JPWO2018169094A1 - Method for establishing immortalized human tumor endothelial cells and immortalized human tumor endothelial cells - Google Patents

Abstract

An object of the present invention is to obtain an immortalized human-derived tumor vascular endothelial cell useful for cell-based experiments such as compound screening. The present invention provides a cell suspension derived from a tumor tissue containing tumor vascular endothelial cells by performing cell separation using magnetic beads and optional cell separation by flow cytometry to obtain CD31-positive cells. The cell group containing CD31-positive cells is re-isolated by combining subculture of the separated cell group and flow cytometry, and the genes encoding the SV40T gene, hTERT gene and fluorescent protein are expressed. The present invention relates to a method for establishing an immortalized tumor vascular endothelial cell line, comprising transforming with a virus vector as possible, and selecting transformed cells by a combination of subculture and flow cytometry. ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide a stable and large quantity of tumor vascular endothelial cells useful for screening of a drug specific to tumor vascular endothelial cells or for developing a drug. [Selection diagram] Fig. 1

Description

  The present invention relates to a method for establishing an immortalized human-derived tumor vascular endothelial cell and to an immortalized human-derived tumor vascular endothelial cell.

  Many of the currently widely used methods of treating tumors suppress the growth of cancer cells by taking advantage of the fact that cancer cells are more actively dividing and growing than other normal cells. . However, since these treatments act on normal tissues as well as actively dividing tissues (such as hair roots and bone marrow), side effects such as hair loss and bone marrow suppression are problematic.

  On the other hand, in recent years, angiogenesis inhibition therapy targeting a new blood vessel indispensable for proliferation of cancer cells and the like in tumor tissue has been widely performed in recent years. When a malignant tumor grows, the cancer cells themselves secrete angiogenic factors to induce angiogenesis in order to obtain nutrients and oxygen necessary for the growth of the cancer cells. In addition, blood vessels in the tumor tissue are also used when the malignant tumor metastasizes to other organs or sites. Therefore, a treatment targeting the blood vessels leads not only to suppression of growth but also to suppression of metastasis.

  BACKGROUND ART In recent years, angiogenesis inhibitory therapy typified by vascular endothelial growth factor (VEGF) neutralizing antibody has been used for treatment of various tumors, and the survival rate has been improved. However, since VEGF is an important molecule for physiological angiogenesis in normal tissues, serious side effects such as gastrointestinal bleeding and hemoptysis may sometimes become a problem in addition to hypertension and delayed wound healing due to VEGF inhibition. . The background is that at the time the existing angiogenesis inhibitors were developed, tumor blood vessels were considered to have the same properties as normal tissue blood vessels.

  However, at present, tumor vascular endothelial cells (Tumor endothelial cells) are different from normal vascular endothelial cells (Normal vascular endothelial cells) in that morphological and functional vascular endothelial cells are different from normal blood vessels. Endothelial cell) is known to be different in various points (Non-Patent Documents 1 and 2). In order to minimize the side effects of angiogenesis inhibitory therapy and to develop new angiogenesis inhibitory therapy more specific for tumor blood vessels, the need for drug development that utilizes the specificity of tumor vascular endothelial cells will be recognized (For example, Patent Documents 1 and 2).

  In the development of new drugs, cell-based screening for evaluating a compound library using target cells is generally performed. It is a method to search for substances that have an inhibitory effect on target cells by comprehensive analysis using thousands or hundreds of thousands of compounds, but for that purpose, a suitable cell type and a sufficient number of cells are required. Needs to be secured. In particular, cell selection is very important and has a significant effect on the success of the screening. Therefore, it is important to secure cells that reproduce the pathological condition of the disease targeted for treatment in vitro as much as possible.

  In order to identify a compound having angiogenesis inhibitory activity for the development of a drug specific to tumor vascular endothelial cells, screening using tumor vascular endothelial cells or cells reproducing the characteristics thereof is necessary. However, the yield of tumor vascular endothelial cells that can be separated from tumor tissue is only less than 2% of a single cell population of tumor tissue, and there are various limitations in mass culture of primary vascular endothelial cells as a primary culture. . For example, in the case of a human surgically removed tissue sample, the amount of tissue that can be used for sorting tumor vascular endothelial cells is limited depending on the size of the tumor. In addition, the complexity of separation from the tissue, the cost of expensive culture media, and the like also become problems. Furthermore, in the case of human-derived cells, there is a problem of arrest of proliferation due to senescence (senescence), and it is difficult to secure a sufficient number of cells. On the other hand, there has been no report that an immortalized cell having the characteristics of a tumor blood vessel-derived endothelial cell was obtained.

International Publication WO2011 / 16461 pamphlet International Publication WO2011 / 19065 pamphlet Br J Cancer. 106, 1214-23, 2012 Br J Cancer. 109, 2237-2247, 2013

  An object of the present invention is to obtain an immortalized human-derived tumor vascular endothelial cell line useful for cell-based experiments such as compound screening.

  The present inventors have performed isolation and culture of human-derived tumor vascular endothelial cells under predetermined conditions, and obtained a gene encoding Simian Virus40 large T (hereinafter, referred to as SV40T gene) and a gene encoding human telomere reverse transcription protein ( (Hereinafter referred to as the hTERT gene) was found to be able to immortalize human tumor vascular endothelial cells efficiently, and completed the following invention.

(1) Cell separation using magnetic beads and optional cell separation by flow cytometry from a cell suspension derived from tumor tissue containing tumor vascular endothelial cells to separate a group of cells containing CD31-positive cells Separation process,
A culture step of subculturing a cell group containing the separated CD31-positive cells,
An optional re-separation step of repeating, once or twice, performing flow cytometry on the cell group after the subculture and re-separating the cell group containing CD31-positive cells,
An infection step of infecting the cell group after the culturing step or the re-separation step with a virus vector capable of expressing the SV40T gene, hTERT gene and a gene encoding a fluorescent protein, and subculturing the infected cell group, A method for establishing an immortalized tumor vascular endothelial cell line, comprising a cell selection step of separating a CD31-positive cell group expressing a fluorescent protein by flow cytometry.
(2) The method for establishing an immortalized tumor vascular endothelial cell line according to (1), wherein subculture is performed 1 to 7 times in the culture step.
(3) The method for establishing an immortalized tumor vascular endothelial cell line according to (1) or (2), wherein the exfoliation of cells from the culture substrate at the time of subculture is performed in a plurality of times in the culture step.
(4) The method for establishing an immortalized tumor vascular endothelial cell line according to any one of (1) to (3), wherein the viral vector is infected at an MOI of 300 to 1200 in the infection step.
(5) The method for establishing an immortalized tumor vascular endothelial cell line according to any one of (1) to (4), wherein the subculture is performed two or three times in the cell selection step.
(6) An immortalized tumor vascular endothelial cell line having the SV40T gene and the hTERT gene as recombinant genes and capable of being subcultured at least 50 times or more.
(7) The immortalized tumor vascular endothelium according to (6), wherein the expression of periostin is enhanced as compared to the expression in immortalized normal vascular endothelial cells having SV40T gene and hTERT gene as recombinant genes. Cell line.
(8) The expression of Biglycan and / or Lysyl oxidase is further enhanced as compared to the expression in immortalized normal vascular endothelial cells having the SV40T gene and hTERT gene as recombinant genes. The immortalized tumor vascular endothelial cell line according to 7).
(9) The immortalized tumor vascular endothelial cell line according to any one of (6) to (8), which is derived from a human kidney cancer tissue.
(10) An immortalized human tumor vascular endothelial cell line deposited under International Accession Number NITE BP-02424.
(11) A tumor comprising a step of bringing a test substance into contact with the immortalized tumor vascular endothelial cell line according to any one of (6) to (10) and a normal vascular endothelial cell to compare responses of the cells. A method for screening for a substance that specifically acts on vascular endothelial cells.
(12) The screening method according to (11), wherein the normal vascular endothelial cell is an immortalized cell line.
(13) The immortalized tumor vascular endothelial cell line is an immortalized human tumor vascular endothelial cell line deposited under International Accession No. NITE BP-02424, and the immortalized normal vascular endothelial cell is International Accession No. NITE BP- The screening method according to (12), which is an immortalized human normal vascular endothelial cell line deposited as No. 02425.

  According to the present invention, an immortalized cell line that can be subcultured at least 50 times or more can be established from tumor vascular endothelial cells derived from tumor tissue, and a screening or drug for a drug specific to tumor vascular endothelial cells can be established. It is possible to provide a stable and large amount of required tumor vascular endothelial cells for the development of.

It is a flow cytometry histogram showing the expression of Venus and mCherry in tumor vascular endothelial cells (h-imTEC) and normal renal tissue-derived vascular endothelial cells (h-imNEC) into which a virus vector having an SV40T gene and an hTERT gene has been introduced. . The human skin microvascular endothelial cell line (imHVEC) is a human skin microvascular endothelial cell that has been immortalized by the introduction of the SV40T gene and the hTERT gene. It is a gel photograph which shows the result of having confirmed the expression of SV40T gene and hTERT gene in h-imTEC and h-imNEC by PCR. Human skin microvascular endothelial cells (HMVEC) are negative controls, imMVEC and mouse islet vascular endothelial cells (MS-1) are positive controls. It is a graph which shows the cell population doubling number (Population doubling) of h-imTEC, h-imNEC, and HMVEC. Photomicrographs of h-imTEC (P57), h-imNEC (P58), imHMVEC (P57) and HMVEC (P13) stained with aging-related acidic β-galactosidase (scale bar = 100 μm). HMVEC is a positive control. It is a micrograph showing the appearance of tumor vascular endothelial cells (h-TEC), h-imTEC, normal vascular endothelial cells (h-NEC), h-imNEC, imHMVEC and HMVEC (scale bar = 100 μm). It is a gel photograph which shows the result of having confirmed the expression of CD31 and CD144 in h-imTEC, h-imNEC, imHVEC, and HMVEC by Regular-PCR. HMVEC is a positive control. It is a micrograph which shows the tube formation ability of h-imTEC, h-imNEC, imHVEC, and HMVEC by the Tube formation assay using Matrigel (trademark) (scale bar = 100 micrometers). HMVEC is a positive control. It is a microscope picture which shows the anchorage dependence of h-imTEC and h-imNEC by a soft agar assay (scale bar = 100 micrometers). Human melanoma cell A375SM is a positive control. It is a graph which shows the result of having measured the expression of Biglycan (panel A) and Lysyl oxidase (panel B) in h-imTEC, h-imNEC, and imHVEC by Real-time PCR. It is a graph which shows the cell proliferation ability of h-imTEC and h-imNEC by MTS assay. It is a microscope picture which shows the cell migration ability of h-imTEC and h-imNEC by Scratch assay (scale bar = 100 micrometers). It is a graph which shows the result of having measured the expression of Periostin in h-imTEC, h-imNEC, and imHVEC by Real-time PCR.

A first aspect of the present invention provides a method for separating CD31-positive cells from a cell suspension derived from a tumor tissue containing tumor vascular endothelial cells by performing cell separation using magnetic beads and optional cell separation by flow cytometry. A separation step of separating a cell group containing
A culture step of subculturing a cell group containing the separated CD31-positive cells,
An optional re-separation step of repeating, once or twice, performing flow cytometry on the cell group after the subculture and re-separating the cell group containing CD31-positive cells,
An infection step of infecting the cell group after the culturing step or the re-separation step with a viral vector capable of expressing the SV40T gene, hTERT gene, and a gene encoding a fluorescent protein,
The present invention relates to a method for subculturing an infected cell group, and then establishing an immortalized tumor vascular endothelial cell line comprising a cell selection step of separating the CD31-positive cell group expressing the fluorescent protein by flow cytometry.

  A first aspect of the present invention provides a method for separating CD31-positive cells from a cell suspension derived from a tumor tissue containing tumor vascular endothelial cells by performing cell separation using magnetic beads and optional cell separation by flow cytometry. And a separation step of separating a cell group containing The cell suspension derived from tumor tissue containing tumor vascular endothelial cells in this step is described in Hida et al. (Hida et al Cancer Res., 2004, 64, pp. 8249-8255; this document is incorporated herein by reference in its entirety. Embedded in the method). Specifically, a cell suspension can be prepared by slicing a tumor tissue collected from a patient clinically diagnosed with cancer, performing collagenase treatment, and converting the tumor tissue into a single cell. It is preferable that the cell suspension is treated with a hemolytic agent to prevent contamination of red blood cells and other blood cells.

  In the first aspect of the present invention, in order to suppress contamination of CD31-negative cells, a cell suspension obtained by performing a collagenase treatment or the like is used to prepare a CD31-positive cell using magnetic beads carrying an anti-CD31 antibody. Cell separation is performed. The CD31 antigen, also called Platelet Endothelial Cell Adhesion Molecular-1 (PECAM-1), is a single-chain membrane glycoprotein with a molecular weight of 140 kDa, and is one of the cell surface antigens expressed on vascular endothelial cells. The cell separation operation using magnetic beads may be performed according to a method widely known or widely used by those skilled in the art, and a CD31 macrobead kit from Milteny Biotec (http://www.miltenyibiotec.co.jp) /ja-jp/products-and-services/macs-cell-separation/cell-separation-reagents/endothelial-cells/cd31-microbead-kit-human.aspx) It may be performed according to the protocol of

  Flow cytometry in the separation step is optionally performed on the cell suspension using CD31 positivity as an index. The flow cytometry in the present separation step can be carried out using a commercially available flow cytometer, and the operation conditions are such that the number of cells in the cell group to be treated is 500 cells / second or less, preferably 100 to Preferably, the flow rate is 300 cells / second and the flow rate is 1-2 mL / hour. Under these operating conditions, the number of cells flowing at the point where CD31-positive cells are separated in flow cytometry can be reduced, and the contamination of cells other than vascular endothelial cells during separation can be suppressed. Flow cytometry in this step may not be performed when the content of CD31-positive cells in the cell group after cell separation using magnetic beads is 80% or more. In addition, if the separation by magnetic beads and flow cytometry is repeated in the separation step, the cell viability significantly decreases and it becomes difficult to obtain cultivable cells. Therefore, these operations in the separation step should be performed once. Is preferred.

  The first aspect of the present invention includes a culture step of subculturing a cell group containing CD31-positive cells separated in the separation step. The cultivation may be performed in a suitable culture medium such as an EGM-2MV medium using a culture dish coated with gelatin and fibronectin, for example. The number of subcultures in the culture step can increase the number of cells to such an extent that cell separation and infection by flow cytometry can be appropriately performed in the subsequent re-separation step and infection step, and cause cell senescence. It is appropriately set within a range not to be performed, for example, 1 to 10 times, preferably 1 to 7 times.

  In the first aspect of the present invention, it is preferable that when the cell density reaches 80 to 90% at each passage, the cells are detached and collected from the culture substrate, and then transferred to the next passage. In addition, cell detachment from the culture substrate is performed after trypsin treatment for about 5 minutes and collection of floating cells, and then confirming the number of detached cells and the state thereof, if detachment is insufficient. It is preferable that the trypsin treatment and the cell collection be repeated. Such measures are also effective for suppressing the proliferation of cells other than tumor vascular endothelial cells and reducing damage to tumor vascular endothelial cells.

  The first aspect of the present invention provides an optional re-separation in which the cell group after subculture is subjected to flow cytometry to re-separate the cell group containing CD31-positive cells once or twice. Process. The term “repetition” refers to performing, once or twice, flow cytometry for collecting CD31-positive cells again on the group of CD31-positive cells collected by performing flow cytometry on the cell group after subculture. Say. The re-separation step is preferably performed when the content of CD31-positive cells in the cell group after the separation step is less than 80%, and the re-separation can increase the content of CD31-positive cells to 80% or more. . The operating conditions in the flow cytometry are the same as the operating conditions in the separation step. If the number of cells obtained after performing the flow cytometry once in the re-separation step is small, one to five subcultures may optionally be performed before further flow cytometry. When the content of CD31-positive cells after the separation step is 80% or more, the re-separation step may not be performed.

  The content of CD31-positive cells in the cell group after the separation step is often less than 50%, and it is virtually impossible to obtain transformed tumor vascular endothelial cells even when infected with a viral vector. Confirmed by the present inventors. It is speculated that tumor vascular endothelial cells had a lower proliferative capacity than those of other cells mixed in the cell suspension. It is presumed that the proliferation of tumor vascular endothelial cells is driven by the proliferation of other mixed cells. The method according to the first aspect of the present invention comprises the steps of appropriately combining cell separation by magnetic beads, cell separation by flow cytometry, subculture, and re-separation by flow cytometry to obtain CD31 in a cell group infected with a viral vector. One of the features is that the above problem is solved by increasing the content of positive cells to 80% or more.

  In addition, by infecting a cell group having a high content of CD31-positive cells with a viral vector before senescence occurs, a cell group containing many CD31-positive cells without aging can be obtained. By performing the cell selection step described below on this cell group, obtaining a cell group not containing senescence, in which the content of CD31-positive cells transformed by introducing the SV40T gene and the hTERT gene is 80% or more, is obtained. Is also a feature of the present invention.

  The first aspect of the present invention has a cell group after the culture step or the re-separation step in which the content of CD31-positive cells is 80% or more, so that the genes encoding the SV40T gene, hTERT gene, and fluorescent protein can be expressed. And an infection step of infecting the virus vector. Both the SV40T gene and the hTERT gene, when transfected into non-proliferating primary cells isolated from a living body, give the cells the ability to grow indefinitely in vitro, ie, immortalize the cells. The gene is a gene widely known and used by those skilled in the art. In addition, fluorescent proteins are particularly limited as long as they can be expressed in mammalian cells and can separate cells expressing fluorescent proteins by flow cytometry using the fluorescence as an index. There is no. Examples of fluorescent proteins include GFP, CFP, EGFP, YFP, DsRed, mCherry, Venus, and the like. These fluorescent protein-encoding genes can be appropriately recombined from commercially available fluorescent protein expression vectors and used.

  The type of the virus vector is not particularly limited as long as it can introduce the SV40T gene, the hTERT gene, and the gene encoding the fluorescent protein so that it can be expressed. Examples of the virus vector that can be used in the present invention include a recombinant retrovirus vector, a recombinant adenovirus vector, and a recombinant adeno-associated virus vector. Among them, a recombinant retrovirus vector is preferred, and a recombinant lentivirus vector is particularly preferred.

  Production of the recombinant virus vector uses a recombinant virus vector plasmid deficient in at least one of the genes encoding proteins required for virus packaging, and any packaging cells capable of replenishing the deficient protein. Thus, it can be prepared. For example, human kidney-derived HEK293 cells, mouse fibroblasts NIH3T3, and the like are used as packaging cells.

  The method for introducing the virus vector plasmid into the packaging cells is not particularly limited, and can be performed by a known gene introduction method such as a calcium phosphate method, a lipofection method, or an electroporation method. The production of the virus vector in the present invention can be performed within the ordinary skill of a person skilled in the art, including the construction, packaging and gene transfer operations of a recombinant virus vector.

  Preferred viral vectors in the first aspect of the invention are SV40T and hTER genes, such as, for example, RPLsin-SV40 Antigen-IRES-mCherry (addgene) and CSII-CMV-hTERT-IRES2-Venus (RIKEN BRC). And a recombinant lentiviral vector having a gene encoding a fluorescent protein.

  In the infection step according to the first aspect of the present invention, it is preferable to infect the virus vector with a MOI (Multiplicity of Infection) of 300 to 1200, preferably 500 to 1000. MOIs other than those described above tend to cause cell damage and decrease infection efficiency. The infection time is preferably about 10 to 14 hours, particularly 12 hours, since the introduction efficiency tends to decrease when the infection time is 6 hours and the cell damage tends to increase when the infection time is 24 hours.

  The first aspect of the present invention comprises a cell selection step of subculturing the infected cell group, and then separating the fluorescent protein-expressing CD31-positive cell group by flow cytometry. The medium for subculture in this step, the cell density adjustment, and the cell detachment operation by trypsin treatment are preferably performed in the same manner as in the culture step, and the operation conditions in flow cytometry are the same as the operation conditions in the separation step. It is preferable to carry out. The number of subcultures in this step is appropriately set within a range in which the number of cells can be increased to such an extent that cell separation by flow cytometry can be appropriately performed, for example, 1 to 5 times, preferably 2 to 3 times. Is done many times. By this cell selection step, it is possible to obtain a cell group in which the content of CD31-positive cells transformed by introducing the SV40T gene and the hTERT gene is 80% or more.

  Regarding the transformed cells prepared through the above steps, confirmation of cell growth ability in an appropriate medium, morphological characteristics of cells, expression of characteristic genes, and other cell biological characteristics By confirming the above, establishment of an immortalized tumor vascular endothelial cell line can be confirmed.

  The cell line established by the method according to this aspect has the ability to grow in EGM-2MV medium and to be capable of being subcultured for at least 50 generations or more under appropriate culture conditions. Aging has the property that substantially no aging is observed.

  In addition, the cell line stably expresses CD31 and CD144, which are markers of vascular endothelial cells, has the tube forming ability generally possessed by vascular endothelial cells, and is derived from a separately established normal tissue. The characteristics of tumor vascular endothelial cells, such as higher growth rate and higher migration ability, and increased expression of biglycan and Lysyl oxidase as compared to cells obtained by immortalizing normal vascular endothelial cells of These cells also have Non-patent Documents 1 and 2).

  As described above, the cell line established by the method according to the first embodiment of the present invention can be concluded as an immortalized tumor vascular endothelial cell. The first aspect of the present invention can also be expressed as a method for producing an immortalized tumor vascular endothelial cell, comprising the above-mentioned steps.

  A second aspect of the present invention relates to an immortalized tumor vascular endothelial cell line having the SV40T gene and the hTERT gene as recombinant genes and capable of being subcultured at least 50 times or more.

  Such a cell line can be produced by the method of the present invention according to the first aspect. The cells according to this aspect have the ability to grow in a suitable medium and be capable of being subcultured at least 50 times or more under appropriate culture conditions, during which senescence of the cells is virtually not observed. In addition to these properties, it stably expresses CD31 and CD144, which are markers of vascular endothelial cells, has the ability to form lumens generally possessed by vascular endothelial cells, and further has a growth rate and migration ability. It has properties such as high vascular endothelial cells. Furthermore, preferably, the expression of Periostin is higher than that of immortalized normal vascular endothelial cells, and more preferably the expression of Biglycan or Lysyl oxidase in addition to Periostin is higher than that of immortalized normal vascular endothelial cells. Preferably, the expression of Periostin, Biglycan and Lysyl oxidase is higher than that of immortalized normal vascular endothelial cells.

The immortalized tumor vascular endothelial cell line of this embodiment can be represented as an immortalized tumor vascular endothelial cell line having the following features (1) to (6).
(1) Having SV40T gene and hTERT gene as recombinant genes (2) Capable of at least 50 or more subcultures (3) Demonstrating tube forming ability in tube formation assay using Matrigel (registered trademark) (4) Biglycan expression is enhanced (5) Lysyl oxidase expression is enhanced (6) Periostin expression is enhanced

  The expression of Biglycan, Lysyl oxidase, or Periostin is enhanced when the expression level of these genes or proteins in the immortalized tumor vascular endothelial cell line is expressed in the immortalized normal vascular endothelial cells. It means larger compared to the amount.

  The immortalized normal vascular endothelial cells used as a comparison target of the expression level are preferably derived from the same tissue as the immortalized tumor vascular endothelial cell line, but may be commercially available immortalized normal vascular endothelial cells (imHVEC). In addition, since immortalized normal vascular endothelial cells may change in properties such as gene expression due to excessive passage, immortalized normal vascular endothelial cells used as a comparison object may have a small number of passages. preferable.

  The measurement of the expression level of the gene or protein of Biglycan, Lysyl oxidase or Periodin employs various methods known to those skilled in the art that can measure the expression level of the gene or protein whose base sequence or amino acid sequence is known. Can be done. A typical method is a PCR method using a nucleic acid consisting of a base sequence designed based on a known base sequence of Biglycan, Lysyl oxidase or Periostin gene as a primer, in particular, a real-time PCR method, a DNA microarray method, and the above-described factor protein. An immunological method using an antibody specific to, particularly ELISA.

  The present inventors separated tumor vascular endothelial cells from a tumor site of a kidney removed from a kidney cancer patient with prior consent and normal vascular endothelial cells from a normal site of the same kidney. By applying the method according to the embodiment, an immortalized tumor vascular endothelial cell line and an immortalized normal vascular endothelial cell line were established. Further, each cell line was transferred to the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation, Room 2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba, Japan, on February 16, 2017. The strain was deposited in Japan under the accession number NITE P-02424 (indication of identification: h-imTEC_RCC01), and the normal vascular endothelial cell line was deposited in Japan under the accession number NITE P-02425 (indication of identification: h-imNEC_RCC01). In addition, each of the above cell lines was converted to the International Depositary Center for Patented Microorganisms, National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Room No. 2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba, Japan, as of January 30, 2018. A request for transfer to an international deposit is made, and the immortalized tumor vascular endothelial cell line is referred to as accession number NITE BP-02424 (indication of identification: h-imTEC_RCC01), and the normal vascular endothelial cell line is referred to as accession number NITE BP-02425 (indication of identification). : H-imNEC_RCC01).

  In a third aspect of the present invention, the international deposit number NITE BP-02424 (indication of identification: h-imTEC_RCC01) was deposited internationally at the Patent Microorganisms Depositary, the National Institute of Technology and Evaluation, which is an international depository organization. It relates to an immortalized tumor vascular endothelial cell line.

  Because immortalized tumor vascular endothelial cell lines can grow virtually indefinitely in vitro, they are needed for the development of new angiogenesis inhibitory therapies specific for tumor blood vessels, especially for the development of drugs specific for tumor vascular endothelial cells. It enables stable and large-volume supply of tumor vascular endothelial cells, and can be used for cell-based screening using the same cell line. For example, the response of cells upon contacting an immortalized tumor vascular endothelial cell line with a normal vascular endothelial cell, particularly an immortalized normal vascular endothelial cell, typically a suitable response comprising the test substance It acts specifically on tumor vascular endothelial cells by comparing changes in cell morphology, changes in proliferation ability, biochemical reactions, induction of apoptosis, changes in gene expression, etc. when cells are cultured in a medium. It becomes possible to screen substances.

  In particular, immortalized cell lines established from tumor vascular endothelial cells and normal vascular endothelial cells isolated from the same individual, that is, from the same organ of one patient, are vascularized as a combination of cell lines having the same genetic background. It can be a useful research material in the development of angiogenesis inhibition therapy or in the screening or development of a drug specific to tumor vascular endothelial cells. The present invention provides a method for screening for a substance that specifically acts on tumor vascular endothelial cells, comprising a step of bringing a test substance into contact with an immortalized tumor vascular endothelial cell line and a normal vascular endothelial cell and comparing the response of each cell. Is provided as a fourth aspect. In particular, normal vascular endothelial cells are preferably immortalized cell lines, and thus a combination of the two deposited cell lines can be used as a useful cell line in the screening method according to the present invention.

  For example, by comparing the response of both cells when a test substance is added to the two deposited cell lines growing in an appropriate medium, the cells can specifically act on immortalized tumor vascular endothelial cell lines. It is possible to efficiently search for a substance that can suppress the proliferation or cause apoptosis, and to increase the so-called high throughput in such a search. In particular, a substance capable of specifically inhibiting the growth of an immortalized tumor vascular endothelial cell line or causing apoptosis is expected to be usable as an antitumor agent or a tumor metastasis inhibitor.

  The following examples illustrate the invention in more detail.

1) Isolation and culture of vascular endothelial cells Under the approval of Hokkaido University's voluntary clinical research and the ethical guidelines for medical research on humans, we obtained preoperative written consent from patients diagnosed with renal cancer clinically. Then, a kidney cancer tissue and a normal kidney tissue were collected from the surgically resected tissue.

  Each of the collected tissues was minced in a Hanks buffer (HBSS: Gibco (registered trademark) Thermo Fisher Scientific) using a surgical scissor, transferred to a 50 mL conical tube, and then transferred to a type II collagenase (Collagenase Type II: Gibco (Gibco)). (Registered Trademark) Thermo Fisher Scientific) and DNase I (Roche) were added, and the mixture was stirred at 37 ° C for 1 hour to prepare a cell suspension.

  A tube was placed on ice, and an inactivated fetal bovine serum (FBS: Hyclone (registered trademark) Thermo Fisher Scientific) was added in an amount equal to the cell suspension to stop the action of collagenase.

  The cell suspension was transferred to another new 50 mL tube through a 100 μm mesh size cell strainer (Falcon®, Corning), and then centrifuged at 1,000 rpm at 4 ° C. for 5 minutes to precipitate the cells. The cells were washed twice with HBSS containing 2.5% FBS, centrifuged at 1,000 rpm at 4 ° C. for 5 minutes, and the supernatant was removed. After suspending in HBSS containing 2.5% FBS, the suspension was transferred through a 70 μm mesh size cell strainer (Falcon (registered trademark), Corning) to another new 50 mL tube, and then centrifuged at 1,000 rpm at 4 ° C. for 5 minutes. Cells were allowed to settle.

  After removing the supernatant, 20 mL of a hemolytic agent (Lysing Buffer: BD Biosciences) was added and suspended, and reacted at room temperature for 15 minutes. After centrifugation at 1,000 rpm at 4 ° C. for 5 minutes, the cells were washed twice with MACS buffer (degassed 0.5% BSA, buffer containing 2 mM EDTA: Miltenyi Biotec). After suspending in MACS Buffer, FcR Blocking Reagent (Miltenyi Biotec) was added and reacted at 4 ° C. for 10 minutes.

  After adding anti-human CD31 magnetic beads (Anti-human CD31 MicroBeads: Miltenyi Biotec) and reacting at 4 ° C for 30 minutes, the cells were washed with MACS Buffer and centrifuged at 1,000 rpm at 4 ° C for 5 minutes. The LS Column (Miltenyi Biotec) was set on the magnet of the MACS manual cell separator, and the MACS Buffer was added three times for calibration, and then a 15 mL tube was set below the column.

  500 μL of the cell suspension suspended in MACS Buffer was added to the column, and the cells were washed three times with 3 mL of MACS Buffer and collected as CD31 negative cells. The column was removed from the magnet, and 5 mL of MACS Buffer was added, and the magnetically labeled CD31-positive cells remaining in the column were collected by physically extruding them into another 15 mL tube with an attached syringe.

  When the CD31 positive rate of the collected cell group was less than 80%, further separation by flow cytometry was performed. In the separation operation by flow cytometry, by setting the number of cells in the solution to 300 cells / second or less and setting the flow rate to the lower limit (1 mL / hour), the point at which CD31-positive cells are separated in flow cytometry is improved. CD31-positive cells were separated while reducing the number of flowing cells and avoiding contamination of cells other than vascular endothelial cells.

The CD31-positive cells derived from the renal cancer tissue and the non-cancerous normal kidney tissue collected in this manner were cultured on a culture dish coated with 1.5% gelatin (SIGMA-ALDRICH) and 10 μg / mL fibronectin (Corning). And subcultured once or twice in an EGM-2 MV medium (Lonza) containing 15% FBS in a 5% CO ₂ incubator at 37 ° C. to proceed to the infection step.

  When the content of CD31-positive cells after the separation step is less than 80%, the cells are subcultured 1 to 7 times under the above-described conditions, and then re-separated by flow cytometry under the above-mentioned conditions, and then 80% or more. Vascular endothelial cells, which are CD31-positive cells having a purity of, were isolated.

  In the above subculture, the cells were subcultured at a cell density of 80 to 90% so that vascular endothelial cells were not driven out by other cells such as contaminating cancer cells and fibroblasts. For trypsin treatment to detach cells from the culture dish, trypsin was quenched 5 minutes after the start of the trypsin treatment, and the detached cells were collected. If a large number of unpeeled cells remained, additional trypsin was added. The treatment was performed while observing the peeled state.

2) Preparation of Virus Solution As lentivirus expression vectors, CSII-CMV-hTERT-IRES2-Venus (provided by Dr. Kenkichi Masutomi and Dr. Genichiro Ishii) and RPLsin-SV40 Antigen-IRES-mCherry (Addgene) # 58993) was used. For lentivirus preparation, pCAG-HIVgp (RIKEN BioResource Center # RDB4394) and pCMV-VSV-G-RSV-Rev (RIKEN BioResource Center # RDB4393) are used in combination with the above plasmids as packaging plasmids. did.

Subconfluent of HEK293T cells (RIKEN # RCB2202) was cultured for about 24 hours were seeded about ^{1 × 10 6 / 4mL 10%} FBS -containing DMEM / dish in Type I collagen-coated dish having a diameter of 60 mm (IWAKI), sub It was confluent. In a microtube, 200 µL of OPTI-MEM (Thermo Fisher Scientific), 2 µg of pCAG-HIVgp, 2 µg of pCMV-VSV-G-RSV-Rev, 4 µg of the above-described expression vector, and the transfection reagent FuGENE (registered trademark) HD TransfectionRegionRegionRegion The mixture was mixed and allowed to stand at room temperature for 15 minutes to prepare a DNA solution.

  After a DNA solution was added to the HEK293T cells and cultured for 12 hours, the culture supernatant was removed by suction, replaced with fresh DMEM containing 10% FBS, and further cultured for 48 hours. The virus-containing culture supernatant was collected, and the suspension cells and the like were removed through a 0.45 μm filter (Corning), followed by centrifugation at 6,000 × g at 4 ° C. for 16 hours. The supernatant was removed, suspended in EGM-2MV medium, and stored at -80 ° C. The titer of the virus was measured using the qPCR Lentivirus Titration Kit (abm).

3) Gene introduction into vascular endothelial cells The vascular endothelial cells separated in 1) were cultured to about 30% confluence using EGM-2MV medium, and then EGM-2MV containing the virus solution of 2) prepared at MOI of 500 to 1000. The medium was changed and the cells were infected for 12 hours.

  After infection, the culture supernatant was aspirated off and replaced with fresh EGM-2MV medium to grow vascular endothelial cells. After subculture twice or three times, only positive cells are separated by FACSAriaII using Venus and mCherry fluorescence and CD31 as an index, whereby vascular endothelial cells into which a viral vector has been introduced (derived from kidney cancer tissue). Cells were designated as h-imTEC, and cells derived from non-cancerous normal kidney tissue were designated as h-im NEC). After the selection, the cells were subcultured 2 to 5 times. If the CD31 positive rate after the culture was less than 80%, reselection by FACS AriaI was performed. The purity of the transfected cells was confirmed by analyzing the fluorescence intensity of mCherry and Venus (FIG. 1).

4) Confirmation of expression of SV40 and hTERT Total RNA was extracted from h-imTEC and h-im NEC using ReliaPrep RNA Cell Miniprep System (Promega). Reverse Tra Ace, RT Buffer (ToYoBo), Oligo dT primer, Random primer (Hokaido System Science) and cDNA obtained from dNTP mixture (Takara) were synthesized by reverse transcription from 1 μg of RNA and dNTP mixture (Takara). The sequences of the primers used are shown below.

SV40:
forward 5'-GGTGGGTTAAAGGAGCATGA-3 '(SEQ ID NO: 1)
reverse 5'-CAACTCCAGCCATCCATTCT-3 '(SEQ ID NO: 2)
hTERT:
forward 5'-GCATCAGGGGCAAGTCCTAC-3 '(SEQ ID NO: 3)
reverse 5'-CCAACAAGAAATCATCCACCAA-3 '(SEQ ID NO: 4)
human GAPDH:
forward 5'-ACAGTCAGCCGCATCTTCTT-3 '(SEQ ID NO: 5)
reverse 5'-GCCCAATACGACCAAATCC-3 '(SEQ ID NO: 6)

  PCR was performed using GoTaq (registered trademark) Green Master Mix (Promega), initial incubation (95 ° C for 2 minutes), heat denaturation (95 ° C for 30 seconds), annealing (58 ° C for 20 seconds), and extension reaction (72 ° C for 60 seconds). Second), and GAPDH was used as an internal standard. The amplified product was subjected to electrophoresis using 2% agarose gel (Promega), and each band was confirmed using ethidium bromide (SIGMA-ALDRICH) under ultraviolet light. As a result, expression of SV40T and hTERT was confirmed in h-imTEC and h-imNEC (FIG. 2).

5) Confirmation of passage number h-imTEC and h-imNEC were adjusted with an EGM-2MV medium to a density of 5.0 × 10 ³ cells / well, and each was seeded on a 24-well plate. After culturing for 2 days, the medium was replaced and culturing was continued for another 2 days. Cells were collected with Trypsin-EDTA solution (SIGMA-ALDRICH), and the number of cells was counted. Population doubling was calculated as Population doublings = log (number of cells at the end of culture / number of cells at the start of culture) / log2.

  The cell number was measured every four days, and the cell population doubling number was calculated. As shown in FIG. 3, in h-imTEC and h-imNEC, it was confirmed that the cell proliferation was maintained even when the number of passages exceeded 50 (P50). Was. In the vascular endothelial cells not transfected, the cells did not proliferate around P15.

6) Avoidance of cell senescence After culturing h-imTEC and h-imNEC to sub-confluence, the culture supernatant was removed and washed with PBS. A Fixation Kit attached to the Senescence Detection Kit (senescence associated β-galactosidase (SA-β-Galactosidase: SA-β-Gal) activity, Abcam) was added for 15 minutes at room temperature with Fixative Solution attached. . After washing twice with PBS, the cells were stained at 37 ° C. using Staining Solution Mix (containing X-Gal) attached to Kit, and observed with an inverted microscope (FIG. 4).

  In HMVEC, β-galactosidase staining was observed at passage number 13, whereas h-imTEC and h-imNEC were not stained at passage number 58, suggesting that the aging phenomenon was avoided.

7) Maintenance of vascular endothelial cell traits FIG. 5 shows a photograph of the morphology of h-imTEC and h-imNEC observed under a microscope. Although slightly elongated compared to the morphology before gene introduction, no significant change was observed.

8) Expression of CD31 and CD144 PCR using the following primers was performed on h-imTEC and h-imNEC under the same conditions as in 4) above, and the expression of CD31 and CD144, which are markers of vascular endothelial cells, was analyzed. .

human CD31:
forward 5'-GACCAGGTGAAAGACTGAACC-3 '(SEQ ID NO: 7)
reverse 5'-TGCAGATATACGTCCCACTGTC-3 '(SEQ ID NO: 8)
human CD144:
forward 5'-CGTGTTCGCCATTGAGAGGC-3 '(SEQ ID NO: 9)
reverse 5'-ACGGACGCATTGAACAACCG-3 '(SEQ ID NO: 10)

  Both h-imTEC and h-imNEC expressed CD31 and CD144 (FIG. 6).

9) Tube forming ability A 96-well plate was coated with a Growth Factor Reduced Matrigel (Corning), incubated at 37 ° C for 30 minutes, and then suspended in 5% FBS-containing EBM-2 (Lonza). ImTEC and h-imNEC were seeded at a density of 1.0 × 10 ⁴ cells / well. After incubation at 37 ° C. for 9 hours, tube formation was observed with an inverted microscope (FIG. 7).

  Both h-imTEC and h-imNEC formed a lumen, suggesting that the introduction of the SV40T antigen and the hTERT gene did not affect properties as vascular endothelial cells.

10) Confirmation of anchorage dependence 2% Agar (BD) was diluted with pre-warmed EGM-2MV medium to prepare 0.9% Agar, 1.2 mL was added to a 6-well plate, and gelled at room temperature. This produced Soft agar. h-imTEC and h-imNEC are suspended in EGM-2MV medium, mixed with 2% Agar, adjusted to a final concentration of 0.36%, seeded on Soft agar, and incubated at 37 ° C for 2 to 3 weeks. Thereafter, the cells were observed with an inverted microscope (FIG. 8).

  Cancer cell A375SM, which is a positive control, forms a colony on Soft agar, whereas neither h-imTEC nor h-imNEC forms a colony, confirming that the cell is anchorage-dependent.

11) Gene expression profile Real-time PCR was performed on h-imTEC and h-imNEC at passages 9 and 13 using KAPA SYBR Fast qPCR Kit (Nippon Genetics) and reported as tumor vascular endothelial cell markers. Biglycan (Yamamoto et al., Br. J. Cancer, 2012, Maishi et al., Sci. Rep., 2016) and Lysyl oxidase (Expression of Osawa et al., Br. J. Cancer, 2013). confirmed. The sequences of the primers used are shown below.

human Biglycan:
forward 5'-GGCCATCCATCCAGTTTGGCAACTAC-3 '(SEQ ID NO: 11)
reverse 5'-CTGGCTTAGCTTCCTGGCTCTG-3 '(SEQ ID NO: 12)
human Lsyl oxidase (LOX):
forward 5'-CGACCCTTACAACCCCTACA-3 '(SEQ ID NO: 13)
reverse 5'-CAGGTCTGGGCCTTTCATAA-3 '(SEQ ID NO: 14)

  Real-time PCR is performed by performing 40 cycles of initial incubation (95 ° C. for 30 seconds), heat denaturation (95 ° C. for 1 second), annealing (58 ° C. for 5 seconds), and extension reaction (58 ° C.), and CFX Manager (Bio-Rad). The amplification product was quantified by. GAPDH was used as an internal standard, and the relative ratio was compared and examined by the ΔCt method (FIG. 9).

  Biglycan expression of h-imTEC at passage 9 was approximately 20-fold higher than h-imNEC. In addition, the expression of Lysyl oxidase was significantly increased in h-imTEC than in h-imNEC.

12) MTS assay h-imTEC and h-imNEC suspended in EGM-2MV medium were seeded at a density of 1.0 × 10 ³ cells / well in a 96-well plate. After incubation at 37 ° C. for 0, 24, 48 and 72 hours, 20 μL of MTS reagent (CellTiter96 Aqueous One Solution Cell Proliferation Assay: Promega) was added to each well, and the absorbance was measured after incubation at 37 ° C. for 1 hour (FIG. 10). As a result, it was confirmed that h-imTEC has a higher growth rate than h-imNEC.

13) Scratch Assay h-imTEC and h-imNEC (1.0 × 10 ⁵ ) suspended in EGM-2MV medium were seeded in a 12-well plate, and incubated at 37 ° C. to reach confluence. The cell layer at the center of each well was scratched at the tip of a pipette tip, cultured for 12 hours, and the cells that had migrated to the scratched portion were observed with an inverted microscope (FIG. 11).

  h-imTEC migrated faster into the gap than h-imNEC, suggesting that it retains properties similar to non-immortalized tumor vascular endothelial cells.

14) Analysis of gene expression profile Total RNA was extracted from h-imTEC and h-imNEC at passages 18 and 19 using ReliaPrep RNA Cell Miniprep System (Promega), and KAPA SYBR Fast qPCR Kit (Nihon Genetics) And Periostin expression was measured by Real-time PCR using primer DNAs having the following nucleotide sequences. Β-actin (ACTB) was selected as the internal standard.

β-actin
forward 5'-TTACAGGAAGTCCCTTGCCATCC -3 '(SEQ ID NO: 15)
reverse 5'-AAGCAATGCTATCACCTCCCCTG -3 '(SEQ ID NO: 16)
Periodin:
forward 5'-GCACCTTCAAAGAAATCCCCGT -3 '(SEQ ID NO: 17)
reverse 5'-AGTGTGGGTCCTTCAGTTTTGAT -3 '(SEQ ID NO: 18)

  Real-time PCR is performed by performing 40 cycles of initial incubation (95 ° C. for 30 seconds), heat denaturation (95 ° C. for 1 second), annealing (58 ° C. for 5 seconds), and extension reaction (58 ° C.), and CFX Manager (Bio-Rad). The amplification products were quantified by the above method, and the relative ratios were compared and examined by the ΔCt method (FIG. 12).

  The expression of Periostin in h-imTEC at passage number 18 was more than 6-fold higher than in h-imNEC. Gene expression profile analysis using DNA microarray (Affymetrix GeneChip Human Gene 2.0 ST Array) confirmed that the expression of Periostin in h-imTEC was 30 times or more that of h-imNEC.

  The method for establishing an immortalized tumor vascular endothelial cell line of the present invention, the method for screening a substance that specifically acts on an immortalized tumor vascular endothelial cell line and a tumor vascular endothelial cell, all target tumor vascular endothelial cells. It can be used in search, development, etc. of therapeutic agents for tumors.

SEQ ID NO: 1 SV40 forward primer SEQ ID NO: 2 SV40 reverse primer SEQ ID NO: 3 hTERT forward primer SEQ ID NO: 4 hTERT reverse primer SEQ ID NO: 5 hGAPDH forward primer SEQ ID NO: 6 hGAPDH reverse primer SEQ ID NO: 7 hCD31 forward primer SEQ ID NO: 8 hCD31 reverse primer SEQ ID NO: 9 hCD144 forward primer SEQ ID NO: 10 hCD144 reverse primer SEQ ID NO: 11 hBiglycan forward primer SEQ ID NO: 12 hBiglycan reverse primer SEQ ID NO: 13 hLOX forward primer SEQ ID NO: 14 hLOX reverse primer SEQ ID NO: 15 β-actin forward primer SEQ ID NO: 16 β-actin reverse primer Mer SEQ ID NO: 17 Periostin forward primer SEQ ID NO: 18 Periostin reverse primer

Claims (13)

Separation step of performing cell separation using magnetic beads on a cell suspension derived from a tumor tissue containing tumor vascular endothelial cells, and optional cell separation by flow cytometry to separate a cell group containing CD31-positive cells ,
A culture step of subculturing a cell group containing the separated CD31-positive cells,
An optional re-separation step of repeating, once or twice, performing flow cytometry on the cell group after the subculture and re-separating the cell group containing CD31-positive cells,
An infection step of infecting the cell group after the culturing step or the re-separation step with a virus vector capable of expressing a gene encoding Simian Virus40 large T, a gene encoding human telomere reverse transcription protein and a gene encoding a fluorescent protein, and A method for establishing an immortalized tumor vascular endothelial cell line, comprising the step of subculturing an infected cell group and then separating the CD31-positive cell group expressing the fluorescent protein by flow cytometry.   The method for establishing an immortalized tumor vascular endothelial cell line according to claim 1, wherein the subculture is performed 1 to 7 times in the culture step.   The method for establishing an immortalized tumor vascular endothelial cell line according to claim 1 or 2, wherein in the culturing step, cells are detached from the culture substrate at the time of subculture in a plurality of times.   The method for establishing an immortalized tumor vascular endothelial cell line according to any one of claims 1 to 3, wherein the viral vector is infected at an MOI of 300 to 1200 in the infection step.   The method for establishing an immortalized tumor vascular endothelial cell line according to any one of claims 1 to 4, wherein subculture is performed two or three times in the cell selection step.   An immortalized tumor vascular endothelial cell line having a gene encoding Simian Virus40 large T and a gene encoding human telomere reverse transcriptase protein as recombinant genes and capable of at least 50 or more subcultures.   The expression of periostin is enhanced as compared to the expression in immortalized normal vascular endothelial cells having as a recombinant gene a gene encoding Simian Virus40 large T and a gene encoding human telomere reverse transcription protein. 7. The immortalized tumor vascular endothelial cell line according to 6.   Furthermore, the expression of Biglycan and / or Lysyl oxidase is expressed in immortalized normal vascular endothelial cells having as a recombinant gene a gene encoding Simian Virus 40 large T and a gene encoding human telomere reverse transcription protein. The immortalized tumor vascular endothelial cell line according to claim 7, which is enhanced in comparison.   The immortalized tumor vascular endothelial cell line according to any one of claims 6 to 8, which is derived from a human kidney cancer tissue.   An immortalized human tumor vascular endothelial cell line deposited under International Accession Number NITE BP-02424.   A method specific to tumor vascular endothelial cells, comprising a step of contacting a test substance with the immortalized tumor vascular endothelial cell line according to any one of claims 6 to 10 and a test substance to compare responses of the cells. For screening substances that act chemically.   The screening method according to claim 11, wherein the normal vascular endothelial cell is an immortalized cell line.   An immortalized tumor vascular endothelial cell line is an immortalized human tumor vascular endothelial cell line deposited under International Accession Number NITE BP-02424, and immortalized normal vascular endothelial cells are deposited under International Accession Number NITE BP-02425. The screening method according to claim 12, wherein the immortalized human normal vascular endothelial cell line has been used.

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