High Throughput Assay for Cancer Cell Growth Inhibition

Abstract

A high-throughput, anchorage-independent assay is described, which screens compounds for inhibition of cancer cell growth. The assay utilizes a three-dimensional matrix or semi-solid media transfected with the subject compound, and enables live colony growth determination and imaging.

1 - 17 . (canceled) 
     
     
         18 . A high throughput method for identifying a cancer causing gene comprising:
 obtaining at least one slide comprising an upper surface;   coating the upper surface of the at least one slide with a base layer, said base layer comprising a first matrix and one or a plurality of spots on or in the base layer at separate distinct locations, wherein the one or plurality of spots comprise one or a plurality of active agents which are capable of altering by either inhibiting or increasing expression of a specific target gene, wherein each of the one or the plurality of spots has a diameter of about 65 μm to about 3.0 mm;   adding a second matrix for mixing with, or for layering atop of, a plurality of cancerous cells, and for forming a top layer on top of the base layer, wherein the cancerous cells uptake the plurality of active agents; wherein the thickness of at least one of said first matrix and said second matrix is about 0.1 mm to about 1.0 mm;   detecting colonies of living cancerous cells;   determining the spots on which cancerous cell growth is either inhibited or increased;   identifying the active agent that corresponds with the spot on which cancerous cell growth is altered;   relating the specific active agent with the gene whose expression the active agent influenced; and   determining the gene whose expression inhibits growth of cancerous cells, wherein the first matrix supports anchorage-independent cell growth and wherein the second matrix limits cell migration and allows three-dimensional cell growth.   
     
     
         19 . The method of  claim 18 , wherein the at least one slide is selected from glass slides, polymer slides, plastic slides, polystyrene slides, quartz wafers, and combinations thereof. 
     
     
         20 . The method of  claim 18 , wherein the one or a plurality of active agents are selected from DNA, RNA, siRNA, shRNA, antibodies, small molecules, proteins, peptides, peptidomimetics, pharmaceutical compositions, drugs and combinations thereof. 
     
     
         21 . The method of  claim 18 , wherein the first matrix and the second matrix are each independently selected from soft agar, agarose, hydrogels, methylcellulose alginate hydrogel, polyvinyl alcohol-hydrogel, collagen vitrigel, poly(2-hydroxylmethacrylate) hydrogels, PVP/PEO hydrogels, copolymers of 2-methacryloyloxyethyl phosphorylcholine, and combinations thereof. 
     
     
         22 . The method of  claim 18 , wherein a physical barrier separates the one or a plurality of spots, the physical barrier selected from the group consisting of at least one well comprised of at least one orifice formed within the slide, and optionally a removable member which contains at least one orifice, wherein the at least one orifice separates the spot placed within the at least one orifice from any other spots surrounding the at least one orifice. 
     
     
         23 . The method of  claim 18 , wherein the target gene is a human gene. 
     
     
         24 . The method of  claim 18 , wherein at least one of the one or a plurality of active agents targets one or more genes. 
     
     
         25 . The method of  claim 18 , wherein at least one of the one or a plurality of active agents targets one or more expressed gene. 
     
     
         26 . The method of  claim 18 , wherein the one or a plurality of active agents target every known human gene. 
     
     
         27 . The method of  claim 18 , wherein the target gene is a cancer gene. 
     
     
         28 . The method of  claim 18 , wherein detecting colonies of living cancerous cells is accomplished-over time.