Culture Based Screening Assay and Methods of Use Thereof to Identify Agents Which Modulate Tumor Development, Invasion and Differentiation

Abstract

A 3D organotypic culture which phenocopies aggressive, invasive cancer and methods of use thereof are provided.

1 - 19 . (canceled) 
     
     
         20 . A method of in vitro screening of a compound comprising
 a) incubating an organotypic culture comprising an artificial stroma including a mixture of collagen and human fibroblasts isolated from a tissue of interest, said stroma being overlayed with primary keratinocytes, said keratinocytes comprising nucleic acids encoding at least one activated oncogene and at least one inactivated tumor suppressor gene, said culture exhibiting enhanced cell migration and invasion in an artificial extracellular matrix relative to control keratinocytes which lack said nucleic acids in the presence and absence of said compound, and observing the effect of said compound upon the migration and invasive characteristics of said keratinocytes relative to cells which are not incubated with said compound.   
     
     
         21 . The method of  claim 20 , further comprising observing the effect of said compound on cultures which do not contain said nucleic acids. 
     
     
         22 . The method according to  claim 20 , further comprising seeding a malignant cell on an artificial stroma to create a positive control model of tumor formation, tumor invasion or tumor-stroma interaction. 
     
     
         23 . The method of  claim 20 , wherein said method comprises contacting said culture with a cellular migration and invasive process altering agent and assessing the effect of said compound on said culture in the presence of said agent. 
     
     
         24 . The method of  claim 23 , wherein said agent is an MMP-9 inhibitor. 
     
     
         25 . The method of  claim 23 , wherein the agent in an AKT inhibitor. 
     
     
         26 . The method of  claim 20 , further comprising determining whether said compound modulates the expression level of at least one cell adhesion molecule selected from the group of N-cadherin, melanoma cell adhesion molecule, periostin and cadherin11. 
     
     
         27 . The method of  claim 20 , further comprising determining whether said compound modulates the expression level of fibroblast hepatocyte growth factor. 
     
     
         28 . The method according to  claim 20 , wherein said compound is screened for toxicity to human tissue, and wherein said observing step comprises observing the effects of said compound on the morphology and life span of said keratinocytes thereby identifying a compound which reduces the life span of the cells or has a negative impact on the morphology of said cells. 
     
     
         29 . A method of in vitro screening of a compound comprising contacting an organotypic culture as claimed in  claim 20 , with said compound wherein said cells of interest are obtained from a tumor isolated from a human patient and observing the effect of said compound upon said migration and invasive characteristics of said cells relative to tumor derived organotypic cultures which are not contacted with said compound. 
     
     
         30 . The method of  claim 29 , wherein said method comprises contacting said culture with a cellular migration and invasive process altering agent and assessing the effect of said compound on said culture in the presence of said agent. 
     
     
         31 . A genetic signature associated with cells exhibiting increased invasiveness, comprising nucleic acids encoding N-cadherin, melanoma cell adhesion molecule, periostin and cadherin 11, said nucleic acids exhibiting differential expression in invading versus non-invading cells as set forth in Table 1. 
     
     
         32 . An assay kit for determining the presence a genetic signature encoding proteins associated with increased tumor cell migration and invasion in a biopsy sample of esophageal cells, comprising antibodies immunologically specific for at least two proteins selected from the group consisting of cadherin 2, type 1, N cadherin, melanoma cell adhesion molecule, periostin, osteoblast specific factor, cadherin 11, type 2, OB-cadherin, claudin 1, claudin 4 claudin 7, occludin, one of said at least one protein or antibody being detectably labeled. 
     
     
         33 . The kit of  claim 32  comprising antibodies immunologically specific for N-cadherin, melanoma cell adhesion molecule, periostin and cadherin11. 
     
     
         34 . The assay kit according to  claim 32 , wherein said antibodies are immobilized on a solid support. 
     
     
         35 . The assay kit according to  claim 32  further comprising said proteins obtained from normal tissue for use as a control sample. 
     
     
         36 . A method for identifying patients at increased risk for metastatic esophageal cancer comprising obtaining a biological sample from said patient and assessing said patient for the presence of at least one of the molecules present in the genetic signature of  claim 31 . 
     
     
         37 . The method of  claim 36 , wherein said biological sample is sera from said patient and said molecule is periostin. 
     
     
         38 . The method of  claim 37 , wherein said periostin is detected using antibodies immunologically specific therefor and an immunocomplex formed between said periostin and said antibody is detected. 
     
     
         39 . The method of  claim 37 , wherein said periostin is detected using nucleic acids that hybridize to nucleic acids encoding said periostin, said nucleic acids comprising a detectable label. 
     
     
         40 . The method of  claim 37 , wherein all of the proteins encoded by the nucleic acids of the genetic signature are detected.