CN105308177A

CN105308177A - Individualized high purity hepatocellular carcinoma stem cells, methods and use of the same

Info

Publication number: CN105308177A
Application number: CN201480025747.0A
Authority: CN
Inventors: N.贾布里尔; C.T.弗里德里克森; A.科恩富思
Original assignee: California Stem Cells Inc
Current assignee: NeoStem Oncology LLC
Priority date: 2013-03-07
Filing date: 2014-03-06
Publication date: 2016-02-03
Also published as: JP2016510756A; KR20150139855A; AU2014225575A1; AU2014225575A8; WO2014138455A1; EP2964754A1; EP2964754A4; CA2903212A1

Abstract

The disclosure provides cancer stem cells, for use in stimulating immune response against a cancer, such as hepatocellular carcinoma (HCC). Methods for preparing and purifying the cancer stem cells are provided.

Description

Personalized high purity hepatocellular carcinoma stem cell, the method for described stem cell and purposes

the cross reference of related application

The application requires the rights and interests of the U.S. Provisional Patent Application 61/774,517 that on March 7th, 2013 submits to according to 35U.S.C. § 119 (e).The application is also the part continuation application of International Application Serial No. PCT/US2013/053850 that on August 6th, 2013 submits to, described international application requires according to 35U.S.C. § 119 (e) U.S. Provisional Patent Application 61/683 that on August 15th, 2012 submits to, the rights and interests of the U.S. Provisional Patent Application 61/718,643 that on October 25th, 477 and 2012 submits to.The full content of described application is incorporated herein by reference.

Technical field

The present invention relates to hepatocellular carcinoma stem cell, be derived from the method for the immunogenic composition of described stem cell and manufacture and the described stem cell of use.

Background technology

In noumenal tumour, the cell of less per-cent has the ability causing the tumour with homologue's heterogeneity as parent's tumour.These cells are called as cancer stem cell, and are also referred to as tumour initiator cell or cancer initiator cell.Cancer stem cell can be defined by one group of characteristic.First, it has the ability making autosynthesis.The second, it can set up new tumour when transplanting.3rd, it can be characterized as being the tumour cell of dormancy or slowly circulation (cell cycle).4th, its may be cause tumour to chemotherapy or radiotherapeutic resistivity reason.5th, it depends on that maintaining it regenerates and the specific microenvironment producing the ability of more differentiated progenitor cells, and wherein said environment maintains the undifferentiated state of cancer stem cell.This microenvironment can comprise mesenchymal cell stem cell, organize the inoblast and endotheliocyte that are associated.The ability forming spherule when cultivating in vitro is another feature that specific cells can be contributed to be identified as cancer stem cell.Even if cancer stem cell non-limiting definition can regenerate the complete heterogeneity of parent's tumour and the cell also grown continuously after repeatedly going down to posterity.

Particular cancers stem cell population may be anything superfluous or useless source, and may be the source of the cancer return of having treated.In addition, cancer stem cell subgroup in the tissue may restart growth cycle when being exposed in some signal and produce the cell can rebuilding tumour.The subenvironment of cancer stem cell is dormancy, until correct intracellular signaling triggers enter proliferating cycle again.Entering signal can derive from local event again, as wound, cell injury, microorganism attack (virus, bacterium or fungi), or is mediated by tropic growth factors, cytokine or cell-cell communication.In addition, hormone can regulate the stem cell in tissue specificity subenvironment.The defect of stem cell subenvironment or sudden change may cause the disturbance of function above.Anything superfluous or useless may be produced by this kind of disturbance, and these disturbances comprise the random mutation of the control affecting cell cycle.The sudden change of cancer is caused to change between individuals.Between those people of cancer suffering from a type (as a patient with breast cancer contrasts another patient with breast cancer) and between dissimilar cancer (as liver cancer contrast melanoma) observe this kind of variability.

The therapy gone out for cancer development comprises the method relating to autoimmune reaction.These methods comprise the autologous tumor cell using fresh collection, and described autologous tumor cell solution coalescence is deployed into vaccine carrier.Another kind method is dendritic cell vaccine, wherein dendritic cell autologous tumor lysate pulse.Another method carries out external modification with galactose polymer to the tumour cell of patient, and modified tumor cell injection is got back in described patient, wherein the tumour cell of semi-lactosi mark is easier is absorbed by antigen presenting cell (APC), increases antitumor reaction thus.These shortcomings relating in the method for autoimmunity therapy are when the block tumour of use or block tumour antigen are as low antigen signal to noise ratio during immunostimulant.Major part tumour cell is quite differentiation and with normal cell as the tumour cell of vascular components, reticular tissue, differentiation, non-living cell and non-viable non-apoptotic cell and normal host tissues mix.Cancer stem cell only represents the smaller portions of tumor mass, to have more in aggressive tumour sometimes maximum 4%, is the most commonly less than 1%.Therefore, when using block tumour as antigenic source, immune response is the cell for more breaking up, thus allows stem cell to escape attack and may cause recurrence or the transfer of tumour.

Another kind of new therapy is the single targeting antibodies for the normal antigen expressed more galore in cancer.Specific antigens targeted therapies (as anti-CD133, anti-EpCAM, anti-CD44, anti-CD13 etc.) is undiscerning, and affects normal cell and cancer cells, thus causes the huge undesirable action to patient.

Summary of the invention

Disclose hepatocellular carcinoma (HCC) cancer stem cell (CSC), HCC-CSC clone herein and be used for the treatment of the immunogenic composition comprising HCC-CSC loading type dendritic cell of hepatocellular carcinoma.

Especially, provide a kind of method for the preparation of the colony of hepatocellular carcinoma (HCC) cancer stem cell (CSC) herein, described method comprises: obtain HCC sample; Make the cell dissociation of described sample, and the cell dissociated described in vitro culture in the substratum determined at composition on non-adhesive substrate, the substratum that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via mitogen-activated protein kinase (MAPK) path, forms HCC-CSC spherule thus; At least about two or more in the following biomarker of cell expressing of 80% in wherein said HCC-CSC spherule colony: alpha fetal protein (AFP), EpCAM, Ov1 and OV6.In another embodiment, express in following biomarker further at least about the cell of 80% in described HCC-CSC spherule colony one or more: CK7, CK19 and E-cadherins.In another embodiment, in described HCC-CSC spherule colony at least about two or more in the following biomarker of cell expressing of 90%: AFP, EpCAM, Ov1 and OV6.

In another embodiment, described method is included in further in the substratum that adhesivity substrate is determined at composition and cultivates described HCC-CSC spherule, the substratum that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of early stage HCC-CSC thus, at least about two or more in the following biomarker of cell expressing of 80% in wherein said early stage HCC-CSC colony: Nanog, Sox2, Oct3/4 and c-kit.In another embodiment, express in following biomarker further at least about the cell of 80% in described early stage HCC-CSC colony one or more: EpCAM, E-cadherins, Sox7, Sox17, Fox2A, Ov1, OV6, CD133 and CD90.In another embodiment, in described early stage HCC-CSC colony at least about two or more in the following biomarker of cell expressing of 90%: Nanog, Sox2, Oct3/4 and c-kit.

In another embodiment, described method is included in further in the substratum that adhesivity substrate is determined at composition and cultivates described HCC-CSC spherule, the substratum that wherein said composition is determined contains serum and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of mixing HCC-CSC thus, at least about two or more in the following biomarker of cell expressing of 80% in wherein said mixing HCC-CSC colony: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.In another embodiment, in described mixing HCC-CSC colony at least about two or more in the following biomarker of cell expressing of 90%: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.

In another embodiment, described method is included in further in the substratum that adhesivity substrate is determined at composition and cultivates described HCC-CSC spherule, the substratum that wherein said composition is determined contains serum origin and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of (EMT)-HCC-CSC that embryo changes to mesenchymal cell thus, at least about two or more in the following biomarker of cell expressing of 80% in wherein said EMT-HCC-CSC colony: NCAM, Slug/Snail and Twist.In another embodiment, express in following biomarker further at least about the cell of 80% in described EMT-HCC-CSC colony one or more: AFP, N-cadherins, CD44 and vimentin.In still another embodiment, one or more at least about in the following biomarker of cell expressing of 90% in described EMT-HCC-CSC colony: NCAM, Slug/Snail and Twist.

In another embodiment, described method is included in further in the substratum that adhesivity substrate is determined at composition and cultivates described HCC-CSC spherule, described mixing HCC-CSC or EMT-HCC-CSC, the substratum that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of early stage HCC-CSC thus, at least about two or more in the following biomarker of cell expressing of 80% in wherein said early stage HCC-CSC colony: Nanog, Sox2, Oct3/4 and c-kit.In another embodiment, express in following biomarker further at least about the cell of 80% in described early stage HCC-CSC colony one or more: CK7, CK19 and E-cadherins.In still another embodiment, one or more at least about in the following biomarker of cell expressing of 90% in described early stage HCC-CSC colony: Nanog, Sox2, Oct3/4 and c-kit.

In another embodiment, described method is included in further in the substratum that adhesivity substrate is determined at composition and cultivates described HCC-CSC spherule, described early stage HCC-CSC or EMT-HCC-CSC, the substratum that wherein said composition is determined contains serum origin and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of mixing HCC-CSC thus, at least about two or more in the following biomarker of cell expressing of 80%: AFP in wherein said mixing HCC-CSC colony, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.In another embodiment, in described mixing HCC-CSC colony at least about two or more in the following biomarker of cell expressing of 90%: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.

In another embodiment, described method is included in the substratum that adhesivity substrate is determined at composition further cultivates described HCC-CSC spherule, described early stage HCC-CSC or mixing HCC-CSC, the substratum that wherein said composition is determined contains serum origin and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of EMT-HCC-CSC thus, at least about two or more in the following biomarker of cell expressing of 80% in wherein said EMT-HCC-CSC colony: NCAM, Slug/Snail and Twist.In another embodiment, express in following biomarker further at least about the cell of 80% in described EMT-HCC-CSC colony one or more: AFP, N-cadherins, CD44 and vimentin.In still another embodiment, one or more at least about in the following biomarker of cell expressing of 90% in described EMT-HCC-CSC colony: NCAM, Slug/Snail and Twist.

In one embodiment, the substratum that described composition is determined is any substratum described in table 2; Come from any substratum of the combination of table 2 and table 3; Come from any substratum of the combination of table 2, table 3 and table 4; Or come from any substratum of combination of table 2 and table 4.

In one embodiment, described somatomedin is one or more in fibroblast growth factor (FGF), Urogastron (EGF) or activin A.In another embodiment, described FGF is basic FGF (bFGF).In still another embodiment, the substratum that described composition is determined is not supplemented with activin A.In another embodiment, the substratum determined of described composition is with the agonist effectively preventing the amount of HCC stem cell Spontaneous Differentiation to be supplemented with activin A.In still another embodiment, described activin A antagonist is follistatin or the antibody with activin A specific binding.

In another embodiment, described substratum is not supplemented with antioxidant.In another embodiment, described antioxidant is superoxide-dismutase, catalase, gsh, putrescine or beta-mercaptoethanol.In still another embodiment, the culture medium supplemented that described composition is determined has gsh.

In another embodiment, described adhesivity substrate is configured to adhere to anchorage dependence cell (as inoblast), and collects described cell.In another embodiment, described non-adhesive substrate is ultralow adhesivity polystyrene surface.In still another embodiment, described adhesivity substrate comprises the surface being coated with the protein being rich in RGD tri-peptide motif.

Also provide a kind of colony of purified HCC-CSC cell herein, it is prepared by any one in method disclosed herein.In a certain embodiment, described purified HCC-CSC cell is HCC-CSC spherule, early stage HCC-CSC, mixing HCC-CSC or EMT-HCC-CSC.

Also provide a kind of HCC-CSC clone herein, it is prepared by any one method in method disclosed herein.In a certain embodiment, described purified HCC-CSC cell is HCC-CSC spherule, early stage HCC-CSC, mixing HCC-CSC or EMT-HCC-CSC.

In one embodiment, provide a kind of immunogenic composition herein, it comprises the dendritic cell by the tumour antigen ex vivo activation being derived from purified HCC-CSC cell colony disclosed herein or HCC-CSC clone.In another embodiment, described tumour antigen comprises the cell extract of described purified HCC-CSC cell or described HCC-CSC clone.In another embodiment, described tumour antigen comprises the lysate of described purified HCC-CSC cell or described HCC-CSC clone.In another embodiment, described tumour antigen comprises complete purified HCC-CSC cell or the intact cell from described HCC-CSC clone.

In another embodiment, described complete HCC-CSC cell is endowed non-proliferative.In another embodiment, described intact cell is endowed non-proliferative by radiation.In still another embodiment, described intact cell is endowed non-proliferative by being exposed in core linking agent by described cell.

In another embodiment, described immunogenic composition comprises pharmaceutically acceptable carrier and/or vehicle further.In another embodiment, described immunogenic composition comprises adjuvant further.In another embodiment, described adjuvant is rHuGM-CSF.

In still another embodiment, described immunogenic composition comprises dendritic cell and HCC-CSC cell.In another embodiment, described purified HCC-CSC cell or described HCC-CSC clone are HCC-CSC spherule, early stage HCC-CSC, mixing HCC-CSC or EMT-HCC-CSC form.

Also provide a kind of method for the treatment of hepatocellular carcinoma in experimenter in need, it comprises and gives immunogenic composition disclosed herein to described experimenter.In one embodiment, described immunogenic composition gives with multiple dosage form, and each dosage comprises about 5-20 × 10 ⁶individual cell.In another embodiment, described dosage comprises about 10 × 10 ⁶individual cell.In another embodiment, described dosage gives weekly once, continues 2-5 dosage, then monthly gives once, continues 3-6 dosage.In still another embodiment, described experimenter accepts the immunogenic composition of 6-10 dosage.

Also provide a kind of in the immunoreactive method of experimenter's moderate stimulation in need for hepatocellular carcinoma, it comprises and gives immunogenic composition disclosed herein, HCC-CSC disclosed herein or HCC-CSC clone disclosed herein to described experimenter.

Immunogenic composition disclosed herein, HCC-CSC disclosed herein or HCC-CSC clone disclosed herein purposes in the medicine for the preparation for the treatment of hepatocellular carcinoma is also provided.

Immunogenic composition disclosed herein, HCC-CSC disclosed herein or HCC-CSC clone disclosed herein is also provided to be used for the treatment of the purposes of hepatocellular carcinoma.

Accompanying drawing explanation

Fig. 1 is from the tumour of excision (solid line boxes and arrow) or hepatocellular carcinoma (HCC) stem cell (HCC-CSC) is separated, increases and is gathered the schema of the process of glomeration body from the small sample (dashed rectangle and arrow) of such as pin biopsy.After generation spherule, the path producing HCC-CSC subgroup is common path.

Fig. 2 is the schematic diagram of spherule.

Fig. 3 A and 3B describes the spherule of different shape and size.

Fig. 4 describes the HCC stem cell being in the amplification phase.

Fig. 5 describes the cell be attached on ultralow adhesivity substrate.

Fig. 6 describes the insensitive HCC stem cell of activin A.These cells grow with compact colony, and by normal host tissues (inoblast) around.The tumour cell more broken up is eliminated by the existence of activin A.

Fig. 7 describes by being exposed to the HCC stem cell to have the growth of specific compact colony to stem cell selected in activin A.

Fig. 8 describes the enrichment HCC culture with the embryonic stem cell sample colony containing little autosynthesis cell.

After Fig. 9 is depicted in and is seeded on adhesivity substrate when seven days, dissociated by the enzymatic of the pin biopsy from HCC tumour and the cell (phase contrast 10 ×) that produces.

Figure 10 is depicted in the intensive Colony forming of typical case (phase contrast 10 ×) after the Growth of Cells 14-21 days of Fig. 9.

The higher proliferation HCC stem cell line (phase contrast 10 ×) set up after Figure 11 is depicted in and repeatedly goes down to posterity.

Figure 12 A description core redyes (two benzimide) to the HCC clone of alpha fetal protein (AFP) in stained positive, confirms HCC identity (falling to penetrating fluorescence, 20 ×).Figure 12 B is depicted in for Figure 12 A cell in the red channel image of AFP dyeing.Figure 12 C describes the blue channel image being used for two benzimide.

Figure 13 A description has the cell that high per-cent nerve cell adhesion molecule (NCAM) is dyeed, and confirms that the early-stage cancer progenitor cell phenotype in the culture established selects (comprising core to redye, two benzimide) (falling to penetrating fluorescence, 40 ×).Figure 13 B is depicted in for Figure 13 A cell in the red channel image of NCAM dyeing.Figure 13 C describes the blue channel image being used for two benzimide.

Figure 14 A describes to be labeled as positive HCC clone for CD44, and described CD44 has specific invasiveness mark to the cancer stem cell with high metastatic potential.Cell is also redyed with core, two benzimide dyeing (falling to penetrating fluorescence, 40 ×).Figure 14 B is depicted in for Figure 14 A cell in the green channel images of CD44 dyeing.Figure 13 C describes the blue channel image being used for two benzimide.

Figure 15 A describes epithelial cell in the EMT-HCC-CSC clone using two benzimide core to redye and changes mark vimentin and Slug/Snail to mesenchymal cell.Figure 15 B is depicted in for Figure 15 A cell in the red channel image of Slug/Snail dyeing.Figure 15 C is depicted in for Figure 15 A cell in the green channel images of vimentin.Figure 15 D describes the blue channel image being used for two benzimide.

Embodiment

The invention provides a kind of cell colony obtained from human hepatocytes cancer (HCC) tumour, described cell colony forms primarily of high purity cancer stem cell.In embodiments, the purity of cell colony is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% cancer stem cell.These cancer stem cells are hepatocellular carcinoma progenitor cells, and the ability that there is continuous autosynthesis and break up to certain level.The invention still further relates to a kind of method that generation is derived from the purified colony of the stem cell of HCC, for the antigenic source being used as cancer autoimmunity therapy further.

Also contain test and screening embodiment.The present invention uses high purity HCC stem cell population to carry out genetic analysis to identify the uniqueness change driving individualized medicine allocation.The invention provides a kind of novel cell lines through external modification, wherein this immunostimulation feature of modifying enhancing HCC.HCC clone is the improvement to the similar techniques using rough antineoplastic agents, because it provides excellent antigen signal to noise ratio.Clone lacks pollutent cell colony (as inoblast), and described cell colony can change or weaken external application.Exemplary cells system of the present invention is also for the manufacture of the medicine being used for the treatment of HCC.

As used herein, term " is derived from " any method containing when being derived from the peptide of one or more cancer cells and obtain described peptide from the colony of cancer cells or cancer cells.Cancer cells can such as destroy by clarifixator or by osmotic bursting, produces crude extract.The peptide of crude extract, oligopeptides and polypeptide can be exposed to dendritic cell, then by peptide described in described dendritic cell process.Term " is derived from " also contains complete cancer cells, wherein said cancer cells is alive, or wherein said cancer cells has metabolic activity by radiotreatment, or wherein said cancer cells has metabolic activity with nucleic acid crosslinking agent process and therefore still comprises described peptide." be derived from " mixture also comprising cancer cells fragment, free cancer cell proteins matter and the cancer cells (therefore it be derived from cancer cells) through overshoot.

" give " its be applied to the mankind, Mammals, mammalian subject, animal, veterinary science experimenter, placebo subjects, research experimenter, experimental subjects, cell, tissue, organ or biofluid time refer to that (but being not limited to) makes exogenous part, reagent, placebo, small molecules, medicament, therapeutical agent, diagnostic reagent or composition contact described experimenter, cell, tissue, organ or biofluid etc." give " to refer to such as treatment, pharmacokinetics, diagnosis, research, placebo and experimental technique.Give the interior therapeutic that can refer to the mankind or animal subjects.Cell process is contained makes reagent exposing cell, and makes reagent contacting with fluid, wherein said fluid and cells contacting." give " also to contain by reagent, diagnosis, bonding composition or carry out external and a kind of cell of ex vivo treatment by another kind of cell.

" significant quantity " contains (but being not limited to) can improve, reverse, relax, prevent or at least one symptom of diagnostic medicine symptom or illness or the amount of sign.Unless in addition clear and definite or specified by context, otherwise " significant quantity " is not limited to the minimum quantity being enough to realize results needed, is also not limited to the optimal amount being enough to realize results needed.

Or the seriousness of disease or illness can be measured by clinical parameter and treat in order to prevention, the ability treating or relax described disease or illness (realizing results needed) by biomarker, and implicit any restriction.Biomarker comprises blood counting; Metabolite level in serum, urine or celiolymph; Tumour cell counts; Cancer stem cell counts; Tumor levels.Tumor levels can react assessment level (ResponseEvaluationCriteriaInSolidTumors by noumenal tumour; RECIST) criterion measures (people (2009) Eur.J.Cancer45:228-247 such as Eisenhauer).Presentation markup contains the expression of the genetic expression of mRNA or gene amplification, the expression of antigen and polypeptide.Clinical parameter comprises progresson free survival phase (PFS), 6 months PFS, without disease survival time (DFS), occurs the time (TTP) be in progress, the time (TDM) occurring remote transfer and total survival rate, and does not imply any restriction.

Composition through " mark " directly or indirectly detects by spectroscopy, photochemistry, biological chemistry, immunochemistry, isotropic substance or chemical process.For example, the marker be suitable for comprises ³²p, ³³p, ³⁵s, ¹⁴c, ³h, ¹²⁵i, stable isotope, epitope tag fluorescence dye, electron-dense reagent, substrate or enzyme, such as use in enzyme immunoassay those, or fluorette(is disclosed in the U.S. 6,747, in 135, the wherein disclosed full content about fluorette is incorporated herein by reference).

Therefore, the open colony for the preparation of the purified spherule of cancer stem cell or be derived from the method for single cell preparation of spherule herein, described method comprises and obtains HCC biopsy; Make the cell dissociation of described biopsy; The cell dissociated described in vitro culture in the substratum that substrate is determined at composition, the somatomedin that the culture medium supplemented that wherein said composition is determined has at least one to work via mitogen-activated protein kinase (MAPK) path, to obtain colony or the single cell preparation of the purified spherule of HCC stem cell.What in the colony of purified spherule or single cell, the cancer stem cell of at least 80% was expressed in following biomarker is one or more or all: ATP is in conjunction with box subfamily G member 2(ATP-bindingcassettesub-familyGmember2; ABCG2; Gene pool accession number AAG52982.1), α-fetoprotein (AFP), CD133, CD44, CD90, Cyfra21-1 (CK19), CK7 (CK7), c-kit, E-cadherins, epithelial cell adhesion molecule (EpCAM; Gene pool accession number NP_002345.2), jaw frame A2(forkheadboxA2; FoxA2), Hepatocyte nuclear factor 4 α (hepatocytenuclearfactor4alpha; HNF4a), Ki-67, Nanog(gene pool accession number NM_024865.2, NP_079141.20), N-cadherins, nerve cell adhesion molecule (NCAM; CD56), Oct3/4(gene pool accession number NP_002692.2; NP_976034.4; NP_001167002.1; NP_068812.10), Ov1, OV6, Slug(SNAI2)/Snail(SNAI1) (Slug/Snail), Sox17, Sox2(gene pool accession number NM_003106.3, NP_003097.1), Sox7, Twist and vimentin.The schema of the cell colony disclosed in formation presents in FIG.

As used herein, term " spherule " refers to the cancer stem cell sphere aggregates formed by cultivating cancer cells in serum free medium.The ability forming spherule is the feature of cancer stem cell.

In certain embodiments, in HCC-CSC spherule colony at least about two or more in the following biomarker of cell expressing of 80% or all: AFP, CK7, CK19, EpCAM, E-cadherins, Ov1 and OV6.In other embodiments, in HCC-CSC spherule colony at least 80% the following biomarker of cell expressing in two or more or all: AFP, CK7, CK19, EpCAM, E-cadherins, Ov1 and OV6.In another embodiment, in described HCC-CSC spherule colony at least 80% the following biomarker of cell expressing in two or more or all: AFP, EpCAM, Ov1 and OV6.

Also providing package is containing the colony of purified spherule of cancer stem cell, and in wherein said purified spherule colony, to express in following biomarker one or more or all for the described cancer stem cell of at least 80%: ABCG2, AFP, CD133, CD44, CD90, CK19, CK7, c-kit, E-cadherins, EpCAM, FoxA2, HNF4a, Ki67, Nanog, N-cadherins, NCAM(CD56), Oct3/4, Ov1, OV6, Slug/Snail, Sox17, Sox2, Sox7, Twist and vimentin.

Spherule colony can increase into the one in three kinds of different subgroups further by change culture condition (as substratum composition and substrate).The feature of block tumour, spherule, early stage, mixing and EMT colony presents in Table 1.

table 1. for being produced the general introduction of the condition of HCC cell colony by block HCC tumour

* CSC=cancer stem cell; * EMT=embryo-mesenchymal cell changes

In addition, as disclosed in table 1, can by change substratum and condition cause HCC-CSC spherule, early stage HCC-CSC, mixing HCC-CSC or EMT-HCC-CSC obtain early stage HCC-CSC, mix in HCC-CSC or EMT-HCC-CSC colony any one.

In one embodiment, on adhesivity substrate, at activin A, HCC spherule is cultivated further to obtain the colony (being called as " in early days " HCC-CSC colony in this article) with minicell under the existence of FGF and serum free medium (Selective agar medium), it has the feature of embryonic stem cell, and in described early stage HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more or all: EpCAM, E-cadherins, Nanog, Sox2, Sox7, Sox17, Oct3/4, Fox2A, Ov1, OV6, c-kit, CD133 and CD90.In another embodiment, in early stage HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more or all: EpCAM, E-cadherins, Nanog, Sox2, Sox7, Sox17, Oct3/4, Fox2A, Ov1, OV6, c-kit, CD133 and CD90.In another embodiment, in early stage HCC-CSC colony at least 90% the following biomarker of cell expressing in two or more or all: Nanog, Sox2, Oct3/4 and c-kit.

In another embodiment, on adhesivity substrate, FGF, EGF and containing the existence of blood serum medium (amplification culture medium) under cultivate further HCC spherule to obtain the colony mixed with individual layer, wherein cell has heterogeneous form.These cells are called as " mixing " HCC-CSC colony in this article, it has mixing differentiation overview, and in described mixing HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more or all: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2, HNF4a and ABCG2.In another embodiment, in early stage HCC-CSC colony at least 90% the following biomarker of cell expressing in two or more or all: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2, HNF4a and ABCG2.

In still another embodiment, on adhesivity substrate, FGF and containing the existence of blood serum medium (amplification culture medium) under cultivate further HCC spherule to obtain the individual layer of fusiformis or irregular shape cell, it is called as mesenchymal cell sample HCC-CSC or " EMT-HCC-CSC " (embryo changes the cancer stem cell of [EMT] to mesenchymal cell) in this article.In this colony, spherule once went through EMT process, it is characterized in that at least one in epithelial cell marker CK7, CK19, EpCAM and E-cadherins or all expression deletions.As used herein, the expression deletion of biomarker refers to undetectable expression, or at 40%(or less) cells, at 30%(or less) cells, at 20%(or less) cells or at 10%(or less) cells.In addition, the feature of EMT process is that at least one or all expression of mesenchymal cell mark Slug/Snail, Twist, CD44, NCAM, N-cadherins and vimentin are increased to the cell of in the colony of expressing object biomarker at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

In one embodiment, in EMT-HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more or all: NCAM, Slug/Snail and Twist.In still another embodiment, in EMT-HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more or all: AFP, NCAM, N-cadherins, Slug/Snail, Twist, CD44 and vimentin.In still another embodiment, in EMT-HCC-CSC colony at least 90% the following biomarker of cell expressing in two or more or all: AFP, NCAM, N-cadherins, Slug/Snail, Twist, CD44 and vimentin.

In some embodiment of cell colony, one or more in the biomarker indicated by described cell expressing.In other embodiments, in the biomarker indicated by described cell expressing two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more.1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or 25 in the biomarker again described in other embodiment indicated by cell expressing.

Single cell, cell colony or the biomarker of cell colony being arranged in ad hoc structure (as individual layer or spherule) express can by the polypeptide form of biomarker as described in measuring or as described in the expression of mRNA form of biomarker measure.Expression of polypeptides can be measured by applying marking antibody, and expression of nucleic acid can be measured by hybridization technique, and it is that technician is available.And the biomarker of non-polypeptide or nucleic acid (as oligosaccharides or small molecule metabolites) also can be measured by the available method of technician.

Also provide a kind of colony of HCC-CSC spherule or be derived from the single cell of described colony, wherein the per-cent of the cell of expressing K i-67 is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

Also provide a kind of colony of HCC-CSC spherule or be derived from the single cell of described colony, the per-cent of wherein expressing the cell of AFP is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

Also provide a kind of colony of HCC-CSC spherule or be derived from the single cell of described colony, the per-cent of wherein expressing the cell of NCAM is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

Also provide a kind of EMT-HCC-CSC colony, the per-cent of wherein expressing the EMT-HCC-CSC of Slug/Snail is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

Also provide a kind of EMT-HCC-CSC colony, the per-cent of wherein expressing the EMT-HCC-CSC of CD44 is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

Also provide a kind of EMT-HCC-CSC colony, the per-cent of wherein expressing the EMT-HCC-CSC of N-cadherins is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

Also contain a kind of colony of HCC-CSC spherule herein or be derived from the single cell of described colony, one, two, three wherein in biomarker CK7, CK19, EpCAM, E-cadherins and ABCG2 or the expression of four can not detect, or at 40%(or less) cells, at 30%(or less) cells, at 20%(or less) cells or at 10%(or less) cells.

Any combination of (colony of such as spheroid or single cell) above is also provided, wherein the per-cent of the cell of expressing K i67 is at least 30%, the per-cent of expressing the cell of CK19 is less than 40%, the per-cent of expressing the cell of NCAM is at least 80%, and the per-cent of expressing the cell of CK7 is less than 10%.

Also disclose from the liver neoplasm sample of all size (1mg is to some grams) herein and obtain the pure method through being separated HCC stem cell population.Tumor sample can be fresh or freezing, dissociated by machinery and/or enzymically treat, or directly cultivate when having bottom line mechanical debris.

Also open herein, non-adhesive substrate is any biocompatible materials with antibiont attachment characteristic or the coating with antibiont attachment characteristic (reducing cell gathering on wetted surface) be coated on common culture surface.Coating can use coating agent (as aminosilane) to apply.If there is non-adhesive or antibiont tack substrate, this substrate so can be used to continue about 0-25 days, as 0-21 days, 5-20 days, 5-10 days, 10-20 days, or any time section between zero and 25 days.

In another embodiment of method using adhesivity substrate, described adhesivity substrate can be rich in RGD(Arg-Gly-Asp) substrate (such as, collagen protein, gelatin, MATRIGEL) of three peptide motifs.Adhesivity substrate is configured to anchorage dependence cell adhesion and collects the surface of described cell.In addition, described substrate can be to be configured to this as fibroblastic anchorage dependence cell adhesion and to collect the adhesivity substrate of described cell.RGD peptide also can be grafted on polymerizability main chain, and described polymerizability main chain is as polystyrene, hyaluronan, poly(lactic acid) or its combination.Main chain can carry protein-polysaccharide further.Protein-polysaccharide can carry somatomedin, as fibroblast growth factor (FGF), Urogastron (EGF), activin A or follistatin.

Non-adhesive substrate can make culture fast and efficiently concentrating cancer stem cell.Non-adhesive substrate can use, can start immediately to make the purifying of HCC-CSC providing enough large sample (example is with the tumour of modus operandi excision) time.If sample minimum (as syringe needle aspirate or peritoneal lavage thing) and non-adhesive are cultivated infeasible, so adhesivity can be used to cultivate to primary amplification, then on non-adhesive substrate, carry out purification step, under adhesivity condition, carry out another amplification subsequently.In FIG (dotted line and square frame) hereafter describing substituting treatment process in detail.

Cultivate in embodiment at some, adhesivity substrate provides the first stage to cultivate, then on non-adhesive substrate, carries out subordinate phase cultivation.Also be provided on non-adhesive substrate and carry out first stage cultivation, then on adhesivity substrate, carry out subordinate phase cultivation.Stage can be such as half a day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days etc. or its any scope, as 2-4 days or 8-10 days etc.In addition, circulation can repeat, and as adhesivity is cultivated, then non-adhesive is cultivated, then adhesivity cultivation etc.In another embodiment, circulation can repeat, and as non-adhesive is cultivated, then adhesivity is cultivated, then non-adhesive cultivation etc.

In another embodiment, the somatomedin that the culture medium supplemented that composition is determined has at least one to work via mitogen-activated protein kinase (MAPK) path.In one embodiment, somatomedin is one or two or its analogue in FGF and EGF.In one embodiment, FGF is Prostatropin (bFGF).In another embodiment, the culture medium supplemented that composition is determined has activin A.In another embodiment, the substratum that composition is determined is not supplemented with activin A.The also open substratum determined with the composition effectively preventing the amount of HCC stem cell Spontaneous Differentiation to be supplemented with activin A agonist.

Also provide a kind of HCC stem cell line each patient to uniqueness obtained from patient's study of primary hepatic, described clone (a) carrying has the feature of autosynthesis and versatility and the stem cell of differentiation capability; And (b) in most cells (as more than 50%), carry unique gene group cancer mark.

Nucleic acid, gene product, polypeptide and peptide fragment are contained in the present invention, and wherein identity reasonably can be established separately through trivial name.Also contain based on the nucleic acid of specific gene storehouse accession number, gene product, polypeptide and peptide fragment, wherein said nucleic acid, polypeptide etc. have with the sequence iden of the nucleic acid, polypeptide etc. at least 50% of described gene pool number, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, the sequence iden of at least 99% or 100%, wherein biochemical function or physiologic function share at least in part or have nothing to do with function alternatively.

There is provided a kind of wherein with in composition disclosed herein in the immunoreactive method of experimenter's moderate stimulation to cancer.The immune response stimulated comprises CD4 ⁺t cell response, CD8 ⁺t cell response and B cell response in one or more.In certain embodiments, CD4 ⁺t cell response, CD ⁺t cell response or B cell are replied and can being analyzed by ELISPOT, being analyzed by intracellular cytokine dyeing (ICS), analyzed by the tetramer or measuring by producing according to assay method detectable antigens specific antibody known to persons of ordinary skill in the art.Immune response can comprise the survival time, and as 2 years total survival rates (OS), and wherein said 2 years total survival rates were at least 60%.Immune response in patient terminal used in also can being tested by oncological clinical is assessed, and described terminal comprises goal response (RECIST criterion), total survival rate, progresson free survival phase (PFS), without disease survival time, occurs time, 6 months PFS, 12 months PFS etc. that shift at a distance.

Herein also openly by the dendritic cell that HCC stem cell or the antigen that is derived from described HCC stem cell stimulate in vitro, it is in hepatocellular carcinoma therapy.Contain herein and comprise the immunogenic composition that in vitro load has the dendritic cell of (being exposed to) HCC-CSC, as vaccine composition.In certain embodiments, dendritic cell and tumour cell are from identical human experimenter, although dendritic cell and HCC cell are also in scope of the present invention from the embodiment of different experimenter.

Dendritic cell load can have HCC tumor-cell antigen, described antigen comprise the full cell of HCC tumour cell, cell lysate, cell extract, through the cell of overshoot or any protein derivatives.Dendritic cell vaccination Immunogenic Compositions can be prepared, and given to human experimenter by one or more route of administration as known to persons of ordinary skill in the art.

In certain embodiments, HCC-CSC cell with dendritic cell load before through overshoot or otherwise process to prevent cell fission.The replacement scheme of radiation comprises the fissional nucleic acid crosslinking agent of prevention.Also providing a kind of uses HCC stem cell population as disclosed to be used as the method for the antigenic source for autoimmunity therapy, such as wherein by radiant (such as, γ, UV, X), temperature (such as, hot or cold) or chemistry (such as, cell growth inhibition, aldehyde, alcohol) method or its combination make HCC stem cell inactivation.In other embodiments, HCC stem cell is used as the antigenic source of dendritic cell ex vivo activation.

The invention provides prepared HCC cell, provide load to have the DC of prepared HCC cell, and providing package is containing the immunogenic composition (or vaccine) of the dendritic cell of the HCC cell prepared by load.When not implicit any restriction, immunogenic composition of the present invention can comprise that load has the DC of HCC spherule, load has the DC of the cell colony comprising spherule, load has and is derived from spherule and the DC of the cell colony increased on adhesiveness surface before load is on DC, load has the DC of the spherule carrying out homogenization or sonication before load is on DC, load has the amplifying cells colony carrying out homogenization or sonication before load is on DC DC etc.In other embodiments, DC load has early stage HCC-CSC, mixing HCC-CSC or EMT-HCC-CSC.

A kind of HCC-CSC colony that can stimulate effective immune response for the cell of expressing at least one HCC specific antigens is also disclosed herein, wherein said HCC-CSC colony contacts with at least one dendritic cell, by in described dendritic cell body or ex vivo treatment, and wherein in described experimenter, there is effective immune response in response to described at least one dendritic cell to the administration of experimenter in wherein said HCC-CSC colony.

The immunostimulation amount of disclosed composition is that (but being not limited to) realizes following amount: make ELISPOT analytical results increase measurable amount, ICS analytical results is made to increase measurable amount, tetramer analytical results is made to increase measurable amount, antigen-specific CD4+T cell colony in blood is made to increase measurable amount, antigen-specific CD8+T cell colony in blood is made to increase measurable amount, or wherein increase at least 10% compared with suitable contrast time, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 doubly, 2.0 doubly, 3.0 times etc.Suitable contrast can be that the wherein non-load of dendritic cell has HCC cell or non-load to have the reference composition of the peptide being derived from HCC cell.

The present invention also provides medicine, reagent, test kit (comprising diagnostic kit), and wherein said medicine, reagent and test kit comprise dendritic cell (DC), antibody or antigen.Also be provided for the method for the composition comprising at least one dendritic cell and at least one antigen; The method formed for stimulating antibody; For stimulating the method for antibody dependent cellular cytotoxicity (ADCC); For stimulating the method for CDC; And for determining patient compliance, for determining patient selection under clinical trial or general medicine treatment situation or getting rid of criterion and for predicting method to the reaction of described medicine or reagent and test kit.Pharmaceutical composition of the present invention, reagent and methods involving contain CD83 Protein-Positive Dendritic Cells, and wherein CD83 is by inducing with through IFN-γ process or undressed cancer cells load.In in CD83 of the present invention, described CD83 is induced at least 2%, at least 3%, at least 4%, 6%, 7%, 8%, 9%, 10% etc.On the other hand, CD83 in wherein dendritic cell is got rid of not by the DC reagent that can induce with detecting with IFN-γ load or DC methods involving.

In one embodiment, provide a kind of test kit, it comprises all reagent for generating HCC-CSC spherule, early stage HCC-CSC, mixing HCC-CSC and/or EMT-HCC-CSC according to method cause tumor sample disclosed herein; And/or for characterizing the reagent of described HCC-CSC spherule, early stage HCC-CSC, mixing HCC-CSC and/or EMT-HCC-CSC; And for generating and/or characterize the specification sheets of described HCC-CSC spherule, early stage HCC-CSC, mixing HCC-CSC and/or EMT-HCC-CSC.In another embodiment, test kit additionally or alternatively comprises for separating of dendritic cell, for dendritic cell described in HCC-CSC load and/or reagent from DC-HCC composition to experimenter and specification sheets for giving.

tumor sample process

Hepatocellular carcinoma of the present invention (HCC) stem cell population can derive from the fresh or freezing sample of patient tumors.Tumor sample can be the biopsy or the lavation thing that contain tumor tissues.From irrigating solution, HCC stem cell is separated from pin biopsy neutralization.

Can be about 7.4(+/-0.6 at pH) universal buffering substratum in transport tumor sample, described universal buffering substratum is as RPMI, DMEM, F12, Williams or combination, and it contains protein source (as animals or humans serum) with the concentration of 0% to 100% or contain albumin or containing the macromole guaranteeing physiology osmotic pressure with the concentration of 0% to 0.5%.Natural or artificial macromolecular example is (but being not limited to) hyaluronan, dextran, polyvinyl alcohol.Microbiotic (as penicillin (penicillin), Streptomycin sulphate (streptomycin), gentamicin (gentramicyn)) and anti-mycotic agent can be used in the medium (as amphotericin B, FUNGIZONE (LifeTechnologies, Carlsbad, CA) optional combination provide antimicrobial property also to reduce the risk during transportation polluted.

Tumor sample can keep below metabolic activity state by substratum temperature is reduced to 2 DEG C to 30 DEG C, therefore allows the rate of surviving before treatment to continue the limited time (between 0 and 72 hour).Packaging (such as, Thermoinsulating packaging) can be used to guarantee, and correct temperature during transportation controls.

Subsequently by use sharp-pointed blade or tissue grinder apparatus to carry out fragment that solid tumor tissue to be processed into little (being less than 1mm in any dimension) by mechanical dissociation.

To dissociate further processing entities tissue optionally by enzymatic.Multiple enzyme may be used for being separated single cell.Can successfully use non-specific proteolysis enzyme, as trypsinase and stomach en-.The specific enzymes that can to use with MIN cell membrane damage in disclosed method be target, comprises Collagenase, Dispase, Proteinase, bone marrow serine or its combination.Deoxyribonuclease (DNAse) may be used for making the free DNA degradation from cell debris, and described cell debris is the reason causing the non-required viscosity of cell preparation.After dissociating, by washing out the cell in suspension from excessive enzyme and fragment via 50-100 μm of screen cloth leaching, and in buffer saline (PBS, HBSS) or cell culture medium repeated centrifugation.

cell culture condition and spherule produce

Single cell suspension as described above is transferred in the culture condition promoting the separation of stem cell, amplification and differentiation and/or Normocellular suppression.This is by physical condition, unanimously the realizing of chemical environment and manipulation.

Cell suspending liquid is exposed in non-adhesive (antibiont tack) substrate not allowing cellular attachment.Mature cell normally anchorage dependence, and to be eliminated fast when not providing correct adhesivity substrate.Antibiont tack substrate can adopt commerical prod, as ultralow adhesivity flask (Corning, Corning, NY); There is the polymkeric substance (polyvinyl, polyethylene, polypropylene, fluoropolymer) of native hydrophobic characteristic; Or there is the coating of natural carbohydrate polymers (as agar, starch etc.).

Cancer stem cell will be assembled and/or increase into the spherule form (Fig. 2) containing high purity cancer stem cell with clone.Show the cancer stem cell aggregate culture with the spherule structure that can easily identify of all size in figures 3 a and 3b.Mature cell is separated keeping and non-adhesive.Can usage variance gravity separation, by allow simply timing vertical sedimentation or in short-term low-force centrifugal (being less than 100 × G) select the larger spherule from single cell.Described system of selection is designed to below realization: (a) eliminates and be generally ripe Normocellular anchorage dependence cell; B () promotes the clonal expansion of the ripe little aggegation block of stem cell in the end of anchorage independence or spherule; C () promotes the local autocrine activity produced due to the clonal expansion of described stem cell; (d) eliminate and originated by the autocrine of the activin A of normal fibroblast or hepatocytes secrete.

Cell is that the cell surface proteins owing to being called as integrin (integrin) causes with forming the part ability of spheroid.The same preferendum integrin that cell surface is expressed guarantees that the cell of identical type " keeps together ".Spheroid is directly formed by the enzymic digestion thing as single cell suspension when cultivating and just starting, or can be formed by freezing sample or existing attachment culture at any time.The inoculation of enzymic digestion thing produces this spherical form being incorporated to the cell with particular surface property.

For example, inoblast is not incorporated in spherule, and removes from culture during gravity charging.Used medium lacks the molecule promoting to adhere to, to prevent from not having with the non-specific coalescent of the cell addicted to characteristic and to prevent from adhering to culture vessel surface.This kind of cell adhesion molecule (CAM) sees in animals or humans serum usually.Therefore, the culture media composition of serum-free is suitable for cultivating non-adhesive spherule.

In serum free medium is cultivated, the supplement of substratum can comprise support growth and maintain or cell physiological and function other needed for any hormone, nutrient, the minerals and vitamins of aspect needs.In some instances, can by adding or regulating the amount of the somatomedin (as FGF family and EGF) with mitogenic activity stimulate and maintain stem cells hyperplasia.

The spheroid (spherule) (comprising cancer stem cell spheroid) of cell can by means of fixing and with By Labeled Antibodies Staining, then carrying out the sign of biomarker expression aspect with confocal microscopy.Biomarker also can pass through other immuno-chemical method, and such as flow cytometry is measured.Spheroid can such as with the suspension obtained by fresh or with being suitable for preparing with the cell of adherent cell form growth.The form of spheroid (such as large and irregular contrast is small and fine and close) can be subject to the impact that substratum is selected.

In another embodiment, adhering to cell colony in antibiont tack coating can based on by Protein Kinase B (AKT) and focal adhesion kinases (focaladhesionkinase; FAK) abnormal activation of sound hedgehog (sonichedgehog) intracellular signaling that intracellular signaling mediates is separated.These phenomenons can by by as metalloprotease enzyme or for the enzyme (trypsinase/Collagenase) that dissociates induce film modifiedly to strengthen.This kind of cell colony may be associated with rapid multiplication and invasive tumour.Fig. 5 describes to be attached and the representative cell colony of the ultralow adhesiveness surface that increases.Method that is normal or abnormal activation for assessment of sound hedgehog intracellular signaling is that those of ordinary skill in the art can with known.

substratum used in cell cultures

The substratum determined for separating of the composition of HCC stem cell promotes cell survival rate, and through allocating for selection especially.Substratum is rich in carbohydrate and lipid, but has the protein (0.1%-3% albumin or 1%-5% serum) of minimum tolerance.It contains the total calcium being no more than 1.5mMol, not containing inorganic iron compound; On the contrary, iron is combined completely with the transporter of such as Transferrins,iron complexes.Substratum has excessive required and nonessential amino acid and required lipid (alpha-linolenic acid and linolic acid) (table 4).Optionally, substratum not containing activin A, and can contain activin A receptor blocking agent, as follistatin.In addition optionally, substratum does not contain antioxidant, as superoxide-dismutase (SOD) or catalase; But containing thiol antioxidants (thiolicantioxidant), as gsh.

As in table 2 describe, substratum based on basic components, as being supplemented with protein (in some formula), DMEM, F12, Williams, RPMI, Lebovitz of amino acid, antioxidant, high energy substrate (glucose, semi-lactosi, L-glutaminate), VITAMIN (B12), hormone (Triiodothyronine, Regular Insulin) and somatomedin (FGF, EGF).

Protein can be concentration is the albumin of 0.1%-0.5%, the foetal calf serum (FBS) of 0.5%-20%.Protein can be replaced by the macromole of concentration in 0.1% to 0.5% scope (as dextran, hyaluronan, polyvinyl alcohol).The composition of this kind of substratum is listed in table 2, table 3 and table 4.Supplement to be added in substratum and mixing for feeder cell culture.

table 2.for the basic medium composition option of cancer stem cell

table 3.lineage stem cells supplement (50mL unit is used for rehydration in 1L basic medium)

table 4.lipid mixt

Component	Concentration: μ g/mL
		Linolenic acid	10
Linolic acid	10
		Tocopherol acetate	50
Cholesterol	100

Lipid mixt is made by using PluronicF68, phosphatidylcholines, Tween80, cyclodextrin or its o/w emulsion combined.

Substratum can by one Wednesday sky schedule (such as, Monday-Wednesday-Friday) replace, if or amplification be fast, so more frequently, such as every other day or every day replace.Continuously feeding or micro-batch of charging bio-reactor can be used in the amplification phase.

Substratum contains the somatomedin worked via MAPK path, as FGF and EGF.The concentration of these somatomedins can between 0.1 to 100ng/mL, and about 10ng/mL changes usually.

In one embodiment, substratum is supplemented with FGF with about 0.1 to 100ng/mL, about 0.5-50ng/mL, about 1-40ng/mL, about 2-30ng/mL, about 3-20ng/mL, about 5-15ng/mL, about 6-14ng/mL, about 7-13ng/mL, about 8-12ng/mL, about 9-11ng/mL or about 10ng/mL.In other embodiments, FGF is present in substratum with about 5ng/mL, about 6ng/mL, about 7ng/mL, about 8ng/mL, about 9ng/mL, about 11ng/mL, about 12ng/mL, about 12ng/mL, about 14ng/mL or about 15ng/mL.

In another embodiment, substratum is supplemented with EGF with about 0.1 to 100ng/mL, about 0.5-50ng/mL, about 1-40ng/mL, about 2-30ng/mL, about 3-20ng/mL, about 5-15ng/mL, about 6-14ng/mL, about 7-13ng/mL, about 8-12ng/mL, about 9-11ng/mL or about 10ng/mL.In other embodiments, EGF is present in substratum with about 5ng/mL, about 6ng/mL, about 7ng/mL, about 8ng/mL, about 9ng/mL, about 11ng/mL, about 12ng/mL, about 12ng/mL, about 14ng/mL or about 15ng/mL.

One or two the substratum be not supplemented with in superoxide-dismutase (SOD) or catalase is also provided.Use antioxidant can have front or negative results.The tolerance of cancer stem cell to free radical and sugar decomposition metabolism is more much higher than normal cell.Therefore, in suboptimum substratum (high-concentration metabolite as in the replacement of high-density, infrequently substratum, substratum), first most probable eliminates normal sensitive cells.By not comprising antioxidant in the medium, can select probably to derive from cancer knurl, having more the cell colony of resistance than normal cell.Therefore, in certain embodiments, in substratum, add antioxidant (as catalase and SOD inhibitor), and in other embodiments, in described substratum, omit these compounds.

In an alternative method, activin/follistatin can be used to be separated pole early-stage cancer stem cell.The interpolation of activin A can select activin A resistance HCC stem cell subgroup.Be illustrated in Fig. 6,7 and 8 being exposed in activin A the early stage HCC stem cell colonies continuing to obtain after 7-14 days.This subgroup with have more rodent HCC form and comparatively early (less differentiation) cancer stem cell be associated.Follistatin is for blocking activin A acceptor and preventing the Spontaneous Differentiation of HCC stem cell, especially when there is cell (as inoblast and the normal liver cell) of a large amount of endogenous excretion activin A.If cell is insensitive or be in and can in the high purity HCC stem cell population of endogenous excretion follistatin, so use follistatin without effect to activin A.

Activin A is originally as the protein of transforming growth factor-beta (TGF-β) superfamily member.When interpolation or when comprising in the medium, activin contributes to maintaining stem cell versatility and autosynthesis.But activin A promotes the maturation and the differentiation that are easy to last mature hepatocytes and the cancer cells accepted.Therefore, initial target carrys out rapid amplifying tumour in vitro by producing correct autocrine environment in culture, and it also maintains the propagation of cancer stem cell.Although activin A can select the cancer stem cell subgroup that extremely end is ripe, in view of the extremely low concentration of hepatic cancer stem cell on the whole, amplification greatly will be postponed in early stage the applied this kind of condition of manufacture.For example, " rapid amplifying " causes having in culture vessel the substratum of apparent consumption sign (change of such as pH) and the amplification of cell number obvious every day higher (reflected by converging of increasing).

It for rapid amplifying, preferably omits and does not add activin A, because will slow down culture growth.For some application, pay close attention to and obtain extremely early stage stem cell population, and use activin A will select described cell colony.Therefore, in one embodiment, cause the amplification containing activin A and comprise the first composition through culturing cell activated by activin A to experimenter, then be separated the insensitive cell of activin A in without the culture of activin A, and comprise this second composition through culturing cell without activin A to described experimenter.

In one embodiment, substratum is supplemented with activin A with about 0.01 to 10ng/mL, about 0.05-9ng/mL, about 0.1-8ng/mL, about 0.5-7ng/mL, about 1-6ng/mL, about 1-5ng/mL.In other embodiments, activin A is present in substratum with about 0.5ng/mL, about 0.7ng/mL, about 0.9ng/mL, about 1ng/mL, about 1.25ng/mL, about 1.5ng/mL, about 1.75ng/mL, about 2ng/mL, about 2.25ng/mL, about 2.5ng/mL, about 2.75ng/mL, about 3ng/mL, about 3.5ng/mL, about 4ng/mL, about 4.5ng/mL, about 5ng/mL, about 6ng/mL, about 7ng/mL, about 8ng/mL, about 9ng/mL or about 10ng/mL.

Also open wherein culture medium supplemented has the embodiment of activin A antagonist, and described antagonist is as (but being not limited to) follistatin or the antibody with activin A specific binding.

In another embodiment, substratum is supplemented with follistatin with about 0.1 to 100ng/mL, about 0.5-50ng/mL, about 1-40ng/mL, about 2-30ng/mL, about 3-20ng/mL, about 5-15ng/mL, about 6-14ng/mL, about 7-13ng/mL, about 8-12ng/mL, about 9-11ng/mL or about 10ng/mL.In other embodiments, follistatin is present in substratum with about 5ng/mL, about 6ng/mL, about 7ng/mL, about 8ng/mL, about 9ng/mL, about 11ng/mL, about 12ng/mL, about 12ng/mL, about 14ng/mL or about 15ng/mL.

The increase that the combination of mitogen (such as, FGF/EGF), activin A and adhesivity substrate can cause normal cell (as inoblast or stellate cell) to breed.Therefore, be rich in environment or " matrix (stroma) " what be made up of the cell (such as, inoblast, stellate cell) with nourishing or parcel characteristic the condition producing and promote activin A insensitive HCC stem cell or progenitor cells amplification extremely in early days.Little by little observe in the process of cell cultures a few days to a few weeks subsequently, HCC colony is along matrix development and replace described matrix (Fig. 7 and 8) in space.For the combination that the substratum in this method is the formula described in table 2,3 and 4.

Relation is there is at FGF, EGF with between activin A and " extremely early stage HCC cancer stem cell ".FGF and EGF causes the HCC stem cell under any differentiation state (comprising extremely early stage HCC stem cell) propagation.When activin A is in cell culture medium, activin A is allowed (permission) exclusively breeds the insensitive extremely early stage HCC stem cell of activin A.If HCC stem cell becomes responsive, so propagation will be stopped by activin A or reduce.

Be not common to the insensitivity of FGF and EGF, and there is not natural blocker.Can be mediated by follistatin the insensitivity of activin A, described follistatin is the natural blocker of activin acceptor.Follistatin can by same tumour cell or by the emiocytosis around tumour.Activin A is usually by the emiocytosis around tumour, and therefore encircling cell (suppression) and described tumour (promoting amplification) are likely depended in the amplification of tumour.The acceptor (feature of extremely early stage undifferentiated carcinoma disease stem cell) lacking activin A can prevent surrounding tissue from controlling tumour.

In-vitro culture medium will containing embryonic stem cell sample colony.These colonies can by stroma cell around, described stroma cell can be normal fibroblast, the tumour cell that changes of the tumour cell of differentiation or mesenchymal cell.This kind of culture represents in the figure 7.

The invention provides the method for the preparation of HCC-CSC, wherein the total incubation time time needed for manipulation of centrifugal and sedimentation (comprise as changed substratum, inoculate) be less than five months, be less than four months, be less than three months, be less than two months, be less than one month, be less than 150 days, be less than 120 days, be less than 90 days, be less than 60 days, be less than 30 days or be less than 150 days (+/-20 days), be less than 120 days (+/-20 days), be less than 90 days (+/-20 days), be less than 60 days (+/-20 days), be less than 30 days (+/-20 days).In exclusiveness embodiment, the present invention can get rid of and meets following any method for the preparation of cancer stem cell and by any cancer stem cell colony prepared by described method, wherein manipulates the required time to be greater than one of time limit disclosed above (time-frame).Also provide by the time be in adherent cultures of one of time limit instruction above.This time be in non-adhesive culture as one of time limit above is also provided.In addition, the adherent cultures that is in identified by one of time limit is above provided to neutralize the assembly time be in non-adhesive culture.

epithelial cell is to the transformation (EMT) of mesenchymal cell

Knownly derive from epithelial tumor regression or be instead divided into mesenchymal cell state.Epithelial phenotype is immotile, contributes to being limited to the volume growth of tumour of tissue of rising, and usually more breaks up.When EMT occurs, cell obtains movability, and produces adjacent tissue infiltration and remote transfer.The cell changed also obtains stem-like cell phenotype, has copying and breaks up the ability to cause new tumour (transfer) in the host tissue with (primary) tumoral character that rises.By EMT, tumour cell obtains other immunosuppression capability, Teat pipette and radioresistance.

Culture media composition and physics system of selection promote external EMT phenomenon.The colony using EMT to change is prophylaxis of tumours recurrence as immunogenic advantage.The antigenicity of EMT cancer cells can make immunity system to identify and destroy the mobile cancer cells causing transfer.In the process of transfer, these cells move with pole low number, and inoculation host tissue, reverts to epithelial cell phenotype (MTE transformation), grow and form the new tumour with the feature similar with primary tumor.Cause the necessary condition of external EMT to be form spherule in serum free medium, stimulate with bFGF, be seeded in subsequently containing RGD(Arg-Gly-Asp) on the adhesivity substrate of peptide motif (such as, collagen protein, gelatin etc.).

EMT-HSC-CSC subgroup is obtained by cultivating HCC spherule or early stage HCC or mixing HCC under the culture condition described in such as table 1 and Fig. 1.

As used herein, term " HCC-CSC " generally can refer to HCC-CSC spherule, early stage HCC-CSC, mixing HCC-CSC or EMT-HCC-CSC.

hCC-CSC is obtained from little tumour source (pin biopsy)

When sample cell number is less, use the alternative method that HCC stem cell is selected.For example, after enzymatic dissociates, a small amount of viable cell obtained from tumour is less than 10 × 10 ⁶individual viable cell.For purposes of the present invention, contrast with the sample obtained from the tumour of excising (it is not considered as comparatively small sample and weight is usually at least 0.5 to 5-10 gram), refer to the sample obtained from such as pin biopsy or Core biopsy compared with small sample.Core biopsy 18 or 16 or 14 specification syringe needles carry out, and produce 5-50mg sample.The relatively new program being called as vacuum assisted biopsy also uses 11 specification syringe needles and vacuum auxiliary device (VAD) to carry out.11 gage probes and vacuum auxiliary device match and usually obtain each core sample of 94mg.14 specification syringe needles and vacuum aided obtain 37mg usually, but only obtain 17mg when matching with automatic biopsy gun.Depicted in figure 1 in this alternative method, before or after dissociating, the cell obtained from tumor sample is transferred to containing being rich in RGD(Arg-Gly-Asp) compound (such as, collagen protein, gelatin or MATRIGEL) adhesivity substrate in, and under being in the existence of selection described herein (serum-free) substratum.Described system of selection is designed to the local autocrine activity that (a) promotes to increase due to described stem cell clone with the amplification of the initial clones of the indivedual cancer stem cells existed compared with low number and (d) promotion.

Adhesivity substrate is the protein being rich in RGD, as collagen protein or gelatin.Substrate can build by described protein or peptide being attached on various material (as polystyrene polycarbonate, cyclic olefine copolymer or glass).RGD peptide can be grafted on polymerizability main chain (as hyaluronic acid, poly(lactic acid) and combination).This base polymer can use the carrier end of somatomedin (as proteoglycan) (such as, heparin sulfate, chondroitin sulfate, keratin sulfate etc.) to strengthen further.

Cell culture surface can directly use or use the coating agent as amino containing silane to be used.Coating has adhesivity characteristic (substrate) to cell and is applied to the compound on growth container material top.It can be natural compounds, as collagen protein or gelatin, and can by have mentioned group/end have more synthetic polymkeric substance to build.Coating agent (glue, as silane) may be used for the adhesivity improving coating and culture vessel material (such as glass).If silane contains required group or end group, so can be used alone.

Use this method and formulation, a large amount of cell can be obtained in the relatively short time period.From several milligrams, tissue samples is (as contained 10 ³to 10 ⁶the pin biopsy of individual cell) culture can increase to about 10 in 3-4 week ⁸individual cell.

HCC the amplification of stem cell culture and the generation of subgroup

As another feature of stem cell, HCC-CSC can Immortalization and amplification.Amplification cultivation on adhesivity substrate represents in the diagram.

In addition, HCC-CSC can partially or completely break up.If remove expansion of stem cells condition, so propagation can be slowed down or stop to HCC stem cell, and form is become the cell type more broken up with character mutation.Form can become flat, class epithelium or have the starlike of multinuclear, and it is the feature of more mature hepatocytes or stellate cell.

Adherent cultures can dissociate and be transferred in non-adhesive (antibiont tack) condition to remove anchorage dependence noble cells in single cell suspension.After 2-3 days, stem cell tends to assemble and increases into comparatively globule with cloning, and described spherule can be separated with single cell based on differential subsidence.Spherule can to inoculate in adhesivity condition and to breed further.If culture is occupied by noble cells or normal cell (as inoblast), so this method will make described culture stem cell content be purified to 90%-99% stem cell content from 1%-30%.Described method can repeat to recover the many number of times required for stem cell purity.

Compared with the size of globule generally between 0.1mm and 2mm.With regard to each cell number compared with globule, size distribution is generally between 10 cells and 10,000 cell.Can be spherical or avette compared with the shape of globule, and also can occur (Fig. 2 and 3) by agglomerated form that is spherical or oval structures.

Patient-specific HCC-CSC clone may be used for the genome mutation causing knurl sex reversal when being identified in compared with the healthy tissues from same patient.Genome mutation can not be expressed in each stage of differentiation.Some Function protein or transcription factor are only temporarily express, and can during maturing disappear, and generation is out of shape but is had the cell of normal protein matter.Identifying the cell colony of expressing sudden change to greatest extent and this colony be exposed in immunity system can be use cancer stem cell as the major advantage of immunotherapy antigenic source.

By identifying genome mutation, the individualized preparation for cancer therapy can be produced, such as small molecules, DNA sequence dna, sense-rna or combination.

This kind of clone can be further used for producing the sifting motion cultivation plate (such as 96 holes) for drug discovery.Multiple pedigrees from various patient can combine the variability solved between individuality in single culture plate.

Hepatocellular carcinoma cancer stem cell can keep rising some characteristics of tissue, as secretory protein, somatomedin and hormone (functioning tumour).In view of the immortal feature of clone, these characteristics can be adopted produce " self " protein (such as albumin, transforming growth factor (TGF), Regular Insulin, glycemic element (glucagon), DOPA etc.) that may be used for same patient.Cell can be incorporated in small-sized biological reactor, and collects secretory product, and purifying also stores to use same patient.This method is particularly advantageous, is the immune resistivity that patient will not develop as more traditional biological analogue.

loaded dendritic cell

The indivedual HCC-CSC clones obtained from patient may be used for producing the antigen for immunotherapy.The advantage of purified stem cell line is used to be good signal to noise ratio.More mature cells from tumour can have can cover antigenic compensatory mechanism, and can can't help immune system recognition.As antigenic source, HCC-CSC can use by the form of the form of the form of living, mitotic division inactive form, non-live or fragmentation.Various method may be used for modified cells for optimum antigen-exposed: radiant (such as, γ, UV, X), temperature (such as, hot or cold) or chemistry (such as, cell growth inhibition, aldehyde, alcohol) or combination.

In exemplary enforcement, reagent for dendritic cell activated (DC) and method are contained in the present invention, and it uses one or more immunological adjuvants, as toll sample acceptor (toll-likereceptor; TLR) agonist, such as CpG-oligonucleotide (TLR9), Imiquimod (TLR7), poly-(I:C) (TLR3), glucopyranosyl lipid A (TLR4), murein (TLR2), flagellin (TLR5); And adjuvant, such as, as CD40 agonist, CD40L; Or cytokine, interferon-γ, prostaglandin E2 etc.See such as United States Patent (USP) 7,993,659; US7,993,648; US7,935,804, the full content about activation DC disclosed in wherein each is incorporated herein by reference.The present invention is contained with the one or more ex vivo treatment DC in adjuvant reagent above, or additionally or alternatively, gives described adjuvant to human experimenter, animal subjects or veterinary science experimenter.

Immunity system contains cellular immunity, humoral immunity and complementary reaction.Cellular immunity comprises and relates to dendritic cell, CD8 ⁺t cell (cytotoxic T cell; Cytotoxic lymphocyte) and CD4 ⁺the cell of T cell (helper cell) and the network of event.Dendritic cell (DC) obtains polypeptide antigen, and wherein these antigens can from the outside acquisition of described DC or by the inner side biosynthesizing of infectious organisms at described DC.DC process polypeptide, produce length be about ten amino acid whose peptides, described peptide is transferred to form mixture in I class MHC or II class MHC, and described mixture is shuttled back and forth move to described DC surface on.When the DC with I class MHC/ peptide complex contacts CD8 ⁺during T cell, result is described CD8 ⁺the activation of T cell and propagation.About the effect of II class MHC, when the DC with II class MHC/ peptide complex contacts CD4 ⁺during T cell, result is described CD4 ⁺the activation of T cell and propagation.Although " can activate " described T cell to the dendritic cell of T cell antigen-presenting, the T cell of activation may not set up effective immune response.CD8 ⁺the effective immune response of T cell usually needs to stimulate DC in advance by one or more in multiple interaction.These interact to comprise and make CD4 ⁺t cell directly contacts DC(by means of making described CD4 ⁺the CD40L contact DCCD40 acceptor of T cell) or make one of TLR agonist toll sample acceptor (TLR) directly contacting dendritic cell.

Humoral immunity refers to B cell and antibody.B cell becomes and changes plasmocyte into, and described plasmocyte expression and secretion antibody.The difference of B progenitor cells (na veBcell) is its not presentation markup CD27, and antigen-specific b cells expresses CD27.Secreted antibody can subsequently with reside in the tumour antigen on tumor cell surface and be combined.Result is that the cell that infects or tumour cell become and be marked with antibody.When the cell of antibody and infection or tumour cell in conjunction with, in conjunction with antibody-mediated cell or the tumour cell killing infection, wherein killing is undertaken by NK cell.Although NK cell be not configured for T cell be configured to for identifying that the mode of target antigen carrys out identification specificity target antigen, the ability that NK cell is combined with antibody constant region makes NK cell can kill the cell being marked with antibody specifically.The Fc that the identification of NK cell antagonist is combined with antibody Fc portion by (in NK cell) is receptor-mediated.Such killing is called as antibody dependent cellular toxicity (ADCC).NK cell also can kill the cell not relying on ADCC mechanism, and wherein this killing needs the expression of I class MHC to lack in target cell or deficiency.

When not wishing by any specific mechanism constraint, the present invention is contained and is given cancer stem cell antigen or give the dendritic cell that load has cancer stem cell antigen, the generation of the one or more antibody in cancer stem cell antigen described in wherein said antigenic stimulation specific recognition, and wherein said antibody-mediated ADCC.Phrase load has antigen to refer to, and dendritic cell is caught viable cell, catches non-viable non-apoptotic cell, catches dead cell, catches polypeptide or is caught the ability of peptide etc.

The present invention is contained and is caught by cross presentation.Also contain the antigen presenting cell using not dendritic cell, as scavenger cell or B cell.

" delayed hypersensitivity " technology may be used for distinguishing the immune response relating generally to cellular immunity or relate generally to humoral immunity.The positive signal indicator cells reaction of delayed hypersensitivity.

The invention provides the composition and method that make tumour cell inactivation, it is such as undertaken by radiation, nucleic acid crosslinking agent, polypeptide linker or these combination.Crosslinked is that two of polymer molecule chains are attached by bridge, and described bridge is formed by being engaged the element of some carbon atom in described chain, group or compound by main chemical bond.Be cross-linked and occur in the material be made up of the polypeptide chain of the disulfide linkage joint by relating to two cysteine residues at occurring in nature; as in Keratin sulfate or Regular Insulin; trivalent pyridinoline and the pyrroles of ripe collagen protein are cross-linked; and crosslinked in clot, it relates to covalency ε-(γ-glutamyl) lysine cross-links of the γ-carboxyl-between amine groups and the epsilon-amino of lysine residue in glutamine residue.

Be cross-linked and can realize to manually in protein, by interpolation chemical substance (linking agent) or by carrying out high energy radiation to described polymkeric substance.The crosslinked enhancing immune response and complete Inhibit proliferaton with fixing agent and thermoinducible gathering are shown.May be used for making the protein cross on HCC-CSC surface and the material therefore preventing HCC-CSC from breeding includes, but is not limited to 10% neutral formalin damping fluid, 4% paraformaldehyde, 1% glutaraldehyde, 0.25-5mM suberoyl imidic acid dimethyl ester (dimethylsuberimidate), ice-cold 100% acetone or 100% methyl alcohol.In addition, 1% glutaraldehyde and the combination of 4% paraformaldehyde in 0.1M phosphate buffer soln can also be used.

Show that formaldehyde and glutaraldehyde both induce the activation of 1 type and 2 type t helper cells.Exactly, also show that thermoinducible antigen aggregation strengthens the interior initiation of body of cytotoxic T lymphocyte.Cause the combination of antigen and dendritic cell to increase by the antigen cross-linking of 3,3'-dithiobis (propionic acid sulfosuccinimide ester), and crosslinked antigen process via the proteasome path for antigen presentation.In addition, the fixing hepatocellular carcinoma tumor cell of formalin in clinical trial, and without propagation evidence.

In one embodiment, fix whole HCC-CSC with linking agent, and subsequently used as the antigenic source combined with dendritic cell.

In another embodiment, the nucleic acid of cell is made to be cross-linked.Exemplary core dialkylaminobenzoic acid agent is Beta-alanine, N-(acridine-9-base), 2-[two (2-chloroethyl) is amino] ethyl ester.Usually with ultraviolet (ultraviolet; UVA) Exemplary cross linking agents (as psoralene (psoralen)) of radiating composite has and DNA is cross-linked but the not modified ability of retaining protein.For example, nucleic acid target compound can be 4'-(4-amino-2-oxa-) butyl-4,5', 8-trimethylpsoralen (S-59).Can with 150 μMs of psoralene S-59 and 3J/cm ²uVA light (FX1019 radiation devices, BaxterFenwal, RoundLake, IL) makes cell inactivation.Use the inactivation of S-59 and UV light to be called as photochemical treatment, wherein treatment condition can be conditioned or titration one-tenth makes the DNA be cross-linked reach to prevent cell fission completely but wherein to the degree that the damage of polypeptide (comprising polypeptide antigen) minimizes.Can by cell suspension in the 5mL physiological saline containing 0,1,10,100 and 1000nM psoralene S-59.Sample can at roughly 2J/cm ²dosage under carry out UVA radiation.Each sample can be transferred in 15mL test tube subsequently, centrifugal and remove supernatant liquor, and use 5mL brine subsequently, centrifugal and remove supernatant liquor, and final precipitation is suspended in 0.5mL physiological saline.See United States Patent (USP) 7,833,775 and 7,691,393, the wherein disclosed full content about cell inactivation is incorporated herein by reference.

For any cell preparation with linking agent process, splitting ability can by those skilled in the art by cultivating or cultivate at least one week, at least two weeks, at least three weeks, at least surrounding, testing at least five weeks, at least two months, at least three months, at least four months etc. in standard medium.Cell fission can be assessed by disclosing karyomit(e) and disclosing the dyeing whether cell fission occur.Cell fission also can be measured by counting cell.Therefore, the cell number in culture plate keeps stable within the period of two weeks, one month or two months etc., the conclusion that cell cannot divide can reasonably be drawn.

In one embodiment, subcutaneous (SC) gives dendritic cell vaccination Immunogenic Compositions.In other embodiments, each dosage, in the scope of about 500 ten thousand-2 thousand ten thousand loading type DC, repeats with the form of a series of 6-10 dosage.In certain embodiments, every five days, weekly, every 10 days, give dosage week about or every two weeks, continue two, three, four, five or six dosage, then every two weeks, every three weeks, often surrounding, every month, every five weeks or every 6 weeks give dosage, continue the extra dose of two, three, four, five or six dosage, to reach 6-10 dosage altogether.In one embodiment, front four injections give weekly, continue one month, and within one month subsequently, once give rear 4 injections.In an alternative embodiment, administration continues 3 weeks weekly, within one month subsequently, once continues 5 months, to reach 8 administrations altogether.

Each dosage comprises about 5-20 × 10 ⁶individual loading type DC, about 5-17 × 10 ⁶individual loading type DC, about 6-16 × 10 ⁶individual loading type DC, about 7-15 × 10 ⁶individual loading type DC, about 7-14 × 10 ⁶individual loading type DC, about 8-13 × 10 ⁶individual loading type DC, about 8-12 × 10 ⁶individual loading type DC or about 9-11 × 10 ⁶individual loading type DC.In further embodiment, each dosage comprises about 8 × 10 ⁶individual loading type DC, about 9 × 10 ⁶individual loading type DC, about 10 × 10 ⁶individual loading type DC, about 11 × 10 ⁶individual loading type DC or about 12 × 10 ⁶individual loading type DC.The mixture of remaining HCC-CSC that loading type DC comprises DC and do not absorbed by described DC.The dosage given comprises the mixture of these cells, and described dosage reflects this mixture.

In another embodiment, loading type DC is given together with pharmaceutically acceptable carrier or vehicle.Pharmaceutically acceptable vehicle described herein (such as mediator, adjuvant, carrier or thinner) is that those skilled in the art knows and the public can easily obtain.Preferably, pharmaceutically acceptable carrier or vehicle are to the chemically inert carrier of loading type DC or vehicle, and are under conditions of use without carrier or the vehicle of harmful side effect or toxicity.

The selection of vehicle or carrier will partly decide by specific therapeutic composition and by the ad hoc approach being used for giving described composition.Preparation described herein is only exemplary, and not in any limiting sense.

On physiology, acceptable carrier is usually water-based pH buffered soln.On physiology, the example of acceptable carrier includes, but is not limited to physiological saline; Solvent; Dispersion medium; Cell culture medium; Aqueous buffer solution, as phosphoric acid salt, Citrate trianion and other organic acid; Antioxidant, comprises xitix; Lower molecular weight (being less than about 10 residues) polypeptide; Protein, as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, as polyvinylpyrrolidone; Amino acid, as glycine, glutamine, l-asparagine, arginine or Methionin; Monose, disaccharides and other carbohydrate, comprise glucose, seminose or dextrin; Sequestrant, as EDTA; Sugar alcohol, as mannitol or Sorbitol Powder; Form the relative ion of salt, as sodium; And/or nonionogenic tenside, as TWEEN, polyoxyethylene glycol (PEG) and PLURONICS.

In some example embodiments, give immunostimulant (boost) adjuvant (GM-CSF) with each dosage simultaneously.In certain embodiments, cell dosage is suspended in the carrier containing GM-CSF.In alternative exemplary embodiment, give GM-CSF immunity enhancement adjuvant, but give together with not single with each dosage.In other exemplary, there is not GM-CSF immunity enhancement adjuvant.

In the case of unrestricted, make dendritic cell (such as, autologous or allogeneic dendritic cells) and the cancer stem cell antigen contact in cell lysate, sour elutriant, cell extract, partially purified antigen, the antigen of purifying, the antigen be separated, partially purified peptide, the peptide of purifying, the peptide be separated, synthetic peptide or its any array configuration.Subsequently to experimenter, the experimenter such as suffering from HCC or the contrast experimenter without HCC give described dendritic cell.In an exemplary embodiment, make dendritic cell contact by one or more in following approach, inject or give: in subcutaneous, intraperitoneal, tubercle in (intranodal), intramuscular, intravenously, nose, suck, oral, by being applied to intestines inner chamber etc.In addition, immunogenic composition directly can be administered to the site of tumour or transfer.

Embodiment

embodiment 1

be separated by syringe needle aspiration biopsy and increased HCC cancer stem cell

Hepatocellular carcinoma (HCC) tumor sample is heterogeneous in histology, is made up of the cancer cells more or less broken up and normal essence, matrix and vascular cell.The object of method presented herein is separated and increases to be derived from the colony of the cancer stem cell of less HCC sample.In addition, these cells are for the preparation of the autotherapy being used for the treatment of HCC and prevention HCC recurrence.

Program and reagent are designed to maintain quintessential stem cells, and do not maintain differentiated hepatocellular, courage epithelial cell, vascular endothelial cell, smooth muscle cell or fibroblasticly in vitro retain/breed.Omit the typical component described in document, as reflunomide and serum; Replace the basal medium formulation with specified proportion amino acid and VITAMIN using and be supplemented with protein.

Have in the pathologic region that the patient of liver neoplasm, macroscopic view identifies from the diagnosis of informed consent and obtain syringe needle aspiration biopsy.Biopsy is transferred in unopened container in transport medium immediately, and delivers in organized processing facility under controlled temperature (4 DEG C-8 DEG C).Biopsy is made to dissociate 30 minutes in Collagenase IV solution (4mg/mL) subsequently.After centrifugation, gained cell suspending liquid is transferred to and is coated with in the polystyrene culture flask of 0.1% gelatin, and allow it to increase about 2-4 week.Within every two days, feed culture with fresh culture, described substratum by for stem cell and the basic components being supplemented with protein/growth factor mixture form.Formula is reproduced in table 2,3 and 4.In addition, in the nursing time, mixture comprises the Prostatropin (bFGF) of 10ng/mL and the EGF of 10ng/mL.

Firm cultivate establish after and when culture reach 60%-100% converge time, with proteolytic ferment (trypsinase, TrypLE), culture is dissociated, and to going down to posterity for 2-6 time subsequently, inoculates on larger surface with the ratio of 1:2 to 1:10.Subsequently cell is seeded in imaging chamber, and with to hepatocellular carcinoma, there is specific antibody staining.

Culture starts slow growth (Fig. 9) at first day, after about 14-21 days, identifies colony recombination structure (Figure 10).Enzymatic dissociates and to go down to posterity the individual layer (Figure 11) obtaining robust proliferation culture to these colonies.Dissociate at the period Collagenase IV that goes down to posterity and prevent from establishing monolayer culture thing.

After 2 to 6 times go down to posterity, with the transformation (EMT) mark (Slug/Snail, Twist) to mesenchymal cell of the cytokeratin (Ck19, Ck7) of immunocytochemistry mode analysis of cells system, tumour-specific markers (alpha fetal protein matter [AFP], ABCG2), adhesion molecule (EpCAM, NCAM, CD44), proliferation marker (Ki67) and epithelial cell.

As shown in the results, although hepatic cancer mark AFP express changeably (Figure 12 A-C and table 5), NCAM(also referred to as nerve cell adhesion molecule, CD56) existence with high proliferation index (as shown in by Ki67, table 5) see (Figure 13 A-C, table 5) in the sample of 100%.NCAM be have with addicted to the different adhesive molecules addicted to characteristic, it allows cell and cell adhesion and explains the wetting capacity of tumour.Previous research shows that the fresh from operated situation of only 8.3% is positive to NCAM.Therefore, culture condition causes the positive of HCC-CSC to select and enrichment.The strikingly disappearance of epithelial cell marker (EpCAM, cytokeratin), shows that the HCC cell of more phenotypic differentiation is not maintained by culture condition.The malignant disease that the existence instruction of NCAM in primary HCC increases and transitivity possibility.

table 5.immunity-cytochemistry from the tumor sample of pin biopsy characterizes

Score: cell positive %:0=is negative; 1=1%-25%; 2=26%-50%; 3=51%-75%; 4=76%-100%.The reactivity of classification on strength grade: 1=is slight; 2=moderate; 3=height.

Surface antigen CD44(hyaluronan acceptor) there is known association with tumour cell movability and transfer characteristics.In analyzed sample, most cells (more than 90%-100%) is positive (Figure 14 A-C) to CD44.

The existence of Slug/Snail and vimentin mark shows that the disappearance of epithelial cell marker (cytokeratin, EpCAM) is caused by the transition process of epithelial cell to mesenchymal cell, and during described process, tumour cell obtains tumor stem cell characteristic (CD44, NCAM) (Figure 15 A-D).

Serum-free culture based formulas, substrate and propagation method that this data display presents cause selection and/or the transformation of the cancer stem cell progenitor cell of the original mixed cancer colonies obtained in small pinhead biopsy.The amplification phase is accelerated after can going down to posterity at 1-2 time containing the amplification in blood serum medium (0.5%-5%) and breeding; But may cause inoblast or myoblastic non-required propagation with based on the substrate (gelatin, RGD peptide) of collagen protein and the combination of somatomedin (as FGF and EGF), described inoblast or sarcoplast may retain during selecting period.

embodiment 2

produce loading type dendritic cell composition

Antigenic source is the autologous tumor cell of the autosynthesis cell from the continuous propagation being derived from patient's fresh tumor tissue.These cells have the feature of tumor stem cell.Operation and pathology environment institute if having time in, strictly observe sterility scheme to dispose biopsy to guarantee that sample is aseptic.

Pathologist obtains flesh tissue from the biopsy of patient tumors.Use sterile scalpel and tweezers, sample cut into 10mm section and transfer in the transport test tube containing transport medium, process is to avoid sample dry rapidly.After excision within 48, by overnight courier, sample is transported in described manufacturing facility.

In manufacturing facility, in clean room, sample is dissociated into single cell suspension, and is placed in the cell culture condition being designed to make HCC-CSC enrichment and propagation.During process tumor sample, eliminate normal cell, as lymphocyte, stroma cell and reticular tissue.After completing amplification and purification step, made the propagation HCC-CSC(tumour cell of enrichment by radiation, TC) inactivation (apoptosis, it contributes to making antigen-exposed in antigen presenting cell), and be placed in gas phase liquid nitrogen storage tank.Depend on quantity and the quality of tumor sample, this process may need up to eight weeks.

Once tumour cell product is by quality-guarantee, patient is apprised of the program (usual six lift sequences) that will carry out being called as Leukapheresis.This process need filtering blood is to collect peripheral blood monocyte (PBMC).By overnight courier, collected PBMC being transported in manufacturing facility, being further purified with the adverse current density centrifugation by being called as elutriation.Elutriation is to make the process of the cell enrichment that can become antigen presenting cell or dendritic cell by other lymphocyte purifying monocytes.In order to generate dendritic cell, the monocyte of institute's elutriation is cultivated six days with cytokine GM-CSF together with interleukin 4 (IL-4).

At the 6th day, from keep in cold storage, shift out purified tumour cell product, thaw and combine 18-24 hour with dendritic cell.This process produces " antigen load " of DC.Final product is DC completely, maybe can be considered as allowing containing the TC(through overshoot of some remnants) and be called as DC-TC.Collect the dendritic cell/tumour cell mixture of combination, freezen protective to keep the survival rate of dendritic cell, and is stored in gas phase liquid nitrogen.

, under gas phase liquid nitrogen condition, described batch to be transported in treatment facility after discharging autogenous cell therapy product at finished goods matter check analysis.After arrival, under cell therapy product being stored in gas phase liquid nitrogen condition, until prepare administration.

embodiment 3

test with the active specific immunotherapy I phase using the autologous dendritic cell of the autologous tumor stem cell pulse through overshoot to carry out in the viral hepatitis type b positive patient suffering from hepatocellular carcinoma

Because micrometastatic disease can not detect when diagnosing, hepatocellular carcinoma is seldom cured by standard treatment.In addition, HCC usually with hepatitis B virus (hepatitisBvirus; HBV) infection is associated, and it makes patient not have liver transplantation qualification.Working load has the active specific immunotherapy (activespecificimmunotherapy of the autologous dendritic cell of the antigen from autologous tumor stem cell; ASI) be associated with long-term surviving result promising in the patient suffering from metastasis melanin tumor, although get rid of the patient suffering from HBV from those tests.This ASI method is worth assessing in other tumor type (comprising HCC).Excision is a part for the standard treatment for many patients, is provided for the tumour source generating autologous tumor cell system thus.Although the overt toxicity during ASI does not study with previous clinical (1 phase and 2 phase melanomas) is associated, in theory, ASI " can disperse " endogenous immune response for HBV, and therefore causes viral infection exacerbates.For this reason, in the patient suffering from HCC and HPB, be necessary to carry out I phase safety testing before consideration II phase efficacy test.

method:the qualified criterion of key of patient enrolment comprise previously untreated can carry out excision primary hepatocellular carcinoma (the independent pathology of >5cm or multiple>=3cm pathology), HBV medical history, be not liver transplantation candidate, Leukapheresis can be carried out, candidate carries out Liver Channel ductus arteriosus Chemoembolization (hepatictrans-catheterarterialchemoembolization; TACE), Qi-Pu Shi classification (Child-PughRating) is A, ECOG0-1 and enough blood counting, kidney and liver function (comprising albumin>=3.5mg/dL), and allows transaminase to raise at most 3 times.

The cell suspending liquid produced by collagenase digestion tumor sample 30-60 minute transfer to be supplemented with in ultralow adhesivity flask (Corning) in the table 2 of 10ng/mLbFGF and 10ng/mLEGF, 3 and 4 substratum and to maintain 14 days, to produce the clone of propagation HCC-CSC spherule.

Formed by 100 to roughly 10, the spherule of 000 cell composition, and carry out purifying when charging by otherness gravity.Spherule is transferred in the flask being coated with 0.1% gelatin subsequently, and allow it to adhere to and propagation.Again through the period in 3-4 week, to be dissociated by enzymatic and every 3-7 days is seeded on larger surface culture is increased gradually.In this stage, substratum is supplemented with 5% foetal calf serum (FBS) in addition.When finally gathering, in the radiator of cobalt-60 source, cell is exposed in 100Gy radiation.At each occurrence, radiation efficiency is confirmed with non-proliferative analysis.

Dendritic cell (DC) is derived from the peripheral blood monocyte (PBMC) obtained from the patient identical with tumor sample by Leukapheresis.Subsequently by HCC-CSC(TC) cultivate spend the night (18 to 24 hours) to produce DC/HCC-CSC composition together with DC, freezen protective is carried out to described composition.Meanwhile, patient accepts the TACE of a course for the treatment of, and brings into use DC/TC composition after 6-8 week.When treating, DC/TC composition being thawed and is suspended in 500 μ g rHuGM-CSF (granulocytemacrophagecolonystimulatingfactor; GM-CSF) to carry out subcutaneous injection weekly in, continue 3 weeks, and monitor patient toxicities, then continue 5 weeks.

Result: collect tumour from 18 patients, and establish clone by these 18 clinical samples.Phenotype through the HCC-CSC characterized is included in table 6.Various level and the amount of the abundant existence of HCC-CSC phenotype AFP and NCAM, hyperproliferative (Ki67) ability and other specific marker (EpCAM, CK19, CK7, ABCG2) define.In all research samples, confirm the existence of AFP, described AFP is the uniquely tagged being recommended in Clinical practice in liver malignant diseases at present at present.These data show that the positive of HCC-CSC is selected to be characterized by the existence of AFP and NCAM in the clone being derived from patient, and show that epithelial cell is to the transition process of mesenchymal cell when epithelial cell marker (EpCAM and cytokeratin) low expression.

table 6. the immunocytochemistry successfully producing the tumor sample of HCC-CSC clone characterizes

Eight patients treat with DC/TC composition, and all eight people accept 3 injections of plan.The patient treated comprises 1 male sex and 7 women.Median age is 56.5 years old, and scope is 37 to 73.When treating, the ECOG of all patients is 0.Injection all has well tolerable property, does not have significant acute toxicity.As by comprise complete quantitate virus that quantitative HBVDNA analyzes test prove there is not the increase of liver transaminase or hepatitis B virus mass formed by blood stasis.Be not recorded to toxicity that is serious or threat to life (rank 4 or 5).

Conclusion: use the treatment of the autologous DC/TC composition be separated from the HCC be associated with viral hepatitis type b not to be associated with the deterioration of viral hepatitis type b.

Except as otherwise noted, otherwise all numbers being expressed as component, character (as molecular weight), reaction conditions etc. used be in the specification and claims interpreted as all being modified by term " about " in all cases.As used herein, term " about " and " roughly " mean in 10% to 15%, preferably in 5% to 10%.Therefore, be contrary unless indicated, otherwise the numerical parameter of setting forth in specification sheets and appended claims may depend on attempt to be obtained by the present invention needed for character and the approximation changed.Minimally, and do not attempt restriction equivalent principle to the application of Claims scope, each numerical parameter should at least be explained by applying the generally technology of rounding up according to the number of reported significant figure.Although numerical range and the parameter of setting forth broad range of the present invention are approximations, the numerical value of setting forth in specific examples is as far as possible accurately report.But any numerical value is inherently containing some error that must be caused by the standard deviation found in other thermometrically value of its point.

Unless indicate in addition herein or obvious and contradicted by context, otherwise the term " (a/an) " that (when especially at following claims) is used under description situation of the present invention, " described " and similar indicator should be interpreted as containing odd number and plural number.Describing of value scope herein is only intended to serve as the shorthand method individually mentioning each independent value be in described scope.Unless instruction in addition herein, otherwise each indivedual value is incorporated in this specification sheets, as it is individually enumerated in this article.Unless instruction or in addition obvious and contradicted by context in addition herein, otherwise all methods described herein can be undertaken by any suitable order.The use of any and all examples provided herein or exemplary language (such as, " as "), only intends to illustrate the present invention better, and does not cause restriction to the otherwise required scope of the invention.Any language in this specification sheets should not be interpreted as indicating the key element of any failed call to be required for putting into practice for the present invention.

The grouping of substituting key element of the present invention disclosed herein or embodiment should not be construed as restriction.Each group member can individually or to mention and requirement with any array configuration of other member in described group or other key element of finding herein.Expection, the one or more members in group can be included in group or by group for the reason of convenience and/or patentability and delete.When any this kind of comprise or delete occur time, this specification sheets be considered to as with revising containing group, therefore meet the written description of all Ma Kuxi groups (Markushgroup) used in appended claims.

There is described herein certain embodiments of the present invention, comprise the present inventor known for carrying out optimal mode of the present invention.Certainly, after reading the above description, the change of embodiment described by these will become apparent for those of ordinary skill in the art.The present inventor expects that those skilled in the art adopt this kind of change in due course, and the present inventor intend be different from herein specifically described alternate manner to put into practice the present invention.Therefore, the present invention includes as by applicable law allow, in appended claims all modifications of theme that describes and equivalent.In addition, unless instruction herein in addition or in addition obviously and contradicted by context, otherwise the present invention contain key element as described above with its any combination of likely version.

Specific embodiments disclosed herein can use in detail in the claims by ... composition or substantially by ... composition language limits further.Time in for claims, and though as submit to or add according to amendment, transitional term " by ... composition " gets rid of unspecified any key element, step or composition in claims.The scope of claims to be limited to specified material or step and not to affect in fact those materials or the step of fundamental sum novel feature by transitional term " substantially by ... composition ".The embodiment of the present invention of requirement like this describes inherently or clearly and is implemented in herein.

In addition, at this specification sheets a large amount of referenced patent and printed publication in the whole text.Each mode individually quoted in full in reference quoted above and printed publication is incorporated herein.

Finally, embodiment of the present invention disclosed herein should be understood and principle of the present invention is described.Other amendment that can adopt is in scope of the present invention.Therefore, unrestricted as an example, alternate configuration of the present invention can be utilized according to content of teaching herein.Therefore, the invention is not restricted to as the content of accurately showing and describe.

Therefore, although shown and described and point out to be applied to the basic novel feature of the present invention of illustrative embodiments of the invention and/or its aspect, should understand those skilled in the art can carry out when not departing from the spirit of the present invention and/or claims its exemplary, disclosure and in form and details various omissions, reconfigure and replace and change.For example, intend to make to carry out identical function in fact with same way in fact to be clearly in scope of the present invention to all combinations of those key elements and/or method steps of realizing identical result.In addition, will be appreciated that the structure and/or key element that to be combined with any disclosed form or embodiment and to show and/or describe and/or method steps can as general design alternative situation to be incorporated in any form that other discloses or describe or advises or embodiment.Therefore, be intended that and do not limit the scope of the invention.All this kind of amendments are all intended to be in the scope of appended claims.

All open, the patent quoted in this specification sheets, patent application, reference and sequence table are all incorporated herein, as being set forth in completely herein in this mode quoted.

There is provided summary to meet 37CFR § 1.72 (b), with the character and the main points that allow reader to determine rapidly technology disclosure.Described summary is submitted to when having following understanding: it is explained not being used in or limits scope or the implication of claims.

Claims

1. an immunogenic composition, it comprises the dendritic cell by the tumour antigen ex vivo activation being derived from purified hepatocellular carcinoma (HCC) cancer stem cell (CSC) colony.

2. immunogenic composition according to claim 1, wherein said tumour antigen comprises the cell extract of described HCC-CSC.

3. immunogenic composition according to claim 1, wherein said tumour antigen comprises the lysate of described HCC-CSC.

4. immunogenic composition according to claim 1, wherein said tumour antigen comprises complete HCC-CSC.

5. immunogenic composition according to claim 4, wherein said intact cell is endowed non-proliferative.

6. immunogenic composition according to claim 5, wherein said intact cell is endowed non-proliferative by radiation.

7. immunogenic composition according to claim 4, wherein said intact cell is endowed non-proliferative by being exposed in core linking agent by described cell.

8. the immunogenic composition according to claim arbitrary in claim 1 to 7, it comprises pharmaceutically acceptable carrier and/or vehicle further.

9. the immunogenic composition according to claim arbitrary in claim 1 to 8, it comprises adjuvant further.

10. immunogenic composition according to claim 9, wherein said adjuvant is rHuGM-CSF.

11. immunogenic compositions according to claim arbitrary in claim 1 to 10, wherein said composition comprises by the dendritic cell that activates and HCC-CSC.

12. immunogenic compositions according to claim arbitrary in claim 1 to 11, wherein said HCC-CSC is HCC-CSC spherule form.

13. immunogenic compositions according to claim arbitrary in claim 1 to 11, wherein said HCC-CSC is early stage HCC-CSC form.

14. immunogenic compositions according to claim arbitrary in claim 1 to 11, wherein said HCC-CSC is in mixing HCC-CSC form.

15. immunogenic compositions according to claim arbitrary in claim 1 to 11, wherein said HCC-CSC is EMT-HCC-CSC form.

16. 1 kinds of methods for the treatment of hepatocellular carcinoma in experimenter in need, it comprises the immunogenic composition giving according to claim arbitrary in claim 1 to 15 to described experimenter.

17. methods according to claim 16, wherein said immunogenic composition gives with multiple dosage, and each dosage comprises about 5-20 × 10 ⁶individual cell.

18. methods according to claim 17, wherein said dosage comprises about 10 × 10 ⁶individual cell.

19. methods according to claim arbitrary in claim 16 to 18, wherein said dosage gives weekly once, continues 2-5 dosage, then monthly gives once, lasting 3-6 dosage.

20. methods according to claim arbitrary in claim 16 to 19, wherein said experimenter accepts the immunogenic composition of 6-10 dosage.

The purposes of 22. immunogenic compositions according to claim arbitrary in claim 1 to 15 in the medicine for the preparation for the treatment of hepatocellular carcinoma.

23. immunogenic compositions according to claim arbitrary in claim 1 to 15 are used for the treatment of the purposes of hepatocellular carcinoma.

24. 1 kinds of methods for the preparation of the colony of hepatocellular carcinoma (HCC) cancer stem cell (CSC), described method comprises:

Obtain HCC sample;

Make the cell dissociation of described sample, and

The cell dissociated described in vitro culture in the substratum that non-adhesive substrate is determined at composition, the substratum that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via mitogen-activated protein kinase (MAPK) path, forms HCC-CSC spherule thus;

In wherein said HCC-CSC spherule colony at least 80% the following biomarker of cell expressing in two or more: alpha fetal protein (AFP), EpCAM, Ov1 and OV6.

25. methods according to claim 24, in wherein said HCC-CSC spherule colony, to express in following biomarker further one or more for the cell of at least 80%: CK7, CK19 and E-cadherins.

26. methods according to claim 24, in wherein said HCC-CSC spherule colony at least 90% the following biomarker of cell expressing in two or more: AFP, EpCAM, Ov1 and OV6.

27. methods according to claim 24, it comprises further:

Adhesivity substrate in the substratum determined at composition cultivate described HCC-CSC spherule, the substratum that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of early stage HCC-CSC thus, in wherein said early stage HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more: Nanog, Sox2, Oct3/4 and c-kit.

28. methods according to claim 27, in wherein said early stage HCC-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: EpCAM, E-cadherins, Sox7, Sox17, Fox2A, Ov1, OV6, CD133 and CD90.

29. methods according to claim 27, in wherein said early stage HCC-CSC colony at least 90% the following biomarker of cell expressing in two or more: Nanog, Sox2, Oct3/4 and c-kit.

30. methods according to claim 24, it comprises further:

Adhesivity substrate in the substratum determined at composition cultivate described HCC-CSC spherule, the substratum that wherein said composition is determined contains serum and is supplemented with the somatomedin that at least one works via described MAPK path, thus formed mixing HCC-CSC colony, in wherein said mixing HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.

31. methods according to claim 30, in wherein said mixing HCC-CSC colony at least 90% the following biomarker of cell expressing in two or more: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.

32. methods according to claim 24, it comprises further:

Adhesivity substrate in the substratum determined at composition cultivate described HCC-CSC spherule, the substratum that wherein said composition is determined contains serum origin and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of (EMT)-HCC-CSC that embryo changes to mesenchymal cell thus, in wherein said EMT-HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more: NCAM, Slug/Snail and Twist.

33. methods according to claim 32, in wherein said EMT-HCC-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: AFP, N-cadherins, CD44 and vimentin.

34. methods according to claim 32, in wherein said EMT-HCC-CSC colony at least 90% the following biomarker of cell expressing in one or more: NCAM, Slug/Snail and Twist.

35. according to the method in claim 24,30 or 32 described in arbitrary claim, and it comprises further:

Adhesivity substrate in the substratum determined at composition cultivate described HCC-CSC spherule, described mixing HCC-CSC or EMT-HCC-CSC, the substratum that wherein said composition is determined is serum-free and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of early stage HCC-CSC thus, in wherein said early stage HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more: Nanog, Sox2, Oct3/4 and c-kit.

36. methods according to claim 35, in wherein said early stage HCC-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: CK7, CK19 and E-cadherins.

37. methods according to claim 35, in wherein said early stage HCC-CSC colony at least 90% the following biomarker of cell expressing in one or more: Nanog, Sox2, Oct3/4 and c-kit.

38. according to the method in claim 24,27 or 32 described in arbitrary claim, and it comprises further:

Adhesivity substrate in the substratum determined at composition cultivate described HCC-CSC spherule, described early stage HCC-CSC or EMT-HCC-CSC, the substratum that wherein said composition is determined contains serum origin and is supplemented with the somatomedin that at least one works via described MAPK path, thus formed mixing HCC-CSC colony, in wherein said mixing HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.

39. according to method according to claim 39, in wherein said mixing HCC-CSC colony at least 90% the following biomarker of cell expressing in two or more: AFP, CK7, CK19, EpCAM, E-cadherins, Nanog, FoxA2HNF4a and ABCG2.

40. according to the method in claim 24,27 or 30 described in arbitrary claim, and it comprises further:

Adhesivity substrate is cultivated in the substratum determined at composition described HCC-CSC spherule, described early stage HCC-CSC or mixing HCC-CSC, the substratum that wherein said composition is determined contains serum origin and is supplemented with the somatomedin that at least one works via described MAPK path, form the colony of EMT-HCC-CSC thus, in wherein said EMT-HCC-CSC colony at least 80% the following biomarker of cell expressing in two or more: NCAM, Slug/Snail and Twist.

41. methods according to claim 40, in wherein said EMT-HCC-CSC colony, to express in following biomarker further one or more for the cell of at least 80%: AFP, N-cadherins, CD44 and vimentin.

42. methods according to claim 40, in described EMT-HCC-CSC colony at least 90% the following biomarker of cell expressing in one or more: NCAM, Slug/Snail and Twist.

43. methods according to claim arbitrary in claim 24 to 42, the substratum that wherein said composition is determined is any substratum described in table 2.

44. methods according to claim arbitrary in claim 24 to 42, the substratum that wherein said composition is determined is any substratum of the combination coming from table 2 and table 3.

45. methods according to claim arbitrary in claim 24 to 42, the substratum that wherein said composition is determined is any substratum of the combination coming from table 2, table 3 and table 4.

46. methods according to claim arbitrary in claim 24 to 42, the substratum that wherein said composition is determined is any substratum of the combination coming from table 2 and table 4.

47. methods according to claim 24, wherein said somatomedin is one or more in fibroblast growth factor (FGF), Urogastron (EGF) or activin A.

48. methods according to claim 25, wherein said FGF is basic FGF (bFGF).

49. methods according to claim arbitrary in claim 24 to 48, the substratum that wherein said composition is determined is not supplemented with activin A.

50. methods according to claim arbitrary in claim 24 to 49, the substratum that wherein said composition is determined is with the agonist effectively preventing the amount of HCC stem cell Spontaneous Differentiation to be supplemented with activin A.

51. methods according to claim 27, wherein said activin A antagonist is follistatin or the antibody with activin A specific binding.

52. methods according to claim arbitrary in claim 24 to 51, wherein said substratum is not supplemented with antioxidant.

53. methods according to claim 52, wherein said antioxidant is superoxide-dismutase, catalase, gsh, putrescine or beta-mercaptoethanol.

54. methods according to claim arbitrary in claim 24 to 51, wherein said culture medium supplemented has gsh.

55. methods according to claim arbitrary in claim 24 to 54, wherein said adhesivity substrate is configured to anchorage dependence cell adhesion and collects described cell.

56. methods according to claim 55, wherein said anchorage dependence cell is inoblast.

57. methods according to claim arbitrary in claim 24 to 54, wherein said non-adhesive substrate is ultralow adhesivity polystyrene surface.

58. methods according to claim 57, wherein said adhesivity substrate comprises the surface being coated with the protein being rich in RGD tri-peptide motif.

The colony of 59. 1 kinds of purified HCC-CSC cells, it is prepared by the method according to claim arbitrary in claim 24 to 58.

60. colonies according to claim 59, wherein said purified HCC-CSC cell is HCC-CSC spherule.

61. colonies according to claim 59, wherein said purified HCC-CSC cell is early stage HCC-CSC.

62. colonies according to claim 59, wherein said purified HCC-CSC cell is mixing HCC-CSC.

63. colonies according to claim 59, wherein said purified HCC-CSC cell is EMT-HCC-CSC.

64. 1 kinds of HCC-CSC clones, it is prepared by the method according to claim arbitrary in claim 1 to 29.

65. HCC-CSC clones according to claim 64, wherein said HCC-CSC is HCC-CSC spherule.

66. HCC-CSC clones according to claim 64, wherein said HCC-CSC is early stage HCC-CSC.

67. HCC-CSC clones according to claim 64, wherein said HCC-CSC is mixing HCC-CSC.

68. HCC-CSC clones according to claim 64, wherein said HCC-CSC is EMT-HCC-CSC.

69. 1 kinds, in the immunoreactive method of experimenter's moderate stimulation in need for hepatocellular carcinoma, comprise the immunogenic composition from claim arbitrary in claim 1 to 15 to described experimenter, the HCC-CSC cell according to claim arbitrary in claim 59 to 63 or the HCC-CSC clone according to claim arbitrary in claim 64 to 68 that give according to.

The 70. HCC-CSC cells according to claim arbitrary in claim 59 to 63 or the purposes of the HCC-CSC clone according to claim arbitrary in claim 64 to 68 in the medicine for the preparation for the treatment of hepatocellular carcinoma.

The 71. HCC-CSC cells according to claim arbitrary in claim 59 to 63 or the HCC-CSC clone according to claim arbitrary in claim 64 to 68 are used for the treatment of the purposes of hepatocellular carcinoma.