KR20150139855A - Individualized high purity hepatocellular carcinoma stem cells, methods and use of the same - Google Patents

Abstract

The disclosure provides cancer stem cells that are used to stimulate an immune response against cancer, such as HCC. A method for preparing and purifying cancer stem cells is provided.

Description

[0001] INDUSTRIAL APPLICABILITY [0002] INDUSTRIAL APPLICABILITY [0001] The present invention relates to an isolated high purity hepatocellular carcinoma stem cell,

relation Cross-references to applications

This application claims the benefit of U.S. Provisional Patent Application No. 61 / 774,517, filed March 7, 2013, at 35 U.S.C. Claim under §119 (e). This application is also a continuation-in-part of International Application No. PCT / US2013 / 053850 filed on August 6, 2013, which is a continuation-in-part of U.S. Provisional Patent Application No. 61 / 683,477 filed on August 15, 2012, No. 61 / 718,643, which is hereby incorporated by reference in its entirety, Claim under §119 (e). The entire contents of all of which are incorporated herein by reference.

Field of disclosure

This disclosure relates to hepatocellular carcinoma stem cells, immunogenic compositions derived therefrom, and methods of manufacture and uses thereof.

In solid tumors, a small percentage of cells have the capacity to initiate tumors of the same histologic heterogeneity as the maternal tumor. These cells are termed cancer stem cells and are also known as tumor-initiated or cancer-initiated cells. Cancer stem cells can be defined by clusters of characteristics. First, it has capacity to update itself. Second, it can establish new tumors when implanted. Third, it can be characterized as a dormant or slowly cycling (cell cycle) tumor cell. Fourth, it may be responsible for the resistance of the tumor to chemotherapy or radiation therapy. Fifth, it relies on a unique microenvironment that renews and maintains its ability to cause more differentiated progenitor cells, and the environment maintains the undifferentiated state of cancer stem cells. Such microenvironment may include mesenchymal stem cells, tissue-associated fibroblasts, and endothelial cells. The ability to form spheroids by in vitro culture is another feature that can contribute to the identification of specific cells with cancer stem cells. One non-limiting definition of cancer stem cells is a cell that can regenerate the entire heterogeneity of the maternal tumor and grow continuously after multiple transitions.

A specific cancer stem cell population can be a source of neoplasms and a source of recurrence of cancer that has been cured. In addition, a subset of cancer stem cells within the tissue can produce cells that can restart the growth cycle and reestablish the tumor when exposed to a signal. Cancer stem cell crevices are dormant until appropriate signal transduction triggers re-entry in the proliferation cycle. The re-entry signal may originate from local events such as trauma, cellular damage, microbial attack (viruses, bacteria or fungi), or may be mediated by local growth factors, cytokines or intercellular communication. In addition, hormones can regulate stem cells in tissue-specific interstitial spaces. Defects or mutations in stem cell crevices can cause small changes in the function. Neoplasms can result from such small changes, including random mutations that affect the control over the cell cycle. Mutations that cause cancer vary from person to person. Such variability is observed not only between different types of cancer, such as between liver and melanoma, but among people suffering from one type of cancer, such as one breast cancer patient versus another breast cancer patient.

The therapies being developed for cancer include approaches involving autologous immune responses. These approaches include the use of newly harvested autologous tumor cells that are degraded and formulated into vaccine vectors. Another approach is a dendritic cell vaccine, in which dendritic cells are pulsed with autologous tumor lysates. Another approach is an in vitro modification of the tumor cells of a patient with a galactose polymer and is to inject the modified tumor cells back into the patient and the galactose-tagged tumor cells are more easily occupied by the antigen-presenting cells (APC) Thereby increasing the antitumor response. The drawbacks of these approaches involving autologous tissue immunotherapy are low antigenic signal to noise ratios when bulk tumors, or bulk tumor antigens, are used as immunostimulants. The majority of tumor cells are significantly differentiated and mixed with normal cells such as vascular components, connective tissue, differentiated tumor cells, non-viable and ganglionic cells, and normal host tissues. Cancer stem cells merely represent small fractions of tumor bulk, sometimes up to 4%, and most typically less than 1% in more aggressive tumors. Thus, when bulk tumors are used as an antigen source, the immune response is directed against more differentiated cells to allow stem cells to avoid attacks and the possibility of causing tumor recurrence or metastasis.

Another new therapy is a single target antibody against a normal antigen that is expressed more abundantly in cancer. Specific antigen-targeted therapies (e.g., anti-CD133, anti-EpCAM, anti-CD44, anti-CD13, etc.) are non-differentiated and affect normal cells with cancer cells, leading to enormous adverse effects on patients .

summary

Immunogenic compositions comprising HCC-CSC-loaded dendritic cells during the treatment of hepatocellular carcinoma (HCC) cancer stem cells (CSC), HCC-CSC cell line, and hepatocellular carcinoma are disclosed herein.

Specifically, a method for producing a population of hepatocellular carcinoma (HCC) cancer stem cells (CSC) is provided herein, the method comprising: obtaining a sample of HCC; Dissociating the cells of the sample, and culturing the dissociated cells in vitro in a restricted medium on a non-adherent substrate, wherein the restricted medium is a serum-free and mitogen-activated protein kinase (MAPK) pathway Lt; RTI ID = 0.0 > HCC-CSC < / RTI > At least about 80% of the cells in the HCC-CSC sphereoid population express two or more of the biomarker alpha-fetoprotein (AFP), EpCAM, Ov1, and OV6. In another embodiment, at least about 80% of the cells in the HCC-CSC sphere population further express one or more of the biomarkers CK7, CK19, and E-carderine. In another embodiment, at least about 90% of the cells in the HCC-CSC spheroid population express more than one of the biomarkers AFP, EpCAM, Ov1, and OV6.

In another embodiment, the method further comprises culturing the HCC-CSC spleod in a restricted medium on an adherent substrate, wherein the limiting medium is serum-free and comprises at least one growth factor acting through the MAPK pathway Thereby forming a population of early HCC-CSCs and at least about 80% of the cells in the initial HCC-CSC population express more than one of the biomarkers Nanog, Sox2, Oct3 / 4, and c-kit. In another embodiment, at least about 80% of the cells in the initial HCC-CSC population have at least one of the biomarkers EpCAM, E-carderine, Sox7, Sox17, Fox2A, Ov1, OV6, CD133, Lt; / RTI > In another embodiment, at least about 90% of the cells in the initial HCC-CSC population express more than one of the biomarkers Nanog, Sox2, Oct3 / 4, and c-kit.

In another embodiment, the method further comprises the step of culturing HCC-CSC spheroids in a restricted medium on an adherent substrate, wherein the restricted medium comprises at least one growth factor containing serum and acting through the MAPK pathway CSC, and at least about 80% of the cells in the mixed HCC-CSC population are biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Nanog, FoxA2 HNF4a, And ABCG2. In another embodiment, at least about 90% of the cells in the mixed HCC-CSC population express more than one of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Nanog, FoxA2 HNF4a, and ABCG2.

In another embodiment, the method further comprises the step of culturing HCC-CSC spheroids in a limited medium on an adherent substrate, wherein the restricted medium comprises at least one growth medium containing a serum source and acting through the MAPK pathway (EMT) -HCC-CSC population and at least about 80% of the cells in the EMT-HCC-CSC population are biomarker NCAM, Slug / Snail ), And a twist (Twist). In another embodiment, at least about 80% of the cells in the EMT-HCC-CSC population further express one or more of the biomarkers AFP, N-carderine, CD44, and vimentin. In another embodiment, at least about 90% of the cells in the EMT-HCC-CSC population express one or more of the biomarker NCAM, slug / snail, and twist.

In another embodiment, the method further comprises culturing HCC-CSC spheroid, mixed HCC-CSC, or EMT-HCC-CSC in a restricted medium on an adherent substrate, CSC, and at least about 80% of the cells in the initial HCC-CSC population are biomarkers Nanog, Sox2, Oct3 / 4, And < RTI ID = 0.0 > c-kit. ≪ / RTI > In another embodiment, at least about 80% of the cells in the initial HCC-CSC population express one or more of the biomarkers CK7, CK19, and E-carderine. In another embodiment, at least about 90% of the cells in the initial HCC-CSC population express one or more of the biomarkers Nanog, Sox2, Oct3 / 4, and c-kit.

In another embodiment, the method further comprises culturing HCC-CSC spleod, early HCC-CSC, or EMT-HCC-CSC in a restricted medium on an adherent substrate, wherein the restricted medium comprises a serum source CSC, and at least about 80% of the cells in the mixed HCC-CSC population are biomarkers AFP, CK7, CK19, < RTI ID = 0.0 > , EpCAM, E-carderine, Nanog, FoxA2 HNF4a, and ABCG2. In another embodiment, at least about 90% of the cells in the mixed HCC-CSC population express more than one of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Nanog, FoxA2 HNF4a, and ABCG2.

In another embodiment, the method further comprises culturing HCC-CSC spleoid, initial HCC-CSC, or mixed HCC-CSC in a restricted medium on an adherent substrate, wherein the restricted medium comprises a serum source At least about 80% of the cells in the EMT-HCC-CSC population are biomarker NCAM, slug / mouse, Nail, and twist. In another embodiment, at least about 80% of the cells in the EMT-HCC-CSC population further express one or more of the biomarkers AFP, N-carderine, CD44, and vimentin. In another embodiment, at least about 90% of the cells in the EMT-HCC-CSC population express one or more of the biomarker NCAM, slug / snail, and twist.

In one embodiment, the restriction media may be any medium from any of the media set forth in Table 2, any medium from the combination of Table 2 and Table 3, combinations of Table 2, Table 3, and Table 4, Lt; RTI ID = 0.0 > 4 < / RTI >

In one embodiment, the growth factor is at least one of fibroblast growth factor (FGF), epithelial growth factor (EGF), or activin A. In another embodiment, the FGF is a basic FGF (bFGF). In another embodiment, the restriction media are not enriched with actinB. In another embodiment, the restriction medium is supplemented with an agonist of Actibin A in an amount effective to prevent spontaneous differentiation of HCC stem cells. In another embodiment, the antagonist of actin A is a pol statin or an antibody that specifically binds to actin A.

In another embodiment, the medium is not supplemented with an antioxidant. In another embodiment, the antioxidant is superoxide dismutase, catalase, glutathione, putrescine, or beta-mercaptoethanol. In another embodiment, the restriction medium is supplemented with glutathione.

In another embodiment, the adherent substrate is configured to adhere to an adhesion-dependent cell, such as fibroblasts, and collect it. In another embodiment, the non-adherent substrate is an ultra-low adherent polystyrene surface. In another embodiment, the adherent substrate comprises a surface coated with a protein rich in RGD tripeptide motif.

Also provided herein is a population of purified HCC-CSC cells produced by any of the methods disclosed herein. In certain embodiments, the purified HCC-CSC cells are HCC-CSC spleoid, early HCC-CSC, mixed HCC-CSC, or EMT-HCC-CSC.

Also provided herein are HCC-CSC cell lines produced by methods for any of the methods disclosed herein. In certain embodiments, the purified HCC-CSC cells are HCC-CSC spleoid, early HCC-CSC, mixed HCC-CSC, or EMT-HCC-CSC.

In one embodiment, provided herein are immunogenic compositions comprising dendritic cells activated in vitro by a tumor antigen derived from purified HCC-CSC cells or HCC-CSC cell lines as disclosed herein. In another embodiment, the tumor antigen comprises cell extracts of purified HCC-CSC cells or HCC-CSC cell lines. In another embodiment, the tumor antigen comprises a purified HCC-CSC cell or a lysate of a HCC-CSC cell line. In another embodiment, the tumor antigen comprises intact, purified HCC-CSC cells or intact cells from an HCC-CSC cell line.

In another embodiment, intact HCC-CSC cells are provided non-proliferative. In another embodiment, the intact cell is provided non-proliferatively by irradiation. In another embodiment, the intact cell is provided non-proliferative by exposure of the cell to a nuclear crosslinking agent.

In another embodiment, the immunogenic composition further comprises a pharmaceutically acceptable carrier and / or excipient. In another embodiment, the immunogenic composition additionally comprises adjuvants. In another embodiment, the adjuvant is a granulocyte macrophage colony stimulating factor.

In another embodiment, the immunogenic composition comprises dendritic cells and HCC-CSC cells. In another embodiment, the purified HCC-CSC cell or HCC-CSC cell line is in the form of HCC-CSC spleoid, early HCC-CSC, mixed HCC-CSC, or EMT-HCC-CSC.

Also provided is a method of treating hepatocellular carcinoma in a subject in need thereof, the method comprising administering to the subject an immunogenic composition as disclosed herein. In one embodiment, the immunogenic composition is administered in a plurality of doses, each dose containing about 5-20 x ¹⁰ cells. In another embodiment, the dose comprises about 10 x ¹⁰ cells. In another embodiment, the dose is administered weekly for 2-5 doses, then monthly for the next 3-6 doses. In another embodiment, the subject receives from 6-10 doses of the immunogenic composition.

Also provided is a method of stimulating an immune response in a subject in need thereof for hepatocellular carcinoma, the method comprising administering to the subject an immunogenic composition as described herein, HCC-CSC disclosed herein, or HCC-CSC cell line disclosed herein .

Also provided is the use of the immunogenic compositions disclosed herein, the HCC-CSCs disclosed herein, or the HCC-CSC cell lines disclosed herein during the treatment of hepatocellular carcinoma.

Also provided is the use of the immunogenic compositions disclosed herein, the HCC-CSCs disclosed herein, or the HCC-CSC cell lines disclosed herein during the treatment of hepatocellular carcinoma.

Figure 1 is a flow chart of a method for isolating proliferation and harvesting of hepatocellular carcinoma (HCC) stem cells (HCC-CSC) from resected tumors (solid boxes and arrows) or from small samples such as needle biopsies (boxes and arrows with dashes) Fig. After the occurrence of spleens, the pathway that creates the HCC-CSC subpopulation is a common pathway.
Figure 2 is a schematic depiction of steroids.
Figures 3A and 3B illustrate spoils of various shapes and sizes.
Figure 4 shows HCC stem cells in the proliferation step.
Figure 5 shows cells attached to an ultra-low adhesion substrate.
Figure 6 shows HCC stem cells that are non-sensitive to actin A; These cells grow in adherent colonies and are surrounded by normal host tissues (fibroblasts). More differentiated tumor cells are removed by the presence of actin A.
Figure 7 depicts HCC stem cells selected by exposure to actin A growing in adherent clonus characteristic of stem cells.
Figure 8 shows a concentrated HCC culture with embryonic stem cell-like colonies containing small, self-renewing cells.
Figure 9 shows cells resulting from dissociation by enzyme of needle biopsy from HCC tumors (phase contrast 10x) for 7 days after a flat culture on an adherent substrate.
Figure 10 shows a typical dense colony formation 14-21 days after growth of the cells of Figure 9 (phase contrast 10x).
Figure 11 shows an established, highly proliferating HCC stem cell line after multiple transitions (phase contrast 10x).
Figure 12a shows HCC cell line staining positive (surface fluorescence, 20x) against alpha-fetoprotein (AFP) with nuclear counterstaining (bisbenzimide), confirming HCC identity. Figure 12b shows the cells of Figure 12a in a red channel image for AFP staining. Figure 12C shows a blue channel image for bisbenzimide.
Figure 13a shows cells with a high percentage of neuronal cell adhesion (NCAM) staining, confirming early cancer cell phenotype selection in established cultures (including nuclear control staining, bisbenzimide) (surface fluorescence, 40x). Figure 13b shows the cells of Figure 13a in a red channel image for NCAM staining. 13C shows a blue channel image for bisbenzimide.
14A shows HCC cell line labeled positive for a marker of CD44, invasive potential, specific for cancer stem cells with high metastatic potential. Cells are also stained with nuclear counterstaining, bisbenzimide (surface fluorescence, 40x). Figure 14b shows the cells of Figure 14a in the green channel image for CD44 staining. 13C shows a blue channel image for bisbenzimide.
Figure 15a shows the epithelial-mesenchymal transcription marker vimentin and slug / snail in the EMT-HCC-CSC cell line, in a bisbenzimide nuclear counterstaining stain. Figure 15b shows the cells of Figure 15a in a red channel image for slug / snail staining. Figure 15c shows the cells of Figure 15a in a green channel image for visentin. 15D shows a blue channel image for bisbenzimide.

The present disclosure provides a population of cells obtained from human hepatocellular carcinoma (HCC) tumors consisting predominantly of high purity cancer stem cells. In an embodiment, the purity of the cell population 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 cells. These cancer stem cells are hepatocellular carcinoma ancestral cells and have some level of capacity for continuous self-renewal and differentiation. The disclosure also relates to a method for producing a purified population of HCC-derived stem cells for further use as an antigen source for autologous tissue immunotherapy of cancer.

Examples of testing and screening implementations are also included. This disclosure uses a high purity HCC stem cell population for genetic analysis to identify unique changes that provide a personalized drug formulation. The present disclosure provides novel cell lines that are modified in vitro , and these modifications enhance the immunostimulatory properties of HCC. HCC cell lines are an improvement to similar techniques using crude tumor preparations, as it provides excellent antigenic signal-to-noise ratios. The cell line is free of contaminating cell populations, such as fibroblasts, which can alter or reduce in vitro application. Exemplary cell lines of this disclosure are also used for the manufacture of a medicament for treating HCC.

As used herein, the term " derived from " in the context of a peptide derived from one or more cancer cells includes any method of obtaining a peptide from a population of cancer cells or cancer cells. Cancer cells can be destroyed, for example, by homogenization or osmotic rupture, resulting in crude extracts. The peptides, oligopeptides, and polypeptides of the crude extract may be exposed to dendritic cells, which in turn may lead to the processing of the peptide by dendritic cells. The term " derived from " also encompasses intact cancer cells, wherein the cancer cells are alive, the cancer cells have been treated with irradiation but are still metabolically active, or the cancer cells have been treated with a nucleic acid cross- And thus still includes peptides. Derived from cancer cell debris also includes a mixture of cancer cell debris, cancer-free cell protein, and irradiated cancer cells, which are then derived from cancer cells.

"Administration" means that the subject, cell, tissue or organism, as applied to a human, mammal, mammalian subject, animal, somatic subject, placebo subject, Refers to the contact of an exogenous ligand, reagent, placebo, small molecule, drug, therapeutic agent, diagnostic agent, or composition to an organ, organ, or biological fluid. "Administration" can refer, for example, to therapeutic agents, pharmacodynamics, diagnosis, investigation, placebo, and experimental methods. Administration may refer to in vivo treatment of human or animal subjects. Treatment of the cells involves contact of the reagents with the cells, as well as contact of the reagents with the fluid, and the fluid contacts the cells. "Administration" also includes in vitro and in vitro treatment of cells, for example, by reagents, diagnostics, binding compositions, or by another cell.

An "effective amount" includes, but is not limited to, an amount that improves, reverses, alleviates, prevents, or diagnoses at least one symptom or indication of a medical condition or disorder. &Quot; Effective amount " is not limited to an optimal amount sufficient to limit the minimum amount sufficient to achieve the desired result and achieve the desired result, unless explicitly or otherwise indicated by context.

The severity of the disease or disorder may be measured by a biomarker or by clinical parameters without implying any limitation, as well as the ability of the treatment to achieve, prevent, cure, or curtail the disease or disorder . Biomarkers include blood levels, serum, urine, or metabolite levels in CSF, tumor cell levels, cancer stem cell levels, and tumor levels. Tumor levels can be determined by the Response Evaluation Criteria In Solid Tumors (RECIST) criteria (Eisenhauer et al. (2009) Eur. J. Cancer. 45: 228-247). Expression markers include genetic expression of mRNA or gene amplification, expression of the antigen, and expression of the polypeptide. Clinical parameters can be assessed without regard to any limitations, such as progression-free survival (PFS), 6-month PFS, disease free survival (DFS), time to progression (TTP), time to distant metastasis . ≪ / RTI >

A "labeled " composition is detectable, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, isotopic, or chemical methods. For example, useful labels include, for example, enzyme- , or fluoro leg comprises ^{a, 32 P, 33 P, 35} S, 14 C, 3 H, 125 I, stable isotopes, an epitope tag fluorescent dye, electron dense reagent, substrate, or enzyme, as used in (fluorettes) (As disclosed in U.S. Patent No. 6,747,135, which is hereby incorporated by reference for all that it discloses regarding fluoret).

Thus, a method of making a single cell preparation of cancer stem cells, a population of purified spoloids, or a steroid, is disclosed herein, which method comprises obtaining a biopsy of HCC, dissociating the cells of the biopsy step comprising the step of culturing in vitro a cell dissociation in limiting medium on the substrate, the limiting medium is the mitogen activated to yield a single cell preparation of the group of the purified spheroid, or HCC stem cell protein kinase ( ≪ / RTI > MAPK) pathway. At least 80% of the population of refolded spheroids or cancer stem cells in a single cell contains the biomarker ATP-binding cassette and G member 2 (ABCG2; gene bank accession number AAG52982.1), alpha-fetoprotein (AFP) (EpCAM; gene bank accession number NP_002345.2), a fork head box A2 (FoxA2), and a flank head box A2 (FoxA2) , Nuclear Factor 4 alpha (HNF4a), Ki-67, Nanog (gene bank accession numbers NM_024865.2 and NP_079141.20), N-carderine, NCAM (CD56), Oct3 / Ov1, OV6, Slug (SNAI2) / Snail (Slug / Snail), Sox17, Sox2 (Gene Bank Accession Number NM_003106. 3, NP_003097.1), Sox7, twisted, and visentin. A flow chart of the formation of the disclosed cell population is shown in Fig.

As used herein, the term " spheroid " refers to spherical agglomerates of cancer stem cells formed by the orientation of cancer cells in a serum-free medium. The ability to form spheroids is a hallmark of cancer stem cells.

In certain embodiments, at least about 80% of the cells in the HCC-CSC spoloid population express two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Ov1, and OV6. In another embodiment, at least 80% of the cells in the HCC-CSC sphere population express two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Ov1, and OV6. In another embodiment, at least 80% of the cells in the HCC-CSC spoloid population express two or more, or all, of the biomarkers AFP, EpCAM, Ov1, and OV6.

Also provided is a population of purified spleoids comprising cancer stem cells wherein at least 80% of the cancer stem cells in the population of purified spleids express one or more of the biomarkers: ABCG2, AFP, CD133, NK carderine, NCAM (CD56), Oct3 / 4, Ov1, OV6, Slug / Snail, Epstein-Barr virus (CD44), CD90, CK19, CK7, c-kit, E-carderine, EpCAM, FoxA2, HNF4a, Ki67, Sox17, Sox2, Sox7, Twist, and Vimentin.

The spoloid population can be further propagated into one of three different subpopulations by changing the culture conditions such as the medium composition and substrate. The characteristics of bulk tumors, spleens, early, mixed, and EMT populations are presented in Table 1.

Table 1 . Summary of conditions used to generate HCC cell populations from bulk HCC tumors

Moreover, any of the initial HCC-CSC, mixed HCC-CSC, or EMT-HCC-CSC populations can be tested for HCC-CSC spleod, early HCC-CSC, mixed HCC- CSC, or EMT-HCC-CSC.

In one embodiment, the HCC spheroids are conjugated to actin A, FGF, and serum-free medium (selective medium) to produce colonies with small cells referred to herein as " early " populations of HCC-CSCs having the characteristics of embryonic stem cells ), And at least 80% of the cells in the initial HCC-CSC population were cultured on an adherent substrate, with at least 80% of the cells in the early HCC-CSC population being biomarkers EpCAM, E-carderine, Nanog, Sox2, Sox7, Sox17, Oct3 / 4, Fox2A, Ov1 , OV6, c-kit, CD133, and CD90. In yet another embodiment, at least 80% of the cells in the initial HCC-CSC population are biomarkers EpCAM, E-carderin, Nanog, Sox2, Sox7, Sox17, Oct3 / 4, Fox2A, Ov1, OV6, CD133, and CD90. ≪ / RTI > In another embodiment, at least 90% of the cells in the initial HCC-CSC population express two or more, or all, of the biomarkers Nanog, Sox2, Oct3 / 4, and c-kit.

In another embodiment, the HCC spheroids are further cultured on an adherent substrate in the presence of FGF, EGF, and serum-containing medium (proliferation medium) to produce mixed colonies in a single layer, It has morphology. These cells are referred to herein as a "mixed" population of HCC-CSCs with mixed differentiation profiles, and at least 80% of the cells in the mixed HCC-CSC population are biomarkers AFP, CK7, CK19, EpCAM, 2, < / RTI > or all of the following genes: Herin, Nanog, FoxA2, HNF4a, and ABCG2. In another embodiment, at least 90% of the cells in the initial HCC-CSC population express two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Nanog, FoxA2, HNF4a, and ABCG2 do.

In another embodiment, the HCC spheroids are spindle-shaped or irregularly-shaped (also referred to herein as mesenchymal HCC-CSC or "EMT-HCC-CSC" Lt; RTI ID = 0.0 > FGF < / RTI > and serum-containing medium (proliferation medium) to produce a single layer of cells. In this population, spolide has undergone a process of EMT characterized by loss of expression in at least one or all of the epithelial markers CK7, CK19, EpCAM, and E-carderine. As used herein, the loss of expression of a biomarker may be due to undetectable expression or expression of 40% (or less) of the cells, expression of 30% (or less) of the cells, expression of 20% (or less) of the cells, Gt; 10% < / RTI > (or less) of expression. In addition, the EMT process may comprise 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%, to at least one or all of the mesenchymal marker slug / snail, twist, CD44, NCAM, N-carderine, and visenter.

In one embodiment, at least 80% of the cells in the EMT-HCC-CSC population express two or more of the biomarker NCAM, slug / snail, and twist. In another embodiment, at least 80% of the cells in the EMT-HCC-CSC population comprise two or more of the biomarkers AFP, NCAM, N-carderine, slug / snail, twist, CD44, Lt; / RTI > In another embodiment, at least 90% of the cells in the EMT-HCC-CSC population comprise two or more of the biomarkers AFP, NCAM, N-carderine, slug / snail, twist, CD44, Lt; / RTI >

In certain embodiments of the cell population, the cell expresses one or more of the specified biomarkers. In other embodiments, the cell expresses at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the specified biomarkers. In another embodiment, the cells are cultured in the presence of 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.

Expression of a biomarker by a single cell, a population of cells, or a population of cells located in a specific structure, such as a monolayer or speroid, can be determined by measuring the expression of the polypeptide form of the biomarker or the mRNA form of the biomarker. Polypeptide expression can be measured using labeled antibodies, while nucleic acid expression can be measured by hybridization techniques, which can be used by those skilled in the art. Polypeptides or nucleic acids, such as oligosaccharides or biomarkers that are not small molecule metabolites, can also be measured by methods available to the skilled artisan.

Also provided is a population of HCC-CSC spheroids, or a single cell derived therefrom, wherein the expression rate of Ki-67 expressing cells 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 provided is a population of HCC-CSC spheroids, or a single cell derived therefrom, wherein the percentage of cells expressing AFP is at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , At least 90%, at least 95%, or at least 99%.

Also provided is a population of HCC-CSC spheroids, or a single cell derived therefrom, wherein the percentage of cells expressing NCAM is at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , At least 90%, at least 95%, or at least 99%.

Also provided is a population of EMT-HCC-CSCs wherein the percentage of EMT-HCC-CSC expressing slug / snail is at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , At least 90%, at least 95%, or at least 99%.

Also provided is a population of EMT-HCC-CSCs wherein the percentage of EMT-HCC-CSC expressing CD44 is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% %, At least 95%, or at least 99%.

Also provided is a population of EMT-HCC-CSCs wherein the percentage of EMT-HCC-CSC expressing N-carderin is at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , At least 90%, at least 95%, or at least 99%.

Also included are a population of HCC-CSC spleoids, or a single cell derived therefrom, wherein said expression of biomarkers CK7, CK19, EpCAM, E-carderine, and 1, 2, 3, or 4 of ABCG2 Is undetectable or is expressed at 40% (or less) of the cells, at 30% (or less) of the cells, at 20% or less of the cells, or at 10% or less of the cells.

Also provided is a population of any of the above, for example spheres or single cells, wherein the percentage of cells expressing Ki67 is at least 30%, the percentage expressing CK19 is 40%, and the percentage expressing NCAM is At least 80%, and the percentage expressing CK7 is less than 10%.

Also disclosed herein are methods for obtaining a pure population of isolated HCC stem cells from hepatic tumor samples of varying size (1 milligram to grams). Tumor samples can be fresh, frozen, dissociated by treatment with mechanical and / or enzymatic, or directly cultured with minimal mechanical fragmentation.

As also disclosed herein, a non-adherent substrate is any biocompatible material having anti-biofouling properties or a coating having anti-biofouling properties applied to a common culture surface Reducing accumulation). The coating can be applied using a coating such as amino-silane. If there is a non-adherent or anti-biofouling substrate, the substrate is incubated for about 0-25 days, such as 0-21 days, 5-20 days, 5-10 days, 10-20 days, or between 0 and 25 days For example.

In another embodiment of the method of using an adherent substrate, the adherent substrate is one that is rich in RGD (Arg-Gly-Asp) tripeptide motifs ( e.g. , collagen, gelatin, MATRIGEL®) . An adherent substrate is a surface that is attached to an adhesion-dependent cell and is configured to collect it. In addition, the substrate may be an adherent substrate configured to attach to and collect cell adhesion-dependent cells that are fibroblasts. RGD peptides can also be grafted onto the polymer backbone such as polystyrene, hyaluronan, poly-lactic acid, or a combination thereof. The skeleton may have an additional proteoglycan. The proteoglycan may have growth factors such as fibroblast growth factor (FGF), epithelial growth factor (EGF), actin A or polystatin.

Non-adherent substrates can cause rapid and efficient enrichment of cultures with cancer stem cells. A non-adherent substrate can be used when a sufficiently large sample is provided for an excised tumor with an exemplary surgery, so that purification of HCC-CSC can be started immediately. If the sample is very small, for example, needle aspiration or peritoneal washing, and non-adherent culture is not feasible, the adherent culture may be subjected to initial proliferation, then to a purification step on a non-adherent substrate, It can be used for proliferation. Alternative processing methods are demonstrated in detail in Figure 1 (intermittent line and box) and below.

In some culture embodiments, a first period of incubation is provided on an adherent substrate, and then a second period of incubation is provided on a non-adherent substrate. A first period of incubation is also provided on the non-adherent substrate, and a second period of subsequent incubation is then provided on the adherent substrate. The period may be, for example, one-half, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, Day, 15 day, etc., or any range thereof, such as 2-4 days, or 8-10 days, and so on. In addition, the cycle may be repeated, e. G., By adhesion culture followed by non-adherent culture followed by adhesion culture. In another embodiment, the cycle may be repeated, for example, in a non-adherent culture followed by an adhesion culture, followed by a non-adherent culture.

In another embodiment, the restriction medium is supplemented with at least one growth factor acting through a mitogen activated protein kinase (MAPK) pathway. In one embodiment, the growth factor is one or both of FGF and EGF, or an analogue thereof. In one embodiment, the FGF is a basic fibroblast growth factor (bFGF). In another embodiment, the restriction media is supplemented with Actibin A. In another embodiment, the restriction media are not enriched with actinB. Also disclosed is a restricted medium which is supplemented with an agonist of actin A in an amount effective to prevent spontaneous differentiation of HCC stem cells.

In addition, each patient obtained from a patient ' s primary liver tumor is provided with a unique HCC stem cell line, which has (a) self-renewing and versatile stem cell characteristics and ability to differentiate; (b) has a unique genomic cancerous signature in the majority of cells, e.g., greater than 50%.

The disclosure includes nucleic acids, gene products, polypeptides, and peptide fragments, the identity of which can be substantially established by utility name alone. Also included are nucleic acids, gene products, polypeptides, and peptide fragments, based on the specific gene bank accession number, and nucleic acids, polypeptides, and the like, may have at least partially or biologically, biologically, At least 50% sequence identity, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the shared gene bank number Sequence identity.

Wherein the immune response to the cancer of the subject is stimulated by one of the compositions disclosed herein. The stimulated immune response includes at least one of a CD4 ⁺ T cell response, a CD8 ⁺ T cell response, and a B cell response. In certain embodiments, the CD4 ⁺ T cell response, CD ⁺ T cell response, or B cell response is assessed by ELISPOT assays, by intracellular cytokine staining (ICS) assays, by assays known by those skilled in the art , Tetramer assays, or by detecting antigen-specific antibody production. The immune response may include a survival time, such as 2 years overall survival (OS), and a 2-year overall survival is at least 60%. Immune responses are also used in oncology clinical trials, including objective response (RECIST criteria), total survival, progression-free survival (PFS), disease-free survival, time to distant metastasis, 6-month PFS, 12- Lt; / RTI >

Also disclosed herein are dendritic cells stimulated in vitro with HCC stem cells for use in therapy of hepatocellular carcinoma, or antigens derived therefrom. An immunogenic composition, such as a vaccine composition, is included herein, which comprises dendritic cells loaded (exposed to) HCC-CSC in vitro . In certain embodiments, dendritic cells and tumor cells come from the same human subject, but embodiments in which dendritic cells and HCC cells come from different subjects are within the scope of this disclosure.

The dendritic cells may be loaded into HCC tumor cell antigens that include whole cells, cell lysates, cell extracts, irradiated cells or any protein derivative of HCC tumor cells. A dendritic cell immunogenic composition can be prepared and administered to a human subject by one or more routes of administration as known to those skilled in the art.

In certain embodiments, HCC-CSC cells are irradiated, or vice versa, to prevent cell division before loading into dendritic cells. Alternatives to radiation include the manufacture of nucleic acid crosslinks to prevent cell division. In addition, as a source of antigen for autologous immunotherapy, HCC stem and cells provided a method of using the group, for example HCC stem cell is radiation (e.g., gamma, UV, X) as described above, ( E.g. , hot or cold), or chemical ( e.g. , cell proliferation inhibiting, aldehyde, alcohol) methods, or combinations thereof. In another embodiment, HCC stem cells are used as a source of antigen for in vitro activation of dendritic cells.

The present disclosure provides an immunogenic composition (or vaccine) comprising the dendritic cells that provide the produced HCC cells, provide DCs loaded with the prepared HCC cells, and loaded the prepared HCC cells. Without wishing to imply any limitation, the immunogenic compositions of the present disclosure are loaded with HCC speeloid, loaded into a population of cells containing spleoid, derived from the spleoid prior to loading onto DC, And loaded into a population of proliferated cells that had been homogenized or sonicated prior to loading onto DC and loaded with spheroids that had been homogenized or sonicated prior to loading onto DC, DC. ≪ / RTI > In another embodiment, the DC is loaded with an initial HCC-CSC, a mixed HCC-CSC, or an EMT-HCC-CSC.

Also disclosed herein is a population of HCC-CSCs, which are capable of stimulating an effective immune response against cells expressing at least one HCC-specific antigen, wherein said HCC-CSC population is contacted with at least one dendritic cell the HCC-CSC population is processed in vitro or in vivo by a dendritic cell, an effective immune response is generated at the target in response to at least one administration of the dendritic cells to the subject.

The amount of immunostimulation of the disclosed compositions can include, but is not limited to, increasing the ELISPOT assay results by a measurable amount, increasing the ICS assay results by a measurable amount, increasing the results of the tetramer assay by a measurable amount, 20%, 30%, 40%, 50%, 50%, 50% or more of the specific CD4 + T and / or increasing the blood population of antigen-specific CD8 + T by a measurable amount, , 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2.0-fold, 3.0-fold, A suitable control may be a control composition, and dendritic cells are not loaded into HCC cells or loaded with peptides derived from HCC cells.

The disclosure also provides a kit comprising a medicament, a reagent, and a diagnostic kit, wherein the medicament, reagent, and kit comprise a dendritic cell (DC), an antibody, or an antigen. Also provided are methods of administering a composition comprising at least one dendritic cell and at least one antigen, a method of stimulating antibody formation, a method of stimulating an antibody-dependent cytotoxicity (ADCC), a method of stimulating complement-dependent cytotoxicity, Methods and kits are provided for determining patient compliance, determining patient inclusion / exclusion criteria in the context of clinical trials or routine medical care, and predicting the response to a pharmaceutical or reagent. The disclosed pharmaceutical compositions, reagents, and related methods comprise CD83-positive dendritic cells and CD83 is induced by loading with IFN-gamma-engineered, or untreated, cancer cells. In terms of the CD83 of the disclosure, CD83 is induced by at least 2%, at least 3%, at least 4%, 6%, 7%, 8%, 9%, 10% In another aspect, DC reagents, or DC-related methods are excluded, and CD83 on dendritic cells is undetectable by loading with IFN-gamma.

In one embodiment, all reagents and / or HCC-CSCs that produce HCC-CSC spheroids, initial HCC-CSC, mixed HCC-CSC, and / or EMT-HCC-CSC from a tumor sample according to the methods disclosed herein CSC, mixed HCC-CSC, and / or EMT-HCC-CSC, and a reagent that characterizes the HCC-CSC spleod, the initial HCC-CSC, the mixed HCC-CSC, and / A kit is provided to generate and / or characterize HCC-CSC. In another embodiment, the kit may additionally or alternatively comprise a reagent for isolating dendritic cells and / or loading dendritic cells with HCC-CSC and / or for administering a DC-HCC composition to a subject And instructions.

Tumor sample processing

The hepatocellular carcinoma (HCC) stem cell population of the present disclosure can originate from fresh or frozen samples of patient tumors. The tumor sample may be a biopsy or a wash of the tumor-containing tissue. HCC stem cells are isolated from needle biopsy and wash fluid.

Tumor samples can be obtained at a concentration of about 7.4 (+/- 0.6), such as RPMI, DMEM, F12, Williams, or a protein source such as animal or human serum, at a concentration of 0-100%, or albumin at a concentration of 0-0.5% Can be transported to a comprehensive buffered medium with a combination containing macromolecules ensuring osmotic pressure. Examples of natural or artificial macromolecules include, but are not limited to, hyaluronan, dextran, polyvinyl alcohol. Antibiotics such as penicillin, streptomycin, and gentamicin can be used in any combination with antifungal such as amphotericin B, FUNGIZONE (Life Technologies, Carlsbad, Calif.), In a medium that provides antimicrobial properties and reduces the risk of contamination during transport Can be used.

Tumor samples can be kept below the metabolic activation state by reducing the medium temperature to 2 to 30 째 C, thus allowing viability maintenance for a limited time (between 0 and 72 hours) before processing. Packaging ( e.g. , insulated packaging) can be used to ensure proper temperature control during transport.

The solid tumor tissue is then processed by mechanical dissociation using a sharp blade or tissue grinder device with small, less than 1 mm (for any dimension) fragments.

The solid tissue is further processed by dissociation by enzymes. A variety of enzymes can be used to isolate single cells. Nonspecific proteolytic enzymes such as trypsin and pepsin can be successfully used. Targeting minimal cell membrane damage specific enzymes, including collagenase, dispase, elastase, or a combination thereof, can be used in the disclosed methods. Deoxyribonuclease (DNAse) can be used to degrade free DNA from cell debris responsible for unwanted tackiness of cell preparations. After dissociation, the cells in the suspension are washed from excess enzyme and debris by straining through 50-100 mu m mesh and repeated centrifugation in buffered saline (PBS, HBSS) or cell culture medium.

Cell culture conditions and Speroid  production

The single cell suspension described above is moved under culture conditions that promote isolation, proliferation and differentiation of stem cells and / or inhibition of normal cells. This is accomplished by a combination of physical conditions, chemical environment, and manipulation.

The cell suspension is exposed to a non-adherent (anti-biofouling) substrate that does not allow cell attachment. Adult cells are typically fixation-dependent and rapidly eliminated when a suitable adherent substrate is not provided. The anti-biofouling substrate can be a commercial product such as a low-adhesion flask (Corning, Corning, NY), a polymer with natural hydrophobic properties (polyvinyl, polyethylene, polypropylene, fluoro- polymer) or a natural carbohydrate polymer such as agar, starch And the like.

Cancer stem cells will aggregate and / or clonally multiply into spearoid formation (Fig. 2) containing high purity cancer stem cells. Cultures of cancer stem cell aggregates having various sizes and easily identifiable spoloid structures are shown in Figures 3A and 3B. Adult cells will remain isolated and non-adherent. Differential gravity separation can be used to select larger spheroids from a single cell by simply allowing timely vertical sedimentation or short-time small force centrifugation (less than 100 x G). The described selection method is designed to achieve the following: (a) in general, to remove adherence-dependent cells, which are mature normal cells; (b) promoting clonal proliferation in small clumps or spheroids of established, independent, neonatal stem cells; (c) promoting local self-cleavage activity as a result of clonal expansion of stem cells; And (d) removing the source of autologous secretion of Actibin A secreted by normal fibroblasts or hepatocytes.

The ability of cells to form sphincters is partly due to cell-surface proteins, called integrins. The specific antibody integrin expressed on the surface of the cells ensures that the same type of cells "stay together". The spheroid is formed directly from the enzyme digestion product, which is a single cell suspension at the beginning of the culture, or is formed from an frozen sample or an existing attached culture at any time. Enzymatic digestion product seeding causes this spherical formation to incorporate cells with specific surface characteristics.

Fibroblasts are removed from the culture medium during gravity feed without being incorporated, for example, with sphere. The medium used does not have molecules that promote adhesion to prevent non-specific aggregation of cells that do not have specific antibody characteristics and to prevent adhesion to the culture vessel surface. Such cell adhesion attachment (CAM) is typically found in animal or human sera. Thus, a serum-free medium composition is suitable for the culture of non-adherent spoloids.

In serum-free medium cultures, the supplement material for the medium may include any hormones, nutrients, minerals, and vitamins needed to support growth and maintenance, or other desired aspects of cell physiology and function. In some cases, stimulation and persistence of stem cell proliferation may be achieved by the addition or modulation of mitogenic activity, such as the amount of growth factors that possess the FGF family and EGF.

Spheroids of cells (spheroids), including spheroids of cancer stem cells, may be characterized by biomarker expression by fixing and staining with labeled antibodies, followed by confocal microscopy. Biomarkers can also be measured by other immunochemical methods, such as flow cytometry. The spheroids can be prepared, for example, from suspensions obtained from fresh tumors or from cells adapted to grow into adherent cells. The morphology of the spheres, for example large and irregular versus very small and dense, can be influenced by the choice of medium.

In another embodiment, the cell population attachment to the anti-biofouling coating is due to abnormal activation of sonic hedgehog signaling and focal adhesion kinase (FAK) signaling mediated by protein kinase B (AKT) Or < / RTI > These phenomena can be augmented by the deformation of membranes induced by enzymes such as metalloproteases or enzymes used in dissociation (trypsin / collagenase). Such a population of cells can be associated with rapid proliferative and invasive tumors. Figure 5 shows a representative population of cells attached and proliferated to an ultra-low attachment surface. Methods of assessing normal or abnormal activation of sonic hedgehog signaling are available and known to those skilled in the art.

Medium used for cell culture

Restriction media used to isolate HCC stem cells promote cell survival and are specifically formulated for selection. The medium is rich in carbohydrates and lipids but has minimal amounts of protein (0.1% -3% albumin or 1% -5% serum). It contains less than 1.5 mMol total calcium and does not contain inorganic iron compounds; Rather, iron is fully bound to the transporter, e.g., transferrin. The medium is provided with excess essential and non-essential amino acids and essential lipids (alpha-linolein and linoleic acid) (Table 4). Optionally, the medium may contain no actin A and may contain an actin A receptor blocker such as pol statin. Optionally, the medium also contains a thiol antioxidant such as glutathione, rather than containing an antioxidant such as superoxide dismutase (SOD) or catalase.

The culture medium contains ribozyme, amino acid, antioxidant, energetic substrate (glucose, galactose, L-glutamine) supplemented with base formulations such as DMEM, F12, Williams, RPMI, protein , Vitamin (B12), hormones (thyroid hormone, insulin) and growth factors (FGF, EGF).

The protein may be albumin at a concentration of 0.1-0.5%, folate serum (FBS) at 0.5% -20%. Proteins can be substituted with macromolecules such as dextran, hyaluronan, poly-vinyl alcohol in concentrations ranging from 0.1% to 0.5%. The compositions of such media are listed in Tables 2, 3 and 4. The supplement is added to the medium and mixed to provide cell culture.

Table 2. Basal medium composition selection for cancer stem cells:

Table 3. Systemic stem cell supplement (50 mL units for reconstitution at 1 L of basal medium)

Table 4. Lipid mixing

Lipid mixing is accomplished by an o / w emulsion using Pluronic F68, phosphatidyl choline, Tween 80, cyclodextrin, or a combination thereof

The medium can be replaced as early as three days of weekly planning ( for example , Monday - Wednesday - Friday) or more often, for example , every other day or every day. Continuous feed or micro-batch feed bioreactors may be used in the propagation step.

The medium contains growth factors acting through MAPK pathways such as FGF and EGF. The concentration of these growth factors may vary between 0.1 and 100 ng / mL, typically about 10 ng / mL.

In one embodiment, the media comprises about 0.1 to 100 ng / mL, about 0.5 to 50 ng / mL, about 1-40 ng / mL, about 2-30 ng / mL, about 3-20 ng / FGF is supplemented with about 15 ng / mL, about 6-14 ng / mL, about 7-13 ng / mL, about 8-12 ng / mL, about 9-11 ng / mL, or about 10 ng / mL. In another embodiment, the FGF is selected from the group consisting of about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 11 ng / ng / mL, about 14 ng / mL, or about 15 ng / mL.

In another embodiment, the media comprises about 0.1 to 100 ng / mL, about 0.5 to 50 ng / mL, about 1-40 ng / mL, about 2-30 ng / mL, about 3-20 ng / 5-15 ng / mL, about 6-14 ng / mL, about 7-13 ng / mL, about 8-12 ng / mL, about 9-11 ng / mL, or about 10 ng / mL . In another embodiment, EGF is administered at a concentration of about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 11 ng / ng / mL, about 14 ng / mL, or about 15 ng / mL.

Also provided are media that are not supplemented with one or both of superoxide dismutase (SOD) or catalase. The use of antioxidants may have both positive and negative results. Cancer stem cells are much more resistant to free radicals and glucose metabolism than normal cells. Therefore, at the high concentration of lane culture, such as high density, rare medium rearrangement, and medium metabolism, normal sensitivity cells are most likely to be removed first. By not including the sulfating agent in the medium, a population of cells that are more resistant than normal cells and potentially capable of cancerous origin can be selected. Thus, in certain embodiments, an antioxidant, such as a catalase and an inhibitor of SOD, is added to the culture medium, and in other embodiments, these compounds are omitted from the culture medium.

In an alternative method, the activin / pol statin system can be used to isolate very early cancer stem cells. Addition of actin A can select a sub-population of actin A-resistant HCC stem cells. Colonies of early HCC stem cells obtained after exposure to actin A for 7-14 days are shown in FIGS. 6, 7, and 8. FIG. These subgroups are associated with more aggressive forms of HCC and early (less differentiated) cancer stem cells. Polystatin is used to blockactin A receptors and prevent the spontaneous differentiation of HCC stem cells, particularly when a significant number of cells that endogenously secrete Actibin A, such as fibroblasts and normal hepatocytes, are present. The use of polystatin has no effect if the cells are non-sensitive to actin A or to a population of high purity HCC stem cells where the polastatin can be endogenously secreted.

Actin A is a protein that is a member of the transforming growth factor-beta (TGF-beta) superfamily. When added or included in the culture medium, actibin assists in maintaining stem cell pluripotency and self-renewal. However, Actin A promotes maturation and differentiation of adaptive neonatal hepatocytes and cancer cells. Thus, the initial goal is also rapid in vitro proliferation of tumors that continue to proliferate cancer stem cells by creating an appropriate autocrine environment in the culture. Activin A is able to select a subset of very early cancer stem cells, but such conditions initially applied during manufacture will greatly delay proliferation if very low concentrations of liver cancer stem cells are given in the bulk. For example, " fast growth " is a proliferation that causes medium in culture vessels with obvious signs of consumption (e.g., change in pH) and the number of cells is clearly higher than ever reflected by increased density.

For rapid proliferation, it is preferred that actin A is omitted and not added because it slows the growth of the culture. Interest in some applications is to obtain very early stem cell populations and the use of actin A will select such cell populations. Thus, in one embodiment, actinin A-containing proliferation is initiated and the first composition is administered to a subject comprising an actinin A-activated cultured cell, followed by treatment with an actin A- Followed by the isolation of sensitive cells and the administration of a second composition comprising the non-cultivated cells of Actin A to said subject.

In one embodiment, the media comprises about 0.01 to 10 ng / mL, about 0.05-9 ng / mL, about 0.1-8 ng / mL, about 0.5-7 ng / mL, about 1-6 ng / And reinforced with actin A at 5 ng / mL. In another embodiment, Actibin A has a concentration of about 0.5 ng / mL, about 0.7 ng / mL, about 0.9 ng / mL, about 1 ng / mL, about 1.25 ng / mL, about 1.5 ng / About 2 ng / mL, about 2.25 ng / mL, about 2.5 ng / mL, about 2.75 ng / mL, about 3 ng / mL, about 3.5 ng / mL, about 4 ng / mL, about 4.5 ng / 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL.

Also disclosed are embodiments and the medium is supplemented with an antagonist of actin A, such as, but not limited to, polystatin or an antibody that specifically binds to actin A.

In another embodiment, the media comprises about 0.1 to 100 ng / mL, about 0.5 to 50 ng / mL, about 1-40 ng / mL, about 2-30 ng / mL, about 3-20 ng / 5-14 ng / mL, about 6-14 ng / mL, about 7-13 ng / mL, about 8-12 ng / mL, about 9-11 ng / mL, or about 10 ng / do. In another embodiment, polystatin is administered at a concentration of about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 11 ng / About 12 ng / mL, about 14 ng / mL, or about 15 ng / mL.

The combination of mitogens ( e.g. , FGF / EGF), actin A, and an adherent substrate may cause an increase in proliferation of normal cells such as fibroblasts or astrocytes. Thus, the conditions may include proliferation of very early HCC stem cells or ancestral cells that are non-sensitive to actin A in a rich environment or " stroma " composed by cells with nutrient or encapsulation properties ( e.g., fibroblasts, astrocytes) . Colonies of HCC continue to develop and are progressively observed to spatially displace the stroma over the next few days or weeks of cell culture (FIGS. 7 and 8). The medium used in this method is a combination of the formulations shown in Tables 2, 3 and 4.

There is a relationship between FGF, EGF, and actin A, and " very early HCC cancer stem cells ". FGF and EGF cause proliferation of HCC stem cells in any differentiation state, including very early. When actin A is in the cell culture medium, actin A is exclusively allowed (allowing it) for the proliferation of very early HCC stem cells that are non-sensitive to actin A. If HCC stem cells are sensitive, proliferation will be stopped or reduced by actin A.

The insensitivity to FGF and EGF is not common and there is no natural blocker. Anesthesia to actin A can be mediated by natural blockers of polystin, an actin receptor. Polystatin can be secreted by the same tumor cells or by cells surrounding the tumor. Actin A is typically secreted by cells surrounding the tumor, and thus it is possible that the proliferation of the tumor is dependent on the surrounding cells (inhibition) and on the tumor (promoting proliferation). The lack of receptors for Actin A, a characteristic of very early undifferentiated cancer stem cells, can prevent the control of tumors by the surrounding tissue.

In vitro cultures will contain embryonic stem cell-like colonies. These colonies can be surrounded by stromal cells, which can be normal fibroblasts, differentiated tumor cells, or mesenchymal tumor cells. Such a culture is depicted in Fig.

The present disclosure provides a method of making HCC-CSC and the total incubation time, including the time required for manipulation such as changing media, replating, centrifugation, and precipitation, is less than 5 months, less than 4 months , Less than 3 months, less than 2 months, less than 1 month, less than 150 days, less than 120 days, less than 90 days, less than 60 days, less than 30 days, or less than 150 days (+/- 20 days) -20 days), less than 90 days (+/- 20 days), less than 60 days (+/- 20 days), less than 30 days (+/- 20 days) In the excluded embodiment, the present disclosure may exclude any method of producing cancer stem cells, and any population of cancer stem cells produced by such methods, and the time required for manipulation is determined by the time- Lt; / RTI > Also provided is the time of attachment culture specified by one of said time-frames. Also provided is a time of non-adherent culture which is one of the time-frames. In addition, combined time of attachment culture and non-adherent culture identified by one of the time-frames is provided.

epithelium- Liver  transition( EMT )

Tumors of epithelial origin are known to regress or over-undifferentiate into a mesenchymal state. The epithelial phenotype is irreversible and contributes to the volume growth of tumors confined to the origin tissues and is typically further differentiated. When EMT occurs, cells acquire mobility and produce adjacent tissue infiltration and distant metastasis. Metastasized cells also acquire stem cell-like phenotypes, and their ability to replicate and undifferentiate causes new tumors (metastases) in host tissues that have the characteristics of primary tumors. By EMT, tumor cells gain additional immunosuppressive ability, drug pump and radioactive resistance.

The media composition and physical selection method promote EMT development in vitro . Advantages of using EMT-transferred groups as immunogens are prevention of tumor recurrence. The antigens of EMT cancer cells may be able to recognize and destroy mobile cancer cells that cause the immune system to migrate. In the process of metastasis, these cells migrate to very low numbers, plant host tissues, return to epithelial phenotype (MTE metastasis), and grow and form new tumors with properties similar to primary tumors. The conditions required to induce in vitro EMT are the formation of spoloids in serum-free medium, stimulation with bFGF, followed by an adherent substrate containing RGD (Arg-Gly-Asp) peptide motif ( e.g. , collagen, gelatin, Lt; / RTI >

The EMT-HSC-CSC sub-population is obtained by culturing HCC spleod, or initial HCC or mixed HCC, under culture conditions as described in Table 1 and FIG.

As used herein, the term " HCC-CSC " generally refers to HCC-CSC spheroid, early HCC-CSC, mixed HCC-CSC, or EMT-HCC-CSC.

Tumor (needle Biopsy ) From a small source of HCC - CSC's  purchase

An alternative method for HCC stem cell selection is used when the number of sample cells is small. For instance, the living cell of a small number obtained from the tumor is less than 10x10 ⁶ viable cells after dissociation of the enzyme. For purposes of this disclosure, a small sample refers to a sample obtained, for example, from a needle biopsy or a core biopsy, as opposed to a sample obtained from a resected tumor, which is typically considered a small sample and is at least 0.5 to 5-10 The weight of the gram goes out. Core biopsies are performed with 18 or 16 or 14 gauge needles, resulting in 5-50 mg samples. A relatively new procedure, called vacuum assisted biopsy, is also performed with an 11-gauge needle, Vacuum Assist Device (VAD). An 11 gauge probe paired with a vacuum assist device typically picks up 94 mg from each core sample. A 14-gauge needle with vacuum assist typically picks up 37 mg, but when paired with an automated biopsy gun, only 17 mg is picked up. In this alternative method depicted in FIG. 1, the cells obtained from the tumor sample are treated with RGD (Arg-Gly-Asp) rich compounds ( such as collagen, gelatin or MATRIGEL®) And an optional (serum-free) culture medium as described herein. The described selection methods are designed to (a) facilitate early clonal expansion of individual cancer stem cells present in small numbers, and (d) promote local autocrine activity as clonal proliferation of stem cells.

The adherent substrate is RGD rich in proteins such as collagen or gelatin. The substrate can be constituted by attaching the protein or peptide to various materials such as polystyrene polycarbonate, cyclic olefin copolymer or glass. RGD peptides can be grafted onto the polymer backbone such as hyaluronic acid, polylactic acid and combinations. Polymer, such as that may be increased in addition to the growth factors, for example, proteoglycans (e.g., heparin sulfate, chondroitin sulfate, keratin sulfate, etc.) for the carrier end.

The cell culture surface may be used directly or with the use of a coating agent such as aminosilane. The coating is a compound with an adherence property (substrate) to the cell and is applied on top of the material of the growth vessel. It can be a natural compound such as collagen or gelatin and can be composed of more synthetic polymers with the radical / end mentioned. Coatings (adhesives, such as silanes) may be used to improve adhesion of the coating to the culture vessel material (e.g., glass). The silane can be used alone if it contains the desired radical or terminal group.

In the case of such methods and formulations, multiple cells can be obtained in a relatively short period of time. Starting at a few milligrams, the culture of tissue samples, such as needle biopsies containing 10 ³ to 10 ⁶ cells, can be propagated to about 10 ⁸ cells within 3-4 weeks.

HCC  Growth of stem cell culture and occurrence of subgroup

HCC-CSC is an additional feature of stem cells, which can propagate undifferentiated and proliferate. A propagation culture on the adherent substrate is depicted in Fig.

Moreover, HCC-CSC can be partially or completely differentiated. Once stem cell growth conditions are removed, HCC stem cells can slow or stop proliferation and change morphology and phenotype to more differentiated cell types. Morphology can be flat, epithelial, or stellate with features of multiple nuclear-mature hepatocytes or astrocytes.

Adherent cultures can dissociate in a single cell suspension to remove fixation-dependent differentiated cells and migrate to non-adherent (anti-biofouling) conditions. After 2-3 days, stem cells tend to aggregate and clonally multiply into small spheroids that can be separated from single cells based on differential sedimentation. Spheroids can be re-flat cultured in the adhered state and further propagated. This method will purify the cultured stem cell content to 1-30% to 90-99% stem cell content once the culture has been overtaken by differentiated cells or normal cells such as fibroblasts. The method can be repeated as many times as necessary to restore stem cell purity.

Small spheroids generally have dimensions between 0.1 mm and 2 mm. The size distribution is generally between 10 and 10,000 cells, with respect to the number of cells per small spheroid. The shape of the small spheroids may be spherical or elliptical, and may also occur as a collection of spherical or elliptical structures (Figs. 2 and 3).

The patient-specific HCC-CSC cell line can be used to identify genomic mutations responsible for neoplastic transformation when compared to normal tissue from the same patient. Genomic mutations may not be expressed at all stages of differentiation. Some regulatory proteins, or transcription factors, are only transiently expressed and can disappear during maturation, resulting in malformed cells but having normal proteins. Identification of cell populations that maximize mutations and expose these populations to the immune system may be a major benefit of using cancer stem cells as an antigen source for immunotherapy.

By identifying genomic mutations, personalized formulations can be generated for cancer therapy, such as small molecules, DNA sequences, antisense RNA or combinations.

Such cell lines may be further used to generate screening plates (e. G., 96 wells) for drug discovery. Multiple lines from various patients can be combined in a single plate to resolve variability among individuals.

Hepatocellular carcinoma Cancer stem cells can maintain the secretion of some characteristics of developing tissues such as proteins, growth factors and hormones (functional tumors). These properties can be used given the immortal nature of the cell line to produce a " self " protein that can be used for the same patient (such as albumin, transforming growth factor (TGF), insulin, glucagon, DOPA, have. Cells can be introduced into small bioreactors and separation products that are collected, purified and stored for the same patient use. This method is particularly advantageous in that the patient will not develop immune tolerance, such as more traditional biosimilar.

Loading of dendritic cells

Individual HCC-CSC cell lines obtained from patients can be used to generate antigens for immunotherapy. The advantage of using purified stem cell lines lies in the better signal-to-noise ratio. The more mature cells from the tumor can mask the antigenicity and have a compensatory mechanism that can not be confirmed by the immune system. As an antigen source, HCC-CSC may be used in an active state, inactive by mitosis, or may be non-viable or fragmented. Various methods can be used to modify cells for optimal antigen exposure: radiation ( e.g. , gamma, UV, X), temperature ( e.g. , hot or cold), or chemical ( e.g. , Cell proliferation inhibitory, aldehyde, alcohol) or a combination thereof.

In an exemplary implementation, the present disclosure provides for the use of dendritic cells (DCs) as adjuvants such as CD40 agonists such as CD40-ligands or cytokines, interferon-gamma, prostaglandin E2, as well as one or more immunoadjuvants such as toll ) -Like receptor (TLR) agonists such as CpG-oligonucleotide (TLR9), imiquimod (TLR7), poly (l: C) (TLR3), glucopyranosyl lipid A ), And plasgelin (TLR5). See, for example, U.S. Patent Nos. 7,993,659; U.S. Patent No. 7,993,648; U.S. Patent No. 7,935,804, each of which is incorporated herein by reference for all that it discloses about activating DC. The present disclosure includes the in vitro treatment of DC with one or more of the adjuvant reagents, or alternatively, or alternatively, administration of adjuvants to a human subject, animal subject, or somatic subject.

The immune system includes cellular immunity, humoral immunity, and complement response. Cellular immunity includes a network of cells and events involving dendritic cells, CD8 ⁺ T cells (cytotoxic T cells; cytotoxic lymphocytes), and CD4 ⁺ T cells (helper T cells). Dendritic cells (DCs) obtain polypeptide antigens, which may be obtained from the outside of DCs or biosynthetized inside DCs by the infecting organism. DCs process the polypeptide to produce peptides of about 10 amino acids in length, transfer the peptide to MHC class I or MHC class II to form a complex, and the complexes to and from the surface of the DC. When DCs containing MHC class I / peptide complexes are contacted with CD8 ⁺ T cells, the results are activation and proliferation of CD8 ⁺ T cells. Regarding the role of MHC class II, when DCs containing MHC class II / peptide complexes are contacted with CD4 ⁺ T cells, the results are activation and proliferation of CD4 ⁺ T cells. Although dendritic cells that provide antigen to T cells may "activate" such T cells, activated T cells may not be able to initiate an effective immune response. Effective immune responses by CD8 ⁺ T cells often require pre-stimulation of DC by one or more of a number of interactions. To one of like receptors (TLRs) - these interactions (as Na2 contacting the CD40 ligand of CD4 ⁺ T cells in the DC CD40 receptor), or tall (toll) of dendritic cells CD4 ⁺ T cells in direct contact of the DC RTI ID = 0.0 > TLR < / RTI >

Humoral immunity refers to B cells and antibodies. B cells are transformed into plasma cells, and the plasma cells express and secrete antibodies. Non-contact B cells are distinguished in that antigen-specific B cells express CD27 while they do not express the marker CD27. The secreted antibody can then bind to tumor antigens that reside on the surface of the tumor cells. The result is that the infected cells or tumor cells are tagged with antibodies. In the case of antibody binding to infected cells or tumor cells, the bound antibody mediates the killing of infected cells or tumor cells, and the killing is by NK cells. Although NK cells are not constructed to recognize specific target antigens, the ability of NK cells to bind to the insoluble region of the antibody in a manner such that the T cell is configured to recognize the target antigen is characterized by the ability of the NK cell to specifically It is possible to kill. Recognition of the NK cells of the antibody is mediated by Fc receptors (of NK cells) that bind to the Fc portion of the antibody. This type of killing is referred to as antibody-dependent cell cytotoxicity (ADCC). NK cells can also kill cells independently of the mechanism of ADCC, which requires the expression of MHC class to be lost or deficient in target cells.

Without intending to be bound by any particular mechanism, the disclosure encompasses the administration of cancer stem cell antigens, or the administration of dendritic cells loaded with cancer stem cell antigens, wherein the antigen specifically binds one or more of the cancer stem cell antigens Stimulates the production of an antibody that recognizes, and the antibody mediates ADCC. An antigen loaded phage refers to the ability of dendritic cells to capture viable cells, capture gangrene cells, capture dead cells, capture polypeptides, capture peptides, and the like.

Capture by cross-presentation is covered by this disclosure. Also includes the use of antigen-presenting cells that are not dendritic cells such as macrophages or B cells.

The technique of "delayed type hypersensitivity reaction" can be used to distinguish immune responses that mainly involve cellular immunity or primarily involve humoral immunity. Positive signals from delayed type hypersensitivity reactions indicate cellular responses.

The present disclosure provides compositions and methods, wherein tumor cells are inactivated by, for example, radiation, nucleic acid crosslinkers, polypeptide linkers, or a combination thereof. Cross-linking is the attachment of two chains of polymer molecules by a bridge, which consists of elements, groups, or compounds connecting some carbon atoms of the chain by primary chemical bonds. Crosslinking is the sharing of the keratin or insulin, the pyrrole cross-bridging of the trivalent pyridinol and mature collagen, and the sharing between the epsilon-amino group of the lysine residue and the gamma-carboxy-amine group of the thrombus glutamine residue. Epsilon- (gamma-glutamyl Quot;) < / RTI > lysine cross-linking, as in cross-linking in the thrombus involving lysine cross-linking, by disulfide bonds with two cysteine residues.

Cross-linking can be artificially effected on proteins by adding chemical materials (cross-linking agents) or by receiving high-energy radiation from the polymer. Crosslinking with fixative and heat-induced aggregation not only completely inhibited proliferation but also increased the immune response. Substances capable of preventing the proliferation of HCC-CSC are used as cross-crosslinking proteins on the surface, including but not limited to, 10% neutral-buffered formalin, 4% paraformaldehyde, 1% glutaraldehyde, 0.25-5 mM dimethyl Bersodium 100% acetone or 100% methanol. In addition, a combination of 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer may also be used.

Formaldehyde and glutaraldehyde were both suggested to induce activation of T helper type 1 and type 2 cells. In particular, the heat-induced aggregation of antigens has also been shown to enhance in vivo priming of cytotoxic T lymphocytes. The cross-linking of the antigen by 3,3'-dithiobis (sulfosuccinimidyl propionate) causes an increased binding of the antigen to the dendritic cells and the cross-linked antigen passes through the proteosome pathway for antigen delivery Processed. Moreover, formalin-fixed hepatocellular carcinoma tumor cells were used in clinical trials without evidence of proliferation.

In one embodiment, the entire HCC-CSC is fixed with cross-linking preparation and then used as an antigen source in combination with dendritic cells.

In another embodiment, the nucleic acid of the cell is cross-linked. Exemplary nucleic acid alkylating agents are beta-alanine, N- (acridine-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester. Exemplary cross-linking agents, such as psoralen, often cross-crosslink DNA, in combination with ultraviolet (UVA) irradiation, but have the ability to leave the protein in an unmodified state. For example, the nucleic acid target compound can be 4 '- (4-amino-2-oxa) butyl-4,5', 8-trimethylsoralene (S-59). Cells can be inactivated with 150 μM psoralen S-59 and 3 J / cm ² UVA light (FX 1019 irradiation device, Baxter Fenwal, Round Lake, IL). Inactivation with S-59 by UV light is referred to as photochemical treatment, and the treatment conditions can be adjusted or titrated to cross-linked DNA to such an extent that cell division is completely prevented, but for polypeptides containing a polypeptide antigen Damage is minimized. Cells can be suspended in 5 mL of saline containing 0, 1, 10, 100, and 1000 nM of psoralen S-59. Samples of approximately 2 J / cm ² Dose can be irradiated with UVA. Each sample is then transferred to a 15 mL tube, centrifuged, supernatant removed, then washed with 5 mL brine, centrifuged, supernatant removed and suspended in final pellet in 0.5 mL of brine. See U.S. Pat. Nos. 7,833,775 and 7,691,393, which are incorporated herein by reference for all that it discusses about cell deactivation.

For any cell preparation that is processed into a cross-linking agent, the ability to divide is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 2 months, at least 3 months, at least 4 months, Can be tested by a skilled person by incubating or culturing in a medium. Cell division may be evaluated by a colorant that reveals chromosomes and reveals that cell division is occurring or not. Cell division can also be measured by counting cells. Thus, when the number of cells in the culture plate stably remains for a period of 2 weeks, 1 month, 2 months, etc., the conclusion that the cells can not divide can be considerably reduced.

In one embodiment, the dendritic cell immunogenic composition is administered subcutaneously (SC). In a further embodiment, each dose ranges from about 5 million to 20 million loaded DCs and is repeated with a series of 6-10 doses. In certain embodiments, the dose is administered every 5 days, every other day, every 10 days, every other week, or every third week for 2, 3, 4, 5, or 6 doses, Dose is administered every 2 weeks, every 3 weeks, every 4 weeks, every month, every 5 weeks, or every 6 weeks for 2, 3, 4, 5 or 6 doses. In one embodiment, the first four injections are provided weekly for one month, and then once a month for the next four injections. In an alternative embodiment, administration is once a week for three weeks for a total of eight doses and once a month for the next five months.

Each dose may be about 5-20x10 ⁶ loaded DC, about 5-17x10 ⁶ loaded DC, about 6-16x10 ⁶ loaded DC, about 7-15x10 ⁶ loaded DC, about 7-14x10 ⁶ loaded DC, about 8-13x10 ⁶ loaded DC, about 8-12x10 ⁶ loaded DC, or about 9-11x10 ⁶ loaded DC. In further embodiments, each dose comprises of about 8x10 ⁶ DC loading, the loading of approximately 9x10 ⁶ DC, with approximately 10x10 ⁶ loaded DC, approximately 11x10 ⁶ loaded DC, or about 12x10 ⁶ loaded DC. The loaded DC comprises a mixture of residual HCC-CSC that was not occupied by DC and DC. The doses administered comprise mixtures of these cells and the doses reflect these mixtures.

In another embodiment, the loaded DC is administered as a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable excipients, e. G., Vehicles, adjuvants, carriers or diluents described herein, are well known to those skilled in the art and are readily available to the public. The pharmaceutically acceptable carrier or excipient is preferably one that is chemically inert to the loaded DC and has no deleterious side effects or toxicity under the conditions of use.

The choice of excipient or carrier will be determined in part by the particular therapeutic composition as well as by the particular method used to administer the composition. The formulations described herein are merely illustrative and are not limiting in any way.

Often physiologically acceptable carriers are aqueous pH buffered solutions. Examples of physiologically acceptable carriers include, but are not limited to, saline, solvents, dispersion media, cell culture media, aqueous buffers such as phosphates, citrates, and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin, or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt formation counterion such as sodium; And / or non-ionic surfactants such as TWEEN (TM), polyethylene glycol (PEG), and PLURONICS (TM).

In some exemplary implementations, boost adjuvant (GM-CSF) is given at all doses simultaneously. In certain embodiments, the cell dose is suspended in a carrier that contains GM-CSF. In an alternative exemplary practice, a GM-CSF boost adjuvant is given, but not every single dose. In another exemplary implementation, there is no GM-CSF boost adjuvant at all.

Dendritic cells (e. G., Autologous or allogeneic dendritic cells) may be used to treat a variety of conditions including, but not limited to, cell lysates, acid elution, cell extracts, partially purified antigens, purified antigens, isolated antigens, partially purified peptides, Purified peptides, isolated peptides, synthetic peptides, or any combination thereof with the cancer stem cell antigens. The dendritic cells are then administered to a subject, for example a subject with HCC, or a control subject without HCC. In an exemplary practice, dendritic cells are contacted with one or more of subcutaneous, intraperitoneal, intranodal, intramuscular, intravenous, intranasal, inhalation, oral routes by application to the intestinal lumen or the like, Injected, or administered by them. In addition, the immunogenic composition may be administered directly to the site of the tumor or metastasis.

Example

Example 1

Isolation and proliferation of HCC cancer stem cells from needle aspiration biopsy

Hepatocellular carcinoma (HCC) tumor samples are heterologous, histologically heterogeneous, composed of somewhat differentiated cancer cells, along with normal parenchyma, matrix and vascular cells. The purpose of the method provided herein is to isolate and propagate a population of cancer stem cells derived from small HCC samples. In addition, these cells are used to prepare autologous tissue therapies for the treatment and prevention of recurrence of HCC.

The procedures and reagents are designed to sustain typical stem cells and not to sustain the survival / proliferation of differentiated hepatocytes, biliary epithelial, vascular endothelial cells, smooth muscle cells, or fibroblasts in vitro . Exemplary ingredients described in the literature such as corticosteroids and sera have been omitted; Instead, a basal medium formulation with a specific proportion of protein-fortified amino acids and vitamins was used.

Needle aspiration biopsy was obtained from an agreed-upon patient diagnosed as a liver tumor from a macroscopically confirmed pathological field. The biopsy was immediately transferred to a closed container in the transport medium and delivered to the tissue treatment facility at a controlled temperature (4-8 ° C). The biopsy was then dissociated in a solution of collagenase IV (4 mg / mL) for 30 minutes. The resulting cell suspension was transferred to a polystyrene culture flask coated with 0.1% gelatin after centrifugation and allowed to proliferate for about 2-4 weeks. Every two days, the culture was fed to a fresh medium consisting of a base formulation for stem cells and supplemented with a protein / growth factor mixture. The formulations are reproduced in Tables 2, 3 and 4. In addition, basic fibroblast growth factor (bFGF) at 10 ng / mL and EGF at 10 ng / mL were included in the mixture at the time of feeding.

After establishing robust cultures and when the cultures reached 60-100% density, the cultures were dissociated with the proteolytic enzyme (trypsin) and at a ratio of 1: 2 to 1:10 for the next 2-6 cycles Lt; / RTI > on a large surface. The cells were then plated in imaging chambers and stained with antibodies specific for hepatocellular carcinoma.

The cultures began to grow slowly in the first (Fig. 9) and colony reassembly structure was confirmed after about 14-21 days (Fig. 10). Enzymatic dissociation and migration of these colonies were obtained for a single layer of strongly proliferating cultures (Figure 11). Dissociation during transition to collagenase IV prevented the establishment of monolayer cultures.

After 2 to 6 transitions, the cell lines were treated with cytokaratin (Ck19, Ck7), tumor specific markers (alpha fetoprotein [AFP], ABCG2), splice attachment (EpCAM, NCAM, CD44), proliferation marker - Immuno-cytochemistry was analyzed for mesenchymal transition (EMT) markers (slug / snail, twist).

12A-12C and Table 5), the presence of NCAM (also known as adherence of neuronal cells, known as CD56) (as shown in Table 5, Ki67 (As shown by Figure 13A-13C, Table 5). ≪ tb > < TABLE > NCAM is a junctional attachment with specificity and nonspecific properties that allow cell-to-cell adhesion and account for the ability of the tumor to penetrate. Previous surveys indicate that only 8.3% fresh tumors from the manipulated cases are positive for NCAM. Thus, culture conditions resulted in positive selection and enrichment of HCC-CSC. The loss of epithelial markers (EpCAM, cytokeratin) suggesting that the more differentiated phenotype of HCC cells is not sustained by culture conditions is significant. The presence of NCAM in the primary HCC indicates increased malignancy and metastatic potential.

Table 5 . Immuno - cytochemical characterization of tumor samples from needle biopsy.

Surface antigen CD44, hyaluronan receptors have a known association with mobility and metastatic characteristics of tumor cells. In the analyzed samples, the majority of cells (90-100% or more) are positive for CD44 (Fig. 14A-14C).

The presence of slug / snail and vimentin markers indicates that loss of epithelial markers (cytokeratin, EpCAM) is caused by an epithelial-mesenchymal transition process in which tumor cells obtain tumor stem cell characteristics (CD44, NCAM) 15a-15d).

These data indicate that the provided serum-free medium formulation, substrate and propagation method resulted in the selection and / or metastasis of cancer stem cell ancestral cells from the originally mixed tumor group obtained from the small needle biopsy. Propagation and propagation in serum-containing medium (0.5-5%) can accelerate the proliferation step after 1-2 passes; The combination of a collagen-based substrate (gelatin, RGD peptide) and growth factors such as FGF and EGF may cause unwanted proliferation of fibroblasts or myoblasts that may persist during the selection step.

Example 2

Loaded  Production of dendritic cell compositions

The antigen source is autologous tumor cells from continuously proliferating, self-renewing cells derived from the patient's fresh tumor tissue. These cells have characteristics of tumor stem cells. At all times in surgery and pathology settings, the biopsy is treated with stringent attachment to a sterilization protocol to ensure that the sample is sterile.

The pathologist obtains fresh tissue from the biopsy of the patient's tumor. Using a sterile scalpel and forceps, the specimen is cut into 10 mm slices and transferred to a transport tube containing the transport medium, working quickly to avoid sample drying. Samples are shipped to the manufacturing facility within 48 hours of surgical resection.

In a manufacturing facility, samples are placed in cell culture conditions designed to dissociate into a single cell suspension in a clean room and enrich and proliferate HCC-CSC. During processing of tumor samples, normal cells such as lymphocytes, stromal cells and connective tissue are removed. Upon completion of the proliferation and purification steps, concentrated proliferative HCC-CSC (tumor cells, TC) is inactivated by irradiation (apoptosis facilitating antigen exposure to antigen-presenting cells) and placed in vapor phase liquid nitrogen storage . This process can take up to 8 weeks, depending on the amount and quality of tumor samples.

If the tumor cell product has an assured quality assurance, the patient is notified to undergo a procedure called leukocyte separation and export (usually 6 liters procedure). This process involves the filtering of blood to collect peripheral blood mononuclear cells (PBMC). The collected PBMCs are shipped to the manufacturing facility for further purification by backwash density centrifugation, referred to as purification. Purification is a process in which monocytes are purified from other lymphocytes to enrich for cells that can be turned into antigen presenting cells or dendritic cells. To generate dendritic cells, the purified mononuclear cells are incubated with cytokines GM-CSF and interleukin-4 (IL-4) for 6 days.

On day 6, the purified tumor cell product is removed from cryopreservation, thawed and combined with dendritic cells for 18-24 hours. This process results in an "antigen load" of DC. The final product may be entirely DC or some residual irradiated TC (considered acceptable) and is referred to as DC-TC. The combined dendritic cell / tumor cell mixture is collected and cryopreserved to maintain the viability of dendritic cells and stored in vapor phase liquid nitrogen.

Upon completion of quality control testing and release of autologous tissue cell therapy products, the batch is transported to the treatment facility under vapor-liquid nitrogen conditions. After arrival, the cell therapy products are stored under vapor-liquid nitrogen conditions until they are prepared for administration.

Example 3

Step I Hepatocytes Carcinoma  Stem cells from autologous tissues in patients with hepatitis B-positive Pulsed  Trial of specific immunotherapy by autologous dendritic cells

Hepatocellular carcinoma is rarely cured by standard therapy because of the microscopic metastatic disease that is not detectable in diagnosis. Moreover, HCC is often associated with evidence of hepatitis B virus (HBV) infection, which makes the patient unsuitable for liver transplantation. Activity-specific immunotherapy (ASI), in which autologous dendritic cells are loaded into antigen from autologous tumor stem cells, was associated with promising long-term survival outcomes in patients with metastatic melanoma, but patients with HBV were excluded from such trials. This ASI approach is worth evaluating in other tumor types, including HCC. Surgical resection is part of the standard of care for many patients, thereby providing a source of tumors for the development of autologous tumor cell lines. Although ASI has not been associated with significant toxicity in previous clinical studies (melanoma, stages 1 and 2), theoretically ASI can "switch" the endogenous immune response to HBV and thus lead to worsening of the viral infection . For this reason, Phase I safety trials were warranted prior to consideration of Phase II efficacy trials in patients with HCC and HPB.

Methods: The main eligibility criteria for patient registration were primary untreated hepatocellular carcinoma (> 5 cm isolated lesion or multiple lesions ≥ 3 cm) well tolerated surgical resection, history of HBV not candidates for liver transplantation, leukocyte separation The candidate qualification for liver trans-catheter arterial chemoembolization (TACE), A, the Child-Pugh Rating of ECOG 0-1, and the appropriate blood count, albumin ≥ 3.5 mg / dl, < / RTI > and up to three-fold elevation of the amino transferase.

Cell suspensions resulting from collagenase digestion of the tumor samples for 30-60 minutes are shown in Table 2, Table 2, supplemented with 10 ng / mL bFGF and 10 ng / mL EGF, to produce lines of proliferating HCC- 3 and the super-low adhesion flask (Corning) in the culture medium of Table 4 and maintained in it for 14 days.

Spoloids, composed of 100 to about 10,000 cells, were formed and purified by differential gravity in the feed. Spheroids were then transferred to a 0.1% gelatin coated flask and allowed to attach and propagate. During another 3-4 week period, the culture was gradually proliferated by dissociation with enzyme and flattened on larger surface every 3-7 days. The medium was further supplemented with 5% fetal bovine serum (FBS) at this stage. At the final harvest, the cells were exposed to 100 Gy radiation in a cobalt-60 source radiator. The irradiation efficiency was confirmed by non-proliferative assays in each case.

Dendritic cells (DCs) were derived from peripheral blood mononuclear cells (PBMC) obtained by leukapheresis withdrawal from the same patient as the tumor sample. The HCC-CSC (TC) was then incubated with DC overnight (18-24 hours) to generate a cryopreserved DC / HCC-CSC composition. In the meantime, the patient received one course of TACE and started on the DC / TC composition after 6-8 weeks. At the time of treatment, the DC / TC composition was fused and suspended in 500 pg granulocyte macrophage colony stimulating factor (GM-CSF) for subcutaneous injection every week for 3 weeks and the patient was monitored for toxicity for an additional 5 weeks.

Results : Tumors were collected from 18 patients and cell lines were established from these 18 patient samples. The phenotypes of the characterized HCC-CSC are included in Table 6. The HCC-CSC phenotype was defined by the abundant presence of AFP and NCAM, the ability to greatly proliferate (Ki67), and the different levels and amounts of other specific markers (EpCAM, CK19, CK7, ABCG2). The presence of AFP, the current markers currently recommended for clinical use in liver malignancies, was confirmed in all irradiated samples. This data demonstrates the positive selection of HCC-CSC characterized by the presence of AFP and NCAM on patient-induced cell lines and suggests an epithelial-mesenchymal process based on the low expression of epithelial markers (EpCAM and cytokeratin) .

Table 6 . Immuno-cytochemical characterization of tumor samples successfully obtained from HCC-CSC cell lines.

Eight patients were treated with the DC / TC composition and all eight received the planned three injections. The treated patients included one male and seven women included. The median age was 56.5 years, ranging from 37 to 73. At the time of treatment, all patients had an ECOG of zero. The injection was well tolerated as not having severe acute toxicity. There was no increase in hepatic amino transferase or hepatitis B viremia as evidenced by complete quantitative viral testing, including quantitative HBV DNA analysis. Serious or life threatening (grade 4 or 5) toxicity was not recorded.

Conclusion: Treatment with autologous DC / TC composition isolated from HCC associated with hepatitis B is not associated with worsening of hepatitis B infection.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term " about. &Quot; As used herein, the terms "about" and "about" mean within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the algebraic parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each algebraic parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. The numerical values set forth in the specific examples are reported as precisely as possible, even though the numerical ranges and parameters setting forth the broad scope of the invention are approximations. However, any numerical value inherently contains an error necessarily resulting from the standard deviation found in its respective testing measurement.

It is to be understood that the terms "a", "an", "the" and similar indicia used in the context of describing the invention (especially in the context of the following claims) Unless explicitly stated to the contrary, it should be construed to cover both the singular and the plural. The description of a range of values herein is merely intended to serve as a shorthand method of referring individually to each individual value within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as it is individually quoted herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illuminate the invention and does not pose a limitation on the scope of the invention as otherwise claimed. The language in the specification should not be construed as representing any non-claimed element essential to the embodiments of the present invention.

The grouping of elements or implementations of the invention disclosed herein should not be construed as a limitation. Each group member may be recited and claimed individually or in any combination with other members or other elements of the group found herein. It is expected that one or more members of the group may be included or removed from the group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification shall be deemed to include a group as modified and thus fulfills the written description of all the groups of macros used in the appended claims.

Certain embodiments of the invention, including the best mode known to the inventors of the present invention, are described herein. Of course, variations on these described implementations will be apparent to those skilled in the art upon reading the foregoing description. The inventor expects a skilled person to appropriately use such changes, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. In addition, any combination of the above-described elements within all possible variations thereof is included by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Certain implementations disclosed herein may be further limited in the claims using the language consisting essentially of or consisting of. When used in the claims, whether submitted or supplemented by an amendment, the terminology " comprising " excludes any element, step, or component not listed in the claims. The term " consisting essentially of " encompasses the claims to those that do not materially affect the specified substance or step and the underlying, new feature (s). The embodiments of the invention thus claimed are essentially or specifically described and are possible.

Moreover, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are incorporated herein by reference in their entirety.

Finally, it is to be understood that the embodiments of the invention disclosed herein exemplify the principles of the invention. Other variations that may be used are within the scope of the present invention. Thus, by way of example and not limitation, alternative configurations of the present invention may be used in accordance with the teachings herein. Accordingly, the present invention is not limited to what is shown and described.

Thus, while a radically new feature and / or aspect of the disclosure as applied to an exemplary implementation is shown, described, and pointed out, it will be appreciated that various omissions, It will be appreciated that placement and substitution and variation can be made by those skilled in the art without departing from the spirit of the disclosure and / or claims. For example, all combinations of those elements and / or method steps that perform substantially the same function in substantially the same manner to achieve the same result are expressly intended to be within the scope of the disclosure. In addition, any structure and / or element and / or method steps shown and / or described in connection with any disclosed form or implementation of the invention may be incorporated into any other disclosed or described or proposed form or practice as a general matter of design choice Points should be recognized. Accordingly, it is intended that the scope of the disclosure is not limited. All such modifications are intended to be within the scope of the appended claims.

All publications, patents, patent applications, references, and sequence listings cited herein are hereby incorporated by reference in their entirety as if fully set forth herein.

The summary is provided in accordance with 37 CFR § 1.72 (b) to allow the reader to quickly ascertain the nature and substance of technical content. The summary is submitted on condition that it is not used to interpret or limit the scope or meaning of the claims.

Claims (71)

An immunogenic composition comprising a dendritic cell in vitro activated by a tumor antigen derived from a population of purified HCC cancer stem cells (CSC). 2. The immunogenic composition of claim 1, wherein said tumor antigen comprises a cellular extract of said HCC-CSC. 2. The immunogenic composition of claim 1, wherein said tumor antigen comprises a lysate of said HCC-CSC. 2. The immunogenic composition of claim 1, wherein said tumor antigen comprises intact HCC-CSC. 5. The immunogenic composition of claim 4, wherein the intact cell becomes non-proliferative. 6. The immunogenic composition of claim 5, wherein the intact cell becomes non-proliferative by irradiation. 5. The immunogenic composition of claim 4, wherein the intact cell becomes non-proliferative by exposure of the cell to a nuclear crosslinking agent. 8. The immunogenic composition of any one of claims 1 to 7, further comprising a pharmaceutically acceptable carrier and / or excipient. 9. The immunogenic composition according to any one of claims 1 to 8, further comprising an adjuvant. 10. The immunogenic composition of claim 9, wherein the adjuvant is a granulocyte macrophage colony stimulating factor. 11. The immunogenic composition according to any one of claims 1 to 10, wherein the composition comprises activated dendritic cells and HCC-CSC. 12. The immunogenic composition according to any one of claims 1 to 11, wherein the HCC-CSC is in the form of HCC-CSC spheroids. 12. The immunogenic composition according to any one of claims 1 to 11, wherein the HCC-CSC is in the form of an early HCC-CSC. 12. The immunogenic composition according to any one of claims 1 to 11, wherein the HCC-CSC is in the form of a mixed HCC-CSC. 12. The immunogenic composition according to any one of claims 1 to 11, wherein said HCC-CSC is in the form of EMT-HCC-CSC. A method of treating hepatocellular carcinoma in a subject in need of such treatment comprising administering to said subject an immunogenic composition of any one of claims 1 to 15. 17. The method of claim 16, wherein the immunogenic composition is administered in a plurality of doses, each dose comprising about 5-20 x ¹⁰ cells. 18. The method of claim 17, wherein the dose comprises about 10 x ¹⁰ cells. 19. The method according to any one of claims 16 to 18, wherein said dose is administered monthly for two to five doses, then three to six doses each month. 20. The method of any one of claims 16 to 19, wherein the subject is accommodated from 6 to 10 volumes of the immunogenic composition. . Use of an immunogenic composition according to any one of claims 1 to 15 in the manufacture of a medicament for the treatment of hepatocellular carcinoma. The use of an immunogenic composition according to any one of claims 41 to 15 for the treatment of hepatocellular carcinoma. A method of producing a population of hepatocellular carcinoma (HCC) cancer stem cells (CSC)
Obtaining a sample of HCC;
Dissociating the cells of the sample, and
Culturing the dissociated cells in vitro in a restricted medium on a non-adherent substrate, wherein the restricted medium is augmented with at least one growth factor that is serum-free and acts through a mitogen-activated protein kinase (MAPK) pathway , Thereby forming HCC-CSC spheroids;
Wherein at least 80% of said cells in said HCC-CSC spoloid population express more than one of biomarker alpha-fetoprotein (AFP), EpCAM, Ov1, and OV6. 26. The method of claim 24, wherein at least 80% of the cells in the HCC-CSC spoloid population further express at least one of the biomarkers CK7, CK19, and E-carderine. 27. The method of claim 24, wherein at least 90% of the cells in the HCC-CSC spoloid population express more than one of the biomarkers AFP, EpCAM, Ov1, and OV6. 25. The method of claim 24,
Further comprising the step of culturing said HCC-CSC spheroids in a limiting medium on an adherent substrate,
Wherein said restricted medium is serum-free and is supplemented with at least one growth factor acting through said MAPK pathway thereby forming a population of early HCC-CSCs, wherein at least 80% of said cells in said initial HCC- Marker Nanog, Sox2, Oct3 / 4, and c-kit. 28. The method of claim 27, wherein at least 80% of the cells in the initial HCC-CSC population are infected with one or more of biomarkers EpCAM, E-carderine, Sox7, Sox17, Fox2A, Ov1, OV6, CD133, ≪ / RTI > 28. The method of claim 27, wherein at least 90% of the cells in the initial HCC-CSC population express two or more of the biomarkers Nanog, Sox2, Oct3 / 4, and c-kit. 25. The method of claim 24,
Further comprising the step of culturing said HCC-CSC spheroids in a limiting medium on an adherent substrate,
Wherein the restricted media is supplemented with at least one growth factor containing serum and acting through the MAPK pathway thereby forming a population of mixed HCC-CSCs, wherein at least 80 of the cells in the mixed HCC-CSC population % Expresses two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Nanog, FoxA2 HNF4a, and ABCG2. 31. The method of claim 30 wherein at least 90% of the cells in the mixed HCC-CSC population express two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Nanog, FoxA2 HNF4a, Way. 25. The method of claim 24,
Further comprising the step of culturing said HCC-CSC spheroids in a limiting medium on an adherent substrate,
Wherein said restricted media is supplemented with at least one growth factor containing a serum source and acting through said MAPK pathway thereby forming a population of embryo-transplanted (EMT) -HCC-CSCs, said EMT-HCC- Wherein at least 80% of the cells in the CSC population express two or more of the biomarker NCAM, Slug / Snail, and Twist. 33. The method of claim 32, wherein at least 80% of the cells in the EMT-HCC-CSC population further express at least one of the biomarkers AFP, N-carderine, CD44, and vimentin. 33. The method of claim 32, wherein at least 90% of the cells in the EMT-HCC-CSC population express one or more of the biomarker NCAM, slug / snail, and twist. 33. The method according to any one of claims 24, 30, or 32,
Further comprising the step of culturing said HCC-CSC spleoid, said mixed HCC-CSC, or EMT-HCC-CSC in a restricted medium on an adherent substrate,
Wherein said restricted medium is serum-free and is supplemented with at least one growth factor acting through said MAPK pathway thereby forming a population of early HCC-CSCs, wherein at least 80% of said cells in said initial HCC- Marker Nanog, Sox2, Oct3 / 4, and c-kit. 36. The method of claim 35, wherein at least 80% of the cells in the initial HCC-CSC population express one or more of the biomarkers CK7, CK19, and E-carderine. 36. The method of claim 35, wherein at least 90% of the cells in the initial HCC-CSC population express one or more of the biomarkers Nanog, Sox2, Oct3 / 4, and c-kit. 33. The method according to any one of claims 24, 27 or 32,
Further comprising the step of culturing said HCC-CSC spheroid, said initial HCC-CSC, or EMT-HCC-CSC in a restricted medium on an adherent substrate,
Wherein the restricted media is supplemented with at least one growth factor containing a serum source and acting through the MAPK pathway thereby forming a population of mixed HCC-CSCs and expressing at least one of the cells in the mixed HCC-CSC population 80% express two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Nanog, FoxA2 HNF4a, and ABCG2. 40. The method of claim 39, wherein at least 90% of the cells in the mixed HCC-CSC population express two or more of the biomarkers AFP, CK7, CK19, EpCAM, E-carderine, Nanog, FoxA2 HNF4a, Way. 32. The method according to any one of claims 24, 27 or 30,
Further comprising the step of culturing said HCC-CSC spleoid, said initial HCC-CSC, or mixed HCC-CSC in a restricted medium on an adherent substrate,
Wherein said restricted medium is supplemented with at least one growth factor that contains a serum source and acts through said MAPK pathway thereby forming a population of EMT-HCC-CSCs and at least one of said cells in said EMT-HCC-CSC population 80% express two or more of the biomarker NCAM, slug / snail, and twist. 41. The method of claim 40, wherein at least 80% of the cells in the EMT-HCC-CSC population further express at least one of the biomarkers AFP, N-carderine, CD44, and vimentin. 41. The method of claim 40, wherein at least 90% of the cells in the EMT-HCC-CSC population express at least one of a biomarker NCAM, a slug / snail, and a twist. 43. The method according to any one of claims 24 to 42, wherein the restriction medium is any of the mediums listed in Table 2. 43. The method according to any one of claims 24 to 42, wherein said restriction medium is any medium from the combination of Table 2 and Table 3. 43. The method according to any one of claims 24 to 42, wherein the restriction medium is any medium from combinations of Table 2, Table 3, and Table 4. 43. The method according to any one of claims 24 to 42, wherein the restriction medium is any medium from the combination of Table 2 and Table 4. 25. The method of claim 24, wherein the growth factor is at least one of fibroblast growth factor (FGF), epithelial growth factor (EGF), or actin A. 26. The method of claim 25, wherein the FGF is a basic FGF (bFGF). 49. The method according to any one of claims 24 to 48, wherein the restriction media are not reinforced with actin A. 49. The method according to any one of claims 24 to 49, wherein the restriction medium is supplemented with an agonist of Actin A in an amount effective to prevent spontaneous differentiation of HCC stem cells. 28. The method of claim 27, wherein the antagonist of actin A is a pol statin or an antibody that specifically binds to actin A. 52. The method according to any one of claims 24 to 51, wherein the medium is not reinforced with an antioxidant. 53. The method of claim 52, wherein said antioxidant is superoxide dismutase, catalase, glutathione, putrescine, or beta-mercaptoethanol. 52. The method according to any one of claims 24 to 51, wherein the medium is fortified with glutathione. 54. The method of any one of claims 24 to 54, wherein the adherent substrate is attached to anchorage dependent cells and is configured to collect them. 57. The method of claim 55, wherein the adhesion-dependent cell is a fibroblast. 55. The method of any of claims 24 to 54, wherein the non-adherent substrate is an ultra-low adherent polystyrene surface. 58. The method of claim 57, wherein the adherent substrate comprises a surface coated with an RGD tripeptide motif-enriched protein. A population of purified HCC-CSC cells produced by the method of any one of claims 24 to 58. 60. The population of purified HCC-CSC cells of claim 59, wherein said purified HCC-CSC cells are HCC-CSC spleod. 60. The population of purified HCC-CSC cells of claim 59, wherein said purified HCC-CSC cells are early HCC-CSC. 60. The population of purified HCC-CSC cells of claim 59, wherein said purified HCC-CSC cells are mixed HCC-CSC. 59. The population of purified HCC-CSC cells of claim 59, wherein said purified HCC-CSC cells are EMT-HCC-CSC. 29. An HCC-CSC cell line produced by the method of any one of claims 1 to 29. 65. The HCC-CSC cell line of claim 64, wherein said HCC-CSC is HCC-CSC spe- croid. 65. The HCC-CSC cell line of claim 64, wherein the HCC-CSC is an initial HCC-CSC. 65. The HCC-CSC cell line of claim 64, wherein the HCC-CSC is a mixed HCC-CSC. 65. The HCC-CSC cell line of claim 64, wherein said HCC-CSC is EMT-HCC-CSC. A method for stimulating an immune response to hepatocellular carcinoma in a subject in need of stimulation of an immune response, comprising administering to the subject an immunogenic composition according to any one of claims 1 to 15, Comprising administering to a subject HCC-CSC cell, or a HCC-CSC cell line of any one of claims 64 to 68. Use of the HCC-CSC cell of any one of claims 59 to 63 or the HCC-CSC cell line of any one of claims 64 to 68 in the manufacture of a medicament for the treatment of hepatocellular carcinoma. 63. Use of the HCC-CSC cell of any one of claims 59 to 63 or the HCC-CSC cell line of any one of claims 64 to 68 for the treatment of hepatocellular carcinoma.

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