem cells (for example DMEM/F12). As used herein, the final concentration of basic fibroblast growth factor reaches about 5ng/ml to about 100ng/ml when it is added to the basic medium for tumor stem cells(for example DMEM/F12). As used herein, the final concentration of insulin reaches about lmg/ml to about 50mg/ml when it is added to the basic medium for tumor stem cells (for example DMEM/F12).

The kit mentioned above may further comprise unnecessary animal serum albumin. As used herein, an animal serum (or sera) may be human serum albumin, which may also be used as a terminator for digest enzyme. It may be used at conventional dose in the art, for example, its volume is relative to the volume of said digestive enzyme, and namely it is about 0.1ml to about 10ml. The ratio between the volume of the animal serum and that of digestive enzyme is about 1 : 1 to about 1 :5.

Said antigen composition(s) of the tumor stem cells is obtained from tumor stem cells of the present invention, whose concentration ranges from about 1 x 10⁵ cell/ml to about l xlθ⁸cell/ml, preferably from about l>< 10⁵ to about 5x 10⁷ cell/ml. If the cell concentration is excessively high, it may waste the cells. On the contrary, if the cell concentration is excessively low, it cannot make dendritic cells load with enough antigens.

The basic medium of the dendritic cells of present invention may be any conventional basic medium capable of culturing dendritic cells in the art, for example, RPMI- 1640, and so on. The culture system for dendritic cells comprises dendritic cells and about ImI to about 100ml, preferably about 30ml to about 80ml, of the basic medium for dendritic cells, which is incubated at 37 ⁰C, in a 5% CO₂ incubator. As used herein, said GM-CSF is added to the basic medium for the dendritic cells with the final concentration ranging from about 100IU/ml to about 2500IU/ml, when the antigens of tumor stem cells are loaded on dendritic cells. Said interleukin-4 is added to the basic medium for the dendritic cells with the final concentration ranging from about 100IU/ml to about 1500IU/ml, when the antigens of tumor stem cells are loaded on dendritic cells. Said interferon α is added to the basic medium for the dendritic cells with the final concentration ranging from about 100IU/ml to about 2000IU/ml, when the antigens of tumor stem cells are loaded on dendritic cells. Said tumor necrosis factor α is added to the basic medium for the dendritic cells with the final concentration ranging from about 0.5ng/ml to about 100ng/ml, when the antigens of tumor stem cells are loaded on dendritic cells.

The kit further comprises human serum albumin, wherein based on the total volume of said antigen composition, the content of human serum albumin ranges from about 1 percent weight in volume to about 5 percent weight in volume, preferably, from about 2 percent weight in volume to about 3 percent weight in volume.

In another aspect, this application further provides a treatment for cancer comprising to administer the patient a drug selected from the group consist of antigen composition(s) of the tumor stem cells provide by present invention, the anti-tumor dendritic cells provide by present invention and the anti-tumor dendritic vaccine provide by present invention.

Injection is a preferable administration method. In one embodiment, the administration method of the antigen composition(s) of the tumor stem cells is selected from the group consisting of injection in tumor tissue, intravenous injection, hypodermic injection, and intracutaneous injection. The administration method of anti-tumor dendritic cell vaccine is selected from the group consisting of hypodermic injection or subcutaneous injection within the area of a lymph node. Preferably, the administration method of the anti-tumor dendritic cell vaccine is hypodermic injection or subcutaneous injection within the area of groin or oxter.

Said antigen composition(s) of the tumor stem cells is obtained from tumor stem cells of the present invention, whose concentration ranges from about 1 x 10⁵ cell/ml to about 1 x 10⁸cell/ml, and its effective dosage ranges from about 0.1ml to about 5ml per individual; the anti-tumor dendritic cell vaccine mentioned above comprises about 1 x lO⁵ to about 5xlO⁷/ml of the anti-tumor dendritic cells of the present invention, and its effective dosage ranges from about 0.1ml to about 2ml per individual. Preferably, said antigen composition(s) of the tumor stem cells is obtained from tumor stem cells of the present invention, whose concentration ranges from about Ix IO⁵ cell/ml to about 1 x 10⁸cell/ml, and its effective dosage ranges from about 0.2ml to about 2ml per individual; the anti-tumor dendritic cell vaccine mentioned above comprises about 1 x lO⁵ to about 5 xlO⁷/ml of the anti-tumor dendritic cells of the present invention, and its effective dosage ranges from about 0.5ml to about 2ml per individual.

The above mentioned effective dosage of the antigen composition(s) of tumor stem cell and the anti-tumor dendritic cell vaccine is for one time injection; and one course of treatment includes four times injection, once per week. The antigen composition(s) or the vaccine takes effects at 3 to 7 days after the first time injection. The standard of "taking effects" includes that: the secretion of cytokines such as interleukin-2, interleukm-12, interferon γ and tumor necrosis factor α, and so on, which may enhance the ability of immune response, is increased; the secretion of cytokines such as interleukin-6, interleukin-10, transforming growth factor beta, epidermal growth factor, and so on, which may inhibit the ability of immune response, is reduced; and T lymphocyte subpopulation is changed, i.e.: the positive rate of CD3+, CD4+ and CD8+ T cells increases or improves; the positive rate of CD16+/56+T cell increases; and the positive rate of CD4+, CD25+ and FoxP3+ T cell reduces. After one course of treatment is finished, the administration may be continued per three months and it can go for 2 or A courses of treatment. If necessary, the administration may be continued after 6 or 12 months, as long as the patient is still alive.

The therapeutically effective Standard may fall within the following three classes:

1. Cytokines detection of the patient blood: the secretion of cytokines such as interleukm-2, interleukin-12, interferon γ and tumor necrosis factor α, and so on, which may enhance the ability of immune response, is increased; the secretion of cytokines such as interleukin-6, interleukin-10, transforming growth factor beta, epidermal growth factor, and so on, which may inhibit the ability of immune response, is reduced; and T lymphocyte subpopulation is changed, i.e.: the positive rate of CD3+, CD4+ and CD8+ T cells increases or improves; the positive rate of CD16+/56+T cell increases; and the positive rate of CD4+, CD25+ and FoxP3+ T cell reduces.

2. The detection for markers of tumor cells from a patient:

For example, the expression level of specific cell surface markers such as tumor antigens, e.g., CEA, AFP reduces, which may be tested by the methods of ELISA and chemical luminescence detection and so on.

3. Medical image detection:

For example, CT, NMR and type-B ultrasonic test, they may detect that: the size of the tumor tissue reduces.

Preferably, said normal dendritic cells and the tumor stem cells for preparing an antigen composition are derived from a same individual, which may relieve side effects of immunologic rejection.

The tumor tissues mentioned in the present invention ex vivo isolated from the patient by operation belong to abandoned tumor tissues. Said tumor tissues may not be the tumor tissue isolating from patients' bodies to achieve the present invention deliberately. Moreover, the tumor cells mentioned in the present invention may be cell lines available commercially. And the techniques of cell long-term cryopreservation have been very mature, such as freezing and conserving cells (e.g. cells of umbilical cord blood) in liquid nitrogen, i.e., -196°C. The techniques mentioned above are available completely that well known to those of skill in the art, which can conserve tumor tissues, tumor cell lines and DCs mentioned in the present invention. DCs may be obtained from peripheral blood, bone marrow, umbilical cord blood, and so on.

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Preparative Example 1 :

Isolation and culture of tumor stem cells in cerebral glioma cancer tissue

Tumor tissues were obtained from a male patient of 50 years old, suffering from astrocytoma diagnosed by pathological examination. The procedure was approved by the Local Ethics Committee and the patient provided written informed consent. The tumor tissues are collected during the surgery for removing tumor. The tissues were minced into approximately 1 mm³ pieces with scissors. The pieces were digested with 0.25% pancreatin 0.5ml/g tissue for 5mins, then O.lml/g bovine serum albumin (Hyclone) was used to end digestion. The resultant was centrifuged at lOOOrpm to collect the digested tissues; the supernatant was discarded, and the precipitate was washed for 3 times with lml/g tissue HBSS supplemented with 0.1% dispase (lIU/mg, sigma) and 0.01% DNase. Then, the washed precipitate was incubated with lml/g tissue(precipitate) accutase (Accutase, sigma) at 37°C for 10 min, and then the resultant was centrifuged at lOOOrpm for 5min to collect the cells. The supernatant was discarded and the precipitate (pellet) was resuspended in the DMEM/F12 lml/g tissue. Then the resuspended tissue was squeezed through a mesh (BD) with a mesh size of 40 mm. The filtration of the 40-mm mesh can intercept large cell debris and non-digestion cell agglomerates, and leach cell suspension.

50μl of the above cell suspension (2x 10⁶ cells/ml) were added to a centrifugal tube which contained 20μL FITC-CD 133 and 20μL PE-Nestin (BD company). The tube was laid in 4°C refrigeratory; the cells were stained for 30min, washed three times with ImI 1 xphosphate-bufFered saline (PBS), and then were resuspended with 0.5ml IxPBS. The stained and washed cells were detected their cytotypes by FACSCalibur Flow cytometry. The result showed that: CD 133+ and Nestin+ glioma stem cells were 2.1% of the washed cells.

The rest of the above cell suspension was collected, and said cells were cultivated in Dulbecco's modified Eagle's medium-F12 (DMEM/F12, Hyclone) supplemented with 5ng/ml B27, 40ng/ml EGF, 40ng/ml bEGF, 30mg/ml insulin. The concentration of cell suspension was diluted to 2x 10⁵ cells/ml. Then each 5ml diluted cell suspension was transferred to a T-25 culture flasks (25 cm²), cultured in an atmosphere of 5% CO₂ at 37°C for 24h.

When the cultured cells grew to neural sphere, the medium was discarded; the cells were washed 1 time with 5ml HBSS, and then washing solution was discarded. The washing step removed those suspending cells pieces and non-adherent cells. The fresh DMEM/F12 supplemented with 8ng/ml B27, 30ng/ml EGF, 30ng/ml bEGF, 20mg/ml insulin was added to the cultured cells. Once the adhered cells reach about 90% confluence of the bottom surface of culture flask, they were digested by accutase. Then the passage cells were transferred to a new flask with DMEM/F12 supplemented with 8ng/ml B27, 30ng/ml EGF, 30ng/ml bEGF, and 20mg/ml insulin (The passage cells obtained from one flask may be transferred into several flasks. And the final concentration of the passage cells transferred into a new flask reached 2x 10⁵ cells/ml.). According to the passage condition, the cells were passed further 4^th generation and obtained cells of neural sphere.

One flask of the tumor stem cells at 5 generation was digested by accutase. After being centrifuged, the cells' precipitate was resuspended in ImI IxPBS. Herein the cells were counted under inverted microscope and resuspended to 2><10⁶cells/ml in PBS. 50μl of the diluted cells suspension was transferred into a centrifuge tube containing 20μL FITC-CD133 and 20μL PE-Nestin, stained for 30mins at 4°C, then washed 3 times in ImI PBS, and then resuspended in 0.5ml PBS. The resultant cells were detected their cytotypes by FACSCalibur Flow cytometry. The result showed that: CD133+ and Nestin+ glioma stem cells were 71.1% of the resuspended cells. Preparative Example 2-3 :

According to the method of Preparative example 1, tumor stem cells of colon cancer and breast cancer were prepared respectively, wherein the differences are as followed:

Tissues of tumor were obtained from a male patient of 40 years old, suffering from colon carcinoma diagnosed by pathological examination. The procedure was approved by the Local Ethics Committee and the patient provided written informed consent. The tumor tissues are collected during the surgery for removing tumor.

Tissues of tumor were obtained from a female patient of 39 years old, suffering from breast cancer diagnosed by pathological examination. The procedure was approved by the Local Ethics Committee and the patient provided written informed consent. The tumor tissues are collected during the surgery for removing tumor. Table 2

Preparative Example 4: Isolation of breast cancer stem cells from cell line MCF-7 Breast cancer cell line (MCF-7) was from American ATCC. 50μl of the breast cancer cell suspension (2x 10⁶ cells/ml) were added to a centrifugal tube which contained 20μL FITC-CD44 and 20μL PE-CD24(BD company). The tube was laid in 4°C refrigeratory; the cells were stained for 30min, washed three times with ImI lxphosphate-buffered saline (PBS), and then were resuspended cells with 0.5ml IxPBS. The stained and washed cells were detected their cytotypes by FACSCalibur flow cytometry. The result showed that: no CD44+ and CD24- breast stem cells were detected in the cell suspension.

Breast cancer cell line (MCF-7) purchasing from American ATCC was cultivated in Dulbecco's modified Eagle's medium-F12 (DMEM/F12, Hyclone) supplemented with 5ng/ml B27, 25ng/ml EGF, 40ng/ml bEGF and 30mg/ml insulin, and cultured in an atmosphere of 5% CO₂ at 37°C for 3 days.

When the cultured cells grew to neural sphere, the medium was discarded; the cells were washed 1 time with 5ml HBSS, and then washing solution was discarded. The washing step removed those suspend cells pieces and non-adherent cells. The fresh DMEM/F12 supplemented with 15ng/ml B27, 30ng/ml EGF, 30ng/ml bEGF, 20mg/ml insulin was added to the cultured cells. Once the adhered cells reach about 90 confluence of the bottom surface of culture flask, they were digested by accutase. Then the passage cells were transferred to a new flask with DMEM/F12 supplemented with 8ng/ml B27, 30ng/ml EGF, 30ng/ml bEGF, 20mg/ml insulin (The passage cells obtained from one flask may be transferred into several flasks. And the final concentration of the passage cells transferred into a new flask reached 2x 10⁵ cells/ml.). According to the passage condition, the cells were passed further 4^th generation and obtained cells of neural sphere.

One flask of the tumor stem cells at 5 generation was digested by accutase. After being centrifuged, the cells' precipitate was resuspended in ImI IxPBS. Herein the cells were counted under inverted microscope and resuspended to 2x 10⁶ cells/ml in PBS. 50μl of the diluted cells suspension was transferred into a centrifuge tube containing FITC-CD44 and 20μL PE-CD24, stained for 30mins at 4°C, then washed 3 times in ImI PBS, and then resuspended in 0.5ml PBS. The resultant cells were detected their cytotypes by FACSCalibur Flow cytometry. The result showed that CD44+ and CD24- breast stem cells were 50% of the resuspended cells.

Preparative Example 5-6:

According to the method of Preparative example 4, tumor stem cells of lung cancer and liver cancer were prepared respectively, wherein the differences are as followed: Table 3

Example 1 : Preparation for radiation screened tumor stem cells

Eight T-25 culture flasks were used. Each four T-25 culture flasks were used for culturing tumor stem cells of Preparative example 1 and Preparative example 5 respectively. The tumor stem cells in the culture flasks grew to neural sphere (diameter >100μm).

The radiation source was γ-ray. The total dose of the radiation exposure is OGy, 3Gy, 5Gy and 9Gy. The linear distance between the radiation source of ray and tumor stem cells is 50cm. The dose rate of the radiation exposure is 200cGy/min.

After radiation, the medium was changed into DMEM/F12 (supplemented with 5ng/ml B27, 40ng/ml e EGF, 40ng/ml bEGF and 30mg/ml insulin). Then the cells and medium were incubated in an atmosphere of 5% CO₂ at 37°C for 20 days. Medium was changed every 3 days. After culture, the tumor stem cells were digested with accutase, and then were centrifuged to collect the cells. The precipitate was resuspended in ImI DMEM/F12 and was counted.

Colony formation is used to determine the cell survival rate. Namely, agar solution was prepared by adding 0.6g agar (melting point is 40⁰C) to ImI DMEM/F12 medium, and stored the solution at 4O⁰C. The above screened and resuspended cell suspensions (4x 10 cells/ml, 0.5ml) mixed with 0.5ml said medium agar solution, and added to 6-wells culture plates immediately. After solidification, the cells were incubated in an atmosphere of 5% CO₂ at 37°C for 1 week. The colonies (the size was larger than lmm) were stained with crystal violet and manually counted the cells. The cell survival rate (SR) was calculated as follows: SR=(colony number of OGy-colony number of 3Gy, 5Gy, or 9Gy)/colony number of 0Gyxl00%. Herein cell survival rate by colony forming assay was calculated under different dosages of radiation exposure. A dose-survival curve was obtained for each experiment and used for calculating the survival rate of tumor stem cells after different radiation exposure. The cell survival rate is Y-axis, while the days after radiation exposure is X-axis.

As showed in figures 1-2, tumor stem cells were killed in different grades under different doses rate radiation exposure at 3Gy, 5Gy and 9Gy, but it will grow better after continuing incubation. In one particular embodiment, the stem cells were killed nearly totally after radiation of 9Gy at 15 day( Lower than 20%), but they recovered with better activity after continuing incubation, and the survival rate was similar with non-radiation tumor stem cells. Moreover, the proliferation rate of tumor stem cells after radiation exposure was much higher than that of those of non-radiation. For example, proliferation rate of breast stem cells was 16%/day after 15 days of 9Gy radiation, but proliferation rate of non-radiation breast cancer stem cells was 1%/day all along.

Moreover, T-25 culture flasks (3 flasks) were used for culturing glioma stem cells of Preparative example 1. The tumor stem cells grew in the culture flasks until it becomes neural sphere (diameter >100μm). The cultured cells were screened by radiation exposure of 9Gy γ-ray repeatedly. The achieved result of its survival rates shows in fig.3 which was similar with the above fig.1.

Moreover, each T-25 culture flasks (16 flasks) were used for culturing tumor stem cells of Preparative examples 2-4 and Preparative example 6 respectively(four flasks for each example). Herein repeated the above experimental step, and achieved similar results. After screening by radiation of 3Gy, 5Gy or 9Gy γ-ray, survival rates of tumor stem cells were very low at 15 day(<20%) , but they recovered with better activity after continuing incubation, and their survival rate was similar with that of non-radiation tumor stem cells.

Example 2: Preparation for tumor stem cells that were screened by interval inducement of high concentration.

T-25 culture flasks (1 flask) were used for culturing breast cancer stem cells of Preparative example 3, and digested with accutase, then centrifuged to collect the cells, resuspended the pellet in ImI DMEM/F12, counted and obtained 2x lO⁵ cells/ml cell suspension. a) Incubated 10ml 2x lO⁵/ml breast cancer stem cells in T-25 culture flasks. Once cells reach 70%-^ 80% confluence, 4-Hydroxy Ifosfamid with the final concentration of 5mg/ml was added to the T-25 culture flasks, incubated in an atmosphere of 5% CO₂ at 37°C for 24h. Then the medium with 4-Hydroxy Ifosfamid was discarded, and the cells were washed 2 times in PBS. Incubated the breast cancer stem cells in DMEM/F12 (supplemented with 5ng/ml B27, 40ng/ml EGF, 40ng/ml bEGF and 30mg/ml insulin) until they reaches 70%~80% confluence. b) 4-Hydroxy Ifosfamid with the final concentration of 5mg/ml dissolved in DMEM/F12 medium was added to the T-25 culture flasks again, incubated in an atmosphere of 5% CO2 at 37°C for 24h. Then the medium with 4-Hydroxy Ifosfamid was discarded, and the cells were washed 2 times in 1 xPBS. c) Herein repeated the step b) described above for 4 times, and then the resistant-drug breast cancer stem cells may be obtained.

According to the above method of resistant-drug breast cancer stem cells, tumor stem cells of preparation examples 3, 5, 7 were prepared, wherein the differences were as followed: Table 4

Example3 : Preparation for tumor stem cells that were screened by gradual increase of concentrations of chemotherapeutic agent.

T-25 culture flasks (1 flask) were used for culturing glioma stem cells of Preparative example 1, and digested with accutase, then centrifuged to collect the cells, resuspended the pellet in ImI DMEM/F12, counted and obtained 2x lO⁵ cells/ml cell suspension. a) Incubated 10ml 2xlO⁵/ml glioma stem cells in T-75 culture flasks (75cm²). Once cells reached 80% confluence, tarceva with the final concentration of lmg/ml was added to the T-75 culture flasks, incubated in an atmosphere of 5% CO₂ and 37⁰C for 24h. Then the medium with tarceva was discarded, the cells were washed 2 times in PBS. Incubated the glioma stem cells in DMEM/F12(supplemented with 5ng/ml B27, 40ng/ml EGF, 40ng/ml bEGF and 30mg/ml insulin) until they reached 80% confluence. b) Herein repeated the step a) described above for 4 times, the difference was tarceva with the final concentrations of 2mg/ml, 4mg/ml, 8mg/ml and 10mg/ml. c) added tarceva to the final concentration of 10mg/ml dissolved in DMEM/F12 medium, and incubated in an atmosphere of 5% CO₂ at 37°C for 24h. Then the medium with tarceva was discarded, and the cells were washed 2 times in PBS. Incubated the glioma stem cells in DMEM/F12(supplemented with 5ng/ml B27, 40ng/ml EGF, 40ng/ml bEGF, 30mg/ml insulin ) in an atmosphere of 5% CO₂ at 37⁰C until they reached 80% confluence. d) Herein repeated the step 3) described above for once. e) added DMEM/F12 medium dissolved tarceva to the final concentration of 10mg/ml, and incubated in an atmosphere of 5% CO₂ and 37°C for 24h. Therefore, the resistant-drug glioma stem cells may be obtained, and grow in the tarceva to the final concentration of 10mg/ml steadily.

According to the above method of resistant-drug glioma stem cells, the following tumor stem cells were screened, wherein the differences are as followed: Table 5

Moreover, T-25 culture flasks (1 flask) were used for culturing colon cancer stem cells of Preparative example 2. The tumor stem cells were screened by the final concentration of lmg/ml, 2mg/ml, 4mg/ml, 8mg/ml and lOmg/ml adriamycin repeatedly. The achieved results saw table 7.

Example 4: The anti-oxidative capacity of tumor stem cells was determined with the use of MTT assay in suspension.

Tumor stem cells obtained from examples 2 and 3 were inoculated into a 96-well plate at the concentration of I xIO⁴ cells/ml. The medium was DMEM/F12 (supplemented with 5ng/ml B27, 40ng/ml eEGF, 40ng/ml bEGF and 30mg/ml insulin). After incubation in an atmosphere of 5% CO₂ at 37°C for 24h, the culture solution was discarded. The cells were treated with following chemotherapeutic agents which were dissolved in the fresh medium according to table 6.

Table 6

Each kind of tumor stem cells in table 6 were repeated in 3 wells at each concentration, and untreated tumor stem cells as the blank control. The above cells treated with chemicals were incubated for another 48h. Then, the cells were added with 20μL PBS solution containing MTT (at the final medium concentration of 5mg/ml ) for 4 h, then DMSO lOOμL was added into each well, and they were mixed misce benefor 15 min. A 96-well microtiter plate reader was used to determine A570, wherein the absorptivity is proportional to live cells number, and the value was the mean of 3 wells. The inhibitory rate of tumor stem cells and RF were calculated as follows: inhibition ratio of tumor stem cells=(l-A(test group)/A (control group)) x 100%. Statistical evaluation was performed by using log-linear model to calculate the medium inhibition concentration (IC50) and resistance factor (RF). RF=IC50 (tumor stem cells resistance-screened by chemotherapeutic agents)/ IC50(tumor stem cells without screening). The achieved results show in table 7. Table 7

Example 5: Preparation for tumor stem cells resistance-screened by radiation exposure and a chemotherapeutic agent(s).

According to the methods of example 1-3 and table 8 as followed, tumor stem cells of preparation examples were applied both two kinds of screenings. Table 8

Fig.4 shows a photograph illustrating western blotting of resistant proteins' expression for glioma stem cells of preparation example 1 after being screened by radiation exposure and chemotherapeutic agents. Wherein β-actin expressed during cell proliferation was set as the internal standard. Epithelial growth factor receptor (EGFR)can accelerate tumor proliferation rates, enhance new blood vessel growtbΛinhibit tumor cell apoptosis. EGFR may also enhance the expression of HIF-lα, enhance the tolerance to hypoxia. The expression of EGFR was enhance as the dosage of γ-ray was increased, such as from 5Gy to 9Gy (see Fig.5).

Fig.5 shows a photograph depicting electrophoresis results of the resistant proteins' expression of glioma cerebral glioma stem cells of preparation example 1, breast cancer stem cells of preparation example 3, and liver cancer stem cells of preparation example 6, after being screened by radiation exposure and a chemotherapeutic agent. Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), a key enzyme in energy metabolism, was used as an internal standard here. CD 133 and nestin are protein markers that are expressed specifically in tumor stem cells. All of glioma stem cells in preparation example 1, breast cancer stem cell in preparation example 4 and liver cancer stem cells in preparation example 6 may specifically express CD 133 and nestin after being screened by radiation exposure and chemotherapeutic agents. Glioma indicates a glioma stem cell that was resistance-screened by radiation exposure and chemotherapeutic agents.

Breast cancer stem cell in preparation example 3, colon cancer stem cell in preparation example 2, and lung cancer stem cell in preparation example 5 may specifically express stem cell markers respectively, which is similar to Fig.5. The specific expression products were higher than the corresponding tumor stem cells without screening by radiation exposure and chemotherapeutic agents.

Example 6: Preparation for antigen compositions of resistance-screened tumor stem cells

The tumor stem cells without screening of preparation example 1-6 and those resistance-screened cells of example 1 -3 and 5 were treated by repeated freeze-thaw 5 times in liquid nitrogen and at room temperature (25°C). Repeated freeze-thaw cycle test the situation of cell lysate under inverted microscope. After 5 times of freeze-thaw, cells were completely lysed. The large particles of the lysed solution were removed by centrifugation at 600rpm for 1 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtered solution was stored at -8O⁰C until it was needed.

Moreover, the resistance-screened tumor stem cells of example 1-3 and 5 were incubated in 42°C water bath incubator for 2 hours, and then repeated the above freeze-thaw step. The heat shock proteins were expressed in the antigen composition of tumor stem cells by western blotting.

Example 7: Collection of dendritic cells

Mononuclear cells were prepared from peripheral blood (100 mL) of normal donors using Ficoll-Hypaque density centrifugation. Peripheral blood mononuclear cells (PBMC) were resuspended in RPMI 1640 and were allowed to adhere to 6-wells culture plates (Corning Costar, Bodenheim, Germany). After incubating at 37⁰C under 5% carbon dioxide for 90min, non-adherent cells were collected. The adherent cells were cultured for 7 days with RPMI 1640 contained 5% autologous serum, recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF, 2000 IU/mL) and IL-4 (1500 IU/mL). On the 3rd day, cells were cultured in the fresh medium containing GM-CSF (2000 IU/mL) and IL-4 (1500 IU/mL) by changing medium. Immature DCs were harvested at fifth day; while mature dendritic cells were generated at day 7 in RPMI 1640 by addition of lipopolysaccharide (LPS, O.lμg/mL) and tumor necrosis factor α (TNF-α; 20ng/mL) for another 24 h of culture(Specific antigens (such as antigens obtained from tumor stem cell lysate) were added at day 5).. Phenotypic changes were assessed by flow cytometric analysis.

Example 8 : loading DCs with antigen compositions of resistance-screened tumor stem cells

After 5 days of culturing, 3^x 10⁶ immature DCs were pulsed with the lysate of tumor stem cells (I xIO⁶ cells) for 4h at 37°C with 5% CO₂ in an incubator. Antigen-loaded DCs were harvested by centrifugation (10 min, 1000 rpm), and washed with saline for three times. The DCs were resuspended in saline to the concentration of mature 3xlO⁶ DC/ml. Then 2% human serum albumin was added into the DCs in order to prepare DC vaccine loading with lysate antigens of tumor stem cells.

DCs loading with antigens of resistance-screened tumor stem cells were stained with FITC-CD86, FITC-CD80, PE-CD83, and Percp-CDl lc. The stained cells were detected cytotypes by FACSCalibur Flow cytometry. From left to right, the ratio of breast cancer antigen loaded DCs were 1 :9, 1 :6 and 1 :3. Fig.6 shows that the double positive rates of CDl lc/CD83, CDl lc/CD86 and CDl lc/CD80 have changed evidently.

Effective example 1 : Anti-tumor effects of resistance-screened tumor stem cells in vitro—Specific Cytotoxic T cell (CTL) response to resistance-screened tumor stem cells

Mature dendritic cells loaded with the above antigens were mixed with T-cell at the ratio of 1 :20 in 6-well plate. Herein added the mixture into the RPMI 1640 medium (supplemented with 5% bovine serum, GM-CSF at a final concentration of 800U/ml, IL-4 at a final concentration of 20ng/ml) to incubate for 7days, and half amount of the medium was changed every other day. Then CTL cells were obtained.

Corresponding tumor stem cells as target cells were selected and labeled with ⁵¹Cr. Namely, target cells (2x lO⁶ cells/ml) were treated with 300 μCi ⁵¹Cr in RPMI 1640 medium at 37°C for 2 hours. The target cells labeled with ⁵¹Cr were washed 3 times in PBS, and then the cells were resuspended to 2x 10⁵ cells/ml in RPMI 1640(supplemented with 10% FCS). The target cells labeled with ⁵¹Cr were added into 96-well microtiter plates 2x10⁴ cells (0.1 ml) per well.

The above cytotoxic T cells (effector cells) were respectively added into the wells at certain ratio between effector cells to target cells (E: T ratio = 2.5:1, 5: 1, 10: 1, 20:1 and 40: 1). They were incubated at 37°C for 4h. 75 μl of the supernatant per well was transferred by pipette into an individual tube. All the individual tubes were counted in gamma radiation counter. Specific cytotoxicity ( Cr release of target cells) was calculated as follows:

(CPM test) (CPM spontaneous release)

% specific lysis = ^- ! 00 /ό

(CPM maximal release)- --(CPM spontaneous release)

Wherein counted number per minute (CPM) spontaneous release of label was obtained from negative control (only target cells without effector cells). CPM maximal release of label was obtained from target cells (without effector cells) treated by 2% NP-40 (surface acting agent, Shenghai Sangon). CPM test was obtained by adding effector cells to target cells.

Fig.7 shows results of immune response of cytotoxic T cells(CTL) to the related target tumor stem cells, wherein the CTL cells were obtained by DCs loaded with antigen compositions. Said antigen compositions are prepared with preparation example 1 screened by radiation exposure and a chemotherapeutic agent in table 8, and preparation example 6 screened by radiation exposure of total dose of 9Gy, dose rate of 200cGy/min and the linear distance between said radiation source of ray and tumor stem cells of 50cm, and preparation example 7 screened by the chemotherapeutic agent in table 4. As showed in fig.7, the ability of CTL response to tumor stem cells was enhanced when the E:T ratio was increased. The result of preparative example 1 resistance-screened by both radiation exposure and a chemotherapy agent in table 8 is the best.

Fig.8 shows the comparative photograph illustrating results of immune response of cytotoxic T cells (CTL) to breast cancer stem cells. CTL may be obtained from the tumor stem cells screened by radiation exposure and a chemotherapeutic agent, or from those tumor stem cells without screening. Wherein the CTL cells are obtained by T cells mixed with the DCs loaded with antigen compositions which are prepared with preparation example 3 resistance-screened with radiation exposure and the chemotherapeutic agent(s) in table 8, as well as preparation example 3 without screening. Wherein, RBTSC-DCl was the cytotoxicity of dendritic cells loaded with the antigens of resistance-screened breast cancer stem cells against resistance-screened breast cancer stem cells. BTSC-DC 1 was the cytotoxicity of dendritic cells loaded with the antigens of breast cancer stem cell without screening against resistance-screened breast cancer stem cells. RBTSC-DC 2 was the cytotoxicity of dendritic cells loaded with the antigens of resistance-screened breast cancer stem cell against breast cancer stem cells without screening. BTSC-DC 2 was the cytotoxicity of dendritic cells loaded with the antigens of breast cancer stem cell without screening against breast cancer stem cells without screening.

As showed in fig.8, the cytotoxicity of dendritic cells loaded with the antigens of resistance-screened breast cancer stem cells (RBTSC-DC) against resistance-screened breast cancer stem cells and breast cancer stem cells without screening is higher than that of the dendritic cells loaded with the antigens of tumor cells without screening (BTSC-DC) respectively. The resistance-screened breast cancer stem cells described above were prepared according to table 8 of example 5.

The cytotoxicity of resistance-screened tumor stem cells in example 1-3 and 5 was much higher than of tumor stem cells without screening in preparation example 1-6. Moreover, the cytotoxicity of resistance-screened tumor stem cells in example 1-3 and 5 by heat shock and repeated freeze-thaw step was much higher than that of resistance-screened tumor stem cells in preparation example 1-3 and 5 by only repeated freeze-thaw step.

Effective example 2: Anti-tumor effects of resistance-screened tumor stem cells in vivo

3 xlO⁶ immature dendritic cells, which were in the medium RPM 11640 supplemented with 5% bovine serum, were pulsed with the antigen composition of tumor stem cells (lysed from Ix IO⁶ Of the tumor stem cells, which were not screened or were resistance-screened in example 6), incubated at 37°C in 5% CO₂ incubater for 4 hours. The dendritic cells loaded with antigens were obtained by centrifugation at lOOOrpm for 10 min. The DCs loaded with antigens were washed 3 times with 0.9% saline, were resuspended to I xIO⁶ cells/ml, and were stored at 4°C until it was needed.

Breast cancer stem cells of preparation example 1 were used to establish a mouse model bearing breast cancer (see O'Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumor growth in immunodeficient mice. Nature, 2007, 445(7123): 106-110). Here randomly divided the obtained mice into three groups, i.e. A, B and C group, 10 mice per group. A group was treatment group comprising dendritic cells loaded with the antigens of resistance-screened breast cancer stem cells. B group was comparative group comprising dendritic cells loaded with the antigens of breast cancer stem cells not being resistance-screened (The injection preparation method of B group is same as that of A group). C group was blank group with the same volume of saline. Bearing cancer mice at A and B group were subcutaneously injected with 0.5-2x 10⁶ cells/ml DCs 0.5ml respectively, and another injection was performed half month later. An inhibitory effect on tumor growth was observed after 7 d, 14 d, 21 d, 28 d and 35d by measuring the size of the tumor. C group were subcutaneously injected with 0.5ml saline and the size of the tumor was measured at the same time points.

The inhibitory effect to mouse models bearing cancer of dendritic cells loaded with the antigen of resistance-screened breast cancer stem cells shows in table 9 (mm³, IT±s).

Table 9

Note: Results represent the mean ± SD of DC treatment group, **P « 0.01 versus control group and ^#P < 0.01 versus comparative group.

Moreover, the anti-tumor activity in vivo of resistance-screened tumor stem cells of example 1-3 and 5 is much higher than that of tumor stem cells not being resistant-screened in preparation example 1-6. Moreover, the anti-tumor activity in vivo of resistance-screened tumor stem cells in example 1-3 and 5 obtained by heat shock and repeated freeze-thaw step was much higher than that of resistance-screened tumor stem cells in examples 1-3 and 5 obtained by simple only repeated freeze-thaw step.

Except as may be expressly otherwise indicated, the article "a" or "an" if and as used herein is not intended to limit, and should not be construed as limiting, the description or a claim to a single element to which the article refers. Rather, the article "a" or "an" if and as used herein is intended to cover one or more such elements, unless the text expressly indicates otherwise.

Claims

CLAIMS:

1. A method for preparing a resistance-screened tumor stem cell, characterized in that the method comprises: a step of performing radiation exposure screening and/or chemotherapeutic agent screening to tumor stem cells; wherein said radiation exposure screening is performed by exposing tumor stem cells to a radiation source of ray, wherein said ray is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent screening is performed by making tumor stem cells contact with a chemotherapeutic agent(s); wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide.

2. The method of claim 1, wherein the linear distance between said radiation source of ray and tumor stem cells ranges from about 10cm to about 5m; wherein the total dose of said radiation exposure ranges from about 2Gy to about 1 OGy; wherein the dose rate of said radiation exposure ranges from about 0.4cGy/min to about 1200cGy/min.

3. The method of claim 2, wherein the linear distance between said radiation source of ray and tumor stem cells ranges from about 20cm to about 2m; wherein the total dose of said radiation exposure ranges from about 3Gy to about 9Gy; wherein the dose rate of said radiation exposure ranges from about 2cGy/min to about 600cGy/min.

4. The method of claim 1, wherein said ray is x-ray and/or γ-ray.

5. The method of claim 1, wherein the final concentration of said chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.1 mg/ml to about 100mg/ml; wherein the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about Ih to about 72h.

6. The method of claim 5, wherein the final concentration of said chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 50mg/ml; wherein the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12h to about 36h.

7. The method of claim 1, wherein said alkylating agent is selected from the group consisting of mechlorethamine, cyclophosphamide, isofosfamide, formylmerphalan, sarcoclorin, chlorambucil and thiotepa; wherein said nitrosoureas is selected from the group consisting of carmustine, lomustine, semustine, nimustine, busulfan and estramustine; wherein said anti-metabolite is selected from the group consisting of methotrexate, fluorouracil, tegafur, tegafur-uracil, carmofur, fluorodeoxyuridine, floxuridine, ancitabine, cytosine arabinoside and gemcitabine; wherein said anti-tumor antibiotics is selected from the group consisting of actinomycin D, mitomycin, bleomycin, pingyangmycin, adriamycin, perarubicin, epirubicin, idamycin and mitoxantrone; wherein said herb-chemotherapeutics is selected from the group consisting of vincristine, vinblastine, vindesine, vinorelbine, etoposide, teniposide, hydroxycamptothecine, irinotecan, topotecan, paclitaxel, docetaxel, harringtonine and hydroxyl-paclitaxel; wherein said hormone and incretion chemotherapeutics is selected from the group consisting of prednisone, dexamethasone, testosterone propionate, diethylstilbestrol, flutamide, progestin, megestrol, tamoxifen, estrogen Receptor antagonist, toremifene, aminoglutethimide, formestane, letrozole and gonadotropin releasing hormone antagonist; wherein said antibody repressor is selected from the group consisting of epidermal growth factor inhibitor-gefϊtinib, tarceva, cetuximab and nimotuzumab.

8. The method of claim 1, wherein said chemotherapeutic agent(s) is selected from the group consisting of 4-hydroxyl-cyclophosphamide, gefttinib, tarceva, 5-fluorouracil, doxorubicin, taxol and cisplatin.

9. The method of claim 1, wherein the method comprises: a step of performing radiation exposure screening to tumor stem cells; wherein the linear distance between said radiation source of ray and tumor stem cells ranges from about 20cm to about 2m, wherein the total dose of said radiation exposure ranges from about 3Gy to about 9Gy, wherein the dose rate of said radiation exposure ranges from about 2cGy/min to about 600cGy/min.

10. The method of claim 1, wherein the method comprises: a step of performing chemotherapeutic agent screening to tumor stem cells; wherein the final concentration of said chemotherapeutic agent(s) in medium for tumor stem cells ranges from about 0.5 mg/ml to about 50mg/ml, wherein the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12h to about 36h.

11. The method of claim 1, wherein the method comprises: a step of performing radiation exposure screening and chemotherapeutic agent screening to tumor stem cells; wherein the linear distance between said radiation source of ray and tumor stem cells ranges from about 20cm to about 2m, wherein the total dose of said radiation exposure ranges from about 3Gy to about 9Gy, wherein the dose rate of said radiation exposure ranges from about 2cGy/min to about 600cGy/min; wherein the final concentration of said chemotherapeutic agent(s) in the medium for tumor stem cells ranges from about 0.5 mg/ml to about 50mg/ml, wherein the time for making tumor stem cells contact with a chemotherapeutic agent(s) ranges from about 12h to about 36h.

12. The method of claim 1, wherein said tumor is selected from the group consisting of breast cancer, glioma, lung cancer, brain tumor, nasopharyngeal carcinoma, hepatocellular carcinoma, carcinoma ventriculi, colon cancer, melanoma, osteosarcoma, renal cell carcinoma, prostate cancer, carcinoma of ovary, acute myelogenous leukemia basic, multiple myeloma, pancreatic cancer and metastatic cancer.

13. The method of claim 12, wherein said metastatic cancer is selected from the group consisting of cerebral metastasis tumor, pulmonary metastasis tumor, liver metastasis tumor and jugular metastasis tumor.

14. The method of any of claims 1 to 13, wherein the method further comprises isolating tumor stem cells from the tumor tissues ex vivo and/ or tumor cell lines.

15. A tumor stem cell obtained by the method of any of claims 1 to 14.

16. The tumor stem cells of claim 15, wherein said tumor stem cells specifically express the proteins selected from the group consisting of epithelial growth factor receptor, P-glucoprotein, multidrug-associated protein, protein coded by ataxia-telangiectasia mutation gene (ATM), ataxia-telangiectasia Rad3 related protein and hypoxia inducible factor.

17. A method for preparing an antigen composition of tumor stem cells, which comprises the steps of washing and re-suspending tumor stem cells with saline and then lysing the obtained tumor stem cells, characterized in that said tumor stem cells are the tumor stem cells of claim 15 or 16.

18. The method of claim 17, characterized in that said step of lysing tumor stem cells comprises heat shock and/or repeated freeze-thaw to tumor stem cells; wherein the temperature of said heat shock ranges from about 37°C to about 45°C; wherein the persistence time of said heat shock ranges from about Ih to about 6h; wherein the number of times of said repeated freeze-thaw ranges from 3 times to 5 times; wherein the time interval between two times ranges from about lOmin to about 2h; wherein the temperature of said freezing ranges from about -120⁰C to about -196⁰C; wherein the temperature of said thawing ranges from about 10⁰C to about 37°C.

19. The method of claim 18, characterized in that said step of lysing tumor stem cells comprises heat shock and repeated freeze-thaw to tumor stem cells; wherein the temperature of said heat shock ranges from about 37°C to about 45°C; wherein the persistence time of said heat shock ranges from about Ih to about 6h; wherein the number of times of said repeated freeze-thaw ranges from 3 times to 5 times; wherein the time interval between two times ranges from about 1 Omin to about 2h; wherein the temperature of said freezing ranges from about -120⁰C to about -196⁰C; wherein the temperature of said thawing ranges from about 2O⁰C to about 37⁰C.

20. An antigen composition of tumor stem cells obtained by the method of any of claims 17 to 19.

21. The antigen composition of claim 20, characterized in that said antigen composition is obtained from the tumor stem cells of claim 15 or 16, whose concentration ranges from about I X lO⁵ cell/ml to about 1 X 10⁸cell/ml .

22. The antigen composition of claim 20, characterized in that said antigen composition further comprises human serum albumin; wherein based on the total volume of said composition, the content of human serum albumin ranges from about 1 percent weight in volume to about 5 percent weight in volume.

23. Use of the tumor stem cells of claim 15 or 16 and the antigen composition of tumor stem cells of any of claims 20-22 for the manufacturing of an anti-tumor medicament.

24. A kit for preparing the tumor stem cells of claim 15, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(c) an enzyme(s) for digesting cells;

(d) a cytokine(s); and

(e) operating instruction; wherein said radiotherapeutic radioisotope source is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide; wherein said operating instruction includes a method of any of claims 1-14.

25. The kit of claim 24, wherein said enzyme(s) for digesting cells is pancreatin and /or accutase.

26. The kit of claim 24, wherein said cytokine(s) is selected from the group consisting of B27, N2, epidermal growth factor, basic fibroblast growth factor and insulin.

27. The kit of claim 24, characterized in that said kit further comprises an animal serum (or sera).

28. A kit for preparing the antigen composition of tumor stem cells of claim 20, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(c) an enzyme(s) for digesting cells;

(d) a cytokine(s); and

(e) operating instruction; wherein said radiotherapeutic radioisotope source is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide; wherein said operating instruction includes a method of any of claims 17-19.

29. The kit of claim 28, wherein said enzyme(s) for digesting cells is pancreatin and/or accutase; wherein said cytokine(s) is selected from the group consisting of B27, N2, epidermal growth factor, basic fibroblast growth factor and insulin.

30. The kit of claim 28, characterized in that said kit further comprises an animal serum (or sera).

31. A method for preparing an anti-tumor dendritic cell, which comprises obtaining an antigen composition of tumor stem cells by lysing said tumor stem cells, making normal dendritic cells contact with said antigen composition of tumor stem cells, and then obtaining an anti-tumor dendritic cell loading with the antigens of said tumor stem cells, characterized in that said tumor stem cells are the tumor stem cells of claim 15 or 16.

32. The method of claim 31, characterized in that said antigen composition of tumor stem cells is the any antigen composition of tumor stem cells of any of claims 20-22.

33. The method of claim 31, wherein the normal dendritic cells and the tumor stem cells for preparing an antigen composition are derived from a same individual; or wherein said tumor stem cells are obtained from the culture of the corresponding tumor cell line(s).

34. The method of claim 31, wherein the persistence time for making normal dendritic cells contact with said antigen composition of tumor stem cells ranges from about Ih to about 24h.

35. An anti-tumor dendritic cell obtained by the method of any of claims 31 to 34.

36. A dendritic cell vaccine, which comprises dendritic cells, characterized in that said dendritic cells are the anti-tumor dendritic cells of claim 35.

37. The vaccine of claim 36, wherein said vaccine includes said anti-tumor dendritic cells, whose concentration ranges from about Ix 10⁵ cell/ml to about 5xlO⁷ cell/ml.

38. The vaccine of claim 36, wherein said vaccine further comprises human serum albumin; wherein based on the total volume of said vaccine, the content of human serum albumin ranges from about 1 percent weight in volume to about 5 percent weight in volume.

39. Use of the anti-tumor dendritic cells of claim 35 for the manufacturing of an anti-tumor medicament.

40. A kit for preparing anti-tumor dendritic cells of claim 35, characterized in that said kit comprises:

(a) basic medium for tumor stem cells;

(c) an enzyme(s) for digesting cells;

(e) basic medium for dendritic cells;

(f) granulocyte macrophage colony stimulating factors;

(g) at least one of inter leukin-4, interferon α and tumor necrosis factor; and (h) operating instruction; wherein said radiotherapeutic radioisotope source is selected from the group consisting of X-ray, β-ray, α-ray and γ-ray; wherein said chemotherapeutic agent is selected from the group consisting of alkylating agent, nitrosourea, anti-metabolite, anti-tumor antibiotics, herb-chemotherapeutics, hormone and incretion chemotherapeutics, antibody repressor, asparaginase, natulane, dacarbazine, cisplatin, carboplatin, oxaliplatin, razoxane and hydroxycarbamide; wherein said operating instruction includes a method of any of claim 31-34.

41. The kit of claim 40, wherein said enzyme(s) is pancreatin and /or accutase.

42. The kit of claim 41, characterized in that said kit further comprises an animal serum (or sera).