Methods for identifying markers for early-stage human cancer, cancer progression and recurrence

Abstract

A method is described to identify secreted proteins identified with stages of malignancy of cancer. The proteins are initially identified by trapping them with a fluorescent protein containing vector that can insert in any gene. The secreted proteins are initially identified by their fluorescence. Secreted proteins identifying tumors with specific degrees of malignancy are isolated to determine if they can serve as markers of cancer progression.

1 . A protein identified by a method to identify secreted protein markers for cancer, which method comprises:
 a) transfecting a plurality of human cancer cells having varying degrees of malignancy and which have reverted toward a non-malignant state with an HIV-1 lentiviral-gene-trap vector containing a reporter gene;   b) identifying reporter-expressing cell lines that secrete reporter-linked proteins in vitro;   c) implanting a clone of each of the identified reporter-expressing cell lines in an animal;   d) identifying an implanted reporter-expressing cell line clone which secretes a reporter-linked protein in serum; and   e) identifying a secreted reporter-linked protein from step d) specific for human cancer cells of varying stages of malignancy from low to high.   
     
     
         2 . A library comprising human cancer cells of varying degrees of malignancy, wherein said cells have randomly trapped genes, each containing a reporter gene. 
     
     
         3 . The library of  claim 2 , wherein the reporter gene expresses green fluorescent protein (GFP). 
     
     
         4 . The library of  claim 2 , wherein a subset of the cells contain reporter-trapped genes capable of encoding secreted proteins. 
     
     
         5 . A library of reporter-trapped genes from the library of cells of  claim 4 , wherein the reporter-trapped genes are capable of expressing proteins which are secreted in vitro. 
     
     
         6 . A library of reporter-trapped genes from the library of cells of  claim 4 , wherein the reporter-trapped genes are capable of expressing proteins which are secreted in vivo. 
     
     
         7 . The library of  claim 2 , wherein the reporter-trapped genes are capable of expressing proteins secreted in vitro or in vivo, when the cancer cells have a specific degree of malignancy. 
     
     
         8 . The library of  claim 7 , wherein the cancer cells have a degree of malignancy such that the cells have the ability to invade or metastasize. 
     
     
         9 . The library of  claim 8 , wherein the reporter-trapped genes are capable of expressing proteins that are secreted in vitro and in vivo and are detectable in the serum of rodents transplanted with cells that express the reporter-trapped genes. 
     
     
         10 . A library of isolated genes which are capable of expressing protein markers of cancer progression identified by a method to identify secreted protein markers for cancer, which method comprises:
 a) transfecting a plurality of human cancer cells having varying degrees of malignancy and which have reverted toward a non-malignant state with an HIV-1 lentiviral-gene-trap vector containing a reporter gene;   b) identifying reporter-expressing cell lines that secrete reporter-linked proteins in vitro;   c) implanting a clone of each of the identified reporter-expressing cell lines in an animal;   d) identifying an implanted reporter-expressing cell line clone which secretes a reporter-linked protein in serum; and   e) identifying a secreted reporter-linked protein from step d) specific for human cancer cells of varying stages of malignancy from low to high,   wherein the genes do not contain a reporter gene or a gene-trap vector during specific steps of tumor progression.