Cell culture substrate, cancer cell aggregate and method for manufacturing same using said substrate, and drug screening method using said cancer cell aggregate

Abstract

A method for producing a cancer cell aggregate having certain properties, screening a drug for preventing and/or treating cancer, and determining anoikis resistance in epithelial cancer cells. The method includes culturing cells using a cell culture substrate having a base material and a biocompatible polymer layer. The cell culture substrate has rough sections not covered with the biocompatible polymer layer on the surface of the cell culture substrate. Each of the rough sections has a shape of a spot with a diameter of 20-100 μm, or a groove with a width of 3-30 μm with an end part of the rough section being optionally connected to another rough section. A distance between two adjacent rough sections is at least 10 μm or more, and the rough section has an uneven structure with a height ranging from 20 nm to 200 nm on the surface.

1 - 6 . (canceled) 
     
     
         7 : A cancer cell aggregate, comprising:
 a plurality of adherent cancer cells,   wherein the cancer cell aggregate is isolated and alive and has the following characteristics (a) to (e):   (a) having cell-in-cell structure,   (b) having non-spheroidal morphology,   (c) having membranous expression of α-tubulin on surface,   (d) having morphological polarity, and   (e) having tissue motion polarity.   
     
     
         8 : The cancer cell aggregate according to  claim 7 , further having at least one of the following characteristics (f) to (k):
 (f) having capability to reversibly release and incorporate live cancer cells,   (g) having cilia on surface,   (h) exhibiting filipodia or lamellipodia morphology,   (i) having capability to incorporate dead cells,   (j) having cell debris suction force, and   (k) having phosphatidylserine-positive surface.   
     
     
         9 : The cancer cell aggregate according to claim attached to a cell culture substrate having a three-dimensional structure. 
     
     
         10 : A method for producing the cancer cell aggregate according to  claim 7 , the method comprising:
 culturing adherent cancer cells using a cell culture substrate comprising a base material and a biocompatible polymer layer,   wherein the cell culture substrate includes a plurality of rough sections not covered with the biocompatible polymer layer on the surface of the cell culture substrate,   each of the rough sections has a shape of a spot with a diameter ranging from 20 μm to 100 μm, or a groove with a width ranging from 3 μm to 30 μm, when the shape of the rough section is a groove, an end part of the rough section is optionally connected to another rough section,   a distance between two adjacent rough sections is at least 10 μm or more, and the rough section has an uneven structure with a height ranging from 20 nm to 200 nm on the surface.   
     
     
         11 . (canceled) 
     
     
         12 : A method for screening a drug for preventing and/or treating cancer, the method comprising:
 making the cancer cell aggregate of  claim 7  coexist with a test substance;   observing the cancer cell aggregate for at least one of the following characteristics:   (a) having cell-in-cell structure,   (b) having non-spheroidal morphology,   (c) having membranous expression of α-tubulin on surface,   (d) having morphological polarity,   (e) having tissue motion polarity,   (f) having capability to reversibly release and incorporate live cancer cells,   (g) having cilia on surface,   (h) exhibiting filipodia or lamellipodia morphology,   (i) having capability to incorporate dead cells,   (j) having cell debris suction force, and   (k) having phosphatidylserine-positive surface,   making a comparison with those of the cancer cell aggregate of  claim 7  that is not made to coexist with the test substance; and   determining that the test substance has an anticancer activity when attenuation or loss of the characteristics is more strongly observed in the coexistence with the test substance.   
     
     
         13 . (canceled) 
     
     
         14 : The method according to  claim 12 , wherein the drug is for inhibiting infiltration and/or metastasis of cancer. 
     
     
         15 : The method according to  claim 12 , wherein the drug is for inhibiting and/or releasing immune evasion mechanism in cancer. 
     
     
         16 : A method for determining anoikis resistance in epithelial cancer cells, the method comprising:
 culturing test epithelial cancer cells using a cell culture substrate comprising a base material and a biocompatible polymer layer; and   determining that the epithetical cancer cells have anoikis resistance when the epithetical cancer cells have proliferated without adhering to the cell culture substrate,   wherein the cell culture substrate includes a plurality of rough sections not covered with the biocompatible polymer layer on the surface of the cell culture substrate,   each of the rough sections has a shape of a spot with a diameter ranging from 20 μm to 100 μm, or a groove with a width ranging from 3 μm to 30 μm, when the shape of the rough section is a groove, an end part of the rough section is optionally connected to another rough section,   a distance between two adjacent rough sections is at least 10 μm or more, and the rough section has an uneven structure with a height ranging from 20 nm to 200 nm on the surface.