KR101261884B1 - Composition for Anticancer Containing Umbilical Cord Blood- or Adipose Tissue-derived Stem Cell - Google Patents

Abstract

The present invention relates to a composition for anticancer containing adult stem cells derived from cord blood or adipose tissue.

According to the present invention, umbilical cord blood or adipose tissue-derived adult stem cells can induce apoptosis, inhibit cancer cell growth, inhibit cancer cell metastasis to other organs such as lungs, and effectively treat breast cancer.

Umbilical cord blood-derived adult stem cells (UCB MSC), adipose tissue-derived adult stem cells (AD MSC), breast cancer

Description

Composition for Anticancer Containing Umbilical Cord Blood- or Adipose Tissue-derived Stem Cells

The present invention relates to a composition for anticancer containing adult stem cells derived from cord blood or adipose tissue.

Mesenchymal stromal cells (MSCs) comprise a population of pluripotent progenitor cells that are isolated from various tissue sources, primarily bone marrow (BM). Under appropriate conditions, MSCs can be induced into ectoderm, endoderm and mesoderm cell lines. MSCs have been extensively studied in regenerative medicine because of their ability to differentiate into various functional end systems. MSCs can perform therapeutic functions by incorporating degenerative sites as well as in vitro and in vivo differentiation and transdifferentiation capabilities. MSCs can be considered as cell vehicles for the delivery of therapeutic proteins.

Umbilical cord blood (UCB) MSCs are stem cells present in human UCB. Some studies have revealed the cell's preferred properties, including progenitor status and immune control. Adipose tissue (AD) MSC can be isolated from breast adipose tissue of a woman undergoing breast reduction. AD MSCs showed similar characteristics to BM derived MSCs. UCB and AD MSCs are gaining attention in the clinic because they are economical and easily obtainable materials.

Carcinomas are the result of a well-balanced desmoplastic response. This process is very similar to wound healing and complements various growth factors, cytokines and matrix remodeling proteins. These factors assume that the tumor site is an unheal wound and the MSC can be supplemented by endocrine and paracrine signals secreted from the tumor site, similar to the wound site. Various stromal cells were supplemented during carcinoma formation. Since MSCs are generally present in stromal cells, the transplantation of MSCs into tumor patients should be closely investigated. A strong antitumor effect was observed in direct cell contact, which inhibited Akt activation in Kaposi 'sarcoma cells. However, it has been found that BM MSCs can promote motility, invasion and metastasis of cancer cells. Therefore, the effect by MSC transplantation should be identified during the early stages of cancer formation without apparent clinical changes.

The present inventors have confirmed that injection of umbilical cord blood or adipose tissue-derived adult stem cells at an early stage of cancer formation inhibits the growth of cancer cells and inhibits metastasis to other organs, thereby effectively treating cancer. It was completed.

It is an object of the present invention to provide an anticancer composition containing umbilical cord blood-derived adult stem cells.

The present invention is based on the finding that umbilical cord blood (UCB) or adipose tissue (AD) derived adult stem cells (MSCs) are useful for the treatment of cancer, particularly breast cancer.

In order to investigate whether human UCB or AD MSCs inhibit tumor growth, we first induce the growth of tumors by injecting breast cancer cell lines (MDA-MB-231 cells) into the mammary fat pad of mice. It was. Thereafter, human UCB or AD MSCs were injected intravenously and the tumor volume measured at regular intervals. As a result, it was found that tumor growth was inhibited after transplanting human UCB or AD MSC (see Example 2-1).

In general, breast cancer cells can metastasize to the lungs. We investigated whether UCB and AD MSCs can inhibit metastasis of breast cancer cells to the lung. To this end, UCB and AD MSCs were injected and metastasis index to the lungs was examined after a certain period of time. As a result, both the UCB and AD MSC treated groups had reduced metastasis index to lung by 50% compared to the control group. In histologic examination (H & E staining) results, there was a markedly less metastasis of tumor colonies in the UCB and AD MSC treated groups compared to the control group (see Example 2-2).

In order to track whether intravenously administered MSCs by one embodiment of the invention homing to primary tumor sites and lungs, both UCB and AD MSCs were first labeled with PKH261. Labeled UCB or AD MSCs were then administered intravenously to mice bearing tumors. The administered UCB and AD MSCs were found to be grafted to the primary tumor location and lungs (see Example 2-3).

Immunohistochemistry was performed to investigate the mechanism by which UCB or AD MSCs inhibit tumor growth. To investigate whether apoptosis is involved in the mechanism, the immunoreactivity to cleaved PARP was analyzed. As a result of the analysis, tumor sections of UCB and AD MSC treated groups showed positive immunoreactivity against cleaved PARP, but metastatic lung sections showed rare immune responses to PARP. To further investigate whether human UCB or AD MSCs are involved in PARP cleavage, proteins were extracted from tumors and lungs and subjected to western blotting. As a result, it was found that PARP and caspase-3 increased in the tumor cells of the UCB and AD MSC treatment groups. Increased cleavage of PARP and caspase-3 means that UCB and AD MSC treatment induces apoptosis in tumor tissues (see Example 2-4).

The effects of UCB and AD MSCs were further investigated in the early stages of breast cancer. To this end, breast cancer cell lines were mixed with human UCB and AD MSC and then injected into mouse mammary fat pads and tumor volumes were measured. Tumor formation and progression were inhibited by human USC and AD MSCs. Lung metastasis index was lower in the UCB MSC treated group than in the AD MSC treated group, but both types of stem cells inhibited lung metastasis (see Example 2-5).

Since stem cells are known to promote cancer formation and metastasis, the therapeutic effect of stem cells should be evaluated in early stage cancer models with no clinical symptoms. To this end, human MDA-MB-231 cells labeled with GFP were injected into the mouse mammary fat pad, and on the same day, human UCB or AD MSC was administered intravenously. Tumor formation and progression were inhibited by UCB MSCs, but not by AD MSCs. In addition, the number of pulmonary colonies metastasized in the UCB MSC treated group was smaller than in the AD MSC treated group and the control group.

In conclusion, adult stem cells derived from umbilical cord blood or adipose tissue can induce apoptosis to inhibit cancer cell growth and inhibit cancer cell metastasis, thereby effectively treating cancer, especially breast cancer.

Accordingly, the present invention provides an anticancer composition containing adult stem cells derived from cord blood or adipose tissue.

The compositions of the present invention can be used for the treatment of various diseases or diseases associated with tumors such as gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, colon cancer and cervical cancer.

Adult stem cells, which are the active ingredient of the composition of the present invention, do not express histocompatibility antigen HLA-DR (class II), which is the most important cause of rejection in tissues or organ transplantation, and therefore, rejection problem that is a problem during transplantation. It does not cause or minimize the immune response, and can be used as well as tag derived from self. Preferably, it may be isolated from umbilical cord blood or adipose tissue of a mammal, including a human. Most preferably, the cord blood derived stem cells disclosed in Korean Patent Application No. 10-2009-0023821.

The cord blood stem cells have at least one of the following properties:

(a) exhibits positive immunological properties for the transcription factor c-myc, ZNF281.

(b) adheres to the bottom of the extracellular matrix and proliferates in a linear or spherical cell population between 5 and 30 days after attachment.

(c) shows a cumulative population doubling level (CPDL) of 30 to 45.

(d) show immunological properties of negative for CD14, CD31, CD34, CD45 and HLA-DR.

(e) can differentiate into cells of mesoderm, endoderm and ectoderm.

(f) TIMP-2, TGF-β, RANTES CINC-3, EOTAXIN, GM-CSF, IFN-γ, IL-1b, IL-3, IL-6, IL-8, IL-10, IL12p40, IL13, Secrete at least one cytokine or chemokine selected from the group consisting of IL-16, IP-10, Leptin, MCP-2, MIG, MIP-3a, b-NGFm, sTNFRI, PFGF-bb.

As used herein, the term 'umbilical cord blood' refers to blood collected from a umbilical vein connecting the placenta and fetus of a mammal.

As used herein, the term 'fat tissue' refers to tissues including a plurality of cell types, including adipocytes and microvascular cells. In addition, the adipose tissue includes connective tissue for storing fat.

The umbilical cord blood can be readily collected from the umbilical vein of the donor at birth. More specifically, in the case of normal vaginal delivery, it can be collected from the umbilical vein that is extracted out of the state in which the placenta remains in the uterus after childbirth. Alternatively, in the case of cesarean section, the placenta is also extracted from the umbilical vein after the baby is delivered out of the uterus.

The adipose tissue can be obtained by any method known to those skilled in the art. For example, adipose tissue can be obtained by inhalation assisted liposuction, ultrasonic assisted liposuction and incisional liposuction and a combination thereof.

Adult stem cells can be isolated from the cord blood or adipose tissue obtained above according to methods known in the art. Isolation of the adult stem cells can be separated by any known separation method.

For example, density gradient fractionation, immunoselection, and differential adhesion separation are used.

The method of separating and culturing stem cells from umbilical cord blood can be used in any of the existing methods, including the method of Korean Patent Application No. 10-2009-0023821.

As a method for isolating and culturing stem cells from adipose tissue, any known method can be used.

In one embodiment, adipose tissue is treated with collagenase at a concentration sufficient for degradation and incubated under suitable conditions (temperature and time), followed by centrifugation or other methods known in the art to separate and precipitate suspended fat cells. Adult stem cells are isolated by tissue culture of the interstitial fraction.

Cultivation of adult stem cells isolated from the above may be carried out in a cell culture medium known in the art, for example, but not limited to, DMEM medium, McCoys 5A medium, Eagls'basal medium, CMRL medium, Glasgow minimum essential medium, Ham's F-12 medium, Iscove's modified Dulbecco's medium, Liebovitz 'L-15 medium, RPMI 1640 medium, and the like. In addition, in the present invention, one or more accessory ingredients may be added to the cell culture medium, if necessary, including serum such as fetal bovine serum, horse or human, and antibiotics and antifungal agents for preventing microbial contamination. . In the present invention, it is particularly preferable to use the cell culture medium disclosed in Korean Patent Application No. 10-2009-0023821.

Isolated or cultured stem cells can be stored by methods known in the art until use. In general, stem cells may be cryopreserved after cryoprotection treatment. The cryoprotective treatment includes DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D as known in the art. Cryoprotectants such as sorbitol, i-inositol, D-lactose or choline chloride can be used.

In addition, the pharmaceutical composition of the present invention comprising adult stem cells as an active ingredient may further comprise a pharmaceutically acceptable carrier and diluent. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to stem cells and recipients to be transplanted thereof. Examples of pharmaceutically acceptable diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

The composition of the present invention is preferably formulated in the form of an injection, the route of administration may include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, spinal cavity, intraoral, route However, the present invention is not limited thereto. It may also be formulated for direct intratumoral administration. In addition, the compositions of the present invention may also be applied using a catheter, in the peripheral venous approach, cells may be injected through the catheter in a single mass or in several smaller aliquots. Administration of cells using a catheter may include, for example, intravenous delivery through a standard peripheral venous catheter, a central venous catheter, or a pulmonary artery catheter.

The dosage of the composition of the present invention is determined according to the specific use described by those skilled in the art in consideration of various factors such as the route of administration, the number of treatments, the age, weight, health status, sex, severity of the disease, diet and excretion rate of the subject in need thereof. An appropriate effective amount can be determined.

According to the present invention, umbilical cord blood or adipose tissue-derived adult stem cells can induce apoptosis, inhibit cancer cell growth, inhibit cancer cell metastasis to other organs such as lungs, and effectively treat breast cancer.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1 Materials and Methods

Example 1-1 Cell Lines

Human breast cancer cell line MDA-MB-231 was purchased from ATCC (Manassas, VA, USA). Dulbecco's modified Eagle's containing fetal bovine serum (Gibco), penicillin (100 U / ml; Gibco) and streptomycin (100 μg / ml; Gibco) in which MDA-MB-231 cells were inactivated with 10% heat. medium; Gibco BRL, Grand Island, NY) at 37 ° C. in a CO ₂ atmosphere.

Lentiviruses were prepared using ViraPowerTM Lentiviral Packaging Mix (Invitrogen, Carlsbad, Calif., USA). Lipofectamine 2000 (Invitrogen) was used to transfect SHC003 (vector control) (Sigma, St Louis, MO, USA) with 293FT cells (Invitrogen). The cell culture medium was changed that day after transfection and the supernatants were recovered at 48 and 72 hours after transfection. The supernatant of the virus was filtered at 0.4 μm (Invitrogen). MDA-MB-231 cells were transformed with Turbo-GFP-lentiviruses of 10 multiplicities of infection (MOI). Polybrene (Sigma) was added to the cell culture medium to a final concentration of 6 μg / ml. The cell culture medium was replaced with fresh culture medium on the day after transformation. For selection, puromycin (Sigma) was added to the cell culture medium at a final concentration of 3 μg / ml and left for 3 days.

Example 1-2 Isolation and Culture of Human UCB MSCs

UCB samples were obtained from umbilical cord blood immediately after delivery after maternal consent. The protocol was approved by Boramae Hospital Institutional Review Board (IRB) and Seoul National University IRB. UCB MSCs were obtained by slightly modifying known methods. UCB sample and Hetasep solution (StemCell Technologies, Vancouver, Canada) were mixed at a ratio of 5: 1, and then incubated at room temperature for 30-100 minutes to remove red blood cells. The supernatant was carefully collected and centrifuged at 2500 rpm for 20 minutes at a Ficoll density gradient to obtain mononuclear cells. Washed once or twice with the cells with PBS (phosphate-buffered saline) and, 2 x 10 ⁵ eseo 37 ℃, humid atmosphere of 5% CO ₂ - was inoculated with 2 x 10 ⁶ cell density / cm ^2. Growth media include heparin, ascorbic acid, recombinant human epidermal growth factor (rhEGF), hydrocortisone (hydrocortisone), vascular endothelial growth factor (VEGF), rhFGF-B (recombinant human fibroblast growth factor) D-media (formula no. 78-5470EF) containing ³ -IGF-1 (recombinant long R insulin-like growth factor-1), GA-100 (gentamycin sulfate, amphotericin-B) and 10% FBS (Gibco); Gibco). After 3-4 days of incubation, non-adherent cells were removed and medium was exchanged. The medium was then changed three times a week. Adherent cells formed colonies about 10-14 days after inoculation and then grew rapidly in linear form.

Example 1-3: Isolation and Culture of Human AD MSCs

AD MSCs were isolated from breast adipose tissue obtained during surgery of women who had undergone breast reduction for cosmetic reasons. All courses were pre-approved by SNU IRB. AD MSCs were isolated and incubated according to known methods. The cells were washed with PBS and once or twice, 2 x 10 ⁵ to 0.1 mg / ml in a humidified atmosphere of 5% CO ₂ 37 ℃ - were seeded at a density of 2 x 10 ⁶ cells / cm ^2. Adipose tissue was washed with the same amount of PBS (Gibco) and then digested with mince collagen type I (1 mg / ml; Gibco) at 37 ° C. for 2 hours. The digested tissue was washed with PBS and centrifuged at 1000 rpm for 5 minutes to recover the pellets. The pellet was filtered through a 100 μm nylon mesh (BD Bioscience, San Jose, Ca, USA) to remove cell debris and allowed to stand overnight at 37 ° C. in a 5% humid CO ₂ atmosphere in DMEM supplemented with 10% FBS (Gibco). Incubated. After 24 hours, unattached cells and residual unattached erythrocytes were removed by washing with PBS and the cell medium was exchanged with K-NAC medium. K-NAC medium was modified MCDB 153 medium (keratinocyte-SFM; Gibco-) containing NAC (N-acetyl-L-cysteine; 2 mM; Sigma) and Asc 2P (L-ascorbic acid 2-phosphate; 2 mM; sigma). Invitrogen Corporation). The calcium concentration in the medium was 0.09 mM. Growth factors and hormones added to this medium were recombinant epidermal growth factor (rEGF) (5 ng / mL), bovine pituitary extract (50 µg / mL), insulin (5 µg / mL) and hydrocortisone (74 ng / mL). Was. The medium was changed at 48 hour intervals until the cells reached confluent. When cells reached more than 90% confluence, they were trypsinized and stored in liquid nitrogen or passaged.

Example 1-4: Determination of MSC Homing in Lungs and Tumors

MSCs were labeled with fluorescent dye PKH26 (Sigma) according to the protocol provided by the manufacturer. Lungs and tumors were recovered when killing mice 36 days after tumor cell injection. Samples were washed with ice cold PBS and immediately embedded in OCT compounds (Tissue Tek, Miles, Naperville, IL, USA). 10-micrometer sections were screened for red signal using fluorescence microscopy. All procedures were completed within 24 hours of tissue recovery.

Example 1-5: Animal Experiment

All mouse experiments were performed according to the protocol (SNU-080304-2) approved by the Laboratory Animal Care and Use Committee of Seoul National University. Female NOD / SCID mice were purchased from Korea Research Institute of Bioscience and Biotechnology (Korea). Animals were housed in a pathogen free environment and supplied with sterile diet and water. Cells were washed once or twice with PBS and seeded at a density of 2 × 10 ⁵ −2 × 10 ⁶ cells / cm ² at 0.1 mg / ml in a humid atmosphere of 37 ° C., 5% CO ₂ . The designated number of cells were placed in 100 μl volume of sterile PBS and injected into the fat pad or tail vein of the breast. Six week old NOD / SCID mice were used. Ten mice were used in each group. Tumors were measured twice per week using precision calipers. Tumor volume was calculated as follows: volume = (length x width ² ) / 2. Mice were killed 36 days after tumor cell transplantation.

Example 1-6: Quantification of Lung Metastasis

Mice were killed using CO ₂ asphyxiation and whole lungs were removed. The lungs were washed with ice-cold PBS and placed in ice-cold HBSS (Hank's buffered salt solution; Gibco). The lobes were dissected under brightfield microscopy and irradiated with fluorescence microscopy within 20 hours of incision to prevent autofluorescence of tissues that typically appear 24 hours after necropsy. Lung metastasis was assessed by the number of GFP-positive colonies observed under fluorescence microscopy. Lung metastasis index for each mouse was calculated by dividing the number of GFP-positive colonies observed in the lung by the mass of primary tumor (g).

Example 1-7 Histological Analysis

For histological analysis, paraffin sections were deparaffinized in xylene, rehydrated with a gradient series of alcohols and stained with hematoxlin and eosin (H & E).

Example 1-8: Immunohistochemistry

Immunohistochemical analysis was performed on tissues fixed in formalin and embedded in paraffin. 5-micrometer thick sections were deparaffinized, rehydrated and antigen regenerated. Sections were incubated for 30 minutes in 3% hydrogen peroxide (AppliChem, Darmstadt, Germany) to inhibit endogenous peroxidase activity. Tissue sections were washed with PBS and then incubated with 5% BSA (bovine serum albumin; Sigma) dissolved in PBS for 1 hour at room temperature to block nonspecific binding sites. Primary antibodies against PARP (poly (ADP-ribose) polymerase-1; Cell Signaling, Beverly, MA, USA) were applied to tissue sections at 4 ° C. for 1 hour. The following day, tissue sections were washed and incubated with secondary HRP-conjugated Ab for 1 hour at room temperature. Tissue sections were washed carefully, counterstained with Mayer's hematoxylin (Dako, Carpinteria, Calif., USA) and washed with xylene. Cover slip was sampled using Permount (Fisher Scientific) and slides were observed using light microscopy (Carl Zeiss, Thornwood, NY, USA).

Example 1-9: Western Blot Analysis

After measuring the protein concentration of the homogenized lysate extracted from the tumor sample, 10 μg of protein was isolated using 10% sodium dodecyl sulfate-polyacrylamide gen electrophoresis (SDS-PAGE) and transduced onto a polyvinylidene fluoride (PVDF) membrane. Blot. The blots were blocked with a powdered nonfat milk solution containing 5% 0.05% Tween-20 (5% dissolved in PBS) and then diluted 1: 1000 with 5% blocking solution. Cell Signaling) and rabbit anti-human cleaved Caspase-3 (Cell Signaling) were probed overnight at 4 ° C. Anti-rabbit IgG diluted 1: 5000 was used as secondary antibody. Cleaved PARP bands were detected with an ECL kit (enhanced chemiluminescence; Amersham Bioscience / GE Healthcare, Little Chalfont, UK).

Example 2: Results

2-1. Transplantation of Human UCB and AD MSCs Inhibits Tumor Growth

The properties of AD MSCs as MSCs have already been demonstrated. UCB MSCs expressed MSC surface antigens. MDA-MB-231 human breast cancer cells were transformed by Turbo-GFP. High expression of GFP was observed 3 days after screening with puromycin (data not shown). MDA-MB-231 cells labeled with 2 × 10 ⁶ GFP were then injected into the left breast breast fat pad. Tumors were observed 7 days after transplantation. 1B shows the green fluorescence of tumors produced by MDA-MB-231 cells labeled with GFP. Tumor volume increased to about 200 mm ³ 22 days after tumor cell injection. On day 22, 1 × 10 ⁶ human UCB or AD MSCs were labeled intravenously with PKH26 and then injected intravenously. This process is shown in Figure 1A. Tumor growth was inhibited after transplantation of human UCB or AD MSCs. On day 36, the tumors treated with MSC were significantly smaller than the controls (FIG. 1C).

2-2. Human UCB and AD MSC Transplantation Inhibits Lung Metastasis

Breast cancer cells can metastasize to the lungs. The number of metastases to the lung was compared 14 days after stem cell transplantation. Both UCB and AD MSC reduced the number of metastases to the lung by 50% compared to the control (FIG. 2B). H & E staining was performed on paraffin sections. Morphological differences between the groups are shown in Figure 2C.

2-3. Human UCB and AD MSCs Return to the Lung and Primary Tumor Sites

To track the homing capacity of the transplanted MSCs into the primary tumor site and lungs, both UCB and AD MSCs were labeled with PKH261. Labeled UCB or AD MSCs were administered intravenously to mice bearing tumors. Animals were killed and tissues were recovered 14 days after stem cell transplantation. 10-micron section slides were prepared to detect homing of stem cells (each group n = 5 and 10 sections were prepared for each mouse). As shown in FIG. 3, UCB and AD MSCs were grafted to the primary tumor location and lungs. The number of MSCs was quantified and shown in FIG. 3B.

2-4. Human UCB and AD Induce PARP Cleavage

Immunohistochemistry was performed to investigate the mechanism of tumor growth inhibition. It was assumed that apoptosis signaling would be involved in this process. PARP is considered to be an important protein for maintaining cell survival; It is a major cleavage target of caspase protein. Lungs and tumors were extracted from mice 14 days after stem cell injection. 5-micrometer paraffin sections were prepared (n = 5 in each group and 10 sections were prepared for each mouse). As shown in FIG. 4A, tumor sections exhibited an immunoreactivity positive for cleaved PARP. However, PARP immunoreactivity was rare in lung metastases.

To further investigate whether human UCB or AD MSCs are involved in PARP cleavage, proteins were extracted from tumors and lungs and subjected to western blotting. Increased cleavage of PARP and caspase-3 meant that UCB and AD MSC treatment induced apoptosis in tumor tissue (FIG. 4C). However, the number of human MSCs grafted into the lungs is limited, and cleavage of PARP and caspase-3 in lung samples could not be detected.

2-5. Human UCB and AD MSCs Inhibit Tumor Progression from Early Stage

The effects of UCB and AD MSCs were further investigated in the early stages of breast cancer. This protocol is shown in Figure 5A. Human MDA-MB-231 cells labeled with 1 × 10 ⁶ GFP were mixed with human UCB and AD MSCs and injected into mouse mammary fat pads. Tumor formation and progression were inhibited by human USC and AD MSCs (FIG. 5B). On day 42, the tumor volume in both the UCB and AD MSC transplanted groups was small compared to the control. Tumor volume in the UCB MSC treated group was significantly smaller than in the AD MSC treated group. Lung metastasis index is shown in Figure 5C. The number of colonies in the metastasized lung in UCB MSC treated group was smaller than in AD MSC treated group. However, both types of stem cells inhibited lung metastasis (FIG. 5C).

2-6. Intravenous transplantation of human UCB and AD MSCs is safe in mouse breast cancer models

Potential risks should be carefully assessed before providing stem cells to patients with certain diseases. Intravenous administration has been widely used in cell therapy. Since stem cells are known to promote cancer formation and metastasis, intravenous administration of stem cells should be evaluated in early stage cancer models where there are no clinical symptoms. In the present invention, human MDA-MB-231 cells labeled with 1 × 10 ⁶ GFP were injected into mouse mammary fat pad. On the same day human UCB and AD MSCs were administered intravenously. Tumor formation and progression were inhibited by UCB MSCs, but not by AD MSCs (FIG. 6B). At day 42 the tumor volume in the group transplanted with UCB MSCs was smaller than that of AD MSCs and controls. There was no significant difference between AD MSC treated group and control group. Lung metastasis index is shown in Figure 6C. The number of pulmonary colonies metastasized in the UCB MSC treated group was less than in the AD MSC treated group and the control group. Thus, it did not appear to affect tumor formation and metastasis after administration of AD MSC (FIG. 6C).

Figure 1 shows that human UCB and AD MSC transplantation inhibits tumor growth, (A) shows that GFP-MDA-MB-231 cells injected into the fat pad of the mouse breast produce a green fluorescent tumor (B) shows the protocol and (C) shows that UCB and AD MSCs inhibit tumor growth.

Figure 2 shows that human UCB and AD MSC transplantation inhibits metastasis to the lung, (A) shows that GFP-MDA-MB-231 cells injected into the fat pad of the mouse breast can metastasize to the lung, (B) shows that UCB and AD MSCs can reduce lung metastasis compared to controls, and (C) shows H & E staining results for UCB or AD MSC treated and control groups and tumor colonies are indicated by red circles. It was.

Figure 3 shows that human UCB and AD MSCs return to the lung and primary tumor site, (A) shows that UCB and AD MSCs are grafted to the primary tumor site and lung, where red is UCB labeled PKH26 and AD MSCs are shown, and (B) is a quantitative number of MSCs present in colonies that metastasize to primary tumors and lungs.

4 shows that human UCB and AD MSCs induce cleavage of PARP and caspase-3, (A) shows that cleaved PARP immunoreactivity is seen in tumor sections, but rare in colony sections that have metastasized to the lung (B) shows the quantification of the number of cells that are immunopositive in the tumor section, (C) shows the Western blotting results for the protein extracted from the tumor.

5 shows that human UCB and AD MSCs inhibit tumor progression from an early stage, (A) shows the protocol, and (B) shows that tumor formation and progression are inhibited by human USC and AD MSCs. (C) is a quantitative representation of lung metastasis index.

6 shows that venous transplantation of human UCB and AD MSCs is safe in mouse breast cancer models, (A) depicts this protocol, and (B) in AD MSCs, although tumor formation and progression are inhibited by human USCs. (C) indicates that the number of lung metastasis colonies in the human UCB treated group was lower than in AD MSC or control.

Claims (9)

An anticancer composition comprising isolated cord blood-derived ZNF281 expressing adult stem cells. The method of claim 1, Gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer or cervical cancer. The method of claim 1, The cord blood-derived ZNF281 expressing adult stem cells have at least one of the following characteristics: (a) show positive immunological properties to the transcriptional regulator c-myc; (b) the extracellular matrix adheres to the coated bottom and proliferates in a line or sphere cell colony between 5 and 30 days after attachment; (c) shows a cumulative population doubling level (CPDL) of 30 to 45; (d) exhibit negative immunological properties for CD14, CD31, CD34, CD45 and HLA-DR; (e) capable of differentiating into cells of mesoderm, endoderm and ectoderm; (f) TIMP-2, TGF-β, RANTES CINC-3, EOTAXIN, GM-CSF, IFN-γ, IL-1b, IL-3, IL-6, IL-8, IL-10, IL12p40, IL13, Secrete at least one cytokine or chemokine selected from the group consisting of IL-16, IP-10, Leptin, MCP-2, MIG, MIP-3a, b-NGFm, sTNFRI, PFGF-bb. The method of claim 1, The adult stem cell composition, characterized in that to inhibit the metastasis of cancer cells. The method of claim 1, The adult stem cell is characterized in that the return to the lung and the primary tumor site. The method of claim 1, The adult stem cell is characterized in that to promote the growth inhibition and death of cancer cells through apoptosis or immunomodulation (immune modulation) of breast cancer cells. The method of claim 1, A composition characterized in that it is formulated for intravenous administration or for direct intratumoral administration. delete delete

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