TWI633187B - Cancer initiating cell and use thereof - Google Patents

Abstract

The present invention relates to a cancer-initiating cell comprising an isolated excess of Crox virus expressing Oct-4 and an adenovirus receptor-positive mouse lung stem/progenitor cell. The invention also relates to the use of a tumor-bearing mouse for screening for an anticancer drug, wherein the tumor cell is induced by a cancer-initiating cell, wherein the cancer-initiating cell comprises an isolated Exxax virus exhibiting Oct-4 and Adenovirus receptor-positive mouse lung stem cells/pioneer cells.

Description

Cancer initiation cell and use thereof

The present invention relates to a cancer-initiating cell comprising an isolated excess of Crosic virus and an adenovirus receptor-positive mouse lung stem cell/progenitor cell exhibiting Oct-4 and its use.

Lung cancer is one of the leading causes of cancer-related deaths worldwide, and the overall five-year survival rate is still less than 14%. A growing body of evidence suggests that cancer stem cells, also known as cancer initiating cells (CICs), play a key role in tumor growth and traditional chemotherapy resistance and may lead to cancer metastasis and recurrence. . Cancer stem cells: quotes, hopes and challenges.

Cancer-initiating cells (CICs) can be identified using different in vitro assays and cell markers, such as side population analysis, sphere forming assay, chemotherapy resistance, acetaldehyde dehydrogenase ( Aldehyde dehydrogenase; ALDH) activity and cell marker CD133. However, the use of the above in vitro assay alone did not confirm that the cells tested were indeed cancer-initiating cells. Therefore, specific in vivo assays, such as the animal model's limiting dilution transplantation experiment, were used to confirm the results of the in vitro assay. Unfortunately, the identification of cancer-initiating cells (CICs) often conflicts among different cancer types. Inconsistencies in the identification of cancer-initiating cells (CICs) may result from The cells studied are derived from different cancer cell lines or mature tumors, and the phenotypic or functional heterogeneity of clinical tumor samples may exacerbate the difficulty in identifying cancer-initiating cells (CICs).

Different hypotheses have been proposed today for the interpretation of the formation of cancer-initiating cells (CICs), such as mutations in adult stem/progenitor cells or differentiation of cells to obtain stem cell-like properties; however, cell origin and related cancers The process of development of the initial cells (CICs) remains unclear. In a mouse model of K-ras ^G12D mutation, stem cells at the bronchioalveolar duct junction were detected as potential sources of adenocarcinoma after Cre/lox-regulated activation. Another study demonstrates that Oct-4, mediated by IGF-IR signaling, forms a complex with beta-carotene and Sox-2, which is self-renewing and carcinogenic to cancer-initiating cells (CICs) in lung adenocarcinoma. Play an important role. In addition, in the lung adenocarcinoma cell line A549, both Oct-4 and Nanog can control epithelial-mesenchymal transdifferentiation, regulate tumor initiation ability and promote metastasis. In addition, patients with non-small cell lung cancer who present high amounts of Oct-4 are associated with cancer metastasis and lower survival rates. Although these studies confirm that certain pluripotent genes, such as Oct-4, Sox-2, and Nanog, are closely related to tumor initiation properties, the expression of abnormal pluripotency genes and cancer-initiating cells (CICs) are generated. The relationship is still unclear and needs further clarification.

The present invention relates to a cancer-initiating cell (CICs) comprising an excess of Oct-4 virus and adenovirus receptor-positive mouse lung stem cells/precursor cells (coxsackievirus and adenovirus receptor positive mouse pulmonary stem/). Progenitor cells; CAR ⁺ /mPSCs) (CAR ⁺ /mPSC ^Oct-4_hi ); the present invention also relates to a method for screening a cancer-resistant mouse for anticancer drug, wherein the tumor cell is induced by a cancer-initiating cell (CIC) Wherein the cancer-initiating cells comprise an excess of Exxaxac and an adenovirus-positive mouse lung stem/progenitor cells (CAR ⁺ /mPSC ^Oct-4_hi ) exhibiting ^Oct-4 .

Solid tumors are thought to result from organs containing stem cell populations, including tumors of heterogeneous populations of cancer cells, whose proliferation is significantly different from the ability to form new tumors; differences in tumor formation capabilities, such as breast cancer, have been reported Cells and tumors of the central nervous system. Although most cancer cells have limited ability to divide, recent studies have indicated that a group of cancer cells called cancer stem cells or cancer initiating cells (CICs) have extensive self-renewal and ability to form new tumors. Increasing evidence suggests that the pathways that regulate normal stem cell self-renewal are uncontrolled or altered in cancer stem cells, leading to self-renewing cancer cells expanding and forming tumors.

In the present invention, cancer-initiating cells (CICs) are produced in animal models to gain insight into the nature and characteristics of cancer-initiating cells (CICs), which can aid cancer research to provide insight into the early diagnosis and treatment of lung cancer. In previous studies, mouse lung stem/progenitor cells (mPSCs) were cultured in serum-free primary screening, followed by the use of oxalic acid and adenovirus receptors in culture (coxsackievirus and adenovirus). Receptor; CAR) was isolated by flow cytometry as a positive screening marker. The coxsackievirus and adenovirus receptor positive mouse pulmonary stem/progenitor cells (CAR ⁺ /mPSCs) exhibit the characteristics of stem cells/pioneer cells and can differentiate into the first Type-I pneumocytes and have angiogenic potential. The present invention recognizes Oct-4 positive lung stem cells/pione cells and confirms their sensitivity to SARS coronavirus (SARS-CoV) in vitro. The lung stem cells/pione cells differentiate into alveolar pneumocytes, and the angiogenesis is induced in a 3D gelatin-microbubble scaffold. The present invention demonstrates that CAR ⁺ /mPSCs can be transformed by overexpression of Oct-4 and develop into a typical cancer initiating cell (CICs) phenotype, a type 1 lung cell wall cell derived from CAR ⁺ /mPSCs (type- I pneumocytes) have also been tested. In the experiments described herein, the characteristics of transformed cells are determined by in vitro assays, including cell cycle and telomerase activity assays, sphere forming assays, CD133 assays, acetaldehyde dehydrogenase (aldehyde dehydrogenase; ALDH) activity and chemotherapy resistance test. In addition, in vivo assays include limiting dilution transplantation and tumor metastasis testing of Severe Combined Immunodeficiency Mice (SCID mice) for further study of the characteristics of transformed cells. Endoelial tube formation and chicken chorioallantoic membrane (CAM) assays are used to assess angiogenesis in transformed cells because of their ability to induce angiogenesis and other characteristics of cancer-initiating cells (CICs). Potential.

In the present invention, it was confirmed that an overexpression of the pluripotency transcription factor Oct-4 in CAR ⁺ /mPSCs can produce transformed cells, which are called CAR ⁺ /mPSCs ^Oct-4_hi . These transformed cells have cancer/tumor initiation, chemotherapy resistance, and significant expression of certain pro-angiogenic factors, including angiopoietin (ANGs) and vascular endothelial growth factor (VEGF), and enhancement. The angiogenic potential. In addition, CAR ⁺ /mPSCs ^Oct-4_hi displays endothelial cell markers including CD31, CD105, CD34 and CD144. In addition, CAR ⁺ /mPSCs ^Oct-4_hi actively participates in tumor angiogenesis and activates the ANGs/Tie2 signaling pathway; these findings provide novel insights into the possible origins and genesis of cancer-initiating cells (CICs) and help elucidate the initiation of cancer Cells (CICs) are responsible for the pathways involved in angiogenesis and provide new strategies for anti-angiogenic therapy for lung cancer.

"an" may be used in connection with the word "comprising", and "a" may mean one or more.

Accordingly, the present invention provides a cancer-initiating cell (CIC) comprising an excess of Oct-4 virus and an adenovirus receptor-positive mouse lung stem cell/precursor cell (coxsackievirus and adenovirus receptor positive mouse pulmonary) Stem/progenitor cell; CAR ⁺ /mPSC) (CAR ⁺ /mPSC ^Oct-4_hi ).

The term "cancer initiating cell (CIC)" as used in this specification and the scope of the patent application is interchangeable and is referred to as a solid cancer stem cell. The definition and function of cancer-initiating cells are characterized as a small group of tumors, which can grow indefinitely under appropriate conditions in vitro (have self-renewal ability), and can live only with a small number of cells (<10 ² cells). Tumors form in the body. Other common methods for identifying cancer-initiating cell (CIC) characteristics include morphology, cell surface marker detection, transcriptional profile, and drug response.

The CAR ⁺ /mPSC in excess of Oct-4 represents that Oct-4 expression in CAR ⁺ /mPSC is 10 times higher than that in normal cells. In a preferred embodiment, the amount of Oct-4 in CAR ⁺ /mPSC is 16 times higher than in normal cells. In a more preferred embodiment, the amount of Oct-4 in CAR ⁺ /mPSC is higher than that in normal cells. 20 times. "Normal cells" herein refers to normal CAR ⁺ /mPSC. The amount of expression refers to the amount of expression of a DNA, RNA or protein. In a preferred embodiment, the amount of expression refers to the amount of protein expressed. This protein is obtained by encoding the gene of Oct-4. In a more preferred embodiment, the sequence of the Oct-4 gene consists of SEQ ID NO: 1. In another specific embodiment, the Oct-4 gene is a cDNA of Oct-4.

In another specific embodiment, the CAR ⁺ /mPSC comprises a vector, wherein the vector comprises a nucleic acid sequence encoding the Oct-4 gene. In a preferred embodiment, the sequence of the Oct-4 gene is SEQ ID NO: 1.

In a specific embodiment, the CAR ⁺ /mPSC ^Oct-4_hi exhibits soft agar colony formation, sphere formation, and immortality.

In another specific embodiment, the CAR ⁺ /mPSC ^Oct-4_hi has cancer cell function, wherein the cancer cell function comprises cell proliferation, cell metastasis, cell invasion, or a combination thereof.

In a specific embodiment, the CAR ⁺ /mPSC ^Oct-4_hi has a tumor initiating capacity. In a preferred embodiment, the CAR ⁺ /mPSC ^Oct-4_hi has a tumorigenic capacity, wherein the tumorigenic capacity comprises tumor formation, tumor regeneration, metastatic ability, or a combination thereof. The CAR ⁺ /mPSC ^{Oct-4_hi of the} present invention can grow indefinitely in certain embodiments, and tumors can be generated from <10 ³ cells in vitro. In a preferred embodiment, the CAR ⁺ /mPSC ^{Oct-4_hi grows} indefinitely and produces tumors from <10 ² cells in vitro.

Different biomarkers have been proposed for lung cancer initiation cells, including CD133 expression, aldehyde dehydrogenase (ALDH) activity, and chemotherapy resistance. In a specific embodiment, CAR+/mPSC ^Oct-4_hi exhibits CD133, aldehyde dehydrogenase (ALDH) activity, chemotherapy resistance, and combinations thereof. This chemoresistance means that CAR ⁺ /mPSC ^Oct-4_hi is resistant to chemoradiation or chemotherapy. Therefore, the cancer-initiating cell is a lung cancer-initiating cell. In a specific embodiment, the CAR ⁺ /mPSC ^Oct-4_hi is a lung cancer initiation cell.

In a specific embodiment, the CAR ⁺ /mPSC ^Oct-4_hi has the function of angiogenesis. In a preferred embodiment, the CAR ⁺ /mPSC ^Oct-4_hi has a function of participating in angiogenesis. Therefore, the CAR ⁺ /mPSC ^Oct-4_hi not only has angiogenic potential, but also participates in tumor angiogenesis. In another specific embodiment, the CAR ⁺ /mPSC ^Oct-t_hi has the function of activating the ANG/Tie2 ^signaling pathway to enhance angiogenesis. In a preferred embodiment, CAR ⁺ /mPSC ^Oct-4_hi has the function of activating the Tie2 ^signaling pathway to enhance angiogenesis.

In another specific embodiment, the CAR ⁺ /mPSC ^Oct-4_hi represents a surface marker of endothelial cells, wherein the surface marker of the endothelial cell comprises CD31, CD105, CD34, CD144, or a combination thereof.

The invention also provides the use of a tumor-bearing mouse for screening for an anti-cancer drug, wherein the tumor cell is induced by a cancer-initiating cell (CIC), wherein the cancer-initiating cell (CIC) comprises an excess of Oct-4. Isolated oxacinvirus and adenovirus receptor positive mouse pulmonary stem/progenitor cell (CAR ⁺ /mPSC) (CAR ⁺ /mPSC ^Oct-4_hi ).

In a specific embodiment, the animal is a mouse.

The term "isolated" as used herein refers to a material that is substantially or substantially free of concomitant components found in the natural state.

"Anti-cancer drug" means a composition comprising an active ingredient having antitumor activity, and "antitumor activity" means an effect of inhibiting tumor growth, a tumor cytotoxicity and/or a tumor degenerative effect.

The CAR ⁺ /mPSC in excess of Oct-4 represents that the amount of Oct-4 in CAR ⁺ /mPSC is 10 times higher than that in normal cells. In a preferred embodiment, the amount of Oct-4 expressed in CAR ⁺ /mPSC is 16 times higher than that of normal cells. In a more preferred embodiment, the amount of Oct-4 expressed in CAR ⁺ /mPSC is 20 times higher than that of normal cells. The "normal cell" in this paper is normal CAR ⁺ /mPSC. The amount of expression refers to the amount of expression of a DNA, RNA or protein. In a preferred embodiment, the amount of expression refers to the amount of protein expressed, which is encoded by a gene encoding Oct-4. In a more preferred embodiment, the sequence of the Oct-4 gene is SEQ ID NO: 1.

In another embodiment, the CAR ⁺ /mPSC comprises a vector, wherein the vector comprises a nucleic acid sequence encoding the Oct-4 gene. In a preferred embodiment, the Oct-4 gene sequence is SEQ ID NO: 1.

In a specific embodiment, the cancer initiating cell is a lung cancer initiating cell. In a preferred embodiment, the tumor is a lung tumor. In another specific embodiment, the anticancer drug is a drug that treats cancer initiating cells. In a preferred embodiment, the anticancer drug is an anti-lung cancer drug. In a more preferred embodiment, the anticancer drug is a drug that treats the initiation cells of lung cancer.

The term "anti-cancer" in this article encompasses the treatment and inhibition of cancer. In addition, "anti-cancer" encompasses the treatment and/or inhibition of cancer-initiating cells. The term "treatment" as used herein includes healing, healing, slowing, alleviating, altering, remedying, ameliorating, improving or affecting a disease, a symptom of a disease, or a predisposition to disease. Treating and/or inhibiting cancer-initiating cells can improve, prevent, eliminate or reduce the number of cancer-initiating cells in an individual, or eliminate cancer-initiating cells in an individual, and the like.

In a specific embodiment, the individual is an animal; preferably, the individual is a mammal; more preferably, the individual is a human.

The present invention further provides a method for screening an anticancer drug, comprising: (a) implanting a cancer-initiating cell in an animal, wherein the cancer-initiating cell comprises an isolated oxacinvirus exhibiting an excess of Oct-4 And an adenovirus receptor-positive mouse lung stem cell/precursor cell (CAR ⁺ /mPSC ^Oct-4_hi ), wherein the cancer-initiating cell develops and forms a tumor; (b) administering a drug candidate to the animal; and (c) The effects of drug candidates on tumors containing cancer-initiating cells (CICs) were evaluated.

In a specific embodiment, the anticancer drug is a drug for treating cancer initiating cells. In a preferred embodiment, the anticancer drug is a drug against lung cancer. In a more preferred embodiment, the antibody A cancer drug is a drug that treats the starting cells of lung cancer.

In another specific embodiment, the animal is a rodent, preferably a rat or a mouse.

The CAR ⁺ /mpSCs overexpressing Oct-4 represents an Oct-4 expression in each CAR ⁺ /mPSCs that is 10 times higher than normal cells. In a preferred embodiment, the amount of Oct-4 per CAR ⁺ /mPSC is 16 times higher than that of normal cells. In a more preferred embodiment, the amount of Oct-4 per CAR ⁺ /mPSC is 20 times higher than that of normal cells. In a preferred embodiment, the amount of expression refers to the amount of expression of a DNA, RNA or protein encoded by the gene of Oct-4. In a more preferred embodiment, the sequence of the Oct-4 gene is SEQ ID NO: 1, and in another embodiment, the Oct-4 gene is the cDNA of Oct-4.

In another specific embodiment, each CAR ⁺ /mPSC comprises a vector, wherein the vector comprises a nucleic acid sequence encoding the Oct-4 gene. In a preferred embodiment, the sequence of the Oct-4 gene is SEQ ID NO: 1.

The term "tumor" refers to the respective stages of benign and malignant tumors. The first stage of tumor progression is an increase in the amount of relatively normal cells, which is the stage of proliferation. In several stages of hyperplasia, the cells gradually accumulate and begin to develop abnormal appearance, which is the emergence of abnormal stages of development.

As used herein, a "candidate drug" refers to any molecule, such as a protein or drug, which is essentially capable of inhibiting the growth of tumor cells.

In a specific embodiment, the cancer initiating cell is a lung cancer initiating cell. In a preferred embodiment, the tumor is a lung tumor. For the latter case, the initial assessment of the effect of the candidate drug is, for example, visual assessment of tumor size and severity. This has the added advantage that visual inspection of the tumor allows for an assessment of the immediate and sustained efficacy of the drug. In the case of non-visible tumors, drug efficacy assessment usually requires the examination of the tumor by sacrificing the animal. Tumors can be detected according to standard techniques of the skilled person, including, in addition to visual inspection (for skin damage), histochemistry and immunohistochemistry techniques. Typically, drug candidates are evaluated for their ability to inhibit tumor formation and/or growth developed by transplanted cell lines. This invention further includes It is determined whether the candidate drug is an anticancer drug, which is the result of the drug efficacy evaluation according to the step (c).

Furthermore, the present invention provides a method for screening an anticancer drug, comprising: (1) providing a tumor tissue, the tumor tissue comprising cancer initiating cells (CICs), wherein the cancer initiating cells (CICs) comprise Overexpression of isolated Octavirus and adenovirus receptor-positive mouse lung stem cells/precursor cells (CAR ⁺ /mPSC ^Oct-4_hi ); (2) contacting drug candidates with the tumor tissue; 3) Detection of the effects of drug candidates on tumor tissues.

In a specific embodiment, the cancer initiating cells (CICs) are a lung cancer initiating cell. In a preferred embodiment, the tumor tissue is a lung tumor tissue. In another specific embodiment, the anticancer drug is a drug that treats cancer initiating cells (CICs). In a preferred embodiment, the anticancer drug is a drug against lung cancer. In a more preferred embodiment, the anticancer drug is a drug against lung cancer initiating cells (CICs).

The CAR ⁺ /mPSC in excess of Oct-4 represents that the expression of Oct-4 in CAR ⁺ /mPSC is 10 times higher than that in normal cells. In a preferred embodiment, the amount of Oct-4 in the CAR ⁺ /mPSC is 16 times higher than in normal cells. In a more preferred embodiment, the amount of Oct-4 expressed in CAR ⁺ /mPSC is 20 times higher than that of normal cells. The amount of expression refers to the amount of expression of a DNA, RNA or protein. In a preferred embodiment, the amount of expression refers to the amount of protein expressed by the gene encoding Oct-4. In a more preferred embodiment, the sequence of the Oct-4 gene is SEQ ID NO: 1.

In another embodiment, each CAR ⁺ /mPSC comprises a vector, wherein the vector comprises a nucleic acid sequence encoding an Oct-4 gene. In a preferred embodiment, the sequence of the Oct-4 gene is SEQ ID NO: 1.

In a specific embodiment, detecting the effect of the candidate drug on the tumor tissue comprises observing changes in cancer-initiating cells (CICs) over time, the progression of the cancer, or its biological properties within the tumor tissue. The present invention further comprises a step of confirming whether the candidate drug is an anticancer drug, which is based on the result of the test compound inhibiting effect in the step (3).

The present invention also encompasses a method of screening for a drug candidate for anticancer comprising: (i) collecting a culture fluid containing cancer initiating cells (CICs), wherein the cancer initiating cells comprise an excess of Oct-4. Mouse lung stem cells/precursor cells (CAR ⁺ /mPSC ^Oct-4_hi ) that are positive for oxacinvirus and adenovirus receptor; (ii) extract an exosomal protein from cancer-initiating cells (CICs); (iii) analyzing exosomal proteins; and (iv) comparing the results of the analysis using step (iii) with a drug database to obtain candidate drugs.

In a specific embodiment, the cancer initiating cells (CICs) are a lung cancer initiating cell. In a preferred embodiment, the drug candidate for anticancer is a drug candidate for treating cancer initiating cells (CICs). In a more preferred embodiment, the drug candidate for anticancer is a drug candidate for treating lung cancer initiation cells. In another specific embodiment, the drug candidate for anticancer is a drug candidate against lung cancer.

In another embodiment, the drug database is a DrugBank.

In a specific embodiment, the invention further comprises a step after step (iv), which is step (v), which is to test the cytotoxicity of the candidate drug for cancer-initiating cells (CICs). Sex. If the candidate drug is significantly toxic to cancer-initiating cells (CICs), the drug candidate is an anti-cancer drug or a drug for treating cancer-initiating cells (CICs).

In addition, the present invention provides a method of preparing a population of cancer-initiating cells (CICs) comprising the steps of: (1) providing a vector comprising a nucleic acid sequence encoding an Oct-4 mutual cDNA; (2) transferring the vector Dyeing to the lung stem cell/precursor cell population (CAR ⁺ /mPSCs) of the Crox virus and adenovirus receptor-positive mice, wherein the vector increases the amount of Oct-4 cDNA in excess by increasing the number of copies of the nucleic acid sequence The number of copies normally present in wild-type CAR ⁺ /mPSCs; and (3) the CAR ⁺ /mPSCs group (CAR ⁺ /mPSC ^Oct-4_hi ) that overexpresses ^Oct- 4 in the separation step (2).

In a specific embodiment, the gene sequence of the Oct-4 cDNA is SEQ ID NO: 1.

In another specific embodiment, the Oct-4 cDNA has the function of encoding an Oct-4 protein.

The term "vector" as used herein refers to a nucleic acid molecule having a nucleic acid sequence that is cleavable in a host cell. The vector also comprises a nucleic acid sequence within the vector that allows for ligating the nucleic acid sequence, which nucleic acid sequence also replicates in the host cell. Representative of the vector comprises a plasmid, a cosmid or a viral vector; preferably, the vector is a retroviral vector.

Figure 1 shows the culture, isolation and differentiation of lung stem cells/precursor cells (CAR ⁺ /mPSCs) from saxagvirus and adenovirus receptor-positive mice. (A) Immunofluorescence staining of coxsackievirus and adenovirus receptor positive (CAR) in primary culture epithelial cell population, (i) in white in phase contrast image The dotted line indicates the epithelial cell population, (ii) the CAR is displayed on the cell-cell junctions of the epithelial cell population for the immunofluorescence image, and (iii) is the magnified image of the inner region of the frame of Figure A-ii (scale bar) : 100 μm). (B) Identification and isolation of CAR-positive populations in primary culture using a fluorescent flow cytometry (FACS), referred to as CAR ⁺ /mPSCs. (C) Gene expression profiles of CAR ⁺ /mPSCs were analyzed by polymerase chain reaction (PCR) and real-time PCR. The gene expression of CAR, Oct-4, Sox-2 and Nanog was evaluated. "L" is mouse lung tissue; "CAR ⁺ " is CAR ⁺ /mPSCs; "ES" is mouse embryonic stem cell line (E14). Experimental data are expressed as mean ± standard deviation. (D) is CAR ⁺ / mPSCs differentiation, CAR ⁺ / mPSCs 7 after separated into a first type talent alveolar wall cells (type-I pneumocytes). On day 1, the magnified image shows the isolated cells within the frame. The white dotted line indicates the phase contrast images of the differentiated cells on days 4 and 7. The expression of CAR and type 1 p-type pneumocytes labeled T1α and AQP5 was assessed by immunofluorescence staining. CAR performance can be detected on day 1, and a magnified image in the box shows that CAR is expressed between cell junctions of isolated cells. The CAR disappeared on the 4th and 7th days. The performance of T1α and AQP5 was detected on days 4 and 7. (Scale bar: 100μm)

Figure 2 shows the excess performance of Oct-4 in CAR ⁺ /mPSCs, which is the step of overexpressing Oct-4 in CAR ⁺ /mPSCs. (i) Representative of the phase contrast maps of the primary culture, the enlarged images show the epithelial cell population of mPSCs. (ii) mPSCs were isolated by flow cytometry in accordance with CAR-positive expression in primary cultured cells, and immediately transfected with retroviral vectors encoded with Oct-4 cDNA. (iii) Co-culture of transfected CAR ⁺ /mPSCs with feeder cells on day 2, a cobble-like cell population was observed on day 21, and a magnified image showing the morphology of a single colony. (iv) Separation and amplification of individual communities like cobblestones on day 28. (v) Establishing a cell line for the community comprising C1, E9 and C7. The morphological map of the C1 cell line is representative. (Scale bar: 100μm)

Figure 3 shows the overexpression of Oct-4 in type 1 type I pneumocytes. In CAR ⁺ / a first type of lung-derived mPSCs muramyl cells (type-I pneumocytes), the time period of Oct-4 overexpression of the process shown in FIG. (i) is a phase contrast representative map showing the primary culture medium of the epithelial cell population. (ii) The differentiation of CAR ⁺ /mPSCs into type 1 type I pneumocytes on day 7. (iii) On day 8, type 1 p-type pneumocytes were transfected with retroviral vectors encoding the Oct-4 cDNA. (iv) Proliferation when the transfected cells were added to the feder cell supplement on days 10, 21, 35, and 42. (Scale bar: 100μm)

Figure 4 shows the ultra-high performance of Oct-4 in CAR ⁺ /mPSCs ^Oct-4_hi . (i) Analysis of Oct-4 expression in C1, E9 and C7 cell lines of CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi by Western blotting, "ES" means mouse embryonic stem cell line (E14). (ii) Quantification of Oct-4 performance, data expressed as mean ± standard deviation. ** P < 0.01: compared to CAR ⁺ /mPSCs.

Figure 5 shows the amount of CAR in CAR ⁺ /mPSCs ^Oct-4_hi . (A) is a group analysis of CAR positive in C1, E9 and C7 cell lines by flow cytometry. (B) is the amount of CAR expression in the cell membrane of C1, E9 and C7 cell lines by immunofluorescence staining, labeling CAR under Alex488nm fluorescence, DAPI is the label of the nucleus, and the inserted image is enlarged in a square area. Figure. Scale bar: 100 μm.

Figure 6 shows the phenotypic changes of CAR ⁺ /mPSCs ^Oct-4_hi . (A) is the altered cell cycle distribution showing a representation of the cell cycle of the C1 cell line and CAR ⁺ /mPSCs. (B) shows the proliferation ability of the C1, E9, and C7 cell lines of CAR ⁺ /mPSCs ^Oct-4_hi , which shows the growth curves of C1, E9, and C7 cell lines of CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi . (C) is the telomerase activity of CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi cell lines, and evaluated the performance of C1, E9 and C7 cell lines at ^{12th, 20th} and 50th generations, CAR ⁺ stands for CAR ⁺ /mPSCs cells, H stands for thermal deactivation.

Figure 7 shows the tumorigenic ability of the CAR ⁺ /mPSCs ^Oct-4_hi C1 cell line. (A) Tumor production of a C1 cell line was performed in Severe Combined Immunodeficiency mice (SCID mice). 1x10 ⁶ cells of C1 cell line (quantity = 6) or CAR ⁺ /mPSCs (quantity = 4) were mixed with matrigel and subcutaneously transplanted into the back of severe combined immunodeficient mice. Tumor growth was monitored with a calipers gauge. (i) After 28 days, a tumor derived from the C1 cell line was observed, the arrow was the position where the C1 cell strain was injected, and the arrow was the position of the CAR ⁺ /mPSCs injection. Further examination after tumor removal. (ii) The tumor growth curve calculated based on data collected daily after injection, and the data of individual tumor measurements are expressed as mean ± standard deviation. (B) Representative image of a tumor derived from a C1 cell strain of hematoxylin-eosin staining (H&E stain). (i) Cells showing a high nuclear to cytoplasmic ratio. (ii) is a magnified image in the box of Figure Ai. (iii) is a tumor cell having a high mitotic rate (indicated by an arrow). (iv) is a magnified image in the box of Figure A-iii. (Scale bar: 100μm)

Fig. 8 shows an immunohistochemical examination of a tumor derived from a C1 cell line, which represents a representative map of an immunohistochemical map of a tumor derived from a C1 cell line. (A) is the performance of Oct-4 and CAR. (B) for oncogene activation, including phosphorylation-Src (phospho-Src; p-Src), phospho-β-catenin (p-β-catenin), c-myc, and cell cycle Protein D1 (cyclin D1). (C) Expression of diagnostic markers for human adenocarcinoma, including TTF1, NAPSA, CK7, and CK-HMW. The inserted image is a magnified image inside the frame. (Scale bar: 100μm)

Figure 9 shows the in vitro tumorigenic phenotype of CAR ⁺ /mPSCs ^Oct-4_hi . (A) is a soft agar colony formation assay. CAR ⁺ /mPSCs, CAR ⁺ /mPSCs ^Oct-4_hi C1, E9, C7 and A549 cell lines were soft agar culture, and the cell population was photographed and quantified after two weeks. (B) is a sphere formation assay. ^{CAR + / mPSCs, CAR + /} mPSCs Oct-4_hi C1, E9, C7 and A549 cells for spheroid formation assay, two spheres _{(seconda r y spheres) (>} 70μm) 10 days in pictures and quantified. (Scale bar: 100μm)

Figure 10 shows in vivo tumorigenic and metastatic ability of CAR ⁺ /mPSCs ^Oct-4_hi C1 cell line (A) is xenograft tumor formation in vivo. (i) is a tumor growth curves, different concentrations of C1 cell lines ^{(105, 104, 103} and ¹⁰² cells) were injected subcutaneously into SCID mice, tumor diameter in a specified time after injection Measured with a caliper, the data is presented as mean ± standard deviation. (ii) The tumor was excised, photographed, and measured on the 28th day after the injection (scale bar: 1 cm). (B) For metastatic tumor nodule formation, C1 cell line (3×10 ⁵ cells) was injected intravenously in the tail of severe combined immunodeficient mice, with CAR ⁺ /mPSCs as the control group, and lungs were recorded after 5 weeks. Metastatic tumor nodule formation (indicated by arrows). Mice injected with C1 cell line showed extensive hemorrhage and nodule formation after hematoxylin-eosin staining (H&E staining), and a magnified image of C1 cell line showed nodules in the lung tissue (scale bar: 100 μm) (C) Kaplan-Meier survival curves (quantity = 10) of mice injected with CAR ⁺ /mPSCs and C1 cell lines.

Figure 11 shows the ^{characteristics} of putative cancer initiating cells (CICs) in CAR ⁺ /mPSCs ^Oct-4_hi . (A) is a flow cytometry analysis of CD133 expression in CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi C1, E9 and C7 cell lines, showing the CD133 positive population of individual cell lines as a percentage. (B) To analyze the activity of aldehyde dehydrogenase (ALDH) by flow cytometry, the results of ALDEFLUOR test of CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi C1, E9 and C7 cell lines are shown. The DEAB-treated samples were negative control groups, and the percentages showed acetaldehyde dehydrogenase (ALDH) positive populations of individual cell lines. (C) is the cell viability of CAR ⁺ /mPSCs ^Oct-4_hi . CAR ⁺ /mPSCs ^Oct-4_hi C1, E9 and C7 cell lines and A549 cells with (i) cisplatin (2.5, 5, 10, ^{25, 50} and 100 μM) and (ii) paclitaxel ( Paclitaxel) (2.5, 5, 10, 50, 100, and 200 nM) was treated for 48 hours and the data was shown as mean ± standard deviation. *P<0.05, **P<0.01: compared to A549. (D) Anti-apoptotic potential of CAR ⁺ /mPSCs ^Oct-4_hi . (i) To analyze the expression of survivin in C1, E9 and C7 cell lines of CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi by Western blotting. (ii) cleavage of caspase-3 in CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi C1, E9 and C7 cell lines treated with 10 μM cisplatin or 10 nM paclitaxel (Cleaved caspase-3; c caspase-3) and the expression of cleaved caspase-9 (caspase-9).

Figure 12 shows the expression of pro-angiogenic factors of CAR ⁺ /mPSCs ^Oct-4_hi . (A) The gene expression of angiogenic factors in CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi C1, E9 and C7 cell lines was analyzed by real-time PCR. ± standard deviation is presented. * P < 0.05, ** P < 0.01: compared to CAR ⁺ /mPSCs.

Figure 13 shows the angiogenic potential of CAR ⁺ /mPSCs ^Oct-4_hi . (A) is a chicken embryo urrioallantoic membrane (CAM) test of CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi C1, E9 and C7 cell lines. (i) A representative image of a photomicrograph after 72 hours of cell transfer, the arrow indicating the branch point of the blood vessel. (ii) To determine the angiogenic potential based on the number of branch points, a sample of matrigel alone was used as the background value; vascular endothelial growth factor (VEGF) (10 ng) was used as a positive control group. Data are expressed as mean ± standard deviation. *P<0.05: compared to the group of Matrigel alone; **P<0.01: compared with the CAR ⁺ /mPSCs group. (B) CD31 expression measured by immunohistochemical staining for CAR ⁺ /mPSCs ^Oct-4_hi C1, E9, C7 cell lines and A549-derived tumors. (i) is a representative map of various tumors (scale bar: 100 μm). (ii) Quantitative analysis of CD31 expression in tumors using the Tissue Gnostics scan and image analysis software (HistoQuest), data expressed as mean ± standard deviation, ** P < 0.01: compared with A549 tumors .

Figure 14 shows tube formation co-cultured with CAR ⁺ /mPSCs ^Oct-4_hi C1 cell line and SVEC4-10. The conjugated focal microscope image shows the column structure of SVEC4-10 co-cultured with the C1 cell line. SVEC4-10 is a mouse endothelial cell line stained with PKH26; C1 clone sphere or C1 clone cells are labeled with calcein-AM. The column structure was taken after 8 hours of co-cultivation. DAPI is a nuclear marker. (A) is a C1 cell strain sphere co-cultured with SVEC4-10. (B) C1 cell line cells were co-cultured with SVEC4-10.

Figure 15 shows the tube formation assay of CAR ⁺ /mPSCs ^Oct-4_hi . (i) The CAR ⁺ /mPSCs ^Oct-4_hi C1, E9, and C7 cell lines were cultured in Endothelial Cell Growth Medium (EGM) for 7 days, and their column formation was detected, while the endothelial cell growth medium was used. In the CAR ⁺ /mPSCs cultured in the (Endothelial cells growth medium; EGM), no column formation was observed, the tube network was stained with calcein-AM, and the fluorescence microscope was used to record 8 hours. . (ii) is the column forming ability, which is determined by the length of the column, and the data is expressed as mean ± standard deviation, ** P < 0.01: compared with CAR ⁺ /mPSCs. (Scale bar: 100μm)

Figure 16 shows that CAR ⁺ /mPSCs ^Oct-4_hi cultured in endothelial cell growth medium (EGM) expresses surface markers of endothelial cells, CAR ⁺ /mPSCs ^Oct-4_hi C1, E9, C7 cell lines in endothelial cell growth medium (EGM) After 7 days of incubation, the performance of endothelial cell-specific surface markers, including CD31, CD105, CD34, and CD144, was analyzed, and the numbers indicate positive expression populations.

Figure 17 shows the angiogenic potential of CAR ⁺ /mPSCs ^Oct-4_hi . (A) Results of endothelial cell antigen expression measured by immunofluorescence staining of C1-GFP cell line-derived tumors. (i) Identification of CD31. (ii) Identification of vWF. (iii) Identification of CD105. The magnified image shows that some GFP-positive cells are involved in angiogenesis (indicated by an arrow), and a portion of GFP-positive cells also exhibit CD31 (marked with an asterisk) (scale bar: 100 μm). (B) is the CD31 expression of the isolated tumors analyzed by flow cytometry for C1-GFP cell lines. The CD31 positive subpopulation representing endothelial cells accounted for 3% of all tumor populations and was found in 18% of CD31 positive endothelial cell subpopulations. GFP performance.

Figure 18 shows the ANGs/Tie2 signal analysis of CAR ⁺ /mPSCs ^Oct-4_hi cultured in endothelial cell growth medium (EGM). (A) Analysis of CAR ⁺ /mPSCs ^Oct-4_hi C1, E9, C7 and CAR ⁺ /mPSCs cell lines cultured in Endothelial cells growth medium (EGM) for real-time PCR Gene expression of angiogenesis-related receptors, including vascular endothelial growth factor receptor 2 (VEGFR2) and Tie2. Data are expressed as mean ± standard deviation, ** P < 0.01: compared to CAR ⁺ /mPSCs. (B) Analysis of signal activation of ANGs/Tie2 of CAR ⁺ /mPSCs ^Oct-4_hi C1, E9, C7 and CAR ⁺ /mPSCs cell lines cultured in endothelial cell growth medium (EGM) by Western blotting, including Tie2, phosphoric acid -Tie2 (phospho-Tie), Angiopoietin 1 (ANG1), Angiopoietin 2 (ANG2), Growth factor receptor bound protein 2 (GRB2), ERK And phosphorylation-ERK (phospho-ERK) performance.

Figure 19 shows that Tie2 kinase inhibitor reduces angiogenesis of CAR ⁺ /mPSCs ^Oct-4_hi . (A) The CAR ⁺ /mPSCs ^Oct-4_hi C1 cell strain was cultured for 7 days in Endothelial cell growth medium (EGM), followed by a tube formation assay to count the number of nodes. Used to verify the formation of the string. After treatment with 2 μM Tie2 kinase inhibitor, the ability to form a column of C1 cell line cultured in Endothelial cell growth medium (EGM) decreased. Data are presented as mean ± standard deviation. ** P < 0.01: compared to the group without Tie2 kinase inhibitor. (B) A chicken embryo chorioallantoic membrane (CAM) test of a C1 cell strain, and the number of blood vessel branch points was used to verify the blood vessel induction of the C1 cell line. Tie2 kinase inhibitor (2 μM) reduced angiogenesis induced by C1 cell lines. Data are presented as mean ± standard deviation. *P<0.05: compared to the group without Tie2 kinase inhibitor. (C) For xenograft tumor production assay, tumor growth of C1 cell line was inhibited by Tie2 kinase inhibitor treatment, Tie2 of 50 mg/kg body weight from day 7 to day 25 The injection was performed once every two days from the abdominal cavity, and the tumor volume was recorded on the 10th to 25th day. Data are presented as mean ± standard deviation, * P < 0.05: compared to the group without Tie2 kinase inhibitor.

The following examples are not limiting and are merely representative of the many aspects and features of the present invention.

Example 1:

Materials and Methods

According to the performance of coxsackievirus and adenovirus receptor positive (CAR-positive), it was isolated from primary cultured cells by fluorescence-activated cell sorting (FACS). Coxsackievirus and adenovirus receptor positive mouse pulmonary stem/progenitor cells (CAR ⁺ /mPSCs) were obtained from coxsackievirus and adenovirus receptor-positive mice. Type I pneumocytes derived from CAR ⁺ /mPSCs and CAR ⁺ /mPSCs were transfected with a retroviral vector containing Oct-4 (SEQ ID NO: 1). High ^-potency cells of Oct-4, the CAR ⁺ /mPSCs ^Oct-4_hi cell line, which utilize Western blot analysis, telomerase repeat amplification assay, flow cytometry Instrumental analysis, soft agar colony formation assay, sphere formation assay, reverse transcription PCR and real-time PCR analysis Gene expression, tube formation assay, and chicken chorioallantoic membrane (CAM) assay. In vivo tumorigenic potential was assessed by Severe Combined Immunodeficiency Mice (SCIDmice) by limiting dilution transplantation experiment and metastasis assay, and tumors were derived for immunohistochemistry. Treatment with immunofluorescence staining.

Cell culture

Human lung adenocarcinoma cell line A549 and mouse axillary lymph node/vascular epithelial cell line were obtained from the Bioresource Collection and Research Center of Taiwan. Line) SVEC4-10 and human embryonic kidney cell line (HEK) 293T, A549, SVEC4-10 and HEK293T cells were cultured in DMEM medium (Dulbecco's modified Eagle medium, Sigma-Aldrich) and 10% fetal bovine serum (FBS) at 37 ° C in a humidified incubator containing 5% carbon dioxide.

Serum-free primary screening and culture of mouse lung stem cells/precursor cells

Neonatal ICR mice (1 to 3 days postpartum) were sacrificed by cervical dislocation, lung tissue was isolated and collected in pre-cooled sum containing penicillin (100 units/mL) and streptomycin (streptomycin, 100 g/mL) of Hank's buffer. The lung tissue was cut into pieces of 1 to 2 mm in diameter and placed in a digested medium containing 0.1% protease type-XIV (Sigma-Aldrich) and 1 ng/mL DNA decomposition. The enzyme-I (DNase-I, Sigma-Aldrich) was placed in MEM (Minimum Essential Medium Eagle) medium at 4 ° C overnight. Next, 10% fetal bovine serum (FBS)/MEM medium was added to neutralize proteolytic enzyme/DNA-degrading enzyme-I, and the tissue suspension was gently mixed several times with a 10 mL pipette. Tissue fragments were filtered through a 100 μm nylon cell strainer, washed and resuspended in MCDB-201 medium (Sigma-Aldrich) supplemented with insulin/transferrin/selenium (ITS) (Invitrogen). in. These cells were cultured at a density of 3×10 ⁵ cells/mL on a cell culture plate coated with collagen-I (Becton Dickinson Biosciences), and after one day of culture, selenium (ITS) and 1 ng/mL were added. Recombinant epidermal growth factors (Invitrogen) MCDB-201 medium was used to regenerate cells. The Pulmonary epithelial colony forms a cell population on the medium on days 10 to 14 of cell confluence. These primary cells were isolated by fluorescent flow cytometry (FACS) for coxsackievirus and adenovirus receptor positive (CAR) positive (CAR-positive) mouse lung stem cells/progenitor cells (mouse) Pulmonary stem/progenitor cells; mPSCs).

Isolation of lung stem cells/precursor cells (CAR+/mPSCs) from Crox virus and adenovirus receptor-positive mice

Cell suspensions from primary cultured cells were analyzed for CAR positive cells by a FACS caliber instrument (Becton Dickinson Biosciences). Briefly, 1 x 10 ⁶ cells were incubated with goat anti-CAR polyclonal antibody (R&D systems) for one hour at 4 °C. After washing, the cells were incubated with Alexa488-conjugated donkey anti-goat IgG antibody (Alexa488-coupled donkey anti-goat IgG, Jackson ImmunoResearch) for one hour at 4 °C. In fluorescence flow cell sorter (FACSAria ^TM, Becton Dickinson Biosciences) evaluation, and data CellQuest ^TM Software (Becton Dickinson Biosciences) analysis. Cells were >90% pure according to CAR positive performance, which was called CAR ⁺ /mPSCs, CAR ⁺ /mPSCs were centrifuged at low speed (1100 rpm, 5 minutes) and resuspended for subsequent use, including Oct-4 transfection and Cell differentiation experiment.

Oct-4 transfection

As previously described, CAR ⁺ /mPSCs were isolated from primary cultured cells by fluorescence flow cytometry (FACS) based on CAR-positive performance. The detailed method is described in the supplementary method. Type I pneumocytes derived from CAR ⁺ /mPSCs and CAR ⁺ /mPSCs were transfected with a retroviral vector encoding Oct-4 (SEQ ID NO: 1). Briefly, the plastid pMXs-mOct-4 (Addgene) and the packaging plasmid (pCMV-gag-pol-PA and pCMV-VSVg) of the retroviral vector were sent using GeneJuice Transfection Reagent (Novagen). In HEK293T cells, the virus supernatant was filtered with a 0.45 μm filter after 48 hours, and 10 μg/mL of polybrene was added. 1×10 ⁴ CAR ⁺ /mPSCs and CAR ⁺ /mPSCs-derived type 1 type I pneumocytes were seeded on each 35 μm cell culture plate and cultured for 16 hours with virus supernatant. . The transfected cells were cultured in mES/MCDB201 (1:1) medium and supplemented with mitomycin C to remove MEF (feeding cells) activity. The cobblestone-like cell population formed on days 18 to 25, and the cell population was manually isolated on day 28 and further expanded on Matrigel (Becton Dickinson Biosciences) medium supplemented with mES/MCDB201 (1:1) to establish C1, E9, C7 cell line. The establishment of the C1-GFP cell line was transfected with a C1 cell line with a reverse transcription vector encoding GFP. Briefly, the retroviral plastid pMXs-puro GFP (Addgene) and the coated plastids (pCMV-gag-pol-PA and pCMV-VSVg) were sent to HEK293T cells by GeneJuice transfection reagent (Novagen), 48 hours later. The virus supernatant was filtered through a 0.45 μm filter and 10 μg/mL of polybrene was added. 1 × 10 ⁴ C1 cell lines were seeded in a 35 mm culture dish and cultured in the virus supernatant for 16 hours. Puromycin (2.5 μg/mL) was added to the culture solution, and after five days, the GFP-positive cell strain was identified and amplified, and it was called a C1-GFP cell strain.

RNA extraction, reverse transcription polymerase chain reaction and immediate polymerase chain reaction

Total RNA was extracted using TRIzol reagent (Invitrogen) and cDNA was synthesized using M-MLV RT (Promega) according to the manufacturer's instructions. Reverse transcription polymerase chain reaction (reverse transcription PCR) was performed using Taq polymerase (Invitrogen) according to the manufacturer's instructions. Real-time PCR was performed using a 7900HT real-time polymerase chain reaction instrument (Applied Biosystems). The primer sequences are listed in Table 1, Glyceraldehyde 3-phosphate dehydrogenase; GAPDH) performance for standardization.

Western blot analysis

Cell lysates were extracted in RIPA buffer (Pierce) and quantified using BCA protein detection reagent (Pierce) according to the manufacturer's instructions. Equal amounts (30 μg) of total protein were synthesized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was separated and transferred to activated polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk, the membrane was incubated with primary antibodies as shown in Table 2. The dots were incubated with a secondary antibody conjugated with horseradish peroxidase and the immune-reacted bands were detected with a enhanced chemiluminescence detection (Millipore).

Flow cytometry analysis

In the CD133 performance analysis, cells were isolated as single cells, washed and suspended in phosphate buffered saline (PBS). Cells were labeled with allophycocyanin (APC) conjugated anti-mouse CD133 antibody (BioLegend) and subsequently analyzed by a fluorescent flow cytometry meter. In the analysis of cell cycle distribution, the cells were cultured on a 6-well culture plate and cultured for 24 hours. After the time, the cells were collected, washed with PBS, and fixed at 70 ° C overnight at -20 ° C. Next, the cells were washed once with PBS and resuspended in PBS containing 200 μg/mL of RNA-degrading enzyme A (RNaseA) and 50 μg/mL of propidium iodide for fluorescence flow sorting. The instrument analyzes the cell cycle distribution. The expression of CD31 and GFP in tumors was determined by tissue isolation kit (Miltenyi Biotec) to isolate the tumor into cell suspension, and the cell suspension was stained with allophycocyanin (APC) conjugated anti-mouse CD133 antibody (BioLegend). This was followed by analysis using a fluorescent flow cell sorting gauge.

Aldehyde dehydrogenase activity test

The aldehyde dehydrogenase (ALDH) activity of the cells was detected using the ALDEFLUOR test kit (StemCell Technologies), which was operated according to the manufacturer's instructions. The cells were suspended in an ALDEFLUOR assay buffer containing BODIPY-aminoacetaldehyde (BAAA) and reacted at 37 ° C for 60 minutes. The cells were treated with an aldehyde dehydrogenase (ALDH) inhibitor (diethylaminobenzaldehyde (DEAB)) and served as a negative control. Propidium iodide staining identified cells that were not viable. A fluorescent flow cytometry meter was used to analyze the acetaldehyde dehydroanthracene (ALDH) activity of the cells by a green fluorescent channel (520-540 nm).

Telomerase repeat amplification assay

Telomerase activity was measured by telomerase repeat amplification (TRAP) assay and cells were homogenized in TRAP lysis buffer. Protein (20 μg) was used in the telomerase reaction with 50 μL of TRAP reaction buffer containing 20 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 63 mM chlorination Potassium, 0.05% Tween-20, 1 mM EGTA, 50 μM deoxynucleotide triphosphate (Pharmacia), labeled TS, ACX and U2 primers each 0.1 μg, 5 × 10 ^-3 Atomo The internal control primer of the attomoles (TSU2), 2 units of Taq DNA polymerase (Invitrogen) and 2 μL of CHAPS extract. After reacting for 30 minutes at 3 ° C, the telomerase elongation product was amplified by PCR under the following conditions: 30 cycles, each cycle containing 30 seconds at 94 ° C, 30 seconds at 60 ° C and 45 seconds at 72 ° C. The reaction mixture was heated to 94 ° C for 5 minutes to deactivate the telomerase, and the amplified product was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then stained with ethidium bromide. Observed under ultraviolet light.

Soft agar cell community formation ability test

The soft agar colony formation assay was performed by inoculating 3 x 10 ³ cells in a 35 mm tissue culture dish containing a layer of 0.35% low melting agarose/ES/MCDB-201. 0.5% low melting agarose / ES / MCDB-201. The whole culture solution was added every two days. After two weeks, the cell population was fixed with 0.05% crystal violet and methanol, and the cell population was photographed and quantified by optical microscopy.

Sphere formation assay

The cells were seeded at a density of 1000 per well in a 24-well ultra low-attachment plate (Corning) and cultured in serum-free DMEM medium supplemented with 2% B27 (Invitrogen), 20 ng/mL. Epidermal growth factor (EGF) and 20 ng/mL basic fibroblast growth factor (basic fibroblast) Growth factor; bFGF) (Invitrogen). After 14 days of culture, the primary spheres were collected by centrifugation, separated by trypsin and resuspended in the culture medium, and secondary spheres (>70 μm) were photographed and quantified after 10 days.

Xenograft tumor assay

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of National Taiwan University College of Medicine. The teratoma formation assay was performed by subcutaneous injection of 1 × 10 ⁶ CAR ⁺ /mPSCs ^Oct-4_hi C1, E9 and C7 cell lines into 8-week male severe combined immunodeficient mice (Severe Combined) In the immunodeficiency Mice; SCID mice); the limiting dilution transplantation experiment is performed on C1 cell lines (10 ⁵ , 10 ⁴ , 10 ³ and 10 ² cells) or CAR ⁺ /mPSCs (10 ⁶ cells). Subcutaneous injection into severe combined immunodeficient mice; in the experiments of C1-GFP cell line and A549-derived tumor, C1-GFP (1 × 10 ⁵ cells) or A549 (1 × 10 ⁶ cells) were injected subcutaneously To severe combined immunodeficient mice. Tumor size was measured once every 3 days with a caliper and the volume (cm ³ ) was calculated according to the following standard formula: length x width ² /2. The tumor was surgically removed and photographed at the end of the experiment. In the tumor metastasis test, 3×10 ⁵ C1 cell lines and CAR ⁺ /mPSCs were injected into the severe combined immunodeficient mice via the lateral tail vein, and the pulmonary metastatic nodules were evaluated by autopsy and histological examination in the fifth week. Kaplan-Meier analysis was used to compare the difference in survival rates between mice injected with C1 cell line and CAR ⁺ /mPSCs.

In the secondary tumor experiment, 1×10 ⁶ C1 cell lines were subcutaneously transplanted for 4 weeks and developed into tumor tissues. The tumor tissues were cut into small pieces and then digested with trypsin-EDTA. The cell suspension was collected in a 100 μm cell strainer. Using differentiated cells FACSAriaII cell sorter to isolate high performance and low performance ^{CAR's, 1x10 5, 10 4, 10} 3 and 1 × 10 ² th CAR high (CAR ^High) and a low CAR (CAR ^Low) cells (Number = 4 / Group) were subcutaneously transplanted into the back of severe combined immunodeficient mice. The formation of secondary tumors for 5 weeks was recorded, and then the CAR performance of secondary tumors was further confirmed.

Immunohistochemistry and immunofluorescence staining

Tumors were fixed in formalin, then dehydrated, embedded in paraffin and sectioned. Tumor sections were subjected to antigen retrieval by microwave irradiation in citrate buffer (10 mM, pH 6.0), and the tumor sections were reacted with primary antibodies at 4 ° C overnight. Immunohistochemical staining was performed by reacting this tumor section with the corresponding HRP-conjugated secondary antibody for one hour at room temperature, using 0.05% 3,3'-diaminobenzidine (3,3'-diaminobenzidine). DAB) was visually observed and the nuclei were counterstained with hematoxylin. The immunofluorescence staining was carried out by reacting the corresponding fluorescent-conjugated secondary antibody for one hour at room temperature, and the nuclei were counterstained with DAPI fluorescent dye. The negative control group was prepared under the same conditions, and the primary antibody was replaced with a control immunoglobulin G (IgG). The antibodies are listed in Table 2. Sections were examined with a microscope (Nikon Eclipse 800), immunohistochemical stained sections were scanned and quantified using a TissueFax Cell Quantitative Analyzer (TissueGnostics GmbH), and the proportion of immunopositive populations was analyzed by HistoQuest software (TissueGnostics GmbH).

IF: immunofluorescence staining; IHC: immunohistochemical staining; WB: Western blotting; FC: flow cytometry; O/N: overnight; RT: room temperature

Cell survival test

3 x 10 ³ cells were seeded into 96-well plates and cultured for 18 hours. Cells were treated with different concentrations of platinum dichloride (cisplatin, Sigma-Aldrich) or paclitaxel. After 48 hours the test WST-1 (Roche) according to manufacturer's instructions cell viability instructions, cell viability was expressed as a percentage of the untreated group, and ₅₀ to give a value IC.

Chicken chorioallantoic membrane (CAM) test

Fertilized chicken eggs were cultured at 37 ° C and 80% humidity. On the 8th day of development, 1 × 10 ⁶ cells were placed on the membrane and transplanted onto the growing chicken embryo urinary chorioallantoic membrane (CAM), day 11 The chicken embryo urinary chorioallantoic membrane (CAM) was fixed with 4% paraformaldehyde and photographed with a stereomicroscope and a digital camera. The branch points were quantified using the NIH Image J software and angiogenesis plug-in.

In vitro CAR ⁺ /mPSCs ^Oct-4_hi tube formation assay

The cells were cultured for 7 days in endothelial cell growth medium (EGM) (Lonza), and the cells were harvested and suspended in DMEM medium supplemented with 2% fetal bovine serum (FBS) to inoculate the basement membrane matrix. (Matrigel), cells were stained with calcein-AM (Invitrogen) after 8 hours, and images were acquired using a fluorescence microscope (Zeiss).

CAR ⁺ /mPSCs ^Oct-4_hi C1 cell line co-cultured with SVEC4-10 to form a column

Spread the basement membrane matrix (Matrigel) in a 35mm Petri dish (Ibidi μ dish) The C1 cell line-derived sphere was labeled with the green fluorescent tracer calcein-AM and mixed with SVEC4-10 cells stained with red fluorescent tracer PHK26 (Sigmla-Aldrich). Counterstained with Hoechst 33342 dye. The cell mixture was seeded in a cell culture plate coated with Matrigel containing DMEM supplemented with 2% FBS and 5% Matrigel. The tube string was recorded by time-lapse immunofluorescence confocal microscopy.

Inhibition of Tie2 kinase inhibitor

The Tie2 kinase inhibitor efficiently, reversibly and selectively targets the ATP binding site of Tie2 kinase with a chemical structural formula of 4-(6-methoxy-2-naphthyl)-2-(4-methylsulfinic acid). 4-(6-methoxy-2-naphthyl)-2-(4-methylsulfinylphenyl-5-(4-pyridyl)-1H-imidazole4-( 6-methoxy-2-naphthyl)-2-(4-methylsulfinylphenyl-5-(4-pyridyl)-1H-imidazole) (CAS number: 948557-43-5) (ab141270, Abcam, Cambridge, MA) The cytotoxicity of the Tie2 kinase inhibitor (2 μM) against the CAR ⁺ /mPSCs ^Oct-4_hi cell line after 24 hours of treatment was 93.2 ± 2.51% survival.

In the tube formation assay, the column formation of CAR ⁺ /mPSCs ^Oct-4_hi C1 cell line cultured in Endothelial cells growth medium (EGM) was treated with 2 μM Tie2 kinase inhibitor for 8 hours. After staining with calcein-AM (Invitrogen), images were acquired with a fluorescence microscope (Zeiss).

In the angiogenesis assay, 1 × 10 ⁶ cells of the CAR ⁺ /mPSCs ^Oct-4_hi C1 cell line were treated with 2 μM Tie2 kinase inhibitor and incubated with the chicken chorioallantoic membrane (CAM) test. On day 11, chicken embryo urinary chorioallantoic membrane (CAM) was fixed with 4% paraformaldehyde and photographed with a stereomicroscope and a digital camera. The branch points were quantified using the NIH Image J software and angiogenesis plug-in.

1 × 10 ⁵ cells of CAR ⁺ /mPSCs ^Oct-4_hi C1 cell line were subcutaneously injected into 8-week male Severe Immunodeficiency Mice (SCID mice). The 50 mg/kg body weight Tie2 kinase inhibitor was administered intraperitoneally every two days on days 10 to 25. The tumor size was measured by a caliper every 2 or 3 days, and the volume (cm ³ ; cubic centimeters) was calculated according to the following standard formula: length × width ² /2. After the end of the experiment, the tumor was surgically removed and photographed.

Statistical Analysis

Quantitative data from at least 3 independent experiments were expressed as mean ± standard deviation (SD), Student's t test was used to compare differences between groups, and Kaplan-Meier analysis was used to obtain survival curves, P < 0.05 is statistically significant.

result

Transfection of Oct-4 to CAR ⁺ /mPSCs for high performance

Although the number of tissue-specific stem cells is small, it is the main reason for maintaining tissue stability. CAR ⁺ /mPSCs have been successfully identified and isolated in previous studies (Figures 1(A) and 1(B)). Compared with the mouse embryonic stem cell line (E14), CAR ⁺ /mPSCs in the polymerase chain reaction (PCR) and real-time PCR analysis of Oct-4, Sox -2 and Nanog have lower performance (Fig. 1(C)). CAR ⁺ /mPSCs showed their potential to differentiate into type 1 type I pneumocytes on day 7, evidenced by the type of squamous cells and type 1 I pneumocytes ) The appearance of T1α and AQP5 (Fig. 1(D)). Therefore, CAR ⁺ /mPSCs have the characteristics of lung-specific stem/progenitor cells, which can be identified by CAR expression and efficiently separated by a fluorescent flow cytometry (FACS).

Transfected with CAR ⁺ /mPSCs and CAR ⁺ /mPSCs-derived type-type pneumocytes by retrovirus to overexpress Oct-4. In this experiment, CAR ⁺ /mPSCs were transfected with Oct-4 (Fig. 2 (i) and (ii)), and feeder cells were supplied on day 2 (Fig. 2 (iii)), cobblestone-like cells. The formation of the community was first observed on the 18th to 25th day. On the 28th day, the well-developed cell community showed bright boundaries. The cells in the community had a high nuclear/cytoplasmic ratio and significant nucleoli. (Fig. 2(iv)), these cells were then picked up and amplified to establish a cell line (Fig. 2(v)). Table 3 shows the frequency of cobblestone-like cell population of the formed derivative of CAR ⁺ / mPSCs transfected into sham control group (sham control) or CAR ⁺ / mPSCs first muramyl lung cells (type-I pneumocytes) to Oct-4 No community formation was observed by transfection.

Data are expressed as mean ± standard deviation

The frequency of cell formation of cobblestone-like cells in CAR ⁺ /mPSCs ranged from 0.05-0.13% (Table 3), and no cell bodies like cobblestone could be observed in the transfection control group (sham control) of CAR ⁺ /mPSCs ( The data is not displayed). Retrovirus transfection of type 1 I-type pneumocytes derived from CAR ⁺ /mPSCs was transfected on day 8, when type 1 type I pneumocytes were well differentiated ( Figure 3 (i), (ii) and (iii)), feeding cells were supplied on day 10 (Fig. 3 (iv)), Oct-4 transfected type 1 type I pneumocytes until No community formation was observed on day 42 after induction. These results show that the overexpression of Oct-4 can induce CAR ⁺ /mPSCs to form a cobblestone-like cell population, and in the cell state, ie stem/progenitor cells stage, but not in the differentiation stage, it seems to be through Oct- 4 Excessive performance induced community formation is critical.

The cobblestone-like cell population is isolated and established as individual cell lines. To assess changes in phenotype, three independent experimental cell lines, named C1, E9, and C7, were taken for further examination. Western blot analysis showed that Oct-4 was highly expressed in C1, E9 and C7 cell lines; therefore, they were called CAR ⁺ /mPSCs ^Oct-4_hi cell line (Fig. 4(i)), Oct-4 The expression levels in C1, E9 and C7 cell lines were 16 to 20 times higher than those of CAR ⁺ /mPSC, and the expression levels of Oct-4 in C1, E9 and C7 cell lines were similar to those of mouse embryonic stem cells (E14), whereas CAR ⁺ / The amount of Oct-4 in mPSCs was low (Fig. 4(ii)). In primary culture, CAR was specifically expressed in lung stem/progenitor cells and was used as a marker for the separation of CAR ⁺ /mPSCs. In the CAR ⁺ /mPSCs ^Oct-4_hi cell line, CAR is expressed in >95% of cells, and these cells lose the ability to differentiate into type I pneumocytes (Fig. 5(A) and (B)), cell cycle analysis showed that the G ₁ -, S- and G ₂ /M- periods of the CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi cell lines were significantly changed (Fig. 6(A)). Table 4 shows the population analysis of CAR ⁺ /mPSCs and CAR ⁺ /mPSCs ^Oct-4_hi C1, E9 and C7 cell lines with G ₁ -, S- and G ₂ /M- periods. The CAR ⁺ /mPSCs ^Oct-4_hi cell line increased significantly in the S- and G ₂ /M- periods.

In addition, the C1, E9 and C7 cell lines were able to multiply for more than 50 generations, and the Doubling time was 23 ± 1 hour (Fig. 6(B)), and the doubling time is shown in Table 5.

Telomerase activity was detected in the 12th, 20th, and 50th generations of C1, E9, and C7 cell lines, but not in CAR ⁺ /mPSCs (Fig. 6(C)). These results demonstrate that the high amount of Oct-4 in CAR ⁺ /mPSCs is sufficient to produce immortal effects such as G ₁ cell cycle progression, proliferative potential and telomerase activity.

CAR ⁺ /mPSCs ^Oct-4_hi shows carcinogenic potential

To assess the ^{CAR +} / mPSCs ^Oct-4_hi cell line as much potential energy, teratoma (teratoma) a C1 formed in the test procedure, E9, cell lines C7, taken C1, 1 × 10 ⁶ cells E9 and Ke cell lines C7 It was injected subcutaneously into severe combined immunodeficient mice (SCID mice). After 20 to 24 days, the teratoma formed approximately one centimeter (Fig. 7(A)), and Fig. 7(B) shows a representative map of the histopathological analysis of the C1 cell line. H&E staining shows that there is no differentiation of ectodrmal, mesodermal and endodermal in tumors; in addition, tumors exhibit features of typical malignant phenotypes, such as high Cell density, small and round immature cell proliferation, high nucleoplasmic ratio of pleomorphic cells, high mitosis ratio (Fig. 7(B)). Immunohistochemical staining can be used to detect Oct-4 and CAR in tumors (Fig. 8(A)). Other activated oncogenes can also be detected in tumors, including phosphorylated-Src, phosphorylated-β-huluo Phospho-β-catenin, c-myc and cyclin D1 (Fig. 8(B)). Diagnostic markers for lung adenocarcinoma, such as thyroid transcription factor-1 (TTF1), Napsin A (NAPSA), cytokeratin 7 (CK7), and cytokeratin heavy molecular weight ( Cytokeratin heavy molecular weight; CK-HMW) can also be detected in tumors (Fig. 8(C)). These data show that CAR ⁺ /mPSCs are carcinogenic after high Oct-4 performance.

The carcinogenic potential of the CAR ⁺ /mPSCs ^Oct-4_hi cell line was then quantified, and the anchorage-independent growth was assessed using a soft agar colony formation. Two weeks later, the C1, E9, and C7 cell lines formed significantly more soft agar cell populations than the human lung adenocarcinoma cell line A549; however, this was not observed in CAR ⁺ /mPSCs. Class of cell-like cells (Fig. 9(A) and Table 6). In order to evaluate the efficiency of secondary sphere formation, C1, E9 and C7 cell lines were cultured without attachment. The secondary sphere formation efficiency of CAR ⁺ /mPSCs ^Oct-4_hi cell line was significantly higher than that of A549 cells, and there was no spheroid formation in CAR ⁺ /mPSCs (Fig. 9(B) and Table 6). Table 6 shows cell community and sphere. Quantitative.

In many experiments, the C1 cell line exhibited the most significant tumorigenic behavior, including the highest rate of proliferation, the highest anchorage-independent colony formation efficiency, and the highest secondary sphere formation efficiency. Therefore, the C1 cell line was selected for the subsequent in vivo tumorigenic experiment. In order to confirm the tumorigenicity of the C1 cell line, a limiting dilution transplantation experiment was performed. The tumor formation ability was 6/6, 5/6, 5/6, which corresponded to 10 ⁵ , 10 ⁴ and 10 ³ C1 cell lines, respectively; in addition, the low concentration of C1 cell line (10 ² ) was sufficient for the average injection. Tumors (4/6) were generated in the last 28 days (Fig. 10(A)-i), and tumor size and morphology were presented in Fig. 10(A)-ii. In contrast, using the CAR ⁺ / mPSCs cell transplantation (10 ⁶ cells) were incubated for 56 days the tumors are still unable to be observed (data not presented). To examine the metastatic potential, 3 × 10 ⁵ C1 cell lines were transplanted through the tail vein of the mouse and allowed to develop for 35 days. Tumor nodule was produced in the lung tissues of all mice injected with the C1 cell line (Fig. 10(B)). The lung tissue stained with hematoxylin-eosin staining showed that extensive bleeding and nodule formation occurred in the transplanted mice of the C1 cell line, but no abnormal lesions were detected in the mice transplanted with CAR ⁺ /mPSCs. Kaplan-Meier survival analysis was used to obtain the survival rate of mice transplanted with C1 or CAR ⁺ /mPSCs cells. The average survival rate of mice injected with C1 cell line was significantly lower than that of transplanted CAR ⁺ /mPSCs (Fig. 10(C)). These results showed that the CAR ⁺ /mPSCs showed a malignant transformation after high-activity of Oct-4, which was confirmed by the tumorigenic potential and tumor initiation ability of CAR ⁺ /mPSCs ^Oct-4_hi cells in vitro and in vivo.

CAR ⁺ /mPSCs ^Oct-4_hi shows the characteristics of lung cancer initiating cells

For the detection of lung cancer-initiating cells, different biomarkers have been proposed, including CD133 expression, aldehyde dehydrogenase (ALDH) activity and chemotherapy resistance. These biomarkers were used to further investigate the characteristics of cancer-initiating cells (CICs) of the CAR ⁺ /mPSCs ^Oct-4_hi cell line. Flow cytometry analysis showed that C1, E9, and cell lines C7, about 17.4-31.7% CD133 ⁺ cells; however, almost impossible to detect the CD133 ⁺ cells (FIG. 11 (A)) in CAR ⁺ / mPSCs in. Aldehyde dehydrogenase (ALDH) activity accounted for 18.4-33.2% of C1, E9 and C7 cell lines, whereas only 0.8% of CAR ⁺ /mPSCs exhibited acetaldehyde dehydrogenase (ALDH) activity (Fig. 11(B)). . Chemotherapy resistance is also a key biological property of cancer-initiating cells, and therefore the chemoresistance of C1, E9 and C7 cell lines for cisplatin and paclitaxel was evaluated. Cell viability is shown in Figure 11 (C). Table 7 shows dichloro diamine platinum (cisplatin) and paclitaxel (paclitaxel) for ^{CAR + / mPSCs Oct-4_hi C1} , E9, IC C7 and A549 cell lines of _50. The IC ₅₀ of cisplatin for C1, E9, and C7 cell lines ranged from 26.4 to 34.7 μM, indicating that the CAR ⁺ /mPSCs ^Oct-4_hi cell line was more resistant to cisplatin than A549. 2 to 3 times higher; and paclitaxel has an IC ₅₀ of 43.2-47.2 nM for C1, E9, and C7 cell lines, which is about 6 times higher than that of A549 (Fig. 11(C)).

Survivin has been well established to inhibit apoptosis and play an important role in conferring chemotherapy resistance to cancer-initiating cells. The present inventors found that survivin expression was significantly higher in C1, E9 and C7 cell lines than CAR ⁺ /mPSCs (Fig. 11(D)). C1, E9 and C7 cell lines showed lower expression of cleaved caspase-3 and cleavage than CAR ⁺ /mPSC after treatment with cisplatin and paclitaxel. Apoptotic protease-9 (cleaved caspase-9) (Fig. 11 (D)-ii). Taken together, these results show that the high amount of Oct-4 expression can drive the transformation of CAR ⁺ /mPSCs and confer properties to their cancer-like cells.

Therefore, the present invention further compares the differences between CAR ⁺ /mPSCs, CAR ⁺ /mPSCs ^Oct-4_hi and A549 cell lines (human lung adenocarcinoma cell lines) (Table 3). Compared to CAR ⁺ /mPSCs, CAR ⁺ /mPSCs ^Oct-4_hi has many characteristics of cancer-initiating cells. A549 is a cancer cell, but not a cancer-initiating cell; soft agar colony, sphere formation, CD133 and acetaldehyde dehydrogenation of CAR ⁺ /mPSCs ^Oct-4_hi compared to A549 cells Enzyme (ALDH) activity has a high performance. In addition, a small amount of CAR ⁺ /mPSCs ^Oct-4_hi (10 ³ cells) can form tumors and have tumor regenerative capacity. These results show that CAR ⁺ /mPSCs ^Oct-4_hi is a cancer-initiating cell.

Table 8, CAR ⁺ /mPSCs, CAR ⁺ /mPSCs ^Oct-4_hi and A549 cell lines

CAR ⁺ /mPSCs ^Oct-4_hi participates in tumor angiogenesis

In previous studies, it was shown that CAR ⁺ /mPSCs can express proangiogenic factors, including vascular endothelial growth factor A (VEGFa), granulocyte colony stimulating factor (GCSF), Vascular cell adhesion molecule 1 (VCAM-1) and basic fibroblast growth factor (bFGF), which can induce endothelial cell tube formation. Therefore, the present invention is intended to assess the angiogenic potential of the CAR ⁺ /mPSCs ^Oct-4_hi cell line. The expression of pro-angiogenic factors of C1, E9 and C7 cell lines of the present invention was found by real-time polymerase chain reaction (Real-time PCR) analysis, including angiopoietin 1 (ANG1) and angiopoietin 2 (angiopoietin). 2; ANG2), vascular endothelial growth factor A (VEGFa), placental growth factor (PLGF), platelet-derived growth factor A (PDGFa), peripheral blood stem cells (granulocyte colony stimulating factor; GCSF), vascular cell adhesion molecule 1 (VCAM-1) and basic fibroblast growth factor (bFGF) were significantly higher than CAR ⁺ /mPSCs (Fig. 12), in order to further confirm the angiogenic potential, we used C1, E9 and C7 cell lines and CAR ⁺ /mPSCs in the chicken chorioallantoic membrane (CAM) test. Compared with the transplantation of CAR ⁺ /mPSCs, when C1, E9 and C7 cell lines were transplanted into chicken embryo chorioallantoic membrane (CAM), extensive angiogenesis was induced (Fig. 13(A)-i)). Quantitative branch points showed that the vascular branch points of C1, E9 and C7 cell lines were significantly increased compared with CAR ⁺ /mPSCs (Fig. 13(A)-ii)), immunohistochemical analysis and quantification of tumors derived from Cl, E9 and C7 cell lines. It was shown that its CD31 positive population was significantly higher than the CD31 positive population of A549-derived tumors (Fig. 13 (B)-i and -ii). These data show that high expression of Oct-4 enhances the angiogenic potential of the CAR ⁺ /mPSCs ^Oct-4_hi cell line.

To further elucidate the functional contribution of CAR ⁺ /mPSCs ^Oct-4_hi in angiogenesis, tube formation of endothelial cells after incubation with C1 cell lines was monitored. The C1 cell line-derived spheres were labeled with a green fluorescent tracer calcein-AM and mixed with SVEC4-10 cells labeled with the red fluorescent tracer PKH26, and co-cultured to form a column. The C1 cell line-derived spheres absorbed SVEC4-10 cells and established a column network (Fig. 14(A)). Cells of some C1 cell lines were found to integrate into the column network of SVEC4-10 cells (Fig. 14(B)). C1, E9 and C7 cell lines were cultured for 7 days in endothelial cell growth medium (EGM) (Lonza) to examine the ability of column formation, and CAR ⁺ /mPSCs were also cultured as control group. C1, E9 and C7 cell lines cultured in endothelial cell growth medium (EGM) exhibited column forming ability, whereas this ability was not observed in CAR ⁺ /mPSCs cultured in endothelial cell growth medium (EGM) (Fig. 15(i) And (ii)). In endothelial cell growth medium (EGM), C1, E9 and C7 cell lines exhibited endothelial cell markers including CD31, CD105, CD34 and CD144 (Fig. 16). These results indicate that CAR ⁺ /mPSCs ^Oct-4_hi not only possesses angiogenic potential, but also participates in tube formation in vitro. To assess the potential of tumor blood vessel formation in vivo, GFP was transfected into C1 cell line (C1-GFP cell line) and transplanted subcutaneously into SCID mice to form Tumor. Endothelial cell antigens in tumors derived from C1-GFP cell lines were detected by immunofluorescence staining, including CD31, vWF and CD105. Some blood vessels do not fully express endothelial cell antigens, and GFP-positive cells (GFP+cell) fuse directly into blood vessels, exhibiting a mosaic-like structure. In addition, some endothelial cells simultaneously expressed endothelial cell antigen and were positive for GFP (Fig. 17 (A)-i, ii and iii). To determine the presence of GFP-positive endothelial cells, tumors were isolated and detected by flow cytometry, and the results were similar to immunofluorescence staining, with 12-18% of the CD31 ⁺ population also being GFP positive (Fig. 17(B)). To examine the association between CAR ⁺ /mPSCs ^Oct-4_hi cell lines and endothelial cells, angiogenesis associated receptors vascular endothelial growth factor receptor 2 was analyzed by real-time PCR. Viral endothelial growth factor receptor 2; VEGF2) and Tie2 gene expression. The specificity of Tie2, a receptor expressed in endothelial cells, was significantly higher in C1, E9 and C7 cell lines cultured in endothelial cell growth medium (EGM) than in CAR ⁺ /mPSCs; vascular endothelial growth factor receptor 2 (VEGF2) There was no significant difference (Fig. 18(A)). Western blot analysis confirmed the involvement of ANGs/Tie2/GRB2/ERK signaling, showing angiopoietin 1 (angiopoietin 1; ANG1) in C1, E9 and C7 cell lines cultured in endothelial cell growth medium (EGM) , Angiopoietin 2 (ANG2), Phosphorylation-Tie2, Growth Factor Receptor Binding Protein 2 (GRB2) and Phosphorylation-ERK were significantly increased relative to CAR ⁺ /mPSCs (Fig. 18(B)). These data show that CAR ⁺ /mPSCs ^Oct-4_hi actively participate in tumor angiogenesis rather than playing a passive role as CAR ⁺ /mPSCs. In addition, Tie2 kinase inhibitors reduce the column formation potential of C1 cell lines cultured in endothelial cell growth medium (EGM) and inhibit the angiogenesis promoted by C1 cell lines in the chicken chorioallantoic membrane (CAM) assay ( Figure 19 (A)). In addition, tube formation of C1 cell line cultured in endothelial cell growth medium (EGM) was not significantly different between antibody and vascular endothelial growth factor A (VEGFA) group and IgG control group. (Fig. 19(B)). In the xenograft tumor assay, a 50 mg/kg body weight Tie2 inhibitor was intraperitoneally injected every 2 days after 7 days of transplantation of the C1 cell line. The Tie2 inhibitor significantly reduced the tumor volume of C1 during the early stage of tumor development (Fig. 19(C)). Therefore, the tumor angiogenesis mechanism proposed by the present invention may be different from the conventional tumor angiogenesis mechanism, and Tie2/ANGs signaling plays an important role in the process of CAR ⁺ /mPSCs ^Oct-4_hi cell line actively participating in tumor angiogenesis.

A person skilled in the art will readily appreciate that the present invention can be easily accomplished with the results and advantages and those present in the present invention. The method of the present invention is representative of the preferred embodiments, which are exemplary and not limited to the field of the invention. Those skilled in the art will be aware of the modifications and other uses therein. These modifications are intended to be within the spirit of the invention and are defined in the scope of the claims.

All patents and publications mentioned in the specification are subject to the general skill of the art in the field of the invention. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication is specifically and individually indicated.

The invention as exemplified herein may be practiced in the absence of any element, or a plurality of elements, limitations, or limitations. The nouns and expressions used are as a description and not a limitation of the description, and are not intended to be used to exclude any nouns and expressions that are equivalent to the features or parts thereof shown and described. Yes, various changes are possible within the scope of the patent application of the present invention. Therefore, it is to be understood that the present invention has been disclosed and described herein in accordance with the preferred embodiments and the features of the present invention. Within the scope.

<110> Lin Taiyuan

<120> Cancer initiation cells and uses thereof

<130> 2726-LTY-TW

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 5958

<212> DNA

<213> Mus musculus

<400> 1

Claims (11)

A cancer-initiating cell comprising an excess of Oct-4 virus and adenovirus receptor positive mouse pulmonary stem/progenitor cell (CAR ⁺ /) mPSC), wherein the amount of Oct-4 exhibited by the isolated CAR ⁺ /mPSC in excess of Oct-4 was more than 10 times higher than the Oct-4 expression of normal CAR ⁺ /mPSC. The cancer-initiating cell of claim 1, wherein the CAR ⁺ /mPSC comprises a vector encoding the Oct-4 gene. The cancer-initiating cell of claim 2, wherein the sequence of the Oct-4 gene is SEQ ID NO: 1. The scope of the patent CAR item 1 of the initial cell cancer, wherein overexpression of the separation of the Oct-4 ⁺ / Oct-4 expression levels of the normal mPSC CAR ⁺ / Oct-4 expression levels of more than 16 times mPSC . A cancer-initiating cell according to claim 1, which is a lung cancer-initiating cell. The cancer-initiating cell of claim 1, which has tumorigenic ability, wherein the tumorigenic ability comprises tumor formation, tumor regeneration, metastatic ability or a combination thereof. The cancer-initiating cell according to claim 1, which has a CD133 expression, Aldehyde dehydrogenase (ALDH) activity, chemotherapy resistance, or a combination thereof. The cancer-initiating cell of claim 1, which has an angiogenic function. The cancer-initiating cell of claim 1, which has a function of participating in tumor angiogenesis. The cancer-initiating cell of claim 1, which exhibits a surface marker of endothelial cells, wherein the surface marker of the endothelial cell comprises CD31, CD105, CD34, CD144 or a combination thereof. The cancer-initiating cell of claim 8, which has a function of enhancing angiogenesis by activating an ANG/Tie2 message transmission pathway.