JP6551855B2 - Human cell line directly produced from primary lesions of lung adenocarcinoma with micropapillary structure and use thereof - Google Patents

Description

  The present invention relates to a human cell line directly produced from the primary lesion of lung adenocarcinoma having a fine papillary structure and use thereof.

  Lung cancer is the cancer with the highest morbidity and mortality in Japan as well as in the world. Among lung cancer, the morbidity rate of adenocarcinoma is the highest, and lung adenocarcinoma having a fine papillary structure among them is easily released from the primary site, causing vascular invasion and lymph node metastasis, and has a poor prognosis (for example, non- See Patent Documents 1 to 3). Therefore, elucidation of the pathology of lung adenocarcinoma having a fine papillary structure and the development of a more effective treatment method are urgently needed, but the current situation is that there are few research materials.

Miyoshi T., et al., Early-stage lung adenonocarcinomas with a micropapillary pattern, a distinct pathologic marker for a significant poor prognosis., Am. J. Surg. Pathol., 27, 101-109, 2003. Hoshi R., et al., Micropapillary clusters in early-stage lung adenonocarcinomas: a distinct cytologic sign of significantly poor prognosis., Cancer, 102, 81-86, 2004. Kamiya K., et al., Histopathological features and prognostic significance of the micropapillary pattern in lung adenocarcinoma., Mod. Pathol., 21, 992-1001, 2008.

  To date, no human cell line has been established which originates from the primary lung adenocarcinoma having a fine papillary structure. It may not be clear whether the cell line not derived from the primary lesion is derived from the target cancer. Accordingly, an object of the present invention is to provide a human cell line that more accurately reflects the properties of lung adenocarcinoma having a fine papillary structure.

The present invention is as follows.
(1) A human cell line derived from the primary lesion of lung adenocarcinoma having a fine papillary structure.
(2) The cell line according to (1), which has a receipt number NITE AP-02155.
(3) A non-human mammal transplanted with the cell line according to (1) or (2).
(4) A method for screening a tumor marker using the cell line according to (1) or (2).
(5) A screening method for an anticancer agent, comprising: bringing a test compound into contact with the cell line according to (1) or (2); and measuring the growth of the cell line.
(6) a step of administering the test compound to the non-human mammal according to (3), and a step of measuring the proliferation of the cell line in the non-human mammal to which the test compound is administered. Screening method for anticancer drug.
(7) A medium for lung adenocarcinoma cells having a fine papillary structure, containing holotransferrin, insulin, progesterone, putrescine and selenite.
(8) A cell line derived from a primary lesion of lung adenocarcinoma having a micropapillary structure, comprising a step of culturing cells derived from a primary lesion of lung adenocarcinoma having a micropapillary structure in the medium described in (7) How to establish.
(9) An additive for medium of lung adenocarcinoma cells having a micropapillary structure, comprising holotransferrin, insulin, progesterone, putrescine and selenite.
(10) A kit for screening a tumor marker, comprising the cell line according to (1) or (2).
(11) A kit for screening an anticancer drug, comprising the cell line according to (1) or (2).

  The present invention can provide a human cell line that more accurately reflects the properties of lung adenocarcinoma having a fine papillary structure.

(A) is a cross-sectional view showing the structure of a cancer tissue having a micropapillary structure and a papillary structure. (B) is the figure which looked at the cut surface in the bb line of (a) from the top. (C) is a cross-sectional view showing the structure of a cancerous tissue having a nipple structure. (D) is the figure which looked at the cut surface in the dd line of (c) from the top. A cell derived from a primary tumor tissue of a lung adenocarcinoma having a fine papillary structure is primarily cultured and is a micrograph of the 25th day (magnification 10 times). It is a microscope picture of Kitasato P21LS cell line (passage number 15 generations) (5 times magnification). It is a microscope picture of Kitasato P21LS cell line (passage number 15 generations) (5 times magnification). It is a microscope picture of the Kitasato P21LS cell line cultured three-dimensionally (passage number 14 generations, magnification 5 times). (A)-(h) is a microscope picture which shows the result of having dye | stained the tissue section | slice of the fine papillary structure part of a lung adenocarcinoma tissue in Experimental example 2 (magnification 20 times). (A)-(h) is a microscope picture which shows the result of having dye | stained the tissue section | slice of the nipple structure part of a lung adenocarcinoma tissue in Experimental example 2 (magnification 20 times). (A)-(h) is a microscope picture which shows the result of having stained the adherent cell part of the Kitasato P21 LS cell line cultured in the dish for cell culture where I type collagen was coated in Experimental example 2 (magnification 20 times) . (A)-(h) is a microscope picture which shows the result of having stained the floating cell part of the Kitasato P21 LS cell line cultured with the type I collagen-coated cell culture dish in Experimental example 2 (magnification 20 times) . (A)-(h) is a microscope picture which shows the result of having dye | stained the Kitasato P21LS cell strain | stump | stock cultured three-dimensionally in Experimental example 2 (magnification 20 times). 10 is a graph showing an experimental result of Experimental Example 3. 10 is a graph showing an experimental result of Experimental Example 4. 10 is a graph showing an experimental result of Experimental Example 4. In Experimental example 6, it is a photograph of the NOD-scid mouse transplanted with Kitasato P21LS cell line. (A) And (b) is a photograph which shows the result of the hematoxylin eosin dyeing | staining of the tissue sample of the tumor tissue originating in Kitasato P21LS cell strain.

  First, the micropapillary structure and the papillary structure will be described. FIG. 1 (a) is a cross-sectional view showing the structure of a cancer tissue having a fine papillary structure and a papillary structure. FIG.1 (b) is the figure which looked at the cut surface in the bb line of FIG. 1 (a) from the top. FIG. 1 (c) is a cross-sectional view showing the structure of a cancerous tissue having a nipple structure. FIG.1 (d) is the figure which looked at the cut surface in the dd line of FIG.1 (c) from the top.

  As shown in FIGS. 1A to 1D, a fibrovascular core is present in a section of cancer tissue having a papillary structure. In contrast, a section of cancer tissue having a micropapillary structure is characterized by the absence of a fibrovascular core.

[Human lung adenocarcinoma cell line with micropapillary structure]
In one embodiment, the present invention provides a human cell line derived from a primary lung adenocarcinoma tumor having a fine papillary structure.

  Heretofore, there has been no established human cell line derived from a primary lung adenocarcinoma tumor having a fine papillary structure. It may not be clear whether the cell line not derived from the primary lesion is derived from the target cancer. Since the cell line of this embodiment is established from the primary lesion of lung adenocarcinoma having a fine papillary structure, it is certain that the cell line is a lung adenocarcinoma cell line having a fine papillary structure. Therefore, it is highly reliable as a cell line that reflects the characteristics of lung adenocarcinoma having a fine papillary structure. The cell line of this embodiment is suitable as a research material for elucidating the pathology of lung adenocarcinoma having a fine papillary structure and developing a more effective treatment. For example, it can be used for elucidation of the growth / progression mode of a tumor having a micropapillary structure, screening for a tumor marker, screening for an anticancer agent, and the like.

  The cell line of this embodiment may be the cell name “Kitasato P21LS” (reception number NITE AP-02155). In addition, the alias of “Kitasato P21LS” is “KU-Lu-MPPt3”, which means the same cell.

  Cells derived from the primary lesion of lung adenocarcinoma were slow to grow in the medium, making it difficult to establish cell lines. On the other hand, as described later in the Examples, the inventors added a serum substitute usually used for serum-free culture and bovine serum to the culture medium, thereby allowing primary lung adenocarcinoma having a micropapillary structure to be primary. For the first time, a cell line (cell name “Kitasato P21LS”, receipt number NITE AP-02155, hereinafter sometimes referred to as “Kitasato P21LS cell line”) was successfully established from the nest.

  As will be described later in Examples, the Kitasato P21LS cell line was confirmed to be an epithelial tumor cell of the lung by immunohistochemical staining. Further, the Kitasato P21LS cell line had both adherent cells and floating cells, and reflected the pathological morphology very strongly. Therefore, according to the Kitasato P21LS cell line, it is possible to individually analyze adherent cells and floating cells that take different forms with one cell line. In addition, as described later in Examples, the Kitasato P21LS cell line had a mutation in exon 19 of epidermal growth factor receptor (EGFR), which is considered to be most effective as a molecular target therapeutic agent. Therefore, the Kitasato P21 LS cell line is also suitable as a target for conducting studies including anticancer drug sensitivity tests.

[Non-human mammal transplanted with lung adenocarcinoma cell line with micropapillary structure]
In one embodiment, the present invention provides a non-human mammal transplanted with a human cell line derived from the primary focus of lung adenocarcinoma having a micropapillary structure. The non-human mammal of the present embodiment is useful as a model for studying lung adenocarcinoma having a fine papillary structure.

  Non-human mammals include, for example, immunodeficient mice. Various research techniques have been established for mice as experimental animals. In addition, since immunodeficient mice do not show rejection even when a human cell line is transplanted, it is highly possible that a cancer tissue can be formed. Examples of immunodeficient mice include SCID mice, NOD-scid mice, NOD / Shi-scid / IL2Rγnull mice (NOG mice), and the like.

  As will be described later in the Examples, the inventors confirmed that the Kitasato P21LS cell line transplanted into NOD-scid mice forms a tumor tissue having a micropapillary-like structure. Therefore, the non-human mammal of this embodiment can be used as an analysis model for a tumor having a micropapillary structure.

[Tumor marker screening method]
In one embodiment, the present invention provides a method for screening a tumor marker using the above human cell line derived from the primary lesion of lung adenocarcinoma having a micropapillary structure. Examples of the screening method of the present embodiment include the following.

(Preparation of tumor-specific monoclonal antibodies)
Using the above human cell line derived from the primary lesion of lung adenocarcinoma having a micropapillary structure as an immunogen, a large number of monoclonal antibodies are produced, and the tumor tissue having a fixed micropapillary structure and the staining property of the above human cell line are used as indicators. And screening for monoclonal antibodies that show specific reactivity with tumor tissue having a fine papillary structure.

  An antigen that such a monoclonal antibody recognizes is considered to be a tumor marker specific to a tumor tissue having a micropapillary structure. The tumor marker can be used for diagnosis of tumor tissue having a fine papillary structure. The monoclonal antibody can be used for diagnosis and treatment of a tumor tissue having a micropapillary structure.

(Screening by two-dimensional electrophoresis)
Proteins are extracted from the above human cell lines derived from the primary lesions of lung adenocarcinoma having a fine papillary structure and from human adenocarcinomas derived from lung adenocarcinoma not having a fine papillary structure, and the two are subjected to two-dimensional electrophoresis. Compare with. Based on the obtained electrophoresis image, identifying a protein having a difference in expression level can be mentioned. Such a protein is considered to be a tumor marker specific for a tumor tissue having a micropapillary structure.

  Here, examples of human cell lines derived from lung cancer that does not have a micropapillary structure include the A549 cell line and the LC2-ad cell line. Further, in the screening method of the present embodiment, in comparison between the above human cell line derived from the primary lesion of lung adenocarcinoma having a fine papillary structure and a human cell line derived from lung line cancer not having a fine papillary structure, For example, a protein having a difference in expression level of 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more may be selected as a tumor marker candidate.

(Analysis of secretome in culture supernatant)
Examples include culturing the above-mentioned human cell line derived from the primary lesion of lung adenocarcinoma having a micropapillary structure in a serum-free medium and specifying the protein in the culture supernatant. For example, the protein in the culture supernatant is precipitated by acetone precipitation or the like, separated by two-dimensional electrophoresis, and the protein spot is analyzed by a mass spectrometer to identify the protein. These proteins are candidate tumor markers specific for tumor tissue having a micropapillary structure.

(Autoantibody analysis using patient sera and cell lines)
Searching for antibodies in the serum of lung adenocarcinoma patients having a micropapillary structure derived from the above human cell lines or cancer patients having a micropapillary structure, and searching for autoantibodies reactive with the above human cell lines It is done.

  For example, two-dimensional electrophoresis of a protein extracted from the above-mentioned human cell line derived from the primary lesion of lung adenocarcinoma having a micropapillary structure or a culture supernatant of the above-described human cell line, React with antibody. Subsequently, the spots of the recognized proteins are analyzed by a mass spectrometer to identify the proteins. The protein to which the antibody in the patient's serum has reacted is a candidate for a tumor marker specific to a tumor tissue having a micropapillary structure.

(Comprehensive analysis by microarray)
A comprehensive gene expression analysis using a microarray is performed on the above human cell line, and a tumor marker specific for a tumor tissue having a micropapillary structure is screened.

  For example, total RNA is extracted from the above human cell lines and lung adenocarcinoma cell lines not having a micropapillary structure, cDNA is synthesized, and comprehensive gene expression analysis is performed by microarray. Genes with different expression in both cell lines or proteins encoded thereby are considered to be tumor markers specific for tumor tissues having a micropapillary structure.

  Whether or not the found gene is a tumor marker specific to a tumor tissue having a micropapillary structure can be confirmed by using, for example, a lung adenocarcinoma tissue sample using an antibody group against a protein encoded by the found gene Immunostaining can be performed by examining whether or not the micropapillary structure site is specifically stained.

  Alternatively, by confirming the reactivity of the above antibody group to the extracted protein of a cell line having a micropapillary structure by Western blotting or the like, the protein encoded by the found gene can be transformed into a tumor tissue having a micropapillary structure. Whether it is a specific tumor marker can be confirmed.

  In addition, the presence of autoantibodies in patient sera can be confirmed by using Western blotting or the like to confirm the reactivity of the cell line with fine nipple structure as the primary antibody and the reactivity of the cell line with fine nipple structure to the extracted protein. You can also check.

[Screening method for anticancer agents]
First Embodiment
In one embodiment, the present invention comprises the steps of contacting a test compound with the above human cell line derived from the primary lesion of lung adenocarcinoma having a fine papillary structure, and measuring the proliferation of the cell line. Provided is a screening method for an anticancer agent.

  For example, adding the compound in the compound library to the medium of the above human cell line and examining the influence on cell proliferation can be mentioned. More specifically, for example, the above human cell line is seeded in a 96-well plate and cultured for about 1 to 5 days in the presence of the compound library. Thereafter, the number of living cells may be analyzed, for example, by coloring by reduction of tetrazolium salt. Compounds that inhibit the growth of the above-mentioned human cell lines are candidates for anticancer agents.

  As the tetrazolium salt, commercially available 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) (MTT) or the like can be used.

Second Embodiment
In one embodiment, the present invention provides a step of administering a test compound to the above-mentioned non-human mammal transplanted with a lung adenocarcinoma cell line having a micropapillary structure, and the above-mentioned method in the non-human mammal to which the test compound is administered. A method for screening an anticancer agent, comprising the step of measuring the proliferation of the human cell line.

  Non-human mammals include those described above. The test compound is administered orally or parenterally to the non-human mammal transplanted with a lung adenocarcinoma cell line having a micropapillary structure. Subsequently, the proliferation of a lung adenocarcinoma cell line transplanted into a non-human mammal is measured to confirm the effect of the test compound. Examples of the method for measuring proliferation of a lung adenocarcinoma cell line include measuring the size (volume, mass, etc.) of cancer tissue derived from the transplanted lung adenocarcinoma cell line. The test compound for reducing the cancer tissue is likely to be usable as an anticancer agent.

[Pulmonary adenocarcinoma cell culture medium and lung adenocarcinoma cell additive]
In one embodiment, the present invention provides a medium for lung adenocarcinoma cells having a micropapillary structure containing holotransferrin, insulin, progesterone, putrescine and selenite.

  In the present specification, “a lung adenocarcinoma cell having a fine papillary structure” means a cell derived from a lung adenocarcinoma that forms a fine papillary structure when cultured. “Lung adenocarcinoma cells having a micropapillary structure” can be referred to as “lung adenocarcinoma cells forming a micropapillary structure”, “lung adenocarcinoma cells having the ability to form a micropapillary structure”, and the like.

  As described later in the examples, the inventors found that a medium containing holotransferrin, insulin, progesterone, putrescine and selenite improves the growth of lung adenocarcinoma cells having a micropapillary structure. In addition, it is difficult to establish a cell line derived from the primary lesion of lung adenocarcinoma having a fine papillary structure using a normal medium or a serum-free medium such as an ACL-4 medium. In contrast, the inventors succeeded in establishing a cell line for the first time by culturing cells derived from the primary lesion of lung adenocarcinoma having a micropapillary structure using the medium of the present embodiment.

  The culture medium of the present embodiment may further contain serum. Examples of serum include bovine serum. Although a medium containing holotransferrin, insulin, progesterone, putrescine and selenite is usually used as a serum-free medium, the present inventors carefully added the serum to the above-described medium to form a fine papillary structure. It has been revealed that the growth of lung adenocarcinoma cells having has improved. Therefore, the medium of the present embodiment is suitable for culturing lung adenocarcinoma cells having a micropapillary structure.

  In one embodiment, the present invention provides an additive for medium of lung adenocarcinoma cells having a micropapillary structure, comprising holotransferrin, insulin, progesterone, putrescine and selenite. The culture medium additive of the present embodiment may further contain serum. Examples of serum include bovine serum.

[Method for establishing cell line derived from primary lung adenocarcinoma having fine papillary structure]
In one embodiment, the present invention comprises a step of culturing cells derived from a primary lesion of lung adenocarcinoma having a micropapillary structure in a medium containing holotransferrin, insulin, progesterone, putrescine and selenite. A method for establishing a cell line derived from a primary lung adenocarcinoma having a structure is provided. Although it is difficult to establish a lung adenocarcinoma cell line having a micropapillary structure, a cell line can be established by this method.

  In the method of this embodiment, the above-mentioned culture medium may further contain serum. Examples of serum include bovine serum.

[Tutorial marker or anti-cancer drug screening kit]
In one embodiment, the present invention provides a screening kit for a tumor marker or an anticancer agent, comprising a human cell line derived from a primary lesion of lung adenocarcinoma having a micropapillary structure.

  Conventionally, research materials useful for elucidating the pathophysiology of lung adenocarcinoma having a fine papillary structure and developing more effective therapies have been scarce. In contrast, since the kit of this embodiment includes a human cell line derived from the primary lesion of lung adenocarcinoma having a fine papillary structure, screening for a tumor marker specific to a tumor having a fine papillary structure, the fine papillary structure It is possible to efficiently carry out screening and the like of anticancer agents effective for cancers having

  The present invention will next be described in more detail by way of experimental examples, but the present invention is not limited to the following experimental examples. In addition, this research has been approved by the ethics committee of Kitasato University School of Medicine under the name of “Analysis of metastasis / invasion mechanism of cancer based on lung adenocarcinoma with fine papillary structure” and has been approved. I have applied for continuation (B09-33).

[Experimental Example 1]
(Establishment of cell lines)
We established a human cell line derived from the primary lung adenocarcinoma having a fine papillary structure. Specifically, first, the primary tumor tissue that was removed by surgery and confirmed histopathologically to be a lung adenocarcinoma having a fine papillary structure was cut into minced pieces finely with scissors. Got. Subsequently, the obtained tissue pieces were cultured (primary culture) in a cell culture dish coated with type I collagen.

  As the medium, RPMI1640 medium is supplemented with 10% bovine serum, and serum substitutes (trade name “N-2 supplement”, Thermo Fisher Scientific Co., Ltd.) usually used for serum-free culture, antibiotics and L-glutamine. The one added was used. N-2 supplement contains human holotransferrin 100 mg / L, insulin 5 mg / L, progesterone 0.0063 mg / L, putrescine 16.11 mg / L and selenite 0.0052 mg / L at final concentrations (concentrations in the medium) Was. As antibiotics, 10 μg / mL gentamicin, 100 U / mL penicillin, and 100 μg / mL streptomycin were added at final concentrations. In addition, L-glutamine was added to a final concentration of 2 mM.

  FIG. 2 is a photomicrograph of cells on day 25 of primary culture (10 × magnification). Sheet-like and raised tufted clumps were formed. Raised cells were harvested at the tip of the chip and seeded directly into new dishes. When adherent cells reached 60-70% confluence, they were detached using trypsin and repeated to pass through to establish a cell line. A human cell line derived from a primary lung adenocarcinoma tumor having an established fine papillary structure was deposited at the National Institute of Technology and Evaluation (2-5-8, Kazusa Tsuji, Kisarazu City, Chiba Prefecture) (reception date: (October 30, 2015, receipt number: NITE AP-02155, cell name “Kitasato P21LS”).

  FIG. 3 is a photomicrograph of Kitasato P21LS cell line (passage number 15) (magnification 5 times). Sheet-like and raised tufted clumps were formed. FIG. 4 is a photomicrograph of another field of view of the Kitasato P21LS cell line (passage number 15) (magnification 5 times). As shown in FIG. 4, a tufted agglomerate that floats without sticking was also observed.

  Subsequently, the Kitasato P21LS cell line was used to perform three-dimensional culture that mimics the extracellular matrix of a living body. More specifically, the Kitasato P21LS cell line cultured up to the 11th generation in a cell culture dish coated with type I collagen was three-dimensionally cultured in the 12th to 14th generations. Three-dimensional culture was carried out using a commercially available plate (trade name “NanoCulture MH pattern low adsorption, 96 well, model“ NCP-LH96-2 ”, Scivax”), and then a 35 mm dish (model “NCD-LH35- 5 ", Scivax).

  FIG. 5 is a photomicrograph of Kitasato P21LS cell line cultured three-dimensionally (passage number 14 generations, magnification 5 times). The Kitasato P21LS cell line was found to form a tufted agglomerate and float in three-dimensional culture. In addition, it was revealed that the Kitasato P21LS cell line formed a tufted agglomerate and floated even in a floating three-dimensional culture using a polymer type scaffolding type and a low adsorption dish.

[Experimental Example 2]
(Immunohistochemical staining of lung adenocarcinoma tissue with fine papillary structure and Kitasato P21 LS cell line)
Immunohistochemical staining of lung adenocarcinoma tissue having a fine papillary structure and Kitasato P21 LS cell line was performed for comparison. More specifically, a fine papillary structure portion and a tissue section of the papillary structure portion of a lung adenocarcinoma tissue, and a formalin-fixed specimen of an adherent cell portion and a floating cell portion of the Kitasato P21LS cell line were prepared from hematoxylin and eosin (HE); epithelium. Antibodies against sex cell markers, cytokeratin, E-cadherin, epithelial cell adhesion molecule (EpCAM) and Mucin 1, cell surface associated (Muc-1); -1); antibody stained for antibodies against N-cadherin and vimentin, markers of epithelial-mesenchymal transformation. In addition, as a lung adenocarcinoma tissue having a fine papillary structure, a tissue derived from Kitasato P21 LS cell line was used.

  The antibodies used were as follows. Anti-cytokeratin antibody (model number "412811", Nichirei Corporation), anti-EpCAM antibody (model number "248 M-14", Cell Marque Corporation), anti-TTF-1 antibody (model number "NCL-L-TTF-1", Leica Corporation) , Anti-Muc-1 antibody (model number “NCL-L-MUC-1”, Leica), anti-E-cadherin antibody (model number “M3612”, Dako), anti-N-cadherin antibody (model number “M3613”, Dako) ), Anti-vimentin antibody (model number “M0725”, Dako company).

6 (a) to 6 (h) are photomicrographs showing the staining results of the tissue section of the fine papillary structure portion of the lung adenocarcinoma tissue (magnification 20 times). Fig.7 (a)-FIG.7 (h) are the microscope pictures which show the dyeing | staining result of the tissue section of the nipple structure part of a lung adenocarcinoma tissue (magnification 20 times). 8 (a) to 8 (h) are photomicrographs showing the staining results of the adherent cell portion of the Kitasato P21LS cell line cultured in a type I collagen-coated cell culture dish (magnification 20 times). 9 (a) to 9 (h) are photomicrographs showing staining results of floating cell portions of Kitasato P21LS cell line cultured in a type I collagen-coated cell culture dish (magnification 20 times).
10 (a) to 10 (h) are photomicrographs showing the staining results of Kitasato P21LS cell line cultured three-dimensionally in the same manner as in Experimental Example 1 (magnification 20 times).

  As a result, the adherent cell part of the Kitasato P21LS cell line shown in FIG. 8, the floating cell part of the Kitasato P21LS cell line shown in FIG. 9, and the three-dimensionally cultured Kitasato P21LS cell line shown in FIG. 10 are cytokeratin, EpCAM, TTF-1, Muc-1, and E-cadherin were positive, vimentin was partially positive, and N-cadherin was negative, and the staining result was the same as that of the primary tissue shown in FIG. These staining results indicate that they are epithelial tumors. From the above results, it was confirmed that the Kitasato P21LS cell line well reflected the properties of lung adenocarcinoma tissue having a fine papillary structure.

[Experimental Example 3]
(Examination of the growth of Kitasato P21LS cell line depending on the medium)
Using a type I collagen-coated cell culture dish (diameter: about 35 mm), Kitasato P21LS cell line was cultured in media having different compositions, and cell proliferation was examined. As the medium, RPMI1640 medium supplemented with 10% bovine serum (hereinafter referred to as “10% FCS medium”), the serum substitute used in Experimental Example 1 (trade name “N-2 supplement”, Thermo Fisher Scientific, RPMI1640 medium (hereinafter referred to as “N2 medium”) and RPMI1640 medium (hereinafter referred to as “10% FCS + N2 medium”) supplemented with 10% bovine serum and “N-2 supplement” were used. .

First, 1 × 10 ⁵ adherent cells of the Kitasato P21 LS cell line were seeded per dish. The 10% FCS medium group had n = 9, the N2 medium group had n = 10, and the 10% FCS + N2 medium group had n = 9. Subsequently, these cells were cultured for 8 days, and then the number of floating cells and adherent cells in each dish was counted to determine an average value and a standard error. In the measurement of the number of cells, only the number of living cells was measured.

  FIG. 11 is a graph showing experimental results. In FIG. 11, "p <0.01" represents that there is a significant difference at a risk factor of less than 1%. As a result, it was revealed that the cell growth of the 10% FCS + N2 medium group was significantly faster than that of the 10% FCS medium group and the N2 medium group.

[Experimental Example 4]
(Examination of proliferation of adherent cells and suspension cells in Kitasato P21LS cell line)
Kitasato P21LS cell line was cultured using 10% FCS + N2 medium in a type I collagen-coated cell culture dish, and the number of adherent cells and floating cells were measured, and the proliferation of each cell was examined.

First, 1 × 10 ⁵ adherent cells of the Kitasato P21 LS cell line were seeded per dish. Subsequently, the number of adherent cells and floating cells were respectively counted every 24 hours from the start of culture, and the average value and the standard error were determined. The number of cells was measured at n = 3 for each measurement point. In the measurement of the number of cells, only the number of living cells was measured. Similar experiments were performed twice. In the second experiment, different lots of bovine serum were used than in the first experiment.

  FIG. 12 is a graph showing the growth curve of the Kitasato P21 LS cell line in the first experiment. In the first experiment, the total doubling time for adherent and floating cells was 74.6 hours. FIG. 13 is a graph showing the growth curve of Kitasato P21LS cell line in the second experiment. In the second experiment, the total doubling time of adherent and floating cells was 59.2 hours.

[Experimental Example 5]
(Analysis of nucleotide sequence of EGFR exon 19)
The nucleotide sequences of exons 18 to 21 of epidermal growth factor receptor (EGFR) were analyzed for the Kitasato P21 LS cell line. Genetic Analyzer 3500Dx (Thermo Fisher Scientific) was used for the analysis of the base sequence. As a result, it has been clarified that the Kitasato P21LS cell line has an E746-A750 deletion mutation in EGFR exon 19 (a deletion mutation of a nucleotide sequence encoding amino acids 746 to 750 of the EGFR amino acid sequence). .

[Experimental Example 6]
(Non-human mammal transplanted with Kitasato P21LS cell line)
5 × 10 ⁶ Kitasato P21LS cell lines were suspended in phosphate buffer (PBS) supplemented with Matrigel (model “354234”, Corning) and injected subcutaneously into NOD-scid mice, which are immunodeficient mice. Whether tumor tissue can be formed was examined.

  FIG. 14 is a photograph of NOD-scid mice 30 days after transplantation of Kitasato P21LS cell line. The arrow in the figure indicates the formed tumor tissue. As a result, it was revealed that a tumor tissue can be formed by transplanting the Kitasato P21LS cell line.

Similar results were obtained when 5 × 10 ⁶ Kitasato P21LS cell lines were suspended in PBS and injected subcutaneously into NOD-scid mice.

  Subsequently, a tissue specimen of the formed tumor tissue was prepared and stained with hematoxylin and eosin. FIG. 15A is a photograph showing the staining result (magnification 2 times), and FIG. 15B is a photograph in which the region indicated by the square in FIG. 15A is enlarged (magnification 10 times).

  As a result, in the formed tumor tissue, a part having a micropapillary-like structure was occasionally found. This result further supports that the Kitasato P21LS cell line is useful as a research material for elucidating the pathogenesis of lung adenocarcinoma having a fine papillary structure.

  The present invention can provide a human cell line that more accurately reflects the properties of lung adenocarcinoma having a fine papillary structure.

Claims (7)

A human cell line generated directly from a primary lung adenocarcinoma tumor having a micropapillary structure , which is Accession No. NITE P-02155 . A non-human mammal transplanted with the cell line according to claim 1 . A method for screening a tumor marker using the cell line according to claim 1 . A method of screening for an anticancer agent, comprising the steps of: contacting a test compound with the cell line according to claim 1; and measuring the growth of the cell line. A method for screening an anticancer agent, comprising: administering a test compound to the non-human mammal according to claim 2; and measuring proliferation of the cell line in the non-human mammal to which the test compound is administered. Method. A kit for screening a tumor marker, comprising the cell line according to claim 1 . A kit for screening an anticancer drug, comprising the cell line according to claim 1 .