Induction of tumor vascular necrosis using fibroblasts

Abstract

Embodiments of the present disclosure relate to methods and compositions relating to the treatment of cancer in an individual using recombinant fibroblasts that have one or more endothelial cell-like activities. The cells are delivered to the tumor microenvironment, where their death results in destabilization of the tumor vasculature. In certain embodiments, the fibroblasts recombinantly express one or more of ETV2, FOXC2, and FLI1.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 184,960, filed May 6, 2021, which is incorporated by reference in its entirety.

[0002] (Technical field) Embodiments of the present disclosure relate to at least the fields of cell biology, molecular biology, and medicine. [Background technology]

[0003] Harnessing the immune system to kill cancer is an area of ​​active research. Immunological control of neoplasms is suggested by: A) evidence of longer survival in patients with various cancers who have a high population of tumor-infiltrating lymphocytes [1-3]; B) the fact that immunosuppressed patients develop cancer at a much higher frequency than non-immunosuppressed patients [4,5]; and C) the clinical effectiveness of cancer immunotherapy in very specific circumstances [6]. While cancer immunotherapy has the potential to remit and control not only primary tumors but also micrometastases, it does have several drawbacks. The most significant drawback is that in many situations immunotherapy is either not feasible or is associated with various toxicities. Various immunotherapies for cancer have been attempted, including a) systemic administration of cytokines, b) gene therapy, c) allogeneic vaccines, d) autologous vaccines, e) heat shock protein vaccines, f) dendritic cell vaccines, g) tumor-infiltrating lymphocytes, h) T cell administration in lymphopenic environments, and i) nutritional interventions. Each approach has important advantages and disadvantages, but none simultaneously meets the criteria of reproducible efficacy, accessibility to large populations, and tumor selectivity / specificity.

[0004] A limitation of many immunotherapeutic approaches to cancer is that tumor antigens are not clearly defined, or if defined, tumors mutate and lose expression of such antigens, or antigen-specific vaccines are only applicable to patients with specific major histocompatibility complex haplotypes. Attempts have been made to circumvent this problem by using autologous vaccines, but this is often an expensive and difficult procedure. Summary of the Invention

[0005] The present disclosure is directed to methods and compositions that are directly or indirectly therapeutic to a recipient individual. In certain embodiments, the therapy comprises cells, including modified cells, provided to an individual in need thereof. In certain embodiments, the cells are modified by the hand of a human before being delivered to the individual. In certain embodiments, fibroblasts are modified and a therapeutically effective amount of the modified cells is administered to an individual with a medical condition, such as cancer.

[0006] In certain embodiments, fibroblasts are exposed to one or more agents and / or conditions that cause the fibroblasts to exhibit more endothelial cell-like activity. In certain embodiments, fibroblasts are exposed to one or more gene products, resulting in more endothelial cell-like behavior than in the absence of such exposure. In various embodiments, fibroblasts are utilized as a means to generate endothelial cells. The "artificial" endothelial cells produced by the disclosed methods enhance the ability of the cells to enter the tumor microenvironment, resulting in the death of the tumor cells. In various embodiments, once the modified fibroblasts enter the tumor microenvironment, they commit suicide via suicide genes or death-inducing genes that cause destabilization of the tumor vasculature. In some embodiments, the individual can be administered any type of alternative therapy that would otherwise be less effective due to conditions in the tumor microenvironment.

[0007] In various embodiments, the fibroblasts express one or more recombinant genes (as opposed to genes endogenous to the fibroblasts) that promote more endothelial cell-like activity. In specific embodiments, the fibroblasts are modified to express one or more of ETV2, FOXC2, and FLI1, such that the modified fibroblasts exhibit one or more characteristics of an endothelial cell and / or a vascular channel. In embodiments in which two or more of the ETV2, FOXC2, and FLI1 genes may or may not be expressed from the same polynucleotide, such as any type of transfected vector, the two or more genes may or may not be expressed from the same polynucleotide, such as any type of transfected vector.

[0008] In various embodiments, the present disclosure provides one or more different methods of creating "artificial" tumor endothelial cells that act as immunogenic compositions, such as "vaccines," to induce immunity against cancer vasculature. In specific embodiments, transarterial chemoembolization (TACE) is utilized as one means of stimulating immunity against cancer endothelium.

[0009] An embodiment of the present disclosure is a method of inducing cell death of tumor cells in an individual, comprising administering to the individual a therapeutically effective amount of a plurality of modified fibroblasts, wherein (I) the fibroblasts express recombinant: (a) one or more endothelial-inducing genes and / or one or more vascular channel-inducing genes (or one or more factors that upregulate them); and (b) one or more suicide- or death-inducing genes (or one or more factors that upregulate them); and (c) optionally one or more immune stimulatory genes; and / or (II) the fibroblasts are cultured in endothelial progenitor cell conditioned medium. In certain embodiments, the fibroblasts express recombinant ETV2, FOXC2, and / or FLI1. In certain aspects, the fibroblasts express recombinant ETV2 and are cultured in a medium comprising an effective amount of one or more of VEGF, EGF, HGF, and IGF-1. In certain embodiments, the fibroblasts express recombinant FOXC2 and are cultured in a medium comprising an effective amount of one or more of VEGF, EGF, HGF, and IGF-1. In certain embodiments, the fibroblasts express recombinant FLI1 and are cultured in a medium comprising an effective amount of one or more of VEGF, EGF, HGF, and IGF-1. In certain embodiments, the endothelial cell progenitor cell conditioned medium is made from pluripotent stem cells differentiated into endothelial progenitor cells. The pluripotent stem cells may be embryonic stem cells, induced pluripotent stem cells, somatic cell nuclear transfer derived stem cells, parthenogenetic derived stem cells, or those differentiated into endothelial progenitor cells by transfection with ETV2, FOXC2, FLI1. In various embodiments, the fibroblasts are transfected with one or more thrombosis-related genes, which are upregulated in response to hypoxia. The thrombosis-related genes may be tissue factor or inhibitor of protein C.

[0010] In certain embodiments, the fibroblasts may be transfected with one or more immunostimulatory genes and induced by the presence of hypoxia. Induction of the immunostimulatory genes may be achieved by placing the genes under the control of the HIF-1α transcription factor. The immunostimulatory genes may be associated with antigen presentation, such as allogeneic MHC molecules. The genes associated with antigen presentation may be xenogeneic MHC molecules. In specific embodiments, the genes associated with antigen presentation may be one or more of HLA B7 molecules, CD80, CD86, and CD40. The immunostimulatory gene may be interleukin 12.

[0011] In various embodiments, the fibroblasts are selected for expression of one or more of CXCR4, CD73, CD74, CD206, and interleukin-3 receptor. The fibroblasts may be allogeneic, allogeneic, or xenogeneic to any recipient.

[0012] In certain embodiments, there is a method for inducing immunogenic cell death of tumor endothelial cells in an individual, comprising: a) transfecting a population of fibroblast cells with one or more endothelial cell-inducing genes and / or one or more vascular channel-inducing genes; c) transfecting the fibroblast cells with one or more suicide or death-inducing genes; d) optionally transfecting the fibroblast cells with one or more immune stimulating genes; and e) administering the fibroblast cells to an individual with cancer. The fibroblast cells may be obtained from one or more tissues selected from the group consisting of: a) dermis; b) bone marrow; c) blood; d) mobilized peripheral blood; e) gums; f) tonsils; g) placenta; h) Wharton's jelly; i) hair follicles; j) fallopian tubes; k) liver; l) milk teeth; m) vas deferens; n) endometrium; o) menstrual blood; and p) fallopian tubes. In certain embodiments, mobilization of peripheral blood is achieved by treatment of the mammal with an effective amount of one or more inhibitors of SDF-1 binding to CXCR4. The inhibitor of SDF-1 binding to CXCR4 can be Plerixafor or BKT140. Mobilization may be induced by exposure to hyperbaric oxygen therapy. Mobilization may be induced by treatment with GM-CSF and / or M-CSF and / or flt-3 ligand. In various embodiments, the fibroblasts are selected for expression of one or more of CXCR4, CD73, CD74, CD206, and interleukin-3 receptor. The fibroblasts may be allogeneic, allogeneic, or xenogeneic to any recipient.

[0013] In certain embodiments, fibroblasts are cultured in endothelial progenitor cell conditioned medium, such as medium made from pluripotent stem cells differentiated into endothelial progenitor cells. The pluripotent stem cells may be embryonic stem cells, induced pluripotent stem cells, somatic cell nuclear transfer-derived stem cells, parthenogenetic stem cells, or those differentiated into endothelial progenitor cells by transfection with ETV2, FOXC2, or FLI1. Fibroblasts can be differentiated into endothelial progenitor cells by transfection with ETV2. Fibroblasts can be differentiated into endothelial progenitor cells by transfection with ETV2 and culturing in a medium containing VEGF. Fibroblasts can be differentiated into endothelial progenitor cells by transfection with ETV2 and culturing in a medium containing EGF. Fibroblasts can be differentiated into endothelial progenitor cells by transfection with ETV2 and culturing in a medium containing HGF. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with ETV2 and culturing them in a medium containing IGF-1. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with FOXC2. Fibroblasts can be differentiated into endothelial progenitor cells by introducing FOXC2 and culturing them in a medium containing VEGF. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with FOXC2 and culturing them in a medium containing EGF. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with FOXC2 and culturing them in a medium containing HGF. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with FOXC2 and culturing them in a medium containing IGF-1. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with FLI1. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with FLI1 and culturing them in a medium containing VEGF. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with FLI1 and culturing them in a medium containing EGF. Fibroblasts can be differentiated into endothelial progenitor cells by transfecting them with FLI1 and culturing them in a medium containing HGF.Fibroblasts can be differentiated into endothelial progenitor cells by transfection with FLI1 and culturing in a medium containing IGF-1.

[0014] In some embodiments, the fibroblasts are transfected with one or more thrombosis-inducing genes or molecules, where the genes are upregulated in response to hypoxia. The thrombosis-related genes or molecules may be tissue factor or inhibitors of protein C. In some embodiments, the fibroblasts are transfected with one or more immunostimulatory genes that can be induced by the presence of hypoxia. In some cases, the induction of the immunostimulatory genes is by placing the genes under the control of the HIF-1α transcription factor. The immunostimulatory genes may be associated with antigen presentation, including antigen presentation that is an allogeneic MHC molecule. The gene associated with antigen presentation may be a xenogeneic MHC molecule. The gene associated with antigen presentation may be an HLA B7 molecule, CD80, CD86, and / or CD40. The immunostimulatory gene may be interleukin-12.

[0015] The features and technical advantages of the present disclosure have been outlined above, rather broadly, in order that the detailed description that follows may be better understood. Additional features and advantages will be described below which form the subject matter of the claims herein. It should be understood by those skilled in the art that the ideas and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. It should also be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as defined by the appended claims. The novel features believed characteristic of the design disclosed herein, both as to organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each figure is provided for the purpose of illustration and description only, and is not intended as a definition of the limits of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] I. Definition example As used herein, "a" or "an" can mean one or more. As used herein in a claim(s), when used in conjunction with the word "comprising", "a" or "an" can mean one or more. Some embodiments of the present disclosure can consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments can be combined.

[0017] The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or unless the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or". For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z can be specifically excluded from an embodiment. As used herein, "another" can mean at least a second or more.

[0018] As used herein, the term "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0019] As used herein, the term "set" means one or more. For example, a set of items includes one or more items.

[0020] As used herein, the phrase "at least one" when used in conjunction with a list of items means that one or more different combinations of the listed items may be used, and only one of the items in the list may be required. An item may be a specific object, thing, step, operation, process, or category. In other words, "at least one" means that any combination or number of items from the list may be used, but not all items in the list are required. For example, and without limitation, "at least one of item A, item B, or item C" means item A; item A and item B; item B; item A, item B, and item C; item B and item C; or item A and item C. In some cases, "at least one of item A, item B, or item C" means, but is not limited to, two of item A, one of item B, and ten of item C; four of item B, and seven of item C; or other suitable combinations.

[0021] As used herein, "substantially" means sufficient to function for the intended purpose. Thus, the term "substantially" allows for minor and inconsequential variations from absolute or perfect conditions, dimensions, measurements, results, etc., as would be expected by one of ordinary skill in the art, but which do not significantly affect overall performance. When used in reference to a parameter or characteristic that is a numerical value or can be expressed as a numerical value, "substantially" means within 10%.

[0022] Throughout this specification, unless the context dictates otherwise, the terms "consisting of," "consist of," and "consisting of" are understood to mean the inclusion of the recited step or element or steps or elements, but not the exclusion of other steps or elements or steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and other elements may not be present. "Consisting essentially of" means including the elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0023] Throughout this specification, references to "one embodiment," "one embodiment," "particular embodiment," "related embodiment," "an embodiment," "additional embodiment," or "further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in the various embodiments.

[0024] "Treatment" or treatment of a disease or condition refers to carrying out a protocol that may include administering one or more drugs to an individual, such as a patient, to alleviate the signs or symptoms of the disease. Desirable effects of treatment include reducing the rate of progression of the disease, improving the disease state, or palliating, remission, or improving the prognosis. Alleviation may occur before or after the signs or symptoms of the disease or condition appear. Thus, "treatment" or "treatment" may include "prophylaxis" or "prevention" of a disease or an undesirable condition. Furthermore, "treatment" or "treatment" specifically includes protocols that do not require complete alleviation of signs or symptoms, do not require a cure, and have only a minor effect on the patient.

[0025] The term "therapeutically effective" as used throughout this application refers to promoting or enhancing the well-being of a subject with respect to medical treatment of the condition, including, but not limited to, reducing the frequency or severity of one or more signs or symptoms of a disease, including breast cancer.

[0026] "Marker" and "biomarker" are used interchangeably to refer to a gene expression product that is differentially present in samples taken from two different subjects, e.g., a sample taken from a subject or patient who has had (or is at risk of having) an ischemic event, compared to a comparable sample taken from a control subject (e.g., a subject who has not had (or is at risk of having) an ischemic event; a normal or healthy subject). Alternatively, the term refers to a gene expression product that is differentially present in one cell population compared to another cell population.

[0027] "Differentially present" refers to the amount or frequency (incidence) of a marker present in a sample taken from a subject compared to a control. For example, a marker can be a gene expression product that is present at a higher or lower level in a blood sample from a risk subject compared to a sample taken from a control subject. Alternatively, a marker can be a gene expression product that is detected at a higher or lower frequency in a blood sample from a risk subject compared to a blood sample from a control subject.

[0028] A gene expression product is "differentially present" between two samples if the amount of the gene expression product in one sample is statistically significantly different from the amount of the gene expression product in the other sample. For example, a gene expression product is differentially present between two samples if it is present in at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% more than it is present in the other sample, or if it is detectable in one sample and not detectable in the other sample.

[0029] As used herein, the terms "antibody" and "antibodies" include monoclonal antibodies, multispecific antibodies, synthetic antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFvs), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), and anti-idiotypic (anti-Id) antibodies (e.g., including anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. In particular, antibodies of the invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to a polypeptide antigen. Immunoglobulin molecules of the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 and IgA.sub.2) or subclass of immunoglobulin molecule.

[0030] An "immunoassay" is an assay in which an antibody is used to specifically bind an antigen (e.g., a marker). Immunoassays are characterized by the use of the specific binding properties of a particular antibody to isolate, target, and / or quantify the antigen. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective response is at least two times the background signal or noise, and more commonly 10 to 100 times or more background. When referring to an antibody, the phrase "specifically (or selectively) binds" or when referring to a protein or peptide, "specifically (or selectively) immunoreacts" refers to a binding reaction that determines the presence of the protein in a heterogeneous population of proteins or other biologics. Thus, under designated immunoassay conditions, a particular antibody will bind to a particular protein at least twice background and will not bind significantly to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.

[0031] As used herein, the terms "affecting expression" and "regulating expression" of a protein or gene should be understood as regulating, controlling, blocking, inhibiting, stimulating, enhancing, activating, mimicking, bypassing, modifying, removing and / or replacing said expression, or in more general terms, intervening in said expression, for example by affecting the expression of the gene encoding that protein.

[0032] II. General and Specific Embodiments Disclosed herein are specific embodiments of methods, means, protocols, and compositions of matter useful for inducing tumor regression, such as by inducing vascular necrosis of tumor-associated blood vessels. In one embodiment, the disclosure provides a method for transfecting fibroblasts with one or more genes, such as one or more death-inducing genes, or means for upregulating same. In a specific embodiment, the gene is a transcription factor (including hypoxia-inducible transcription factor) that upregulates one or more death-inducing genes. In one embodiment, fibroblasts are transdifferentiated into endothelial cells, including tumor endothelial cells, and the differentiated endothelial or endothelial-like cells initiate coagulation and / or complement cascades that lead to selective necrosis of tumor vasculature. Examples of death-inducing genes include TNFα, TNFβ, FAS ligand, TRAIL, and the like.

[0033] In some embodiments, the present disclosure encompasses the use of endothelial targeting using fibroblasts and / or endothelial differentiated fibroblasts as a means of enhancing the efficacy of cancer antigen-specific vaccines and / or inducing vascular necrosis to tumors. In one embodiment, the present disclosure encompasses the administration to an individual of any type of fibroblast differentiated into endothelial cells similar to cancer endothelial cells, including, for example, placenta-derived fibroblasts. Targeting of tumor endothelial cells with vaccines is possible, but is not produced from a standardized source [7-10]. The present disclosure provides an embodiment in which tumors are sensitized to treatment with cancer therapeutics, including at least a cancer vaccine. In a specific embodiment, the cancer vaccine consists of a peptide vaccine, a protein vaccine

[11] , a cellular vaccine, and / or an endogenous vaccine. Without being bound by theory, the cancer endothelial targeting using fibroblast approaches can specifically induce inactivation of tumor endothelium-mediated lymphocyte death, thereby allowing cancer-killing T cells to specifically enter the tumor and mediate tumor cell death. In certain embodiments, the treatment to which the tumor is sensitized consists of any type of adoptive cell therapy, including T cells and / or NK cells engineered to express non-endogenous antigen receptors, including chimeric antigen receptors, T cell receptors, etc.

[0034] In one embodiment, endothelial progenitor cells (EPCs) generated from fibroblasts can be used to stimulate immunity against tumor endothelium. The endothelial cells produced by the methods encompassed herein, in one embodiment, consist of a population of cells that have one or more specific surface markers on each cell or on the majority of the cells. Embodiments include populations of cells having the surface marker CD44

[12] , cells having the surface marker CD13 [13, 14], cells having the surface marker CD90 [12, 15-20], cells having the surface marker CD105 [13, 16, 21-27], cells having the surface marker ABCG2, cells having the surface marker HLA1, cells having the surface marker CD34, cells having the surface marker CD133, cells having the surface marker CD117, cells having the surface marker CD135, cells having the surface marker CXCR4, cells having the surface marker c-met, cells having the surface marker CD31, cells having the surface marker CD14, cells having the surface marker Mac-1, cells having the surface marker CD11, cells having the surface marker c-kit, cells having the surface marker SH-2, cells having the surface marker VE-cadherin, cells having the surface marker VEGFR, and / or cells having the surface marker Tie-2s. EPCs may be treated in a manner that mimics the tumor microenvironment; specifically, they may be cultured under the acidic conditions of the tumor microenvironment, the information of which is incorporated by reference [28-41]. In one embodiment of the present disclosure, endothelial progenitor cells or their products are cultured under conditions in which GCN2 kinase is activated [42, 43], including in particular culture in the presence of uncharged tRNA [44-47], tryptophan depletion [48-50], arginine depletion [51-56], asparagine depletion [57-61], and / or glutamine depletion [62, 63].

[0035] In certain embodiments, endothelial cells are produced from fibroblasts after transfecting the fibroblasts with one or more different immunomodulatory genes, including a combination of genes. In certain embodiments, genes related to immunomodulation are transfected into the cells. In specific embodiments, the genes include one or more interleukins, one or more HLA molecules, one or more costimulatory molecules, and / or one or more adhesion molecules.

[0036] In one embodiment, fibroblasts are transfected with one or more cytokine genes, such as interleukin-12, and subsequently induced to differentiate into endothelial cells, which are then administered systemically or locally to the tumor. Administration of fibroblast-derived endothelial cells, in certain embodiments, allows for the induction of immunity against tumor endothelial cells.

[0037] In another embodiment, one or more immunologically active components are transfected under the control of hypoxia-inducible elements and fibroblast-derived endothelial cells are injected into the tumor to induce an immune response against the hypoxia-inducible elements.

[0038] The present disclosure utilizes compositions and methods for reprogramming somatic cells into angiogenic and / or endothelial cells, both in vitro and in vivo, for use in targeting cancer cells. One embodiment includes a polynucleotide comprising two or more nucleic acid sequences encoding a protein selected from the group consisting of ETV2, FOXC2, and FLI1, for generating a cancer vaccine.

[0039] In some embodiments, fibroblasts are reprogrammed into cancer-treating endothelial cells as previously disclosed in US Publication No. 20200115425, which is incorporated herein by reference. In one embodiment, EPC refers to endothelial colony forming cells (ECFCs), whose progenitor potential was characterized as described (Wu, Y et al., J Thromb Haemost, 2010; 8:185-193; Wang, H et al., Circulation research, 2004; 94:843, and Stellos, K et al., Eur Heart J., 2009; 30:584-593). Briefly, human blood was collected from healthy volunteer donors. All volunteers were free of risk factors for CVD, including hypertension, diabetes, smoking, positive family history of premature CVD, hypercholesterolemia, and were free of wounds, ulcers, retinopathy, recent surgery, inflammatory disease, malignant disease, and medications that may affect EPC dynamics. After dilution with HBSS (1:1), blood was layered 1:1 onto Histopaque 1077 (Sigma-Aldrich Co. LLC, St. Louis, Mo.) and centrifuged at 740 g for 30 min at room temperature. Buffy coat MNC were collected and centrifuged at 700 g for 10 min at room temperature. MNC were cultured on collagen type I (BD Bioscience, San Diego) (50 μg / ml)-coated dishes in EBM2 basal medium (Lonza Inc, Allendale, NJ) supplemented with 2% fetal bovine serum (FBS), EGF (20 ng / ml), hydrocortisone (1 μg / ml), bovine brain extract (12 μg / ml), gentamicin (50 μg / ml), amphotericin B (50 ng / ml), and epidermal growth factor (10 ng / ml). Colonies that appeared between 5 and 22 days of culture were identified as a well-ordered monolayer of cobblestone-like cells. ECFCs that expressed endothelial markers in vitro and had robust proliferation, colony-forming, and angiogenic activity were defined as EPCs (Wang, H et al., Circulation research, 2004; 94: 843 and Stellos, K et al., Eur Heart J., 2009; 30: 584-593). EPCs from stages 4 to 6 were used in the experiments. For simple characterization, to confirm endothelial phagocytic function, EPCs were fixed in 2% paraformaldehyde for 10 min after 1,1-dioctadecyl-3,3,3-tetramethylindocarbocyanine (DiI)-labeled acetylated low-density lipoprotein (acLDL) (Biomedical Technologies, Inc.) in 4-well chamber slides for 1 h and 3 washes for 15 min with PBS. Cells were then incubated with FITC-conjugated UEA-1 (Ulex europaeus agglutinin) (10 μg / ml) (Sigma-Aldrich Corporation, St. Louis, Mo.) for 1 h at room temperature. UEA-1 binds to glycoproteins on the cell membrane and allows visualization of the entire cell. Cell integrity was examined by nuclear staining with DAPI (100 ng / ml). After staining, cells were imaged in high-power fields at 200× magnification under an inverted fluorescence microscope (Axiovert 200, Carl Zeiss, Thornwood, NY) and imaged using the Image Quantification was performed using J software.

[0040] In certain embodiments, EPCs may be identified by means of selecting cells expressing one or more specific genes. In certain embodiments, the cells express one or more genes selected from the group consisting of ADORA1, ADORA2A, ADORA2B, ADORA3, AGTRL1 (APLNR), AMPH, APLN, CCBE1, CDC42, CGNL1, CREBBP, CRIP1, CRIP2, CRIP3, CYB5B, DLL4, DUSP5, EEA1, egr-1, ELK1, ELK3, ELK4 (SAP1), EP300, ERG1 (KCNH2), ETS1, ETS2, EXOC3L, FGD1, FGD2, FGD3, FGD4, FGD5. , FLT1, FST, GATA6, GRRP1, HO-1 (HMOX1), HO-2 (HMOX2), IFNG, IL1A, IL1B, LAMA4, Lamb1-1, LGMN, MMP3, Nos2, PAI1, PHD1, PLVAP, RAB5a, RIN3, ROCK2, SOX18, SOX7, SRF, STAB1, STAB2, STUB1, TFEC, THBS1, THBS2, THBS3, THBS4, THBS5, THSD1, TNFAIP8, and XLKD1 (LYVE1). In specific embodiments, EPCs may be purified from a variety of sources, including peripheral blood, placental cells, umbilical cord blood, umbilical cord, adipose tissue, and / or bone marrow.

[0041] In another embodiment, the EPCs are characterized by expression of at least one gene, and in a specific embodiment, ADORA1, ADORA2A, ADORA2B, ADORA3, AGTRL1 (APLNR), AMPH, APLN, CCBE1, CDC42, CGNL1, CREBBP, CRIP1, CRIP2, CRIP3, CYB5B, DLL4, DUSP5, EEA1, egr-1, ELK1, ELK3, ELK4 (SAP1), EP300, ERG1 (KCNH2), ETS1, ETS2, EXOC3L, FGD1, FGD2, FGD3, FGD4, FGD5, FLT1, FST, GA The tumor expresses at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or all of the genes selected from the group consisting of TA6, GRRP1, HO-1 (HMOX1), HO-2 (HMOX2), IFNG, IL1A, IL1B, LAMA4, Lamb1-1, LGMN, MMP3, Nos2, PAI1, PHD1, PLVAP, RAB5a, RIN3, ROCK2, SOX18, SOX7, SRF, STAB1, STAB2, STUB1, TFEC, THBS1, THBS2, THBS3, THBS4, THBS5, THSD1, TNFAIP8, and XLKD1 (LYVE1). In certain embodiments, EPCs are characterized by expression of one or at least 2, 3, 4, 5, 10, 15, 20, 25, 30, or all of the genes selected from the group consisting of ADORA2A, AGTRL1 (APLNR), APLN, CCBE1, CGNL1, CRIP2, CYB5B, DLL4, DUSP5, ELK3, ERG1 (KCNH2), ETS1, ETS2, EXOC3L, FGD5, GRRP1, HO-1 (HMOX1), HO-2 (HMOX2), LAMA4, Lamb1-1, LGMN, PLVAP, RIN3, ROCK2, SOX7, SOX18, STAB1, STAB2, STUB1, TFEC, THSD1, TNFAIP8, and XLKD1 (LYVE1).In a specific embodiment, the step of increasing the number of activated endothelial progenitor cells comprises increasing the number of activated endothelial progenitor cells in the blood of the subject by expression of ADORA1, ADORA2A, ADORA2B, ADORA3, AGTRL1 (APLNR), AMPH, APLN, CCBE1, CDC42, CGNL1, CREBBP, CRIP1, CRIP2, CRIP3, CYB5B, DLL4, DUSP5, EEA1, egr-1, ELK1, ELK3, ELK4 (SAP1), EP300, ERG1 (KCNH2), or other endothelial progenitor cells in the blood of the subject. , ETS1, ETS2, EXOC3L, FGD1, FGD2, FGD3, FGD4, FGD5, FLT1, FST, GATA6, GRRP1, HO-1(HMOX1), HO-2(HMOX2), IFNG, IL1A, IL1B, LAMA4, Lamb1-1, LGMN, MMP3, Nos2, PAI1, PHD1, PLVAP, RAB5a, RIN3, ROCK2, SOX18, SOX7, SRF, STAB1, STAB2, STUB1, TFEC, THBS1, THBS2, THBS At least one gene selected from the group consisting of 3, THBS4, THBS5, THSD1, TNFAIP8, and XLKD1 (LYVE1), more preferably at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or all of the genes, and optionally ADORA2A, AGTRL1 (APLNR), APLN, CCBE1, CGNL1, CRIP2, CYB5B, DLL4, DUSP5, ELK3, ERG1 (KCNH2), ETS1, ETS2, EX The present invention relates to a method for treating cancer, the method comprising: increasing expression of at least one gene, or at least 2, 3, 4, 5, 10, 15, 20, 25, 30 or all of the genes selected from the group consisting of OC3L, FGD5, GRRP1, HO-1 (HMOX1), HO-2 (HMOX2), LAMA4, Lamb1-1, LGMN, PLVAP, RIN3, ROCK2, SOX7, SOX18, STAB1, STAB2, STUB1, TFEC, THSD1, TNFAIP8, and XLKD1 (LYVE1).

[0042] Generation of EPCs and EPC-derived endothelial cells can occur by culturing EPCs or EPC-derived endothelial cells under conditions similar to the tumor microenvironment. One such condition is exposure to ion concentrations similar to the tumor microenvironment. Tumors are known to have areas of cellular necrosis, which is associated with reduced survival in various cancers. One study showed that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumors, significantly suppressing T cell effector functions. Elevated extracellular potassium concentrations ([K+]e) impair T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programs. Potassium-mediated inhibition of Akt-mTOR signaling and T cell function is dependent on the activity of the serine / threonine phosphatase PP2A. The inhibitory effect of elevated [K+]e is independent of changes in cell membrane potential (Vm) but requires increased intracellular potassium ([K+]i). Thus, increasing potassium efflux in tumor-specific T cells by overexpressing the potassium channel Kv1.3 reduces [K+]i, improves effector function in vitro and in vivo, and enhances tumor clearance and survival in mice with melanoma. In one embodiment, a means of modifying EPCs and EPC-derived endothelial cells to resemble tumor endothelial cells is the use of culture conditions similar to those associated with necrotic tissue

[64] . In some embodiments, EPCs or EPC-derived endothelial cells are cultured under conditions of free adenosine similar to those found in tumor cells. Numerous publications have reported the concentrations found in tumors, some of which are incorporated herein by reference [65-74]. In one embodiment, EPCs or endothelial cells derived therefrom are cultured with one or more enzymes known to locally induce the production of adenosine in a manner similar to that found in the tumor microenvironment. Enzymes or exoenzymes useful in carrying out the methods of the present disclosure include CD39 [75-78] and / or CD73

[79] , which are described in the relevant literature and incorporated herein

[80] .

[0043] Tumor endothelium acts as a barrier to prevent the immune system from attacking cancer. In certain embodiments, tumor endothelium-targeting vaccines are used to reduce or substantially eliminate the ability of tumor endothelium to protect tumors from infiltrating immune cells.

[0044] Tumor endothelium acts as a barrier that prevents the immune system from attacking cancer. In certain embodiments, tumor endothelium-targeting vaccines are used to reduce or substantially eliminate the ability of tumor endothelium to protect tumors from infiltrating immune cells. In addition, quantitative analysis of T cell infiltration in these tumors was performed. FasL expression was observed in all cell lines and 9 of 14 specimens by Western blot and immunohistochemistry. FasL distribution was observed within tumor vasculature. Both FasL expressions were associated with a significant reduction (p<0.05) in T cell infiltration when compared with FasL-negative areas and specimens within the same tumor. Since FasL-expressing tumor endothelial cells induce apoptosis in T cells, the authors suggested that apoptosis induction by FasL expressed on tumor cells and / or vascular endothelium may be one mechanism of T cell exhaustion in astrocytoma tissues

[83] . Thus, it appears that the prevention of T cell entry into tumors is partly mediated by a barrier provided by blood vessels that contain death ligands. The importance of FasL in maintaining immune privilege has also been observed in physiological contexts. For example, immune privilege in the eye [84-89], the intradiscal nucleus pulposus [90,91], the testis [92-106], the blood-brain barrier

[0107] , and the placenta [108-111] is associated with FasL expression. In another study, researchers tried to show that the presence of T cells in TILs, especially the ratio of CD8+ to CD4+ T cell subsets, correlates with tumor prognosis in some tumors, although the significance of such infiltration in gliomas remains controversial. However, gliomas have an extremely low degree of T cell infiltration, making it useful to evaluate factors that reduce the presence of T cells in tumor tissue. Fas ligand, a proapoptotic cell surface protein, may play an important role in the reduction of T cells in tumor tissue. We assessed the level of FasL expression in brain tumor endothelium and correlated this with the relative levels of CD4+ and CD8+ T cell subsets in TILs from brain tumors. CD3+, CD4+, and CD8+ cells in fresh TILs were quantified by flow cytometry.Paraffin-embedded sections of tumors, including meningiomas, gliomas, and extracranial malignancies, were subjected to immunohistochemical staining for FasL and Von-Willebrand factor (Factor VIII) to determine the expression levels of endothelial FasL. FasL expression was higher in aggressive intracranial malignancies compared with more indolent neoplasms and inversely correlated with the CD8+ / CD4+ TIL ratio for all tumor classes combined (ANOVA, p<0.05). Low levels of T cells in TILs and low CD8+ / CD4+ TIL ratios appear to be characteristics of the presence of parenchymal tumors. The high levels of endothelial FasL expression in gliomas, together with the inverse correlation seen between FasL expression and the CD8+ / CD4+ TIL ratio, suggest that FasL may reduce the presence of T cells in brain tumors in a subset-selective manner, thereby contributing to the immune privilege of gliomas.

[0045] Patients with infiltration of T cells and NK cells (tumor-infiltrating lymphocytes (TILs)) have a better prognosis compared to patients without tumor-infiltrating lymphocytes. Thus, in one embodiment, endothelial-targeted vaccines are utilized as a means to enhance the ability of lymphocytes to invade tumors. TILs have been noted in various tumors and have been correlated with better prognosis in certain cancers, such as liver cancer

[0113] , melanoma [114,115], bladder cancer

[0116] , colorectal cancer

[0117] , and ovarian cancer [118,119]. Many tumor immunologists believe that TILs infiltrate tumors and induce tumor eradication, but this does not occur in vivo because immunosuppressive factors secreted by tumors inhibit immune activation. In TIL therapy, tumor masses are surgically removed, TILs are separated from tumor cells by density gradient, lymphocytes are expanded under immunostimulatory conditions in vitro, and the activated killer cells are reinfused into the patient [120,121]. In a mouse model comparing the antitumor effects of TIL therapy with LAK therapy, TIL therapy showed approximately 100-fold greater tumor killing effect [122,123]. The increased tumor killing effect of TILs is likely due to the fact that this therapy activates only lymphocytes that recognize and respond to tumors. In the clinic, results with TILs have been reasonable, with reproducible responses in approximately 20% of melanoma patients

[0124] . A means of enhancing the efficacy of TILs is to enhance their killing ability by transfecting them with cDNA against TNF

[0125] . Thus, in one embodiment of the present disclosure, a vaccine targeting the tumor endothelium is utilized to overcome tumor endothelium-mediated immune evasion and enhance the tumor-killing ability of vaccine-induced T cells.

[0046] Numerous means of stimulating immunity against tumor-associated endothelial cells are known in the art. In one embodiment, growth factors, growth factor receptors, or antigens associated with tumor endothelial cells are selected for vaccine production. Active immunization against tumor endothelium by vaccination against markers found on proliferating or tumor endothelium has provided promising preclinical data. Specifically, it has been reported that immunization against antigens found specifically in tumor vasculature results in tumor regression in animal models. Studies have been reported with the following antigens: survivin [126-128], endosialin

[0129] , xenogeneic FGF2R

[0130] , VEGF

[0131] , VEGF-R2

[0132] , MMP-2

[0133] , endoglin [134,135]. Human clinical trials have been conducted using human umbilical vein endothelial (HUVEC) cells as tumor antigens, with reported responses in patients. In a report of a clinical trial involving 17 patients, Tanaka et al. demonstrated that HUVEC vaccine therapy significantly extended tumor doubling time and inhibited tumor growth in patients with recurrent glioblastoma, and induced both cellular and humoral responses against the tumor vasculature without any adverse events or significant toxicity

[0136] .

[0047] For example, one study described the optimization of an endothelial cell-based vaccine. The authors of this study utilized human umbilical vein endothelial cells (HUVECs) prepared by various methods: 1) paraformaldehyde-fixed HUVECs, 2) glutaraldehyde-fixed HUVECs, 3) HUVEC lysate, and 4) live HUVECs. These four commonly used antigen forms were used to prepare vaccines, named Para-Fixed-EC, Glu-Fixed-EC, Lysate-EC, and Live-EC, respectively. The researchers showed that Live-ECs showed the best tumor growth and metastasis inhibition effect among the four vaccines in both H22 hepatocellular carcinoma and Lewis lung carcinoma models. High titers of anti-HUVEC antibodies were detected in the serum of Live-EC-immunized mice, and the immune serum from the Live-EC group significantly inhibited HUVEC proliferation and tube formation. Furthermore, T cells isolated from Live-EC-immunized mice showed strong cytotoxicity against HUVEC cells, and IFN-γ production was increased and Treg production was decreased in Live-EC-immunized mice. Finally, CD31 immunohistochemical analysis of excised tumors confirmed a significant decrease in vascular density after Live-EC vaccination, consistent with an antitumor effect. Taken together, these results demonstrate that live HUVECs are the most effective antigen form for inducing robust HUVEC-specific antibody and CTL responses. Thus, in one embodiment of the present disclosure, live HUVEC cells are utilized as a vaccine for immune stimulation against tumor endothelial cells, and in a specific embodiment, this immune stimulation results in sensitization of tumors to conventional cancer vaccines that induce T cell or B cell activation. Furthermore, in a specific embodiment, a means of overcoming the immune privileged state of tumor endothelium by means of selectively inhibiting the immune suppressive state of tumor endothelium is encompassed herein. Elimination of the immunosuppressive state can be achieved by inducing killing of the tumor endothelium, but also by blocking inhibitors, proteins and / or peptides found on the tumor endothelium. For example, in one embodiment, vaccination with an immunogen that targets the tumor endothelium can result in antibodies against molecules such as FasL, which block the ability of FasL on the tumor endothelium to induce killing of T cells attempting to infiltrate the tumor.Methods for inactivating immunosuppressive molecules present in tumor endothelium include blocking their function with antibodies, producing coagulation on the tumor endothelial surface, and activating complement on the tumor endothelial surface.

[0048] In certain embodiments, various adjuvants can be added to enhance the immunity of the vaccine, which plays a role in stimulating immunity against tumor endothelium. Various adjuvants are known in the art, including various agonists of Toll-like receptors. In particular, adjuvants such as lipopolysaccharide, an activator of TLR-4, polyIC, a TLR-3 agonist, imiquimod, a TLR-7 agonist, and CpG motifs, such as TLR-9, are included. Other adjuvants useful for implementing at least some methods of the present disclosure include Freund's complete adjuvant, Freund's incomplete adjuvant, BCG, and also loading of antigen-presenting cells. In one embodiment, the adjuvant is cationic liposome-DNA, complex JVRS-100, aluminum hydroxide, aluminum phosphate vaccine, aluminum potassium sulfate adjuvant, Alhydrogel, ISCOM(s), Freund's complete adjuvant, Freund's incomplete adjuvant, CpG DNA vaccine adjuvant, cholera toxin, cholera toxin B subunit liposome, saponin, DDA, squalene-based adjuvant, Etx B subunit, IL-12, LTK63 vaccine mutant adjuvant, Titormax Gold adjuvant, Ribi vaccine adjuvant, Montanide ISA 720 adjuvant, Corynebacterium derived P40 vaccine adjuvant, MPL® adjuvant, AS04, AS02, lipopolysaccharide vaccine adjuvant, muramyl dipeptide adjuvant, CRL1005, Killed Corynebacterium parvum Vaccine adjuvants, Montanide ISA 51, Bordetella pertussis component vaccine adjuvant, cationic liposomal vaccine adjuvant, Adamantylamide dipeptide vaccine adjuvant, Arlacel A, VSA-3 adjuvant, aluminum vaccine adjuvant, Polygen vaccine adjuvant, Adjumer®, algae glucan, Bay R1005, Theramide®, thalidomide, stearyl tyrosine, specol, algamulin, Avridine®, calcium phosphate gel, CTA1-DD gene fusion protein, DOC / AlumComplex, gamma inulin, Gelb adjuvant, GM-CSF, GMDP, recombinant hIFN-γ / interferon-g, interleukin-1β, interleukin-2, interleukin-7, scrabo peptide, Rehydragel LV, Rehydragel HPA, loxoribine, MF59, MTP-PE liposome, muramethide, murapalmitine, D-Murapalmitine, NAGO, nonionic surfactant vesicle, PMMA, protein cocreate, QS-21, SPT (Antigen Formulation), nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G vaccine adjuvant, E. coli heat-labile toxin, LT, amorphous aluminum sulfate hydroxyphosphate adjuvant, calcium phosphate vaccine adjuvant, Montanide Incomplete SeppicAdjuvant, imiquimod, resiquimod, AF03, flagellin, poly(I:C), ISCOMATRIX®, Abisco-100 vaccine adjuvant, albumin-heparin microparticle vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, immunoliposomes containing antibodies against costimulatory molecules, SAF-1, Sendai proteoliposomes, Sendai-containing lipid matrix The vaccine adjuvant is selected from the group consisting of threonyl muramyl dipeptide (TMDP), Ty particle vaccine adjuvant, bupivacaine vaccine adjuvant, DL-PGL (polyester poly(DL-lactide-co-glycolide)) vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, non-toxic variant E112K of cholera toxin mCT-E112K, Matrix-S, and combinations thereof. One type of antigen presenting cell useful as an adjuvant in the practice of the methods of the present disclosure is a dendritic cell. Numerous means of generating dendritic cells have been described in the art. In one embodiment, peripheral blood mononuclear cells (PBMCs) are extracted and then resuspended in 10 ml of AIM-V medium and allowed to adhere to a plastic surface for 2-4 hours. The adherent cells are gently washed with Hank's buffered saline (HBSS) to remove non-adherent cells, and then cultured at 37°C in AIM-V medium supplemented with 1,000 U / mL granulocyte monocyte colony-stimulating factor and 500 U / mL IL-4. Half of the GM-CSF and IL-4-supplemented medium is replaced every other day. Immature DCs are harvested on day 7.

[0049] In some embodiments, the enhancement of endogenous cellular vaccines is achieved by stimulating immunity against tumor endothelium, which aims to sensitize tumors to T cells. In other embodiments, targeting of tumor endothelium is achieved to overcome the ability of endothelium to protect tumors.

[0050] In one embodiment of the present disclosure, endogenous release of tumor antigens is used as a source of tumor antigens. One method of inducing localized tumor cell death is transarterial chemoembolization (TACE), or otherwise defined as transcatheter chemoembolization, which is a clinical procedure used primarily to treat primary and secondary liver cancers. TACE is typically used when standard therapies are ineffective or have proven ineffective. TACE combines the advantages of intra-arterial chemotherapy with the advantage of embolizing the portal vein, which allows tumors to be preferentially "starved" while sparing non-malignant liver tissue. Specifically, intra-arterial administration of chemotherapy to the liver has been demonstrated to result in 10-fold higher intratumoral concentrations compared to administration via the portal vein. This is due in part to the observation that both primary and secondary liver tumors derive their blood supply preferentially from the hepatic artery. Anecdotal evidence suggests that embolization caused by catheter thrombosis during intra-arterial chemotherapy may be beneficial in improving tumor response. This has led some investigators to induce local necrosis using surgical ablation

[0141] or angiographic embolization [142-144]. Unfortunately, this chemotherapy-free approach has had little impact on long-term survival. Thus, the advantage of TACE is that it simultaneously delivers chemotherapy locally to the tumor and embolizes the tumor blood flow, causing local tumor necrosis

[0145] . In some embodiments, these advantages are used in the practice of the disclosed methods, in which TACE is combined with cancer endothelial cell vaccination.

[0051] Stimulation of cancer immunity is, in one embodiment, a result of the release of tumor cell antigens from dying cancer cells. Globally, apoptotic cell death is associated with anti-inflammatory and possibly tolerance, whereas necrotic cell death is recognized by the immune system as a "danger signal" and is associated with immune activation [146-150]. Specific examples of the anti-inflammatory aspect of apoptotic cell death include: IL-10 production by apoptotic monocytes

[0151] , suppression of inflammatory cytokines by apoptotic bodies in vitro [152,153], the observation that administration of apoptotic bodies, but not necrotic bodies, can confer active immunosuppressive properties to macrophages

[0154] , and clinically administered apoptotic blood cells have been demonstrated to be successful in treating inflammation associated with advanced heart failure in a recent phase II trial

[0155] . Conversely, cell necrosis is associated with the release of various innate immune activation signals, such as heat shock proteins [156-158], HMGB1

[0159] , mRNA with endogenous secondary structure

[0160] , and even DNA complexed with endogenous factors such as natural antibodies [161,162]. Thus, induction of cell necrosis by TACE induces the release of tumor antigens, which are picked up by the immune system. Although the release of tumor antigens in such situations has been reported in the literature

[0163] , exploiting this antigen release therapeutically has not been achieved to date. In the case of hepatocellular carcinoma, the tumor itself [164-167] and the host cells infiltrating the tumor are known to be immunosuppressive

[0168] , but the microenvironment in which TACE induces cell necrosis is also usually immunosuppressive. Intrahepatic administration of antigens is known to result in systemic immune deviation to weak cellular immunity

[0169] . For example, administration of donor cells into the hepatic circulation has been shown to prolong donor-specific graft acceptance in various transplantation models [170-174]. The local immunosuppressive effect of the liver is well known to transplant clinicians, and liver transplant recipients require less immunosuppression compared to other organs. Furthermore, in various strain combinations of mice, liver grafts are accepted spontaneously, whereas hearts and kidneys are rejected [175-177].At the cellular level, this is explained by the presence of immature hepatic DCs [178,179], the tolerogenicity of hepatic sinusoidal endothelial cells [180,181], and natural killer T cells predisposed to release IL-4 [182,183]. Based on this, it is speculated that the release of tumor antigens in the hepatic microenvironment leads to a Th2, i.e., immunoregulatory, switch, which not only fails to mount protective immunity against micrometastases, but in some cases may even increase the tumor growth rate, due to the phenomenon of "tumor enhancement" described by Prehn

[0184] . Therefore, it is one objective of the present disclosure to stimulate cell-based Th1 immunity and avoid antibody-based immunity against tumor cells.

[0052] One specific embodiment of the present disclosure includes modifying the TACE procedure to induce a systemic antitumor immunological effect. Specifically, patients are selected to meet the criteria for TACE, including: a) adequate liver function; b) patent portal circulation (as confirmed during the venous phase of abdominal or superior mesenteric angiograms); and c) adequate renal function. Generally, only patients without cirrhosis, and those in Child group A or B, are considered, although other groups may be candidates for the procedure, depending on the experience of the practitioner, as discussed by Shah et al. TACE is performed in a selective or superselective manner. Patients selected to undergo this procedure are given 10 mg of phytonadione intravenously prior to the procedure (intravenous injection should be given slowly). Femoral catheterization and catheter positioning are performed. As a premedication, lorazepam (Wyeth Laboratories, UK) 0.25 mg / kg is given orally 1 hour before surgery to address anxiety. For analgesia, 30 to 40 mg of 1% lidocaine is injected intra-arterially.

[0053] The following components were repeatedly injected and filled into the syringe over a 10-minute period to form an emulsion: 10 mL of Lipiodol Ultrafluid (Mallinckrodt Medical, UK), 5 mL of Omnipaque 300 (Amersham Health, UK; the water-soluble contrast agent helps emulsify the mixture), 50 mg of doxorubicin, and 0.025 mg / m of clinical grade Poly(IC) stabilized with carboxymethylcellulose. 2 to 12 mg / m 2 Concentration of 0.2 mg / m 2 The concentration of 100 mg / kg / day is used. Intra-arterial infusion is performed under direct visualization to prevent reflux into the gastroduodenal and splenic vessels. Embolization is performed using Ultra Ivalon 250-400 μm (Laboratories Nycomed SA). Cefuroxime (750 mg) and metronidazole (500 mg) are administered intravenously three times a day for five days. These antibiotics are administered as prophylaxis against sepsis and liver abscess formation. After administration, the patient is admitted to a high dependency ward and transferred after six hours of rest. Postoperative analgesia is provided if the patient requires it. In addition, ranitidine (H2 antagonist) is administered intravenously three times a day until the patient begins to eat. The patient is discharged home after five days or when the systemic symptoms begin to disappear. Non-contrast and contrast-enhanced CT scans are performed 10-14 days after embolization to monitor the success of the procedure. In addition, alpha-fetoprotein levels are evaluated at six-week outpatient examinations. If the TACE procedure is successful (post-procedure CT scan demonstrates >50% uptake of Lipiodol in the necrotic tumor), embolization is repeated in 6-8 weeks. Immunologic monitoring is performed by assessing interferon alpha production levels at 12, 24, and 72 hours using ELISA. Additionally, DTH, cellular, and antibody responses can be measured using predefined antigens representative of the tumor type.

[0054] In some embodiments of the present disclosure, various chemotherapeutic agents can be used. In particular, chemotherapeutic agents that induce upregulation of costimulatory molecules can be utilized. One example of such an agent is melphalan, which induces expression of CD80 on both tumor cells and non-tumor B cells. Furthermore, a wide variety of chemotherapeutic agents are known in the art. These include: alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylameramines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards, such as chlorambucil, chromafazine, clophosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, and orthopeptidase. Antibiotics such as thromycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU);folate analogues such as denopterin, methotrexate, pteropterin, and trimetrexate;purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calciterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; deformamine. Demecolcine;Diaziquone;Elfornithine;Elliptinium acetate;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamine;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Philamin;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK (registered trademark);Razoxane;Sizofiran;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2''-Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitolactol;Pipobroman;Gacytosine;Arabinoside ("Ara-C");Cyclophosphamide;Thiotepa;Taxanes, such as paclitaxel (TAXOL (registered trademark), Bristol-Myers Squibb Oncology, Princeton, NJ), and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbin; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS 2000; difluoromethylorutin (DMFO); retinoic acid; esperamicin; and / or capecitabine.

[0055] The TACE modification procedure may be utilized as an embodiment. Furthermore, modifications can be made to enhance the efficacy of the mediated antitumor response. In particular, a wide variety of drugs can be administered to the individual prior to TACE treatment to enhance the general immunological state, specifically the function of T cells, NK cells, and NKT cells. One particular modification may involve the administration of antioxidants that can reverse the immunosuppression seen in many cancer patients. Cancer-induced immunosuppression has been well documented in advanced cancer patients with various malignancies [188-195]. The correlation between immunosuppression and poor prognosis has been widely noted [196-198]. There are several known means by which tumors suppress immune responses. For example, various tumor cells have the ability to induce cleavage of the T cell receptor zeta (TCR-ζ) chain through a caspase-3-dependent manner [199, 200]. TCR-ζ is important for signal transduction, so that the host's T cells are unable to respond to tumor antigens. TCR-ζ cleavage was initially reported in tumor-bearing mice [201, 202] and subsequently in patients [203-208]. A correlation between TCR-ζ suppression and suppressed IFN-g production has been reported, suggesting a functional impact

[0204] . TCR-ζ suppression is thought to be due, at least in part, to reactive oxygen radicals generated by: A) inflammatory activity occurring within the tumor (it is well established that there are certain necrotic areas within tumors); B) tumor-associated macrophages; and C) neutrophils activated directly by the tumor or tumor-associated macrophages.

[0056] Tumors are usually associated with infiltration of macrophages, which correlates with tumor stage and is thought to contribute to tumor progression by stimulating angiogenesis [209-211]. Cytokines such as M-CSF

[0209] and VEGF

[0212] produced by tumor-infiltrating macrophages are essential for tumor malignant progression. Indeed, tumors implanted in M-CSF-deficient op / op mice (which lack macrophages) do not metastasize or become vascularized

[0213] . Tumor-associated macrophages have an activated phenotype and release various inflammatory mediators, such as cyclooxygenase metabolites [214, 215], TNF

[0216] , and IL-6

[0217] , leading to increased levels of oxidative stress produced by host immune cells. In addition, tumor-associated macrophages themselves generate large amounts of free radicals, such as NO, OH, and H2O2 [218-220]. High levels of macrophage activation in cancer patients are indicated by high serum levels of neopterin, a tryptophan metabolite associated with poor prognosis

[0221] . In addition to oxidative stress caused by tumor-associated macrophages, the presence of the tumor itself induces systemic changes associated with chronic inflammation. Erythrocyte sedimentation rate, C-reactive protein, and IL-6 are markers of inflammatory stress used to indicate the progression of pathological immune diseases such as arthritis [222, 223]. Interestingly, patients with advanced cancer have all of these inflammatory markers [224-228]. Another marker of chronic inflammation is a decrease in albumin synthesis by the liver, which is also seen in cancer patients and is thought to contribute, at least in part, to cachexia [229, 230]. In addition, fibrinogen D-dimer, an inflammation marker, is also elevated in cancer patients compared to controls [231-233]. Schmielau et al. reported that activated neutrophils circulate in large numbers in patients with various cancers

[0203] . These neutrophils secrete reactive oxygen radicals, such as hydrogen peroxide, which leads to the suppression of TCR-ζ and IFN-γ production. This was demonstrated by co-incubating neutrophils from cancer patients with lymphocytes from healthy volunteers. A significant suppression of TCR-ζ expression was observed.Evidence for the important role of hydrogen peroxide was provided by the fact that the addition of catalase inhibited the downregulation of TCR-ζ. There, a simple method for assessing the number of circulating activated neutrophils is described, in which peripheral blood is drawn from patients, the blood is spun on a density gradient such as Ficoll, and the lymphocyte fraction is collected. The lymphocyte fraction from healthy volunteers contained mainly lymphocytes, whereas the lymphocyte fraction from cancer patients contained both lymphocytes and a large number of neutrophils. The reason these neutrophils are present in the lymphocyte fraction is because their density changes upon activation, and they copurify differently on the gradient. One indication of the importance of activated neutrophils to cancer progression is provided by Tabuchi et al., who showed that removal of granulocytes from the peripheral blood of cancer patients reduced tumor size, but unfortunately, this study was performed on only two patients. As a mechanism to compensate for immune overactivation, inflammatory mediators have immunosuppressive properties. This is best illustrated by the immunosuppression seen after immune overactivation such as septic shock. Following primary sepsis, patients become systemically immunocompromised due to circulating immunosuppressive factors released in response to inflammatory stress. This suppression is called the compensatory anti-inflammatory response syndrome (CARS) and is associated with many opportunistic infections and inactivation

[0235] . The clinical importance of CARS immunosuppression is illustrated by the fact that sepsis survivors show normal T cell proliferation and IL-2 release, whereas deceased individuals show suppressed T cell responses

[0236] . Interestingly, CARS-related immunosuppressive mediators, such as PGE2, TGF-b, and IL-10, have also been linked to cancer-induced immunosuppression

[0237] . The role of oxidative stress in sepsis-induced immunosuppression was recently demonstrated in experiments in which administration of antioxidants (ascorbic acid or n-acetylcysteine) to animals undergoing experimental sepsis blocked immunosuppression

[0238] . Another example showing the potential of antioxidants to stimulate immune responses in inflammatory conditions is that patients with Dukes C and D colorectal cancer were given 750 mg of vitamin E per day for two weeks, which restored IFN-g and IL-2 production.

[0239] Uncontrolled inflammation is a problem seen in sepsis.Although n-acetylcysteine ​​has little clinical benefit as a monotherapy, therapeutic administration of n-acetylcysteine ​​results in suppression of the constitutively activated neutrophils seen in these patients. Administration of n-acetylcysteine ​​to smokers suppresses markers of oxidative stress. Furthermore, oral administration of n-acetylcysteine ​​blocks angiogenesis and inhibits the growth of Kaposi's sarcoma. Thus, a method of preparing a host for TACE treatment includes administration of n-acetylcysteine ​​at a concentration sufficient to reduce tumor-associated suppression of T cell activity. In certain embodiments, such a concentration ranges from 1 to 10 grams per day, e.g., 4 to 6 grams, administered intravenously for a period of time of a type sufficient to normalize the production of IFN-g from PBMCs of cancer patients upon ex vivo stimulation. Those skilled in the art will appreciate that n-acetylcysteine ​​is but one example of a compound suitable for reversing oxidative stress-associated immunosuppression. Many other compounds can also be used, such as ascorbic acid [243-245], coenzyme Q10 in combination with vitamin E and α-lipoic acid

[0246] , genistein

[0247] , or resveratrol

[0248] .

[0057] In some embodiments, dendritic cells are utilized to induce an enhanced immune response following TACE-induced release of antigens. In other embodiments, dendritic cells are administered in proximity to TACE-induced cell death. In one embodiment, dendritic cells are generated from the patient's leukocytes by leukopheresis. Numerous means of leukopheresis are known in the art. In one example, a Frenius Device (Fresenius Com.Tec) is used with the P1Y kit at about 1500 rpm using the MNC program. The flow rate of the plasma pump is adjusted to about 50 mL / min. Various anticoagulants can be used, for example, ACD-A. The inlet / ACD ratio can range from about 10:1 to 16:1. In one embodiment, about 150 mL of blood is processed. The leukopheresis product is then used to initiate dendritic cell culture. To separate peripheral blood mononuclear cells from the leukopheresis product, the mononuclear cells are separated by Ficoll-Hypaque density gradient centrifugation. Monocytes are then enriched by Percoll hyperosmolarity density gradient centrifugation and allowed to adhere to plate culture for 2 hours. Cells are then centrifuged at 500 g to separate the different cell populations. Adherent monocytes are cultured in 6-well plates at 2×106 cells / mL in RMPI medium supplemented with 1% penicillin / streptomycin, 2 mM L-glutamine, 10% autologous cells, 50 ng / mL GM-CSF, and 30 ng / mL IL-4 for 7 days. On day 6, immature dendritic cells are pulsed with tumor endothelial lysate or fibroblast-derived endothelial cells. Pulsing can be performed by incubating the lysate with dendritic cells or by fusing immature dendritic cells with fibroblast-derived endothelial cells. Means of generating hybridomas or cell fusion products are known in the art and include fusion via electrical pulses or stimulation of cell fusion by polyethylene glycol treatment. On day 7, immature DCs are induced to differentiate into mature DCs by culturing them with 30 ng / mL interferon gamma (IFN-γ) for 48 hours. During the process of generating dendritic cells for clinical purposes, microbiological monitoring tests are performed at the start of culture, on day 5, when freezing cells for further use, or before releasing the dendritic cells.Administration of fibroblast-derived endothelial cell-pulsed dendritic cells is utilized as a multivalent vaccine, and after administration, antibody or T cell responses are evaluated for induction of antigen specificity, and peptides corresponding to the stimulated immune response are used in further immunization to focus the immune response. Protocols useful for generating dendritic cells have been previously used to generate immunity against various tumors, including melanoma [249-300], soft tissue sarcoma

[0301] , thyroid [302-304], glioma [305-326], multiple myeloma [327-335], lymphoma [336-338], leukemia [339-346], as well as liver cancer [347-352], lung cancer [353-366], ovarian cancer [367-370], and pancreatic cancer [371-373].

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[0059] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods and steps described herein. As will be readily understood by those skilled in the art from the present disclosure, any currently existing or later developed processes, machines, manufacture, compositions of matter, means, methods or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

Claims

1. 1. A method for inducing cell death of tumor cells in an individual, comprising: The method includes administering to the individual a therapeutically effective amount of a plurality of modified fibroblasts, (I) the fibroblasts express a recombinant: (a) one or more endothelial-inducing genes and / or one or more vascular channel-inducing genes; and (b) one or more suicide-inducing or death-inducing genes; and (c) optionally one or more immune stimulatory genes; and / or (II) fibroblasts are cultured in endothelial progenitor cell conditioned medium; Fibroblasts. (a) the fibroblasts express recombinant ETV2, FOXC2, and / or FLI1; and / or (b) the fibroblasts express recombinant ETV2 and are cultured in a medium containing an effective amount of one or more of VEGF, EGF, HGF, and IGF-1; and / or (c) the fibroblasts express recombinant FOXC2 and are cultured in a medium containing an effective amount of one or more of VEGF, EGF, HGF, and IGF-1; and / or (d) the fibroblasts express recombinant FLI1 and are cultured in a medium containing an effective amount of one or more of VEGF, EGF, HGF, and IGF-1; The fibroblast cell of claim 1.

3. The fibroblast according to any one of claims 1 to 2, The endothelial cell progenitor cell conditioned medium is produced from pluripotent stem cells differentiated into endothelial progenitor cells, wherein: Optionally, the pluripotent stem cells are (i) an embryonic stem cell, or (ii) an induced pluripotent stem cell, or (iii) somatic cell nuclear transfer-derived stem cells, or (iv) stem cells of parthenogenetic origin, or (v) Differentiated into endothelial progenitor cells by transfection with ETV2, FOXC2, and FLI1; Fibroblasts.

4. The fibroblast according to any one of claims 1 to 2, The endothelial cell progenitor cell conditioned medium is produced from pluripotent stem cells differentiated into endothelial progenitor cells, wherein: (a) the fibroblasts are transfected with one or more thrombosis-associated genes, said genes being upregulated in response to hypoxia, wherein the thrombosis-associated genes are optionally Tissue factor, or the thrombosis-related gene is an inhibitor of protein C; and / or (b) the fibroblasts are transfected with one or more immunostimulatory genes, wherein the immunostimulatory genes are optionally Inducible by the presence of hypoxia, or Induction of the immunostimulatory gene is achieved by placing the gene under the control of the HIF-1α transcription factor, and / or (c) the immunostimulatory gene is associated with antigen presentation, wherein the gene associated with antigen presentation optionally comprises: an allogeneic MHC molecule; or a xenogeneic MHC molecule, or one or more of HLA B7 molecule, CD80, CD86, and CD40; Fibroblasts.

5. The fibroblast according to any one of claims 1 to 4, wherein the immune stimulatory molecule is interleukin-12.

6. The fibroblast of any one of claims 1 to 5, wherein the fibroblast is selected for expression of one or more of CXCR4, CD73, CD74, CD206, and interleukin-3 receptor.

7. Fibroblast cells for use in a method of inducing immunogenic cell death of tumor endothelial cells in an individual, said method comprising the steps of: a) transfecting said population of fibroblasts with one or more endothelial cell-inducing genes and / or one or more vascular channel-inducing genes; c) transfecting the fibroblasts with one or more suicide or death-inducing genes d) optionally transfecting said fibroblasts with one or more immunostimulatory genes; and e) administering the fibroblasts to an individual with cancer; Fibroblasts.

8. The fibroblast of claim 7, The fibroblasts are obtained from a tissue selected from the group consisting of: a) dermis; b) bone marrow; c) blood; d) mobilized peripheral blood; e) gums; f) tonsils; g) placenta; h) Wharton's jelly; i) hair follicles; j) fallopian tubes; k) liver; l) milk teeth; m) vas deferens; n) endometrium; o) menstrual blood; and p) fallopian tubes, wherein optionally (a) said mobilization of peripheral blood is achieved by treatment of the mammal with an effective amount of one or more inhibitors of SDF-1 binding to CXCR4, and further optionally, said inhibitor of SDF-1 binding to CXCR4 is Plerixafor or BKT140; or (b) the mobilization is induced by exposure to hyperbaric oxygen therapy; or (c) the mobilization is induced by treatment with GM-CSF; or (d) the mobilization is induced by treatment with M-CSF; or (e) the mobilization is induced by treatment with G-CSF; or (f) the mobilization is induced by treatment with flt-3 ligand; Fibroblasts.

9. The fibroblast of claim 7 or 8, wherein: (a) the fibroblasts are either allogeneic, allogeneic or xenogeneic to the recipient; and / or (b) the fibroblasts are selected for expression of CXCR4, and / or (c) the fibroblasts are selected for expression of CD73, and / or (d) the fibroblasts are selected for expression of CD74, and / or (e) the fibroblasts are selected for expression of CD206, and / or (f) the fibroblasts are selected for expression of interleukin-3 receptor, and / or (g) the fibroblasts are cultured in an endothelial progenitor cell conditioned medium, optionally generated from pluripotent stem cells differentiated into endothelial progenitor cells, further optionally comprising: (i) the pluripotent stem cells are embryonic stem cells, or (ii) the pluripotent stem cells are induced pluripotent stem cells, or (iii) the pluripotent stem cells are somatic cell nuclear transfer-derived stem cells; or (iv) The pluripotent stem cells are differentiated into endothelial progenitor cells by transfection with ETV2, FOXC2, and FLI1. Fibroblasts.

10. The fibroblast according to claim 7, wherein the fibroblast is differentiated into an endothelial progenitor cell by transfection with: (a) ETV2, and / or (b) differentiated into endothelial progenitor cells by transfection with ETV2 and cultured in VEGF; and / or (c) differentiated into endothelial progenitor cells by transfection with ETV2 and cultured in EGF; and / or (d) differentiating into endothelial progenitor cells by transfection with ETV2 and culturing in HGF; and / or (e) Differentiated into endothelial progenitor cells by transfection with ETV2 and cultured in IGF-1; Fibroblasts.

11. The fibroblast according to any one of claims 7 to 10, wherein the fibroblast is differentiated into an endothelial progenitor cell by transfection with: (a) FOXC2, and / or (b) differentiated into endothelial progenitor cells by transfection with FOXC2 and cultured in VEGF; and / or (c) differentiated into endothelial progenitor cells by transfection with FOXC2 and cultured in EGF; and / or (d) differentiated into endothelial progenitor cells by transfection with FOXC2 and cultured in HGF; and / or (e) Differentiated into endothelial progenitor cells by transfection with FOXC2 and cultured in IGF-1; Fibroblasts.

12. The fibroblast according to any one of claims 7 to 11, wherein the fibroblast is differentiated into an endothelial progenitor cell by transfection with: (a) differentiated into endothelial progenitor cells by transfection with FLI1; and / or (b) differentiated into endothelial progenitor cells by transfection with FLI1 and cultured in VEGF; and / or (c) differentiated into endothelial progenitor cells by transfection with FLI1 and cultured in EGF; and / or (d) differentiated into endothelial progenitor cells by transfection with FLI1 and cultured in HGF; and / or (Effect of eFLI1 on differentiation into endothelial progenitor cells and cultured in IGF-1. Fibroblasts.

13. The fibroblast of any one of claims 7 to 12, wherein the fibroblast is transfected with a thrombus-inducing gene, the gene being upregulated in response to hypoxia, and optionally, (i) the thrombosis-related gene is tissue factor, or (ii) the thrombosis-related gene is an inhibitor of protein C; Fibroblasts.

14. The fibroblast of any one of claims 7 to 13, wherein said fibroblast is transfected with one or more immunostimulatory genes, optionally wherein said immunostimulatory genes are inducible by the presence of hypoxia, and further optionally wherein induction of said immunostimulatory genes is by placing said genes under the control of a HIF-1α transcription factor. Fibroblasts.

15. The fibroblast of claim 14, wherein: (a) the immunostimulatory gene is associated with antigen presentation, optionally wherein the gene associated with antigen presentation is (i) is an allogeneic MHC molecule; or (ii) is a xenogeneic MHC molecule; or (iii) an HLA B7 molecule; or (iv) CD80, (v) is CD86, (vi) CD40, and / or (b) the immune stimulatory gene is interleukin-12; Fibroblasts.