Expression profiling of budding cells in colorectal cancer reveals an EMT-like phenotype and molecular subtype switching

Abstract

Background: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.Methods: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.Results: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.Conclusions: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.

A pre-existing population of ZEB2+ quiescent cells with stemness and mesenchymal features dictate chemoresistance in colorectal cancer

“…It is likely that both the stromal and the epithelial part of CMS4 tumors contribute to the establishment of an aggressive phenotype through an interplay of signals orchestrated by TGFβ, resulting in refractoriness to conventional and targeted therapies [53,54]. This hypothesis is corroborated by recent observations showing that budding areas of the tumor, which are in close contact with the surrounding stroma, are characterized by down regulation of proliferation genes, EMT and switching to CMS4 [55].…”

Putative anoikis‐resistant subpopulations in colorectal carcinoma: a marker of adverse prognosis

“…According to our current criteria, buds do not qualify as putative AR structures and hence, the quantitation of the putative AR structures at the front likely does not provide prognostic information. Bulk and front show other biological and structural differences (33). Taken together, we suggest that tumor bulk may be the part of the tumor where biological conditions in the carcinoma cells and the microenvironment allow the emergence of features that mark potential of the cells to resist anoikis.…”

Recommendations for reporting tumor budding in colorectal cancer based on the International Tumor Budding Consensus Conference (ITBCC) 2016

“…Both ITB and PTB are morphologic manifestations of epithelial-mesenchymal transition (EMT). A ZEB1, SNAIL1, TWIST1 positive microenvironment is conductive to the tumor budding phenotype 3 . Additionally, tumor buds show loss of the adhesion molecule E-cadherin and express markers of an activated wnt signaling pathway such as nuclear beta-catenin and APC 4,5 .…”