WO2019151625A1 - Method for preparing cancer stem cell spheroids - Google Patents

Abstract

The present invention relates to a method or a kit for preparing cancer stem cell spheroids and a method for screening a cancer cell resistance treating drug by means of the prepared cancer stem cell spheroids. Cancer stem cell spheroids can be prepared in a simple manner, and the prepared cancer stem cell spheroids can effectively be utilized for screening a cancer cell resistance treating drug.

Description

 [Explanation of invention]

 [Name of invention]

 Cancer stem cell spheroid production method

 Technical Field

 The present invention relates to a method for producing cancer stem cell spheroids or a kit for manufacturing. The present invention also relates to a method for screening a drug for treating cancer cell resistance using the cancer stem cell spheroid prepared by the above method or kit.

 【Technology behind the invention】

 Cancer stem cells (CSCs or tumor-based cell cel ls: TIC) have many similar characteristics with normal stem cells such as self-renewal, endogenous drug resistance and differentiation ability. Since the discovery of cancer cells similar to stem cells in acute myeloid leukemia, there is increasing evidence of the presence of a few cancer stem cells in tumor aggregates that are primarily responsible for tumor recurrence and drug resistance. Therefore, cancer stem cells are attracting considerable attention in cancer research and drug development.

 The cancer stem cells are generally isolated from patient derived tumor tissue based on cancer stem cell surface markers. However, the supply of patient-derived tumor tissue is limited, and since only a small amount of cancer stem cells can be separated, it is not easy to obtain cancer stem cells. Alternatively, attempts have been made to isolate cancer stem cells from existing cancer cell lines, but since cancer cells contain only less than 1-2% of cancer stem cells, it is not practical to secure sufficient amounts of cancer stem cells (Cel l 144, 646-674

(2011)). In addition, the three-dimensional structure of cancer cells can represent the tumor environment better than the two-dimensional monolayer structure, there is a considerable interest in developing a method for promoting the spheroid formation of cancer cells. The spheroids used for drug screening or efficacy testing can be used to insert cells into a hydrophilic UL tr low-attachment (ULA) surface, a concave agarose gel (U-bot tom) or a hole in a hanging-drop cell substrate. It is currently manufactured by the method. However, even the spheroid produced by the above method does not sufficiently contain cancer stem cells. In such a situation, it is necessary to develop a convenient method for producing cancer stem cell spheroids having cancer-forming ability in human cancer cell lines. Therefore, the present inventors endeavored to develop a method for producing cancer stem cell spheroids, and thus a method for producing cancer stem cell spheroids using a cell culture substrate containing a cyclosiloxane polymer and a medium containing albumin 2019/151625 1 »（： 1 ^ 1 {2018/013838

To establish and complete the present invention.

 [Task to be solved]

 One object of the present invention is to provide a composition for inducing cancer stem cells from cancer cells, comprising a cell culture medium containing albumin. Another object of the present invention is to provide a method for producing cancer stem cells from cancer cells, comprising the step of culturing cancer cells using a composition inducing cancer stem cells from cancer cells comprising a cell culture medium containing albumin. .

 Still another object of the present invention is a kit for producing cancer stem cell spheroid, comprising a cell culture substrate, and a composition for inducing cancer stem cells from cancer cells including a cell culture medium comprising albumin, wherein the cell culture substrate is cyclo It comprises a siloxane polymer, the medium is to provide a kit for producing cancer stem cell spheroid, comprising albumin.

Another object of the present invention, (3) preparing a cancer stem cell spheroid by the method for producing cancer stem cell spheroid; (A step of treating the cancer cell resistance treatment candidate material to the cancer stem cell spheroid of step ( ₃₎ ; and ( ₀₎ Cancer stem cell spheroid group and cancer cell resistance treatment candidates treated with the cancer cell resistance treatment candidate of step ( ₀₎ The present invention provides a method for screening a drug for treating cancer cell resistance, comprising comparing the untreated control group.

 [Measures of problem]

 This will be described in detail as follows. In addition, each description and embodiment disclosed in this application may be applied also to each other description and embodiment. That is, all combinations of the various elements disclosed in this application are within the scope of the present application. Moreover, the scope of the present disclosure is not to be limited by the specific description set forth below.

 As one aspect for achieving the object of the present invention, there is provided a composition for inducing cancer stem cells from cancer cells, including a cell culture medium containing albumin.

Another aspect for achieving the object of the present invention, comprising the steps of culturing cancer cells using a composition for inducing cancer stem cells from cancer cells comprising a cell culture medium containing albumin, producing cancer stem cells from cancer cells Provide a way to do it. 2019/151625 1 »（： 1 ^ 1 {2018/013838

As another aspect for achieving the object of the present invention, comprising a cell culture substrate, and a composition for inducing cancer stem cells from cancer cells comprising a cell culture medium comprising albumin, a kit for producing cancer stem cell spheroid, The cell culture substrate comprises a cyclosiloxane polymer, and the medium provides a kit for producing cancer stem cell spheroid, comprising albumin. In another aspect for achieving the object of the present invention, preparing a cancer stem cell spheroid; Treating a cancer cell resistance treatment candidate with cancer stem cell spheroids; And a step of comparing the cancer stem cell spheroid group treated with the cancer cell resistance treatment candidate and the control group not treated with the cancer cell resistance treatment candidate.

 The inventors of the present invention provide a three-dimensional cancer stem cell spheroid, such as an in vivo environment, that has complete cancer stem cell characteristics when cultured cancer cells in a medium containing albumin on a cell culture substrate containing a polymer formed by a cyclosiloxane compound. The invention is provided by elucidating that it can be manufactured in high yield. Hereinafter, the present invention will be described in more detail.

 As one aspect for achieving the object of the present invention, comprising the steps of culturing cancer cells using a composition for inducing cancer stem cells from cancer cells comprising a cell culture medium containing albumin, producing cancer stem cells from cancer cells Provide a way to do it.

 The step of culturing the cancer cells using a composition for inducing cancer stem cells from cancer cells comprising the cell culture medium containing the albumin, cultured cancer cells separated using a composition comprising a cell culture medium containing albumin. In such a step, the culturing may be performed on a cell culture substrate containing a cyclosiloxane polymer.

The term "cancer cell" or "isolated cancer cell" of the present invention may be a cell derived from a human or may be a cell derived from various individuals other than humans, but is not limited thereto. Cells may be included, but are not limited to, both internally and ex vivo, and specifically, the isolated cancer cells may be cells derived from various tissues of human, and may be ovarian cancer, breast cancer, liver cancer, brain cancer, colon cancer, or prostate cancer. ， Cervical cancer, lung cancer, stomach cancer, skin cancer, pancreatic cancer, oral cancer, rectal cancer, laryngeal cancer, thyroid cancer, parathyroid cancer, colon cancer, bladder cancer, peritoneal cancer, adrenal cancer, tongue cancer, small intestine cancer, esophageal cancer, renal cancer, kidney cancer, heart cancer, duodenal cancer, Cancer cells derived from ureter cancer, urethral cancer, pharyngeal cancer, vaginal cancer, tonsil cancer, anal cancer, pleural cancer, thymic cancer, or nasopharyngeal cancer, but are not limited to these, and may be used for the purpose of the present invention. Including, but not limited to, primary cultured cells isolated from cancer tissue via biopsy, or all established cell lines.

 In addition, cancer cell markers can be used to identify the cancer cells. Specifically, the markers include AFP (Alpha-fetoprotein), CA15-3, CA27-29, CA19-9, CA-125, Calci tonin, Calret inin, CD34, CD117, Desmin, inhibin, Myo Dl, NSE (neuronspeci fic enolase), PLAP (pl acental alkal ine phosphatase), and PSA (prostate peptide ant igen) may be used, but are not limited to these.

 As used herein, the term "cyclosiloxane compound" is used to encompass a compound having a cyclosiloxane structure as a basic skeleton and having a functional group (eg, an alkyl group, an alkenyl group, etc.) at its silicon atom. According to one embodiment of the present invention, the cyclosiloxane compound is represented by the following formula (1).

 [Formula 1]

Prize

essence) ;

Alkenyl independently hydrogen or Al 02-10 to 1 mu eunseo and _(where: I 1 popularly least two places is an alkenyl nilim 02-10); 0 2019/151625 1 »（： 1 ^ 1 {2018/013838

Figure 2 independently of one another is hydrogen, 01-10 alkyl, 02-10 alkenyl, halo, metal element, 05-14 heterocycle, 03-10 cycloalkyl or 03-10 cycloalkenyl. As used herein, the term 'alkyl' refers to a straight or branched unsubstituted or substituted saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isobutyl, pentyl or nuclear chambers and the like. 01-010 alkyl refers to an alkyl group having an alkyl unit having 1 to 10 carbon atoms, and when 01-010 alkyl is substituted, carbon number of the substituent is not included.

According to one embodiment of the present invention, 01-010 alkyl herein is 01-08 alkyl, 01-07 alkyl or 01-06 alkyl. As used herein, the term "alkenyl ¹ " refers to a straight-chain or branched unsubstituted or substituted unsaturated hydrocarbon group having a designated carbon number, for example, vinyl, propenyl, allyl, isopropenyl, butenyl / isobutenyl, 1; -butenyl, - pentenyl and ₁₁ - comprises a nuclear hexenyl 02 - 10 alkenyl is, if a substituted alkenyl group means, and 02-10 alkenyl group having an alkenyl unit having 1 to 10 carbon atoms, the substituents Is not included.

According to one embodiment of the present invention, 02-10 alkenyl in the present invention is 02-8 alkenyl, 02-6 alkenyl, 02-5 alkenyl, 02-4 alkenyl or 02-3 alkenyl. According to one embodiment of the invention, at least three of the seedlings 1 is 02-10 alkenyl. According to one embodiment of the invention, the cyclosiloxane has ₁₁ + 1 or ₁₁ + 2 02-10 alkenyl at the seedling position. For example, when II is 2, the compound of formula 1 becomes a cyclotetrasiloxane having 3 or 4 02-10 alkenyl in position. These alkenyl groups are involved in the polymerization reaction.

As used herein, the term `` halo '' refers to a halogen group element and includes, for example, fluoro, chloro, bromo and iodo. As used herein, the term 'metal element' refers to an alkali metal element (ni,

X; 加 ，

肝）, alkaline earth metal element ，

8 _3, 1¾), aluminum group element (Co, Ga, 1: _1, II), tin group element (¾, ¾), money metal element (0 ₁ , show _§ , show ₁₁ ), zinc group element (0 （ 1, 1¾）, rare earth element （,

57-71）, Titanium elements (,

), Banadum tribe elements,, 1 ₃ ), Chromium element elements (0,

¾ ， ，， Manganese element （1 & _1, 70, 1¾） ， Iron family element creation （： ₀ ，）, Platinum group element （如 ， 此, M （1,, II ·,

And elements that make metallic scatter element materials such as actinide elements (89-103). As used herein, the term `` heterocycle '' means a partially or completely saturated monocyclic or bicyclic 5- to 14-membered heterocycle ring. N, 0 and S are examples of heteroatoms. Pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, tetrazole, 1,2,3,5-oxadiadiazole-2-oxide, triazolone, oxa Diazolone, isoxazolone, oxadiazolidinedione, 3-hydroxypyro-2, 4-dione, 5-oxo- 1,2,4-thiadiazole, P.ridine, pyrazine, pyrimidine, indole, Isoindole, indazole, phthalazine, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline and carboline are examples of C5-14 heterocycles.

 As used herein, the term "cycloalkyl" means a cyclic hydrocarbon radical, which includes cyclopropyl, cyclobutyl and cyclopentyl C3-10 cycloalkyl is a cycloalkyl having 3-10 carbon atoms to form a ring structure. Alkyl, and when C3-10 cycloalkyl is substituted, the carbon number of the substituent is not included.

 According to one embodiment of the present invention, in the specification, C1-C10 cycloalkyl is

C1-C8 cycloalkyl, C1-C7 cycloalkyl or C1-C6 cycloalkyl.

 The term "cycloalkenyl" of the present invention means a cyclic hydrocarbon group having at least one double bond, and includes, for example, cyclopentene, cyclonuxene and cyclonusadiene. It means a cycloalkenyl having 3-10 carbon atoms, and when C3-10 cycloalkenyl is substituted, the carbon number of the substituent is not included.

 According to one embodiment of the invention, C2-10 cycloalkenyl is C2-8 cycloalkenyl, C2-6 cycloalkenyl, C2-5 cycloalkenyl, C2-4 cycloalkenyl or C2-3 cycloalkenyl to be.

 According to an embodiment of the present invention, R2 is independently hydrogen, Cl-

10 alkyl or C2-10 alkenyl. According to one specific example, at least two or at least three of R2 may be C1-10 alkyl or C2-10 alkenyl. According to one specific example, the cyclosiloxane may have n + 1 or n + 2 C1-10 alkyl or C2-10 alkenyl at the R2 position.

 According to an embodiment of the present invention, n is an integer of 1-7, an integer of 1-6, 1-

It is an integer of 5, the integer of 1-4, or the integer of 1-3.

According to one embodiment of the invention, the cyclosiloxane compound is 2,4, 6, 8-tetra0: 2-10) alkenyl-2,4, 6, 8-tetra ((： 1- 2019/151625 1 »（： 1 ^ 1 {2018/013838

10) alkylcyclotetrasiloxane, 1,3,5-triti-10) alkyl-1,3,5-tri0: 2_

10) alkenylcyclotrisiloxane, 1,3,5,7-tetra （（： 1-10） alkyl-1,3,5,7-tetra 0 ： 2-10） alkenylcyclotetrasiloxane ， 1,3 , 5,7,9-penta （（： 1-10） alkyl-

1,3,5,7,9-penta0: 2-10) alkenylcyclopentasiloxane, 1,3,5-tri (（： 1_10) alkyl-1,3,5-tri0 ： 2-10） Alkenylcyclotrisiloxane, 1,3,5,7-tetra （（1-10） alkyl-

1.3.5.7 -tetra 犯 2-10） alkenylcyclotetrasiloxane, 1,3, 5, 7,9 -penta

10) Alkyl-1,3,5,7,9-penta 0: 2-10 Alkenylcyclopentasiloxane, 1,3,5-triti-10) Alkyl 1,3,5-tribuy 2 -10) alkenylcyclotrisiloxane, 1,3,5,7 -tetra ratio 1-10) alkyl-1,3,5,7 -tetra ratio 2-10) alkenylcyclotetrasiloxane, 1,3,5 , 7,9-penta （（： 1_10） alkyl—1,3,5,7,9-penta0 ： 2-10） alkenylcyclopentasiloxane, nucleated 1X2-10） alkenylcyclotrisiloxane, octa ᄄ 2_

10) alkenylcyclotetrasiloxane, deca2- 2-10) alkenylcyclopentasiloxane,

2.4.6.8 tetravinyl-2,4,6,8, _tetramethylcyclotetrasiloxane and combinations thereof.

 According to one specific example, the cyclosiloxane compound is 1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane, 2,4,6,8-tetramethyl-2,4,6,8_ Tetravinylcyclotetrasiloxane） 4）, 2,4,6,8,10-pentamethyl-2,4,6,8,10-pentavinylcyclopentasiloxane, 2,4,6,8,10,12- Nucleated methyl-2,4,6,8,10,12-nucleated vinyl cyclohexansiloxane, octa (vinylsilasesquioxane)

2,2,4,4,6,6,8,8,10,10,12,12 -dodecamethylcyclonuclesiloxane, 2,4,6,8-tetra 0: 2-4) alkenyl-2 , 4, 6, 8-tetra （（： 1_6） alkylcyclotetrasiloxane （for example ，

2.4.6.8-tetra ^{"vinyl-2,4,6,8-tetramethyl} cyclotetrasiloxane), 1,3,5-tree band-6) alkyl-1, 3, 5-tri (02-4) alkenyl Cyclotrisiloxane (for example, 1,3,5-triisopropyl-1,3,5-trivinylcyclotrisiloxane), 1,3,5,7 -tetra （-

6) Alkyl- 1,3,5,7-tetrazol2-4) alkenylcyclotetrasiloxane (for example, 1,3,5,7-tetraisopropyl-1,3,5,7-tetravinylcyclo Tetrasiloxane], 1, 3,5,7, 9-penta ((： 1_6) alkyl-1,3,5,7,9-penta 0: 2-4) alkenylcyclopentasiloxane (for example, 1, 3,5,7,9-pentaisopropyl-1,3,5,7,9-pentavinylcyclopentasiloxane), 1,3,5-tri-specific 1-6) alkyl-1,3,5-tri 0: 2-4) Alkenylcyclotrisiloxane (for example, 1,3,5-tri-de-butyl-1,3,5-trivinylcyclotrisiloxane), 1,3,5,7-tetra （ (:One-

6) Alkyl- 1,3,5,7-tetrax-2-4) alkenylcyclotetrasiloxane (for example, 1,3,5,7-tetra-%-butyl-1,3,5,7-tetra Vinylcyclotetrasiloxane), 1, 3, 5,7,9-penta 0: 1-6) alkyl-1,3,5,7,9-penta0-2-4) alkenylcyclopentasiloxane (for example , 2019/151625 1 »（： 1 ^ 1 {2018/013838

1,3,5,7,9-penta- ₃₆ (： -butyl-1,3,5,7,9-pentavinylcyclopenta siloxane), 1,3,5-tri 0: 1-6) alkyl- 1,3,5-tribune2-4) alkenylcyclotrisiloxane (for example, 1,3,5-triethyl-1,3,5-trivinylcyclotrisiloxane), 1,3,5,7 Tetra （(1-6) alkyl-

1,3,5,7-tetrasub-2-4) alkenylcyclotetrasiloxane (for example, 1,3,5,7-tetraethyl-1,3,5,7-tetravinylcyclotetrasiloxane), 1 , 3, 5, 7, 9 -pentati-

6) Alkyl- 1,3, 5,7,9-penta 0: 2-4) Alkenylcyclopentasiloxane (for example, 1,3, 5, 7, 9-pentaethyl-1,3,5, 7 , 9-pentavinylcyclopentasiloxane), nuclear yarn (02-

4) alkenylcyclotrisiloxane (eg nucleated vinylcyclotrisiloxane), octa 0: 2-4) alkenylcyclotetrasiloxane (eg octavinylcyclotetrasiloxane), decax 2-4) alkenyl Cyclopentasiloxane (eg, decavinylcyclopentasiloxane) and combinations thereof.

The term `` cell culture substrate comprising a cyclosiloxane compound '' refers to the case where the polymer formed by the cyclosiloxane is part of a cell culture substrate (eg, a cell culture substrate coated with the polymer). In addition, it can mean that the solid polymer formed by cyclosiloxane can be used as a cell culture substrate, but is not limited thereto. The form of the cell culture substrate is not limited because it is sufficient to provide any space for culturing the cells. For example, the cell culture substrate may be a dish (culture plate), a chalena plate (for example, a microtiter plate such as 6 wells, 24 wells, 48 wells, 96 wells, 384 wells, 9600 wells, micro plates, deep well plates, etc.). ), Flasks, chamber slides, tubes, cell factories, roller bottles, spinner flasks, hollow fibers (1 ₁₀ 11 (L, microcarriers, beads, etc., but are not limited thereto, and if the material having a support, the cell culture The substrate can be used without limitation. For example, plastic (eg, polystyrene, polyethylene, polypropylene, etc.), metal, silicon, and glass can be used as the cell culture substrate.

In addition, the polymer formed by the cyclosiloxane compound is (1) the same kind formed by the polymerization of the same cyclosiloxane compound.

_, (2) formed by polymerization of siloxane compounds in heterogeneous cycles

And (3) used in the sense encompassing both copolymers formed by polymerization of the same or different cyclosiloxane compounds with other monomer compounds. In the present specification, the copolymer is a random copolymer, block copolymer, alternating copolymer 2019/151625 1 »（： 1 ^ 1 {2018/013838

Or graft copolymer, but is not limited thereto. Therefore, according to one embodiment of the present invention, the polymer formed by the cyclosiloxane compound is a homopolymer formed by polymerization of the same cyclosiloxane compound.

 According to another embodiment of the present invention, the polymer formed by the cyclosiloxane compound is a copolymer formed with the first monomer which is the cyclosiloxane compound and the second monomer capable of polymerization with the cyclosiloxane compound.

 According to one specific example, the second monomer is a cyclosiloxane compound different from the first monomer (copolymer formed by the heterocyclosiloxane compound).

 According to another specific example, the second monomer is a compound having a carbon double bond for polymerization with the first monomer. In this case, the first monomer may also have a carbon double bond for polymerization with the second monomer. Such a second monomer compound is, for example, a siloxane having a vinyl group (e.g., nuxavinyldisiloxane, tetramethyldisiloxane, etc.), a methacrylate monomer, an acrylate monomer, an aromatic vinyl monomer (e.g., Vinylbenzene, vinylbenzoate, styrene, etc., acrylamide-based monomers (e.g. isopropylacrylamide, non-dimethylacrylamide, etc.), maleic hydride, silazane or cyclosilazane having a vinyl group (e.g., 2 , 4,6-trimethyl-2,4,6-trivinylcyclosilazane and the like; 03-10 cycloalkane having a vinyl group (e.g., 1,2,4-trivinyl cyclonucleine, etc.), vinylpi Ralidone, 2- (methacryloyloxy) ethyl acetoacetate, 1- (3-aminopropyl) imidazole, vinylimidazole, vinylpyridine, silane having a vinyl group (e.g. allyl Trichlorosilane, acryloxymethyltrimethoxysilane, etc.) and combinations thereof.

 According to another specific example, the second monomer is 1,3,5-trivinyl_ 1,3,5-trimethylcyclotrisiloxane, 2,4, 6,8-tetramethyl-2,4, 6, 8-tetravinylcyclotetrasiloxane (4¾), 2,4,6, 8, 10 -pentamethyl-2,4,6,8, 10 -pentavinylcyclopentasiloxane, 2,4,6,8,10 ， 12-nucleated methyl-2,4,6,8,10,12 nucleated vinyl-cyclonucleic siloxane, octa (vinylsilasesquioxane), and

With 2,2,4,4,6,6,8,8,10,10,12,12-dodecamethylcyclonucleosiloxane

It may be one or more selected from the group consisting of.

Examples of the methacrylate monomers include methacrylate, 2019/151625 1 »（： 1 ^ 1 {2018/013838

Methacrylic acid, glycidyl methacrylate, perfluoro methacrylate, benzyl methacrylate, 2- (dimethylamino) ethyl methacrylate, perfuryl methacrylate, 3,3,4,4,5, 5,6,6,7,7,8,8,9,9, 10, 10, 10-heptadecafluorodecyl methacrylate, nuclear chamber methacrylate, methacrylic ¾: hydride, pentafluorophenylmeta Methacrylate, propazyl methacrylate, tetrahydroperferyl methacrylate, butyl methacrylate, methacryloyl chloride and di (ethylene glycol) methyl ester methacrylate.

 Examples of the acrylate monomers include acrylate, 2- (dimethylamino) ethyl acrylate, ethyleneglycoldiacrylate,

1 ~ large, dog_dodecafluoro heptyl acrylate, inside, inside, dog-dodecafluoroheptyl acrylate, isobornyl acrylate, inside, 111,2} {, L-perfluorodecyl acryl Tetrahydroperfuryl acrylate, poly (ethylene glycol) diacrylate, mono-, dodecafluoroheptyl acrylate and propazyl acrylate.

 The copolymer of the present invention may further include other monomers as comonomers in addition to the monomers mentioned herein.

 According to one embodiment of the invention, the copolymer contains at least 50% or more cyclosiloxane compound. According to one specific example, the copolymer contains at least 60%, at least 70%, at least 80% or at least 90% of the cyclosiloxane compound. This content is based on a flow rate of 1 unit: e), and 90% is the cyclosiloxane contained in the copolymer formed by flowing (flowing) each monomer at a flow rate ratio of 9: 1 (cyclosiloxane compound: other monomers). Cyclosiloxane compound contained in the copolymer formed by flowing the content of the compound, 80% at a flow rate of 8: 1, 70% at a flow rate of 7: 1, 60% at a flow rate of 6: 1 Means the content of.

In addition, the cell culture substrate comprising the polymer may be a cell culture substrate containing a polymer of various thickness. The thickness of the polymer is, for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300 or more, or about 10,000, 5,000, 1,000, 900, 800, 700, 600, 500, 400, 300 or less, or about 10 to 300, 10 to 500, 10 to 1000, 50 To It may be 300 nm, 50 to 500 nm, 50-1000 nm, but is not limited thereto.

 In the method for producing cancer stem cells from cancer cells comprising the step of culturing cancer cells using a composition for inducing cancer stem cells from cancer cells comprising a cell culture medium containing albumin, wherein the cancer stem cells are in the spheroid form Can be. The method may be characterized in that it does not include other genetic manipulations or other compounds known for stem cell proliferation or known to reverse differentiate stem cells from adult cells. The cell culture medium may not include other growth factors except albumin.

 The term "spheroid" refers to a cell aggregate in which three thousand or more single cells form a three-dimensional sphere, more accurately mimicking the structural and physical properties of three-dimensional tissues surrounding cells in the human body. The spheroid is characterized by cancer stem cell spheroids for the purposes of the present invention. Also, the term "cancer stem cell (Cancer stem cel l or Tumor ini t iat) of the present invention. ing cel l) "means cells that have the ability to generate tumors, and the cancer stem cells have characteristics similar to those of normal stem cells. Cancer stem cells are characterized by self-renewal and differentiation, which is characteristic of stem cells in various cell types. Tumors have the ability to develop tumors through their ability to develop tumors, a new species that is distinguished from other populations in tumors by this cancer formation ability. It also causes recurrence and metastasis by generating sheep, and is another property of cancer stem cells, which is drug resistant and resistant to chemotherapy such as the use of anticancer drugs. Only ordinary cancer cells are removed, and cancer stem cells die. Cancer can return again, so research on cancer stem cells is important to cure cancer.

 In addition, cancer stem cell markers may be used to identify the cancer stem cells. The cancer stem cell markers are CD47, BMI-1, CD24, CXCR4, DLD4, GLI-1, GLI-2, PTEN, CD166, ABCG2, 抑 171, 抑 34, CD96, TIM-3, CD38, STR0-1, and CD19. Specifically, CD44, CD 133, ALDH1A1, ALDH1A2, EpCAM,

This can be, but is not limited to, CD90 and LGR5.

Cancer stem cell spheroid production method and kit for producing the present invention, in the production of cancer stem cell spheroid, artificial genetic manipulation As it is not necessary, there is an advantage that cancer stem cells can be produced more easily and quickly.

 In addition, cancer stem cell (CSC) marker genes prepared by the above methods and kits are expressed (Example 6), have drug resistance characteristics by drug release, and have cancer-forming ability in vivo (Example 12). As described above, cancer stem cell spheroids prepared by the methods and kits of the present invention can be used to study cancer stem cells and to screen for therapeutic agents having cancer stem cell characteristics.

 Cancer stem cell spheroid of the present invention may be cultured in a three-dimensional three-dimensional culture, drug resistance, or may be a patient-specific cancer stem cell spheroid isolating cancer cells, but is not limited thereto .

The term "albumin" as used herein constitutes the basic material of a cell together with globulin, and is included in the culture medium of cancer cells plated in the cell culture substrate of the present invention, and the cancer cells are referred to as cancer stem cell spheroids. Materials that can be formed are included without limitation. Albumin of the present invention may be selected from the group consisting of serum albumin (serum albumin), egg white albumin (ovalbumin), lactalbumin (lactalbumin) and combinations thereof, but is not limited thereto. Examples include, but are not limited to, commercial serum serum (SR). Most cells require serum to proliferate, and art ifi cial serum, or serum replacement, can perform the same or similar function as natural serum. The artificial serum or serum replacement agent, which can be used to replace natural serum in cell culture, usually includes albumin. The albumin of the present invention may be added as an albumin alone component, or may be provided as an agent included in a serum substitute, an agent prepared by further adding albumin to a serum substitute, or an agent prepared by further adding albumin to FBS. More preferably, it may be provided as an agent in which albumin is additionally added to the serum replacement agent, but is not limited thereto. Furthermore, the serum albumin is not bovine serum albumin, human serum albumin, and ^"may be selected from but limited to the group consisting of a combination thereof, depending on its origin. In the present invention, it was confirmed that the spheroid prepared using bovine serum albumin expresses cancer stem cell-related markers (Example 2019/151625 1 »（： 1/10 公 018/013838

6) It can be seen that albumin may induce cancer stem cells.

The albumin concentration may be included in the medium at a concentration of 0.101 _§ / 11] 1 to 5001 /. Specifically, the albumin concentration is about 0.1, 0.2, 0.5, 0.6, 1, 1.1, 2, 3, 4, 5, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86, 91, 96, 100, 101, 106, 111, 116, 121, 126, 131,

136, 141, 14 & 1 塔 / 1 or more, or about 500, 450, 400, 350, 300, 250, 200, 199, 195, 190, 175, 170, 150, 149, 144, 139, 134, 129, 124 119, 114, 109, 104, 99, 94, 89, 84, 79, 74, 69, 64, 59, 54, 49, 44, 39, 34, 29, 24, 19, 14, 9, 4, 1.4, 0.9, 0.41 / or less, more specifically

From about 201¾ / to about 2001 /, about 400 塔 / 1111 to about 20 (concentration of about 1/41/1/1111, about 0. ^ / 1111 to about 15011 ^ / 1111,

To a concentration of about 0.5 of about US客three _11¾ / ₁₁₁ from 1 to about 60 _[11客/ ₁₁₁ 1 concentration, about _1/111 1 to a concentration of about beauty years, about ¾ / ₁₁₁ from 1 to about 60 ₁₁退/ ₁₁₁ in 1 concentration, about 1 (¾ _¾ / ₁₁₁ 1 To a concentration of about 60 mg / ml, about 20 mg / ml to about 60 mg / ml, about 40 mg / ml to about 60 mg / ml, about 0.1 mg / ml to about 50 mg / ml, about 0.5 mg / ml concentration of ml to about 50 mg / ml, concentration of about lmg / ml to about 50 mg / ml, about

Concentration of 5 mg / ml to about 50 mg / ml, concentration of about 10 mg / ml to about 50 mg / ml, concentration of about 5 20 mg / ml to about beauty treatment, concentration of about 40 mg / ml to about beauty treatment, about 0.1 mg / ml to about 40mg / ml, about 0.5mg / ml to about 40mg / ml, about lmg / ml to about 40mg / ml, about 5mg / ml to about 40mg / ml, about

Albumin included in the serum 10 replacement (Senim replacement) may be included in the medium at a concentration of 10 mg / ml to about 40 mg / ml, a concentration of about 20 mg / ml to about 40 mg / ml, or a concentration of about 40 mg / tnl It may be included in the medium at a concentration of, but is not limited thereto. More preferably, the albumin concentration may be included in the medium at a concentration of 0.1 mg / ml to 400 mg / ml, or 0.1 mg / ml to 200 mg / ml. More preferably, the albumin concentration may be included in the medium at a concentration of 0.5 mg / ml to 400 mg / ml, 0.5 mg / ml to 200 mg / ml, or 0.5 mg / ml 15 to 100 mg / ml.

 In the present invention, the term `` about '' includes a range of +0.5, ± 0.4, ± 0.3, +0.2, +0.1, etc., and includes all values that are equal or similar to those following the term about, This is not restrictive.

As used herein, the term "culturing" refers to the growth of the cells in a moderately controlled environmental conditions, the culture process of the present invention can be made according to the appropriate medium and culture conditions known in the art _. According to the selected cells, those skilled in the art can easily adjust and use the cells, and specifically, the present invention can be cultured in a medium containing albumin to prepare cancer stem cell spheroids, and for example, serum replacement (SR). 25 may be grown in a medium containing, but not limited to.

Another aspect of the present invention provides a cancer stem cell spheroid prepared by the above production method. The "cancer stem cells ¹ " and "spheroid" are as described above.

Another aspect of the present invention is a kit for producing cancer stem cell spheroid, comprising a cell culture substrate, and a composition for inducing cancer stem cells from cancer cells comprising a cell culture medium comprising albumin 30, the cell culture substrate Silver cyclosiloxane polymer, the medium comprises albumin, provides a kit for producing cancer stem cell spheroid. 2019/151625 1 »（： 1 ^ 1 {2018/013838

The "cell culture substrate containing a cyclosiloxane polymer", "albumin" "cancer stem cells" and "spheroids" are as described above.

 The kit of the present invention can produce cancer stem cell spheroid. The kit may include a cell culture substrate and a medium as a basic configuration, and specifically, the cell culture substrate may be a substrate including a polymer formed by a cyclosiloxane compound, but may produce or culture cancer stem cell spheroids. The substrate is not limited thereto. In addition, the medium may specifically be a medium containing albumin or a medium containing a serum substitute, but is not limited to any medium capable of producing or culturing cancer stem cell spheroid. The kit may further include instructions for producing cancer stem cell spheroids.

Another embodiment of the present invention ( ₃₎ preparing a cancer stem cell spheroid by the method; (A step of treating the cancer cell injury treatment candidate material to the cancer stem cell spheroid of step ( ₃₎ ; and ( ₀₎ Cancer stem cell spheroid group and cancer cell resistance treatment candidates treated with the cancer cell resistance treatment candidate of step ( ₀₎ The present invention provides a method for screening a drug for treating cancer cell resistance, comprising comparing the untreated control group, wherein the "cancer stem cells" and "spheroids" are as described above.

 Comparing the cancer stem cell spheroid group treated with the cancer cell resistance treatment candidate of step (：) and the control group not treated with the cancer cell resistance treatment candidate may include measuring and comparing expression levels of cancer stem cell markers. And, the step of measuring the expression level of the cancer stem cell markers, conventional expression level measurement methods used in the art can be used without limitation, for example, Western blot (6 Ra11 ratio), 此 比 radioimmunoassay , Radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, or protein chip method.

The term "candidate substance" of the present invention is a substance that is expected to be able to treat cancer or a substance that is expected to improve the prognosis, and specifically, to remove cancer stem cells to suppress cancer cell resistance to treat cancer or to improve the prognosis. It may be a substance that can be improved and is not limited so long as it is expected to improve or improve cancer or cancer stem cells directly or indirectly. 2019/151625 1 »（： 1 ^ 1 {2018/013838

It includes all curable probable materials such as genes or proteins. The screening method of the present invention confirms the expression level of cancer stem cell markers before and after administration of the candidate substance, and when the expression level is reduced compared to before administration of the candidate substance, the candidate substance is expected to be a therapeutic agent for cancer stem cell or cancer cell resistance. Can be determined as

 In addition, the above step may further include, but is not limited to, treatment with a drug that is resistant.

 【Effects of the Invention】

 Cancer stem cell spheroid production method and kit for the production of the present invention can easily produce cancer stem cell spheroid, cancer stem cell spheroid prepared from the method and kit can be effectively used for screening drugs for the treatment of cancer cell resistance.

 【Brief Description of Drawings】

FIGS. 3 to 11 show structures of compounds used in various fabrication, FIGS. ¾ to 11 show structures of various cyclosiloxane compounds, and FIG. 1 are diagrams showing a process for forming a cancer-forming spheroid on a specific surface. , _{FIG. 11} is a diagram confirming the conventional 10 ^ and whether or not the arm-forming spheroid formation on a variety of functional, Figure ₁₀ also naeja

A diagram showing the formation of spheroids on a substrate containing various cyclosiloxane compounds.

FIG. ₂₃ is a variety of human cancer _cells! 4½ ^ confirms the formation of spheroids on the surface, and ¾ various

This is a diagram showing what type of spheroid is formed on the surface.

3 is a diagram showing a VIII-1 spectrum of ¥ 404 monomer and 404,

Fig. 3 shows the results of near _80311. FIG. 3 shows water contact angles of uncoated _kgunla , ₁ 404 coated ^ k, uncoated cell culture substrate, and ₁ 404 coated cell culture substrate. ， 3 （1 is not coated

Coated

A diagram showing an image.

4 shows 10, 50, 100, 200, and 300 minus

The figure confirms the formation of spheroids on 4? Coated 10? Phase having a thickness.

Figure ₅₃ is a diagram showing the expression level of 0) 133 and 0044 of the cell culture in a medium containing various kinds of I and myoeul, is also加 This is a drawing confirming the albumin content of 통 through Western blot.

 Figure 6a is an image showing the spheroid formation according to the concentration of BSA contained in serum-free medium (SFM), Figure 6b is a diagram showing the expression level of CD133 according to the concentration of BSA.

 FIG. 7A is a diagram showing the 抑 133 expression level of cells cultured in serum free medium (SFM) containing FBS, SR or BSP at 40 mg / ml in TCP or pV4D4].

 Figure 7b is a diagram showing the spheroid formation of three types of cancer cells cultured in serum-free medium (SFM) containing BSA in pV4D4.

 7C is a graph showing the expression level of CD133, a cancer stem cell marker gene of a spheroid prepared on a substrate including various cyclosiloxane compounds. In FIG. 7C, lg represents pV4D4 and lg cyclosiloxane compounds copolymerized in the x-axis. 133 expression of the cancer stem cell spheroid prepared on the prepared substrate, and CD133 expression of the cancer stem cell spheroid prepared on the substrate copolymerized with pV4D4 and cyclosiloxane compound of Fig., PV4D4 and CD133 expression of the cancer stem cell spheroid prepared on the substrate copolymerized with the cyclosiloxane compound shows the CD133 expression of the cancer stem cell spheroid prepared on the substrate copolymerized with the cyclosiloxane compound of pV4D4 and lj. , Lk is a female line prepared on a substrate copolymerized with pV4D4 and the cyclosiloxane compound of FIG. Lk Cells will showing the 抑 133 expression level of the spheroid, and 11 shows the expression level of a CD133 CSCs spheroid prepared from pV4D4 and cycloheptane the siloxane compound is a copolymer substrate of Fig.

 The figure shows that CD133 expression level was measured after culturing 況 0V3 in a substrate containing a cyclosiloxane polymer according to various albumin concentrations.

 FIG. 7E shows the concentration of albumin in SFM medium at 0, 0.01 mg / ml, 0.1mg / ml, lmg / ml, 10mg / ml, 100mg / ml, 200mg / ml, and 400mg / in substrates containing cyclosiloxane compounds. It is a graph showing the expression level of CD133 of the spheroid formed by culturing cancer cells in a medium supplemented with BSA to ml, according to the concentration of albumin.

 Figure 8a is a diagram showing the form of SK0V3 spheroid prepared using the hanging-drop, U-bottom, ULA and pV4D4.

FIG. 8B shows SK0V3 spheroids prepared on the surface of AV or pV4D4. A diagram showing a laminin expression pattern, in which red represents laminin and blue represents nuclei.

 Figure 8c is a diagram showing the ALDH1A1 mRNA expression level of SK0V3 spheroid prepared using hanging-drop, U-bottom, ULA and pV4D4.

 FIG. 8D shows the 0V3-ssiCSCs (4 and 8 days) prepared on the pV4D4 surface.

Figure showing 0ct3 / 4, Sox2 and Nanog mRNA expression levels.

 9 is a diagram showing the results of wound healing assay (a) and penetration assay (b) of 況 0V3-ssiCSCs prepared on the pV4D4 surface.

 10 is a view confirming the formation of steroids by SK0V3-ssiCSCs and U87MG-ssiCSCs.

 FIG. 11 shows CSC-related marker mRNA expression levels (a and b) and flow cytometry results (c) in SK0V3-, MCF-7-, Hep3Band SW480_ssiCSC spheroids cultured for 4 and 8 days on the pV4D4 surface of FIG. .

 Figures 12a and b show lateral phage group assay results (a) and doxorubicin for SK0V3-ssiCSC, MCF-7-ssiCSC, Hep3B_ss iCSC and SW480-ssiCSC spheroids cultured for 4 and 8 days on the pV4D4 surface; Cell viability (is a figure showing, c is a diagram showing the cell viability of doxorubicin in cells passaged once or twice SWSW-ssiCSCs, d is a drug release ABC of 況 0V3-ssiCSCs prepared by incubation for 8 days Figure showing the mRNA expression level of the transporter gene.

 Figure 13a is a diagram showing the tumor formation process by administering SK0V3-ssiCSC spheroid-derived cells to BABL / c nude mice, b is a diagram showing the liver metastasized tumor, c is a liver metastasized tumor H 湖 staining observed, d is a diagram showing the lesions metastasized liver of BABL / c nude mice injected with SK0V3- ssiCSC spheroid-derived cells, e is a diagram observed by staining the TNC between the tumor metastasis to be.

14 a shows the heat map of the Wnt target gene (n = 46) of the SK0V3-ssiCSC spheroid, b is the expression of DKK1 (day 1, day 4 and 8) and SK0V3-ssiCSCs in SK0V3-ssiCSCs Express levels of expression of AXIN2 and MMP-2 mRNA (day 4 and day 8), c is phosphorylated yo- of SK0V3-ssiCSCs (day 4 and day 8)

FIG. 15 shows TNC expression (a) and DKK1 mRNA expression levels in MCF-7-ssiCSC, Hep3B_ssiCSC, and SW480-ssiCSC spheroids.

 FIG. 16A is a view showing microscopic observation of spheroids formed by culturing cancer cells in a medium containing a cyclosiloxane compound, in which BSA is added to FBS medium.

 Figure 16b is a graph showing the DKK-1 gene expression levels of spheroids formed by culturing cancer cells in a medium containing a cyclosiloxane compound, in which BSA was added to FBS medium, on a Beta-act in (housekeeping gene) basis. to be.

 Figure 16c is a graph showing the DKK-1 gene expression level of the spheroid formed by culturing cancer cells in a medium containing a cyclosiloxane compound, BSA added to the FBS medium, on the basis of GAPDH (housekeeping gene).

 [Specific contents for carrying out the invention]

 Hereinafter, the present invention will be described in more detail through reference examples, comparative examples, and examples. However, these reference examples, comparative examples and examples are intended to illustrate the invention by way of example, and the scope of the present invention is not limited to these reference examples, comparative examples and examples. Reference Example 1: Xenograft Tumor Formation Analysis

Female BALB / c nude mice (6 weeks old) were obtained from Orient Bio Inc. and stored aseptically in animal laboratory at KAIST _. The mice were randomly assigned to any experimental group. All operations were performed under i sof lurme anesthesia and all animal related procedures were reviewed and approved by the Korea Institute of Science and Technology's Animal Care and Use Committee (KAIST-IACUC) for ethical procedures and scientific management. : KA2014-21).

In addition, in order to construct a human ovarian cancer xenograft model, 50% Mart was isolated from the 0D3-ssiCSCs isolated from 2D-cultured control SK0V3 cells or their corresponding spheroids at different concentrations (10 ⁶ to 10 ² cells). Mixed with Rigel (Matr igel, Corning) and then subcutaneously injected into 6 week old female BALB / c nude mice. Tumor formation was monitored for up to 120 days, and tumor formation was recorded when the tumor volume reached about 50 mm ³ . To construct a human breast cancer xenograft model, 6-week old female BALB / c nude mice were injected subcutaneously with ssiCSCs derived from 2D control cells or MCF7-LUC cancer cells at different concentrations (10 ⁷ to 10 ² cells). 50 ii l of three-three oils (Sigma) in which | 3 -estradiol 17- valerate (2.5 ug; Sigma) was dissolved was subcutaneously administered to BALB / c nude mice every 10 days through the throat. To construct a human glioma xenograft model, 50% Matrigel was applied to 2D control U87MG cells, ULA-cultured U87MG spheroid or pV4D4-cultured U87MG-ssiCSC cells at different concentrations (10 ⁶ to 10 ² cells). (Mat ri gel) was mixed and injected subcutaneously into 6 week old female BALB / c nude mice. Tumor formation from MCF7-Luc and U87MG cells was monitored for up to 90 days and recorded tumor formation when tumor volume reached about 50 mm ³ . Reference Example 2: Cell Viability Analysis

SsiCSC spheroids (況 0V3, MCF-7, Hep3B and SW480) prepared from various types of cancer cells were isolated using trypsin (TrypLE Express, Gibco) and the isolated cells were washed twice with D-PBS. The ssiCSCs were plated in 96-well plates (lxlO ⁴ cells / well) and incubated for 24 hours at 37 ^° C. in cell growth medium containing 10% FBS. Thereafter, the medium was removed and fresh medium containing various concentrations of doxorubicin was added to each well and incubated for 24 hours. Thereafter, each well was washed with D-PBS and replaced with a new cell growth medium of 100 yl, followed by incubation for 4 hours with the addition of 10 ul of JST-1 cell proliferation reagent (Roche). Subsequently, absorbance at 450 nm (reference wavelength, 600 nm) was measured using a microplate reader (Molecular Devices). Reference Example 3 Histological Analysis and Immunohistochemistry

 Liver biopsy specimens from BALB / C nude mice inoculated with 2D control or SK0V3-ssiCSC cancer cells were fixed with 10% formalin, embedded with dehydration and paraffin, cut into 5 um thick specimens and placed on slides. . The samples were waxed and stained with hematoxyl in% eosin (H 湖) for histological evaluation under a standard optical microscope (Eel ipse 80i, Nikon).

Liver metastasis after paraffin embedding and sectioning tissue (S ym) It was confirmed by immunohistochemical method. The sectioned liver tissue was sterilized with 10 mM citrate sodium buffer (pH 6.0) for antigen recovery and blocked with PBS containing 5% bovine serum albumin (BSA) and 1% chlorine serum at room temperature (RT). Incubated with rabbit anti-human TNC primary antibody for 1 hour (20yg / ml; cat. No. AB19011; Millipore). After incubation, the slides were washed with D-PBS, incubated with biotinylated anti-rabbit secondary antibody (1: 200; Vector Laboratories) for 30 minutes at room temperature, and then HRP (horseradish peroxidase) for 30 minutes at room temperature. , 1: 500, Vector). The immunoreactive protein was visualized using substrate 3, 3-diaminobenzidine (Vector Laboratories) and counterstained using hematoxylin. Reference Example 4: Western Blot Analysis

2D control SK0V3 cells and SK0V3-ssiCSC spheroids were lysed with RIPA lysis buffer containing Proteinase Inhibition Cocktail (ThermoFisher Scientific) for 30 minutes on ice. Quantify the protein of the lysate using a Bradford Protein Assay Kit (Bio-Rad), and use an equivalent amount of protein (50 ^ to Bolt 4-12% Bis-Tris Plus polyacrylamide gel (ThermoFisher Scientific). The gels were dried blot onto PVDF (polyvinyl idene difluoride) membranes using iBlot2 transfer syst emdhermoF iS Scientific, according to the manufacturer's instructions.

The PVDF membranes were immunoblotted by incubation with Cruz Biotechnology, followed by HRP-bound anti-rabbit IgG secondary antibody (1: 5000, cat. No.31460; Invitrogen) or anti-mouse IgG using standard procedures. (l: 5000, cat. no.31430; Invitrogen) with appropriate incubation with secondary antibody. Proteins were visualized using SuperSignal West Pico Chemi luminescent Substrate (ThermoFisher Scientific) and Chemi Doc MP System (Bio-Rad). Reference Example 5: Flow Cytometry

Flow cytometry was performed as follows. Specifically, in a single layer Cultured 2D control cancer cells and the corresponding ssiCSC spheroids (cultured for 8 days) were trypsinized and then the cells were isolated with buffer [D-PBS with 1% FBSC fetal bovine serum], respectively. SK0V3, MCF-7, Hep3B, and SW480 cancer cells were subjected to APC (allophycocyanin) -conjugated anti-CD133 primary antibody (1: 100; eBioScience), FITC-conjugated anti-CD44 primary antibody (1: 200; BD Biosciences), PE (phycoerythr in) -conjugated anti-CD90 primary antibody (1: 100, MACS; Miltenyi

Biotec), and FITC-conjugated anti-VII 133 primary antibody (1: 100; Miltenyi Biotec) and analyzed using a flow cytometry system (BD Cali bur and BD LSR Fortessa).

 In addition, for side population assays, 2D control cancer cells and ssiCSCs were isolated using trypsin and Hoechst 33342 (DMEM containing 2% FBS and 10 mM HEPES buffer for 90 minutes at 37 ° C). ThermoFisher Scientific). Cells were then washed with HBSS containing 2% FBS and analyzed using a flow cytometry system (BD LSR Fortessa). Flow cytometry data histograms and plots were analyzed using FlowJo software (Tree Star Inc.). Reference Example 6 Live Cell Imaging

 The ssiCSC spheroids were imaged using the LumaScope 620 system (Etaluma), which enables live cell imaging in a standard incubator (humidified 5% carbon dioxide, 37 ° C). Phase contrast images were observed every 2.5 minutes for 24 hours using a 10x objective lens. Reference Example 7: RNA Extraction and mRNA Sequencing

 MRNA was extracted from the SK0V3 spheroid and 2D control SK0V3 cells incubated in pV4D4 coated plates for 8 days using a magnetic mRNA separation kit (NEB) according to the manufacturer's protocol. DNase treatment, as described in the manufacturer's protocol above. Libraries were prepared using the NEXT flex Rapid Directional mRNA-Seq kit (BIO⑴). Each library was sequenced on a HiSeq2500 system using the single-end method (50 bp reads). The sequenced results were STAR aligner (v. 2.4.0) 61 was used to compare with the human genome (Hgl9 version).

In addition, the H0MER software algorithm and DESeq R to investigate DEG I used a package. Heatmaps and MA plots were visualized using the pheatmap and plotMA functions of the R statistical programming language v.3.3.0 (http: / 八 wr-project.org/), respectively. Reference Example 8: Immunostaining Method for Immune Cytochemistry

 The spheroids were fixed by transferring SK0V3 spheroids from ULA plates and pV4D4 plates into 1.5-ml tubes and incubating in 4% paraformaldehyde solution (Sigma) for 30 minutes at room temperature (RT). Incubate the fixed spheroids in Dulbecco's phosphate-buf fered sal ine solution containing 0.25% (w / v) Tr i ton X-OO (Sigma) for 10 minutes at room temperature. After washing, the cells were incubated with D-PBS containing 3% BSA for blocking.

To stain laminin on the spheroid, the immobilized spheroid was incubated with anti-human laminin primary rabbit antibody (1: 100, cat. No.11575; Abeam) at 4 ^° C. for 12 hours. After washing with D-PBS, the obtained spheroids were incubated with rhodamine red_X_conjugated anti-rabbit secondary antibody (1: 500, cat. No. R6394; Invi trogen) for 1 hour at room temperature, and the Incubate with Hoechst 33342 for two minutes.

In addition, for TNC staining, SK0V3 2D control or SK0V3 spheroid was incubated for 12 hours at 4 ° C with anti-human TNC primary rabbit antibody (20 ug / ml, cat. No.AB19011; Mill ipore). After washing with D-PBS, the cells and spheroids were incubated with FITC-conjugated anti-rabbit secondary antibody (1: 500 _/ cat. No. Sc-2012; Santa Cruz) for 1 hour at room temperature. Thereafter, the cells were incubated with Hoechst 33342 for 10 minutes.

cat. no. ab6786; Abeam) and then incubated with Hoechst 33342 for 10 minutes. All fluorescence images were visualized using confocal laser-scanning microscope (LSM 780, Carl Zei ss). Reference Example 9: Statistical Analysis and Data Sources Data are expressed as mean 土 standard deviation (sd). Statistical analysis was performed using the unpaired Student's t_test from GraphPad Prism software (La Jolla). Pvalue <0.05 was considered statistically significant.

 In addition, GSE106848 RNA sequencing data from the Gene Expression Omnibus data store of was used. Example 1 Preparation of Cell Culture Substrate or Cover Glass Comprising Cyclosiloxane Polymer

 1-1: Preparation of PTF Cell Culture Substrate or Cover Glass Through iCVD Process A polymer thin film (PTF) containing a polymer formed by a cyclosiloxane compound was prepared by the following method.

 First, pV4D4 [poly (2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasi loxane) polymer thin film (PTF) was prepared. Specifically, for vaporization of the monomer, ¥ 404 [2,4,6,8-tetravinyl-2,4,6,8_tetramethyl cyclotetrasiloxane (2,4,6,8-tetravinyl-2,4, 6,8-tetramethyl cyclotetrasi loxane) (99%; Gelest) and tert-butyl peroxide (TBPO, 98%; Aldrich) were heated to 70 and 30, respectively. Vaporized V4D4 and TBP0 were introduced into the iCVD chamber (Daeki Hi-Tech Co. Ltd.) at flow rates of 1.5 and 1 standard cmVmin (seem), respectively. The substrate temperature was maintained at 40, the filament temperature was maintained at 200, and the pressure of the iCVD chamber was

It was set to 180 mTorr. The deposition rate of the pV4D4 film is estimated to be 1.8 nm / min. The thickness of the pV4D4 film was monitored in situ using a He-Ne laser (JDS Uniphase) interferometer system. 1-2: Preparation of Cell Culture Substrate Containing Various Cyclosiloxane Polymers

 To prepare cell culture substrates containing various cyclosiloxane compounds, 1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane, 2,4, 6,8-tetramethyl-2,4, 6,8-tetravinylcyclotetrasiloxane (V4D4), 2,4,6,8,10-pentamethyl-2, 4, 6, 8, 10-pentavinylcyclopentasiloxane, 2,4,6,8, 10, 12-nucleus methyl-2, 4, 6, 8, 10, 12-nucle vinyl-cyclonucleus siloxane,

Octa (vinylsilasesquioxane), and 2, 2, 4, 4, 6,6,8,8,10,10, 12,12-dodecamethylcyclonuxasiloxane, the ratio of PV4D4 and 1: 9, respectively Furnace copolymer The substrate was formed. The various cyclosiloxane compounds and chemical structures are shown in FIGS. 1 g to 1.

 Figures lg to 11 show structures of various cyclosiloxane compounds, lg shows structures of 1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane, and lh shows 2,4. The structure of 6,8-tetramethyl_2,4,6,8-tetravinylcyclotetrasiloxane (V4D4) is shown in Figure 2,4,6,8,10-pentamethyl-2,4,6. , 8,10-pentavinylcyclopentasiloxane shows the structure, or 2,4,6,8,10,12-nucleated methyl-2,4,6,8,10,12-nucleated vinyl-cyclonuclear It shows the structure of the siloxane, Figure lk shows the structure of octa (vinylsilasesquioxane), Figure 11

2, 2, 4, 4, 6, 6, 8, 8, 10, 10, 12, 12-shows the structure of dodecamethylcyclonuclesiloxane.

1-3 ： Analysis method

 Fourier Transform Infrared Spectra of V4D4 Monomers and pV4D4 Polymers (FT-

IR) was obtained using 64 average scans and an optical resolution of 0.085 cm ^_1 in normal absorbance mode using an ALPHA FTIR spectrometer (Bruker Opt i cs, USA). Each spectrum was calibrated at baseline and recorded in the 400-4000 cm ^_1 range.

The chemical composition of the pV4D4 PTF surface was analyzed by X-ray photoelectron spectroscopy (XPS; K-alpha, Thermo VG Scient ific Inc.) at an atmospheric pressure of 2.0 x 10 ⁹ mbar. XPS spectra were recorded in the 100-1100 eV range using a monochromatic A1 K a radiation X-ray source with kinetic energy (KE) of 12 kV and 1486.6 eV.

 The surface topography of the 45 x 45 um region was analyzed by atomic force microscopy (AFM; PSIA XE-100, Park Systems) at a scan rate of 0.5 Hz in contactless mode.

Water contact angles for Si wafers, pV4D4 coated Si wafers, tissue culture substrates, and pV4D4 coated substrates were measured using a contact angle analyzer (Phoenix 150; Surface Electro Opt ics, Inc.) by dropping 10 ul of deionized water onto the corresponding surface. Measured. Example 2 Formation of Cancer Cell-Derived Spheroids Using Various Polymer Thin Films (PTFs) 2-1 ： Preparing various human cancer cell lines

 Human ovarian cancer cell line (況 0V3 ， 0VCAR3), human breast cancer cell line (MCF-7, T47D, BT-474), human hepatocarcinoma cell line (Hep3B, HepG2), human glioma cell line (U87MG, U251), human colon cancer cell Lines (部 480, HT-29, HCT116, Caco-2), human lung cancer cell lines (A549, NCIH358, NCI-H460) and human prostate cancer cell lines (22RV1), human cervical cancer cell lines (HeLa), human melanoma cell lines ( A375), and human gastric cancer cell line (NCI-N87) were purchased from Korea Cell Line Bank (KCLB). e-Myco Mycoplasma PCR Detection Kit (iNtRON Biotechnology) was used to confirm that all cancer cells were free of mycoplasma.

2-2 ： Spheroid Formation Method

Cancer cells (lxlO ⁶ ) were seeded on various polymer thin film substrates, and 10% (v / v) serum replacement (ser / replacement: SR, Gibco), 1% (v / v) in a humidified 5% C02 atmosphere at 37 ° C. v) RPMI-1640 medium containing penicillin / streptomycin (P / S, Gibco), and L-glutamine, DMEM (Dulbecco's)

Incubated appropriately in Modi f ed Eagle Medium (MEM) medium or Minimal Essential Medium (MEM) medium.

 Specifically, SK0V3, T47D, BT-474, SW480, HT29, 22RV1, A549, NCI_H358, NCI-N87, 0VCAR3, NCI-H460, and HCT116 cell lines 10% (v / v) SR, 1% (v / v) cultured in RPMI-1640 medium (Gibco) containing P / S, and 25 mM HEPES (Gibco). MCF-7, Hep3B, HeLa, U251, and A375 cell lines were cultured in DMEM containing 10% (v / v) SR and 1% (v / v) P / S (Gibco). HepG2, U87MG, and Caco-2 cell lines were cultured in MEM containing 10% (v / v) SR and 1% (v / v) P / S (Gibco). In addition, the medium was changed every 2-3 days for optimal spheroid growth.

2-3: Confirmation of Spheroidation Specificity of Cyclosiloxane Polymer Thin Films In order to introduce various surface functionalities into cell culture substrates, conventional tissue culture substrates (ti) were prepared from various monomers using an intiated chemical vapor deposit ion (iCVD) process. Polymer thin film (PTFs) libraries were constructed on ssue culture plates (TCP), and the production capacity of cancer-forming spheroids of each PTF was confirmed (FIG. lm). To this end, SK0V3, a human ovarian cancer cell line, was cultured in various PTFs. Tested The chemical structures constituting the PTFs are shown in Figures la to If. FIG. La shows the structure of ethylene glycol diacrylate (EGDMA) and its polymer (pEGDMA), FIG. Lb shows the structure of 1-vinyl imidazole (VIDZ) and its polymer (pVIDZ), and FIG. shows the structure of sobornyl acrylate) and its polymer (pIBA), Figure Id shows the structure of PFDA (1H, 1H, 2H, 2H- perf luorodecyl acrylate) and its polymer (pPFDA), Figure le is GMA ( The structure of glycidyl methacrylate) and polymers thereof (pGMA) is shown, and if is the structure of V4D4 (2,4,6,8-tetraviny 卜 2,4,6,8-tetramethyl cyclotetrasi loxane) and polymers thereof (pV4D4). Is shown.

 As a result, pV4D4 [po ly (2, 4, 6, 8-tetravinyl -2,4,6,8-tetramethyl) prepared from a cyclosiloxane compound polymer

 cyclotetras i loxane)] Only a large number of multicellular spheroids were formed within 24 hours on PTF. In contrast, V 0V3 grown in other phases showed a similar pattern of attachment and spread as cells grown on TCP (FIG. In). In is a view confirming the formation of cancer-forming spheroid on the conventional TCP and various functional PTF. Example 3 Confirmation of Spheroid Formability of Substrates Containing Various Cyclosiloxane Compounds

 In order to check whether a spheroid is formed on the cell culture substrate including various cyclosiloxane compounds, it was confirmed that spheroid was formed after 24 hours by inoculating SK0V3 cells into the cell culture substrate prepared in Example 1-2.

 Specifically, as a result of confirming that the spheroid is formed in the cell culture substrate including the various cyclosiloxane compounds of FIGS.

1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane (Fig.lg), 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (V4D4 ) (Also lh),

2,4,6,8,10_pentamethyl-2,4,6,8,10-pentavinylcyclopentasiloxane (Fig. Li),

2,4,6,8,10,12-nucleated methyl-2,4,6,8,10,12-nucleated vinyl-cyclonuclear siloxane (FIG. Lj), octa (vinylsilasesquioxane) (FIG. Lk) , And

It was confirmed that spheroids were formed on cell substrates including 2,2,4,4,6,6,8,8,10,10,12,12-dodecamethylcyclonuclesiloxane (FIG. 11) (FIG. Lo). Or it). Figures lo to Figure It shows that the spheroid is formed on a substrate containing a variety of cyclosiloxane compounds, Figure lo is a cell culture containing 1,3,5-trivinyl-1,3,5-trimethylcyclotrisiloxane It shows that the spheroid formed on the substrate, Figure lp shows that the spheroid in the cell culture substrate containing 2, 4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (V4D4) Lq shows that spheroids were formed on a cell culture substrate containing 2, 4,6,8, 10-pentamethyl-2,4,6,8, 10-pentavinylcyclopentasiloxane. ， Fig lr

2.4.6.8.10.12-Nucleated methyl-2, 4, 6, 8, 10, 12-shows the formation of spheroids on the cell culture substrate containing the nucleated vinyl-cyclonuclear siloxane, Figure Is is octa (vinylsilases) Shows the formation of spheroids in the cell culture substrate containing quinoxane], FIG.

2.2.4.4.6.6.8.8.10.10.12.12-shows the formation of spheroids in cell culture substrates containing dodecamethylcyclonucleosiloxane. Example 4 Confirmation of Spheroid Formability Using Various Cancer Cell Lines It was confirmed that the PTF containing the cyclosiloxane compound polymer had spheroid-forming ability in other cancer cell lines other than the human ovarian cancer cell line SK0V3.

 As a result, multicellular spheroids (50-300 y m diameter) were formed within 24 hours in most human cancer cell lines, regardless of origin or origin, and showed high efficiency and reproducibility (FIG. 2 a). The morphology of each spheroid varied from grape cluster 'to dense spheres (Fig. 2b), and these results indicate the diversity of the PTF platform. Comparative Example 1: Conventional Spheroid Formation Method

In order to form a spheroid by the conventional method was carried out as follows. Specifically, a hanging-drop 96-well plate (3D Biomatr ix), a U-bottom 96-well plate (SBio), and an ul tra-low-attachment (此 A) 6-well plate (Corning) were used. Cells were seeded at a density of lxlO ⁴ cells / inoculated at a density of 1 to 50 and, 5xl0 ⁴ cells / inoculated at a density of 2ml, 5x10 ^s cells to此A plate in U-bottom plate / 2ml in hanging drop plate. Medium was changed every 2-3 days for optimal spheroid growth. Example 5: Characterization of the prepared cancer stem cell spheroids

5-1 ： Cancer Cell-derived Spheroid Formation Characteristics of Cyclosiloxane Compound Polymer Substrate

 In the spheroid formation process of Example 2-3, each cancer cell was initially attached to the pV4D4 surface, but immediately spontaneously interacted with each other to form a multicellular spheroid. This spontaneous interaction on pV4D4, which is dependent on simple physical or mechanical contact-based binding, is not observed in other spheroid-forming techniques.

 Unlike conventional hydrophilic ULA (u 11 r a 1 ow-at t achment) surfaces, FT_

Characterized by Fourier transform infrared (IR) spectroscopy and X-ray photoelectron spectroscopy (XPS), the pV4D4 PTF surface (Figures 3a and b, Table 2) has a relatively hydrophobic water contact angle of -90 ° (Figure 3c). It has a smooth surface with roughness similar to that of conventional TCPs (Fig. 3d).

 [Table 2]

 Atoms Measured value [%] Theoretical value [% J

C: 5a08 ~ — — W. ^"

0 21,49 20

In addition, pV4D4 was fabricated by depositing pV4D4 on TCP with thicknesses of 10, 50, 100, 200 and 300 nm using He-Ne laser (JDS Uniphase) interferometer system to produce thickness and spheroid formation capability. As a result of correlation, the thickness change of pV4D4 PTFs in the range of 50-300 nm did not affect the spheroid-forming ability at all (FIG. 4). Taken together, it can be seen that in the case of pV4D4, the specific surface functionality (chemical or biological stimulus) present in pV4D4 rather than a mechanical signal induces spheroid formation.

These results suggest that cell culture substrates containing polymers formed by cyclosiloxane compounds can form cancer cells into 3D spheroids with specific properties. 5-2: Morphological analysis of the prepared cancer stem cell spheroids

 First, the characteristics of cancer cell spheroids prepared by incubating in pV4D4 PTF for 4 to 8 days were compared with the spheroids prepared by other conventional spheroid-forming methods prepared in 1-2.

 As a result, SK0V3 cancer cells form one large aggregated spheroid through the hanging-drop method and the U-bottom method, whereas several small spheroids were formed on the ULA and pV4D4 surfaces. It was more uniform and slightly smaller than the spheroids formed in ULA (FIG. 8 a). Immunohistochemical analysis was performed to compare SK0V3 spheroids cultured on the surface of A or pV4D4 for 8 days. Laminin, a major component of the extracellular matrix (ECM), was observed for spheroids cultured on the pV4D4 surface. Although present in this spheroid, in the case of spheroid cultured on the surface of the 此 A, laminin existed only around the spheroid (FIG. 8 b).

 Based on the above results, spheroids prepared by culturing in pV4D4 of the present invention are not cancer cell aggregates such as spheroids prepared using conventional methods, but the ECM-mediated multicellular structures of tumor tissues are repeated in vivo. Shows. The ECM has been shown to play a pivotal role in the development of drug resistance, self-renewal and cancer formation in the tumor microenvironment. Example 6: Preparation of cancer stem cell spheroid using albumin

 6-1: Production of cancer stem cell spheroid

 To form cancer stem cell spheroids, SK0V3 cells (1x106) were inoculated onto a substrate coated with pV4D4, and a 10% (v / v) serum replacement (SR / SR) in a humidified 5% C02 atmosphere at 37 ° C. Gibco), l% (v / v) penicillin / streptomycin (P / S, Gibco), and L-glutamine were incubated appropriately in RPMI-1640 medium. Medium was changed every 2-3 days for optimal spheroid growth and spheroids were obtained. The albumin concentration of serum replacement was more than lmg / ml and was higher than the concentration of albumin in serum of FBSC fetal bovine serum.

6-2 ： Cancer Stem Cell Spheroid Formation through Identification of Gene-related Gene Expression Confirm

In order to confirm that the spheroid prepared in Example 6-1 has the characteristics of cancer stem cells, expression of CSC-related genes was confirmed using qRT-PCR and RT-PCR. As a control, a spheroid formed by the conventional method of Comparative Example 1 was used. ^·

 Specifically, to perform qRT-PCR, total RNA was isolated from 2D-cultured control cancer cells and ssiCSC spheroids according to the manufacturer's instructions. The isolated whole show was mixed with AccuPower RT PreMix (Bioneer) and reverse transcribed into cDNA using Rot or -Gene Q thermocycler (Qiagen). qRT-PCR experiments were performed with 50ng of RNA using a Rot or -Gene Q thermocycler (Qiagen) and KAPA SYBR FAST Universal #CR kit (Kapa Biosystems) according to the manufacturer's instructions.

 In addition, the HyperScript One-step RT-PCR kit (GeneAll Biotechnology Co. Ltd.) was used to analyze the expression level of the cancer stem cell marker genes CD44, CD133, ALDH1A1, ALDH1A2 and EpCAM using RT-PCR. P-actin was used as internal control.

 The sequence of primers for performing the qRT-PCR and RT-PCR are shown in Table 1 below.

[Table 1]

2019/151625 1 »(： 1/10 公 018/013838

As a result, quantitative real-time polymerase chain reaction was found to significantly increase the expression of ALDHIAKaldehyde dehydrogenase 1 family member Al, known as CSC marker, only in SK0V3 spheroids cultured in pV4D4. QRT-PCR) analysis to confirm (Fig. 8c). In addition, SK0V3 prepared by culturing in pV4D4 As compared to the 2D-cultured SK0V3 control grown on TCP, spheroid was found to significantly increase the expression of 0ct3 / 4, Sox2 and Nanog, typical autologous genes (FIG. 8D). The results indicate that the cancer cells in the spheroid has stem cell characteristics.

6-3 ： Confirmation of cancer stem cell induction function of albumin

 To determine whether the characteristics of the cancer stem cell (CSC) of the spheroids were induced by albumin, the following experiment was performed.

First, when various types of FBS and serum replacement (SR) were used, the following experiment was performed to confirm the expression level of the CSC marker gene. Specifically, the expression levels of CSC markers CD133 and CD44 were confirmed by flow cytometry after 6 days of cultured U87MG plated on pV4D4 PTF in FBS and SR of Welgene, Hyclone, and GIBC⑴ for 6 days. In comparison with the three types of FBS, it was confirmed that the expression level of CD133 and 抑 44 was excellent when 況 was added (Fig. 5 a), and the results of comparing the albumin content of FBS and seedlings using nat ive-gel In addition, it was confirmed that SR contains a greater amount of albumin than FBS (Fig. 5b). Based on the above results, it can be seen that SR promotes CSC induction of spheroids due to higher albumin content than FBS. As a result, 5 × 10 ^s U87MG plated on pV4D4 PTF was prepared in serum free medium (SFM) containing FBS and various concentrations of bovine serum albumin (BSA) (0.1, 5, 10, 20, 40, and 80 mg / ml). After culturing for 8 days at, the formation of spheroids was confirmed, and BSA concentrations were 0.1 and 5 through flow cytometry. The expression level of the CSC marker gene (# 133) of cells cultured in serum free medium (SFM) at 10, 20, 40, and 80 mg / ml was confirmed.

 As a result, it was confirmed that the spheroid is formed in the medium containing the BSA,

It was confirmed that CD133, a CSC marker, was expressed (FIGS. 6A and B). In addition, it was confirmed that the expression level of CD133 increased as the concentration of BSA increased. In addition, when using FBS contained in the general cell growth medium, it was confirmed that spheroids are formed, but CD133, which is a CSC marker, is not expressed. That is, CSC markers are expressed in the medium containing more than a certain concentration of albumin, it can be seen that the characteristics of the cancer stem cells, but when the albumin is included in a low concentration of CSC markers do not have the characteristics of cancer stem cells It was confirmed that cancer stem cells are induced by albumin above a certain concentration. 2019/151625 1 »（： 1 ^ 1 {2018/013838

Confirmed.

Also, bang! ^{With 3}

1187¾¾,, (3, and 1) were cultured in the serum-free medium ().

Markerin

0) Expression level of 133 was confirmed by flow cytometry and displayed graphically (Fig. 7 and Fig.).

Based on the above results, it can be seen that albumin may induce cancer stem cells, and when cultured on ₁ 404, incubation with albumin above a certain concentration in serum-free medium 0 may effectively induce cancer stem cells.

6-4 ： Confirmation of cancer stem cell characteristics of spheroids prepared from substrates containing various cyclosiloxane compounds

 In order to confirm that the spheroids prepared on the substrate containing various cyclosiloxane compounds have cancer stem cell characteristics, the expression level of the cancer stem cell marker gene 0) 133 was measured, and the results are shown in FIG. .

Specifically, the copolymer substrates of 4 4 and the six cyclosiloxane compounds of FIGS. Degree

Trivinyl-1,3,5-trimethylcyclotrisiloxane, the degree being 2,4,6,8-tetramethyl_ 2, 4, 6,8-tetravinylcyclotetrasiloxane 0) 4), or 2 , 4,6,8,10-pentamethyl-2,4,6,8,10-pentavinylcyclopentasiloxane, or 2,4,6,8,10,12-nuxamethyl-2,4,6 , 8, 10, 12-nucleated vinyl-cyclonuclear siloxane, 止 is octa (vinylsilasesquioxane), and FIG. 11 is 2,2, 4, 4,6, 6, 8, 8, 10, 10, 12, 12-dodecamethylcyclonuxasiloxane is shown. Each substrate was treated with $ 3 cells to confirm that spheroids were formed after 24 hours, and after 8 days, flow cytometry confirmed that the number of 0) 133 expressed cells increased.

¾ in the X-axis of FIG.

133 expression of the cancer stem cell spheroid prepared from the substrate copolymerized with the cyclosiloxane compound, and 는 expression of the cancer stem cell spheroid prepared from the substrate copolymerized with the cyclosiloxane compound of 4 4 and FIG. 는 represents the amount of 0 133 expression of the cancer stem cell spheroid prepared on the substrate copolymerized with the cyclosiloxane compound of 4 4 and Fig., And the cancer stem cell prepared on the substrate copolymerized with the cyclosiloxane compound of CD133 expression level of the spheroid, lk is the expression of 발현 133 expression of cancer stem cell spheroid prepared on the substrate copolymerized with the cyclosiloxane compound of pV4D4 and Figure lk, 11 is the cyclosiloxane compound of pV4D4 and Figure 11 CD 133 expression of the cancer stem cell spheroid prepared from this copolymerized substrate is shown.

 Therefore, it was confirmed that cancer stem cell characteristics could be induced even when other cyclosiloxane compounds were used in addition to pV4D4. Example 7 Cancer Stem Cell Spheroid Production at Various Albumin Concentrations 7-1: Confirmation of Spheroid Formation at Various Albumin Concentrations

 The medium was prepared by adding BSA so that the concentration of albumin was 0, 0.01 mg / ml, 0. lmg / ml, lmg / ml, 2mg / ml, 5mg / ml, and lOmg / ml in the SFM medium, and the cyclosiloxane compound Cancer cells were cultured on a substrate and a TCP substrate, and the spheroids were formed.

 As a result, as can be seen, the spheroid morphology appeared on the pV4D4 substrate, which is a cyclosiloxane compound, but no spheroid was formed on the TCP substrate. 、

7-2 ： Confirmation of cancer stem cell marker of spheroid

 The medium was prepared by adding BSA so that the concentration of albumin in the SFM medium was 0, 0.01 mg / ml, 0. lmg / ml, lmg / ml, lOmg / ml, lOOmg / ml, 200mg / ml, 400mg / ml. Cancer cells were cultured on a substrate containing a cyclosiloxane compound to determine whether cancer stem cell spheroids were formed.

 As a result, as can be seen in Figure 7e, it was confirmed that the expression level of CD133 changes depending on the albumin concentration.

Taken together, the surface of PV4D4, an example of a polymer formed by cyclosiloxane compounds, provides specific stimuli to activate and deform SK0V3 cancer cells, inducing spheroid formation of cancer cells, and albumin to induce its cancer stem cell properties. Thus, it can be seen that it produces a spheroid containing a considerable amount of CSC-like cells. Therefore, the CSC-like cells were named as surface-stimulated-induced cancer stem cells (ss iCSCs). Example 8: Confirmation of cancer stem cell spheroid formation ability using various cancer cell lines

 In order to confirm the possibility of generalization of the spheroid production method using the pV4D4, ssiCSC spheroids derived from various cancer cell lines were prepared, and CSC-related characteristics were confirmed. Four human cancer cell lines from various tissues were selected for this purpose: SK0V3, MCF-7C human breast cancer), Hep3B (human cancer) and 況 ^ 480 (human colorectal cancer). In addition, the estimated CSC characteristics for each cell line were confirmed using specific surface markers for each cell line: SK0V331-ALDH1A1; MCF-7-CD44 (cluster of di f ferent iat ion 44); Hep3B36-CD90; And SW48037-LGR5 (leucine-r i ch repeat -containing G-prote incoup led receptor 5). In addition, CD133 was used as a general putative CSC marker for all cell lines. Expression of the CSC marker gene was confirmed by qRT-PCR of ssiCSC spheroids cultured on the pV4D4 surface for 4 and 8 days, and the corresponding 2D control and CSC cultured with TCP.

The expression level of genes increased with incubation time, indicating that CSC-like properties are enhanced with culture. _Also,. Reverse transcript ion-PCR (TPCR) analysis showed increased expression of various CSC-related genes in all ssiCSC spheroids compared to 2D cultured control cancer cells (FIG. 11 b).

 Next, the fraction of putative CSC-marker-positive cancer cells in spheroids prepared by incubating for 8 days on the surface of pV4D4 was quantified by flow cytometry. As a result, the ssiCSC spheroids of SK0V3, Hep3B and SW480 showed an approximately 10-fold increase in the expression of cell-type-specific CSC-related surface markers (expressed in gene counts) compared to 2D-cultured controls. CD44 of MCF-7 cells increased less than 10-fold (FIG. 11 c).

These results suggest that ssiCSC spheroids prepared using pV4D4 have similar characteristics to CSCs. Example 9 Wound Healing Assay, Penetration Assay and Spheroid-Formation Analysis of Prepared Cancer Stem Cell Spheroids 9-1 ： Analysis method

 SK0V3 cells were incubated for 8 days on pV4D4-coated substrates. After confirming SK0V3- spheroid formation, the ssiCSC spheroids were separated with trimsin (TrypLE Express; Gibco) and the isolated cells were washed twice with D-PBS.

 The wound heal ing assay was first densely cultured SK0V3 cells and SK0V3-ssiCSCs in a single layer in a 6-well plate and then synchronized the cells for 24 hours in medium containing 1% FBS. The `` wound '' was then made by scraping the cell monolayer uniformly with a standard 200 u 1 pipette tip, removing the washed cells twice with D-PBS and then adding serum-free medium. Migration was observed using a phase contrast microscope (LumaScope 620, Etaluma) immediately after the wound was made (Oh), 12 hours (12h) and 24 hours (24h) after the wound was made.

Invasion assays were performed by first culturing SK0V3 cells and SK0V3_ssiCSCs cells in serum-free medium for 24 hours and then culturing them in a transl chamber (Corning). Plate cells (lxlO ⁵ cells / well) into the upper chamber of a transparent PET membrane (8.0 um pore size) coated with Matrigel (200 yg / ml; Corning) and fill the bottom with medium containing 10% FBS Penetration into the chamber was allowed. Cells were incubated for 24 hours and fixed with 4% formaldehyde (Sigma). Unpermeable cells on the upper chamber of the membrane were removed using a cotton swab. Moving cells on the lower surface of the membrane were stained with Hoechst 33342 (ThefmoF i sher Scient ific) and fluorescence microscopy

(Ecl ipse 80i, Nikon) was used to count the nuclei of infiltrating cells. Infiltration was calculated as the average cell count per five fields of the cornea.

 For spheroid formation assays, SK0V3 cells and 況 0V3-ssiCSCs were identified as B27.

DMEM including (Invi trogen), 20ng / ml EGF (epidermal growth factor, Gibco), lOng / ml LIF (leukemia inhibi tory factor, Invi trogen) and 20ng / ml bFGF (basi cf ibroblast growth factor, .Invi trogen) / F12 (1: 1, Gibco) was incubated. The formation of spheroids was observed through images after 1 and 24 hours using a phase contrast microscope (LumaScope 620; Etaluma).

9-2 ： Results

SK0V3 prepared by incubating in pV4D4 for 8 days in a wound healing assay 2019/151625 1 »（： 1 ^ 1 {2018/013838

Cancer cells isolated from spheroids migrate faster than the control cells cultured 2 [] _ to fill gaps (FIG. 9 _& ), and the cancer cells isolated from the spheroids in a transwell-based infiltration assay were detected. It was confirmed that the gel substrate can penetrate more than the cells (~ 4 times) (Fig. 9, it can be seen that the spheroid prepared by culturing at {404 has improved cell mobility and permeability. Example 10: Confirmation of maintaining 050 characteristics of the prepared cancer stem cell spheroids

 Cancer cells isolated as single cells from 1 (3 cancer stem cell spheroids) prepared by culturing at 404 for 8 days were cultured in a conventional shamk

"Spheroid Formability" was evaluated. ₃₃ shown in the drawing Figure 10比洗₃ confirming whether formed steroid by-additive況(greater比就greater and 11871犯. As can be seen in Figure 10, it shows that the spheroid is formed spontaneously, which means that the spheroid

It shows that it retains its characteristics. Example 11: Confirmation of drug resistance of ₃ GPa

One other important feature is the inherent or acquireable drug resistance to chemotherapeutic agents due to the ability to push the drug out. In this _{regard, ¾ ½ (± 31 ;-(切} 6 aspects based on-populations (3 6-1) 01) 111八)] 1) The control over the assay surface _1-404 incubated for 8 days to prepare spheroid The drug-exhaust capacity of each cancer cell isolated from was confirmed. As a result, it was confirmed that the fraction of drug release-positive cells was significantly increased in ₃₃ cells prepared from four cancer cell lines compared to 21) -cultured control group. Specifically, drug release-positive fractions range from 0% to 13.8% for 況 0 3 cells, from 0.59% to 9.6% for! 1-7 cells, from 0.58% to 9.2% for ¾- ₃ seedlings,

In the case of cells increased by 0.1% to 10% ( _{3 in} Figure 12).

In addition, ₃₃ drug resistance to doxorubicin 0) 0, known as an anticancer agent, was confirmed. Specifically, ₃₃ non-spheroids prepared by incubating at ₁ 404 for 8 days were separated into single cells, and the cells were cultured in a conventional monolayer on a surface of 20 ^, followed by various concentrations of 1） 0） （ Was treated for 24 hours. Cell viability was measured using the

1) of 50 ^ 11 had high resistance to « 12b）. In addition, SK0V3- and SW480-ssiCSCs were completely resistant to Dox, and 480-ssiCSCs showed higher cell viability than cancer cells of the untreated control. In the case of 洲 ^ SO-ssiCSC, drug resistance was maintained even when two passages were carried out on the surface of TCP. Through this, the original cancer cells were transformed into CSC-like cells (Fig. 12c).

 The drug-releasing capacity is known to be mediated by the ATP-binding cassette (ABC) protein family. Therefore, qRT-PCR was used to analyze the expression of the major multiple drug resistance (MDR) genes ABCB1, AB12, ABCB5, ABCC1 and ABCG2 panels in SK0V3_ssiCSC. All five MDR-related genes were found to be highly upregulated in ssiCSCs as compared to the 2D-cultured controls. In particular, the degree of upregulation in the ABCBr and ABCB5 gene was prominent (FIG. 12D). The results of significant upregulation of the MDR gene in ssiCSC correlated with the results of the lateral sieve group assay (FIG. 12A) and the D0X resistance test (FIG. 12B).

 Comprehensive molecular or functional analysis of the ss iCSC spheroids of these four types of cells revealed that when exposed to specific stimuli on the surface of pV4D4, cancer cells strongly express CSC-related genes and have strong drug resistance. The conversion was confirmed. Example 12 In Vivo Cancer Formation of ssiCSC Spheroids

In vivo cancer-forming ability of ssiCSC was confirmed. Specifically, SK0V3-derived ssiCSC spheroids were isolated into single cells and cells of different concentrations (10 ² to 10 ⁶ cells) were mixed with Matrigel and injected subcutaneously into BALB / c nude mice (FIG. 13A). ）. Xenograft tumor formation by cells isolated from the spheroids was monitored for 120 days and compared with 2D TCP-cultured SK0V3 controls (Table 3).

□□□□□□ [Table 3] | Tumor formation and metastasis of SKOV3 in BALB / c nude mice.

Ceil Tumor formation Liver metastasis number ⁶ 20 00 111 ^ 1 551080 20 0001 ^ 1 551080

 100 0/5 0/5 0/5 4/5

 1, 000 0/5 1/5 0/5 4/5

 10,000 0/5 4/5 0/5 4/5

 100,000 0/5 3/5 0/5 5/5

 1,000,000 2/4-0/4-

^& Tumor formation and metastasis were monitored up to 120 days _*

^¾ Afl cells were dissociated into single cells and counted with a hemocytomeier before subcutaneous injeciion. As a result, the 2D control group did not form tumors at doses of 10 ⁵ cells or less (0/5 mice), and it was able to form tumors at 50% frequency (2/4 mice) at 10 ⁶ cell doses. (Table 3). In contrast, ssiCSC-derived cells are _. Very small doses were able to form tumors with a higher frequency than controls. Specifically, tumor formation frequency is 10 for ^{the five} cell capacity (3/5 mice) 60% 10 ⁴ 80% of the capacity of cells (4/5 mice) and 10 ³ 20% of the capacity of the cell (1 / 5 mouse) (Table 3). Considering how difficult it is to obtain xenograft tumors of human ovarian cancer cells (SK0V3) in athymic nucleus mice without using a severe combined immunodeficiency (SCID) mouse, in

It was confirmed that SK0V3-ssiCSC has excellent cancer formation ability.

In addition, a significantly abnormal external metastatic sinter node was found in the liver of ssiCSC-inoculated mice, whereas the liver of mice inoculated with 2D SK0V3 control appeared normal (FIG. 13B). Histological analysis showed that the abnormal livers inoculated with ssiCSC clearly distinguished between normal and tumor sites, resulting in numerous premalignant lesions throughout the tissue, while the metastases in livers of mice inoculated with 2D control cancer cells. It was not seen at all (FIG. 13C). In particular, mice inoculated with 10 ² cell doses of cells derived from 況 0V3-ssiCSC showed high frequency of liver metastasis (4/5 mice) (Fig. 13D, Table 3). It can be seen that it has a greatly improved metastatic capacity and cancer formation ability. The major component of cancer-specific ECM is the metabolic environment Immunohistochemical examination of liver metastases for the expression of the essential component _{tenasin- ᅣ 61133} （： 1 ₁₁ − （^: 110）

It was confirmed that it exists significantly (Fig. 13). Through this, tumor sinter nodes of the liver were injected subcutaneously.

Know that it is due to metastasis _. Can be. Next, the cancer formation ability of ₃₃比 엤 ₃ derived from various cancer cell lines was confirmed. As a result, 1 ^? -7 （¾0 ^? ^ 比 ᄄ ₃ derived from 7- 切 （：） cells and 1] 87¾10 human glioblastoma cells significantly increased cancer-forming ability compared to 20 cultured control cells (Tables 4 and 5).

[Table 4]

 100, ：-0/5

 1, 000 2/5

 10,000 2/5

 100,000 0/5 4/5

 1, 000,000 0/5

 10.000,000. 1/5

 aTumor formation was monitored up to 90 days.

[Table 5] □□□□□□

 Tumor formation of U87MG in BALB / c nude mice.

 Cell number 2D control ULA ssiCSC

 100 0 / S 1/5

 1, 000 0/5 2/5

 10,000 1/4 0/5 3/5

 100,000 2/4

 1,000,000 4/4

«Tumor formation was monitored up to 90 days. Specifically, tumors did not form even when 21) -cultured 0 7-cells were inoculated at a dose of 10 ⁶ cells per mouse, but! 7- (： -deck was inoculated at a dose of 10 ⁵ cells per mouse. In one case, tumors were formed at high frequency (4/5 mice) (Table 4). Similarly

10 ⁴ per mouse Tumors were formed at 60% frequency (3/5 mice) when inoculated with dog cells, whereas no tumors were formed when inoculated with 118 ^ 0 spheroids cultured on 11 surfaces, which was at-and ₁ 404_. The difference in cancer formation ability of cultured spheroids is apparent.

 Taken together, it can be seen that 4½-based can be used as a platform for producing cancer-forming spheroids and can be used for the production of various human xenograft tumor models that are difficult to manufacture in athymic nude mice. Example 13: Confirmation of relationship between cancer formation ability of ssiCSC spheroid and Wnt / p-catenin signaling

To identify cellular and molecular mechanisms associated with stem cell-like characteristics of ssiCSCs, several important signal transduction pathways associated with cancer formation and stem cell properties of CSCs such as Notch, Hedgehog, and Wnt 八 catenin (Wnt / P-catenin) To _. Confirmed.

First, an experiment was performed to determine whether the fct 八 P-catenin signaling pathway is activated in SK0V3-ssiCSCs and the expression of the Wnt target gene (n = 46) is increased. As a result, the expression of 30 genes among 46 Wnt / p-catenin target genes increased by 1.5-fold in 0V3-ss iCSC, and the Di ckkopf-related protein KDi ckkopf-related protein 1, a key inhibitor of the Wnt signaling pathway. : DKK1) was confirmed to significantly reduce the expression (Fig. 14a). In addition, qRT-PCR analysis of SK0V3-ssiCSC spheroid cultured for 1, 4 and 8 days confirmed that the expression of DKK1 mRNA was dramatically reduced (FIG. 14B). P-catenin signaling is activated. In addition, the qRT-PCR result showed that the decrease in DKK1 expression was associated with the expression of Axin2 (axi s inhibit ion protein 2) and MMP2 (matr ix metal loproteinase-2), which are downstream target genes of Wnt 八 (3-catenin signaling). It was shown to be directly related to the increase (FIG. 14B). In addition, qRT-PCR did not show a change in the level of 0-catenin mRNA in ssiCSC spheroids.

It was shown that the catenin protein was significantly reduced (FIG. 14C). In addition, as a result of immunostaining, there is almost no P-catenin in the nucleus of 2D-cultured SK0V3 cells, but ssiCSCs show that 13-catenin migrates to the nucleus (FIG. 14D). 2019/151625 1 »（： 1 ^ 1 {2018/013838

Next, we confirmed the upstream signal that caused a significant decrease in 1) 照 1 in the non-specific spheroid. As a result,

Associated with liver metastases

13 1) Downregulation of 狀 1 activates the ¾ / yon-catenin signaling pathway. therefore,

In order to confirm the association between the 3 and ₃₃ ratio spheroids cultured for 8 days,

Immunostaining. As a result,

Abundantly present throughout the spheroid,

Target 1) downregulated 1¾1, thereby activating the new-catenin signaling pathway (FIG. 14 ₆ ).

Also, from 1-7, ¾- ₃ tables and 31480 spheroids

With a marked decrease in gene expression

(Showed significant expression of FIG. 15 (FIG. 1¾), which was found in other cancer cells.

The same in the manufacturing process

\ ftit 八 [3-shows that catenin signaling is involved.

Putting the above results together,

Activation of the mediated new-catenin signaling pathway shows that the 404 surface is able to convert cancer cells into cancer-forming 050-like phenotypes. Example 14 Cancer Stem Cell Spheroid Formation in FBS Medium with Increased Albumin Concentration

To ensure that the albumin concentration in the seedling medium is above a certain level

Cancer cells were cultivated in the added medium to determine whether cancer stem cell spheroids were formed.

Specifically, 1 ^ 3 cells were cultured on the substrate so that albumin concentration was 5 ₁ /, 10 ₁ / mi 1 in the urine medium. As a control

Medium was used.

As a result, as can be seen in Figure 16

It was confirmed that spheroids were not formed well when the albumin concentration was not added to a predetermined level because it was not added.

Also, the cancer cells

Expression level was measured, and the results shown by the _seed - ₃₀₁₁₁₁ criterion (FIG. 1 加 ，) and 仰 (FIG. 160 criterion,

When cultured in the added medium did not show the characteristics of cancer stem cells, it was confirmed that cancer stem cells are induced only when the albumin concentration is increased by a certain amount by the addition of the show.

Claims

[Claim]

 [Claim 1]

 A composition for inducing cancer stem cells from cancer cells, including albumin and cell culture medium.

 [Claim 2]

 The composition of claim 1, wherein the cancer stem cells are in spheroid form.

 [Claim 3]

 The composition of claim 1, wherein the albumin is included in the medium at a concentration of 0.1 to 500 mg / ml.

 [Claim 4]

 According to claim 1, wherein the albumin is provided as a serum replacement (Serum Replacement), or as a preparation prepared by the addition of the albumin to the serum replacement, or albumin is further added to FBS (Fetal bovine serum) The composition provided as a prepared formulation.

 [Claim 5]

 The composition of claim 1, wherein the albumin is selected from the group consisting of serum albumin, ovalbumin, lactalbumin, and combinations thereof.

 [Claim 6]

 The composition of claim 5, wherein the serum albumin is selected from the group consisting of bovine serum albumin, human serum albumin, and combinations thereof.

 [Claim 7]

 According to claim 1, wherein the cancer stem cells are cancer-specific cancer stem cells are isolated from the cancer cells, the composition.

 [Claim 8]

 The composition according to claim U, wherein the cancer stem cells have one or more characteristics selected from the group consisting of cell mobility, permeability, drug resistance, and cancer formation ability.

 [Claim 9]

 The method according to claim 1, wherein the cancer stem cells are CD47, BMI-1, CD24, CXCR4, DLD4,

With GLI-1, GLI-2, PTEN, 抑 166, ABCG2, CD171, CD34, CD96, TIM-3, CD38, STR0-1, CD19, 抑 44, 抑 133, ALDH1A1, ALDH1A2, EpCAM, CD90, and LGR5 At least one marker selected from the group consisting of is expressed, the composition. 2019/151625 1 »（： 1 ^ 1 {2018/013838

[Claim 10]

 According to claim 1, wherein the cancer cells ovarian cancer, breast cancer, liver cancer, brain cancer, colon cancer, prostate cancer, cervical cancer, lung cancer, stomach cancer, skin cancer, pancreatic cancer, oral cancer, rectal cancer, laryngeal cancer, thyroid cancer, parathyroid cancer, colon cancer, bladder cancer , Peritoneal cancer, adrenal cancer, tongue cancer, small intestine cancer, esophageal cancer, renal cancer, kidney cancer, heart cancer, duodenal cancer, ureter cancer, urethral cancer, pharyngeal cancer, vaginal cancer, tonsil cancer, anal cancer, pleural cancer, thymus cancer or nasopharyngeal cancer Composition derived from.

 [Claim 11]

The method of claim 1, wherein the cancer cells, human ovarian cancer cell line (況 0 3, 0 能 3), human breast cancer cell line (1 -7, 1470, 81-474), human liver carcinoma cell line (¾ _? 3 民 11 印 02）, human glioma cell line 018 11251）, human colon cancer cell line 1480,, -29, 10116, 0300-2）, human lung cancer cell line 549, large 358, 1 inner 460）, human prostate cancer cell The composition (221 1), the human cervical cancer cell line (¾1), the human melanoma cell line 375), and the human gastric cancer cell line 4 주 7) is at least one selected from the group consisting of.

 [Claim 12]

 The composition of claim 1, wherein the composition does not contain any growth factors other than albumin.

 [Claim 13]

 Using a composition according to any one of claims 1 to 12, comprising the step of culturing cancer cells, a method for producing cancer stem cells from cancer cells.

 [Claim 14]

 The method of claim 13, wherein culturing the cancer cells is performed by culturing the cancer cells on a cell culture substrate comprising a cyclosiloxane polymer.

 [Claim 15]

 The method of claim 14, wherein the cell culture substrate comprising the cyclosiloxane polymer has a water contact angle of less than 90 °.

 [Claim 16]

 The method of claim 14, wherein the cyclosiloxane polymer is a homopolymer or heteropolymer comprising a monomer having Formula 1

[Formula 1] 2019/151625 1 »（： 1/10 公 018/013838

 Cool down]

/ \

And (an integer of ₁₁ = 1-8);

 Are independently of each other hydrogen or 02-10 alkenyl, with at least two of which are 02-1 ◦ alkenyl;

Figure 2 independently of each other is hydrogen, 01-10 alkyl, 02-10 alkenyl, halo, metal element, 05-14 heterocycle, 03-10 cycloalkyl or 03-10 cycloalkenyl.

[Claim 17]

The method of claim 16, wherein the compound of formula 1 is characterized by having ₁₁ +1 or ₁₁ +2 02-10 alkenyl at position VII.

 [Claim 18]

 18. The method of claim 17, wherein the cyclosiloxane compound is 2,4,6,8-tetra-2-2-) alkenyl-2,4,6,8-tetra ((: 1_10) alkylcyclotetrasiloxane, 1,3 , 5_tri （（： 1_10） alkyl—1,3,5-tri 比 2-10) alkenylcyclotrisiloxane, 1,3,5,7-tetra ((: 1_10) alkyl-1,3,5, Alkenylcyclotetrasiloxane,

1,3,5,7,9-penta （（: 1-10） alkyl-1，3,5,7,9-penta

10) alkenylcyclopentasiloxane, 1,3,5-tri （（： 1-10） alkyl-1，3,5-tri0 ： 2-

10) alkenylcyclotrisiloxane, 1,3,5,7-tetra （（： 1-10） alkyl-1，3,5,7-tetra 0 ： 2-10） alkenylcyclotetrasiloxane ， 1,3 , 5, 7, 9-penta 犯 1-10) alkyl_ 1,3,5,7,9-penta ratio 2-10) alkenylcyclopentasiloxane, 1,3,5-tri (（： 1-10 Alkyl-

1,3,5-tribune 2-10) alkenylcyclotrisiloxane, 1,3,5,7-tetra （（： 1-10） alkyl-

1,3,5,7-tetra-2-2) alkenylcyclotetrasiloxane, 1,3,5,7,9-penta （（： 1-

10) Alkyl-1,3,5,7,9-penta ᄄ 2-10 Alkenylcyclopentasiloxane, nucleus 0: 2_ 2019/151625 1 »（： 1 ^ 1 {2018/013838

10) alkenylcyclotrisiloxane, octa: 2-10) alkenylcyclotetrasiloxane, deca 1X2-10) alkenylcyclopentasiloxane, 2,4,6,8-tetravinyl-2,4,6,8, _ Tetramethylcyclotetrasiloxane and combinations thereof.

 [Claim 19]

 The method of claim 16, wherein the cyclosiloxane polymer is a heteropolymer of a first monomer having a formula (1), and a second monomer containing a vinyl group,

 The second monomer is a siloxane having a vinyl group, a methacrylate monomer, an acrylate monomer, an aromatic vinyl monomer, an acrylamide monomer, a maleic anhydride, a silazane or a cyclosilazane having a vinyl group, or a vinyl group. Having 03-10 cycloalkane, vinylpyrrolidone, 2-

And (methacryloyloxy) ethyl acetoacetate, 1- (3-aminopropyl) imidazole, vinylimidazole, vinylpyridine, and a silane having a vinyl group.

 [Claim 20]

19. The method of claim 18, wherein the second monomer is 1,3,5-trivinyl-1,3,5_trimethylcyclotrisiloxane, 2,4,6,8-tetramethyl-2,4,6,8_tetra

2, 4, 6, 8, 10-pentamethyl-2,4,6, 8, 10-pentavinylcyclopentasiloxane, 2,4,6,8, 10, 12-nucleus methyl-2,4,6, 8, 10, 12-nucleus vinyl-cyclonucleus siloxane, octa (vinylsilasesquioxane), and

2,2,4, 4,6,6, 8, 8, 10, 10, 12, 12-dodecamethylcyclonuclesiloxane

The method is one or more selected from the group consisting of.

 [Claim 21]

 The method of claim 13, wherein the method for producing cancer stem cell spheroids is such that it does not perform artificial gene manipulation.

 [Claim 22]

 A cell culture substrate comprising a cyclosiloxane polymer, and

 Claim 1 to 12, comprising a composition according to any one of, a kit for producing a spheroid of cancer stem cells.

 [Claim 23]

 Using the composition according to any one of claims 1 to 12, culturing cancer cells to produce a spheroid of cancer stem cells;

Treating the candidate material on the spheroid of the cancer stem cells; 2019/151625 1 »（： 1 ^ 1 {2018/013838

Measuring cancer stem cell viability of the candidate substance treatment group and the candidate substance untreated control group; And

 And comparing the cancer stem cell survival rate of the candidate substance treatment group with the cancer stem cell survival rate of the untreated control group of the candidate substance.

 [Claim 24]

 24. The method of claim 23, further comprising comparing the survival rate and determining the candidate substance as a cancer treatment drug when the cancer stem cell survival rate of the candidate substance treatment group is lower than that of the control group.

 [Claim 25]

 Using the composition according to any one of claims 1 to 12, culturing cancer cells to produce a spheroid of cancer stem cells;

 Treating the spheroid of the cancer stem cells with a drug resistance alleviation candidate of cancer cells together with a cancer cell resistant drug; And

 Comprising the cancer stem cell survival rate of the candidate substance treatment group, comparing the cancer stem cell survival rate of the untreated control group of the candidate substance, Screening method of drug resistance alleviating drug of cancer cells.