title: Presence of Twist1-positive neoplastic cells in the stroma of chromosome-unstable colorectal tumors.
abstract: OBJECTIVE
  Cancer cells undergo an epithelial-to-mesenchymal transition (EMT) to become invasive, allowing tumors to progress. However, there is no direct evidence that human cancer cells undergo an EMT. In mouse cancer cells, up-regulation of transcription factor Twist1 was shown to promote an EMT. We searched the stroma of human colorectal tumor samples for TWIST1-positive cells with a mesenchymal phenotype and neoplastic genotype.
  
  
  METHODS
  We measured the expression of TWIST1 in human colorectal cancer (CRC) cell lines and examined the effects of overexpression or knockdown in vitro and in mice. We used immunohistochemistry to measure levels of TWIST1 in 201 colorectal tumor samples. In 20 samples, immunostaining was combined with fluorescence in situ hybridization analyses. Levels of TWIST1 messenger RNA (mRNA) were measured in blood samples from 15 patients.
  
  
  RESULTS
  TWIST1 was required to maintain the mesenchymal phenotype and invasiveness of the microsatellite-stable CoLo741 cells (which express endogenous TWIST1) and SW480 (expressing transgenic TWIST1). TWIST1 mRNA was not translated in CRC cells with microsatellite instability (HCT116). Syngenic TWIST1-positive colon carcinoma cells (CT26) that invaded tissues surrounding tumors acquired a mesenchymal phenotype. The presence of TWIST1-positive cells in the stroma of human colorectal tumors correlated with microsatellite stability (P = .05), stage IV cancer (P = .02), and disease-free survival time (P < .01). Trisomies of chromosome 7 and/or chromosome 20 were detected in 17 of 20 colorectal tumor samples, each of which contained TWIST1-positive cells with matching chromosomal gains in the tumor stroma (86 of 776 counted cells; 11.1%). No trisomy was observed in TWIST1-negative stromal cells (0 of 1249 cells; P < .001). Levels of TWIST1 mRNA were significantly higher in blood samples from patients with CRC than controls.
  
  
  CONCLUSIONS
  The stroma of human colorectal tumors contains TWIST1-positive cancer cells with mesenchymal phenotypes. Patients with CRC have higher levels of TWIST1 mRNA than healthy individuals.
authors.affiliation: Laboratory of Molecular Gastroenterology, Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Celesti
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Di Caro
authors.forename: Paolo
authors.initials: P
authors.lastname: Bianchi
authors.forename: Fabio
authors.initials: F
authors.lastname: Grizzi
authors.forename: Gianluca
authors.initials: G
authors.lastname: Basso
authors.forename: Federica
authors.initials: F
authors.lastname: Marchesi
authors.forename: Andrea
authors.initials: A
authors.lastname: Doni
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Marra
authors.forename: Massimo
authors.initials: M
authors.lastname: Roncalli
authors.forename: Alberto
authors.initials: A
authors.lastname: Mantovani
authors.forename: Alberto
authors.initials: A
authors.lastname: Malesci
authors.forename: Luigi
authors.initials: L
authors.lastname: Laghi
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000236:Adenoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000368:Aged; D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism*; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D053842:Microsatellite Instability; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009687:Nuclear Proteins / Q000378:metabolism*; D010641:Phenotype; D017154:Stromal Cells; D051879:Twist-Related Protein 1 / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1
keywords: 4′,6-diamidino-2-phenylindole; CEP; CI; CRC; Colon Cancer; DAPI; EMT; EMT-TF; FISH; GFP; MSI; MSS; Metastasis; PBS; Tumor Invasion; cDNA; centromeric enumeration probe; colorectal cancer; complementary DNA; confidence interval; epithelial to mesenchymal transition; epithelial to mesenchymal transition transcription factor; fluorescence in situ hybridization; green fluorescent protein; iFISH; immunofluorescence combined with fluorescence in situ hybridization; mRNA; messenger RNA; microsatellite stable; microsatellite unstable; phosphate-buffered saline