title: Expression profiling of budding cells in colorectal cancer reveals an EMT-like phenotype and molecular subtype switching.
abstract: Tumour budding, described as the presence of single cells or small clusters of up to five tumour cells at the invasive margin, is established as a prognostic marker in colorectal carcinoma. In the present study, we aimed to investigate the molecular signature of tumour budding cells and the corresponding tumour bulk.
  
  
  
  Tumour bulk and budding areas were microdissected and processed for RNA-sequencing. As little RNA was obtained from budding cells, a special low-input mRNA library preparation protocol was used. Gene expression profiles of budding as compared with tumour bulk were investigated for established EMT signatures, consensus molecular subtype (CMS), gene set enrichment and pathway analysis.
  
  
  
  A total of 296 genes were differentially expressed with an FDR <0.05 and a twofold change between tumour bulk and budding regions. Genes that were upregulated in the budding signature were mainly involved in cell migration and survival while downregulated genes were important for cell proliferation. Supervised clustering according to an established EMT gene signature categorised budding regions as EMT-positive, whereas tumour bulk was considered EMT-negative. Furthermore, a shift from CMS2 (epithelial) to CMS4 (mesenchymal) was observed as tumour cells transit from the tumour bulk to the budding regions.
  
  
  
  Tumour budding regions are characterised by a phenotype switch compared with the tumour bulk, involving the acquisition of migratory characteristics and a decrease in cell proliferation. In particular, most tumour budding signatures were EMT-positive and switched from an epithelial subtype (CMS2) in the tumour bulk to a mesenchymal subtype (CMS4) in budding cells.
authors.affiliation: Translational Cell and Tissue Research Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.
authors.forename: Linde
authors.initials: L
authors.lastname: De Smedt
authors.affiliation: Translational Cell and Tissue Research Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.
authors.forename: Sofie
authors.initials: S
authors.lastname: Palmans
authors.affiliation: Translational Cell and Tissue Research Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.
authors.forename: Daan
authors.initials: D
authors.lastname: Andel
authors.affiliation: Translational Cell and Tissue Research Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.
authors.forename: Olivier
authors.initials: O
authors.lastname: Govaere
authors.affiliation: Vesalius Research Center, Vlaams instituut voor Biotechnologie (VIB), Leuven, Belgium.|Laboratory for Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium.
authors.forename: Bram
authors.initials: B
authors.lastname: Boeckx
authors.affiliation: Vesalius Research Center, Vlaams instituut voor Biotechnologie (VIB), Leuven, Belgium.|Laboratory for Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium.
authors.forename: Dominiek
authors.initials: D
authors.lastname: Smeets
authors.affiliation: Vesalius Research Center, Vlaams instituut voor Biotechnologie (VIB), Leuven, Belgium.|Laboratory for Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium.
authors.forename: Eva
authors.initials: E
authors.lastname: Galle
authors.affiliation: Translational Cell and Tissue Research Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.
authors.forename: Jasper
authors.initials: J
authors.lastname: Wouters
authors.affiliation: Bioinformatics Core Facility, Swiss Institute for Bioinformatics, Lausanne, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Barras
authors.affiliation: Bioinformatics Core Facility, Swiss Institute for Bioinformatics, Lausanne, Switzerland.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Suffiotti
authors.affiliation: Laboratory of Hepatology, Department of Clinical and Experimental Medicine, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Dekervel
authors.affiliation: Translational Cell and Tissue Research Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.|Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Tousseyn
authors.affiliation: Translational Cell and Tissue Research Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.|Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Gert
authors.initials: G
authors.lastname: De Hertogh
authors.affiliation: Digestive Oncology Unit, Department of Oncology, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Hans
authors.initials: H
authors.lastname: Prenen
authors.affiliation: Digestive Oncology Unit, Department of Oncology, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Sabine
authors.initials: S
authors.lastname: Tejpar
authors.affiliation: Vesalius Research Center, Vlaams instituut voor Biotechnologie (VIB), Leuven, Belgium.|Laboratory for Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium.
authors.forename: Diether
authors.initials: D
authors.lastname: Lambrechts
authors.affiliation: Translational Cell and Tissue Research Unit, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.|Department of Pathology, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Xavier
authors.initials: X
authors.lastname: Sagaert
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002455:Cell Division / Q000235:genetics*; D049109:Cell Proliferation / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D005812:Genes, Switch / Q000235:genetics*; D006801:Humans; D008297:Male; D000072662:Margins of Excision; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D010641:Phenotype; D046888:Tissue Array Analysis; D059467:Transcriptome