Proteomics profiling of normal and tumoral human colon stem and epithelial cell populations

The colon epithelium is composed of diverse cell types with specific functions, originating from intestinal stem cells (ISCs) which are crucial in various intestinal disorders, including colorectal cancer (CRC). Previous studies on colon epithelial cells (EC), especially ISCs, have shown high expression of proteins like EpCAM (CD326), PGP-1 (CD44), and ALCAM (CD166). However, these studies have limitations in depth and analyte detection. Comprehensive proteomic analysis can offer a deeper insight into the colon epithelium and identify new CRC protein targets. Our goal was to analyze freshly purified ISCs and EC populations from paired normal and CRC samples to depict their proteomes.

Paired healthy and tumoral tissue biopsies from seven CRC patients were dissociated. Single-cell suspensions were sorted into four subsets by flow cytometry: colon ISCs (CD326⁺/CD166⁺/CD44⁺), and three additional EC populations [CD166^bright ECs (CD326⁺/CD166^bright/CD44^-), CD44⁺ ECs (CD326⁺/CD166^-/CD44⁺) and other ECs (CD326⁺/CD166^-/het/CD44^-)]. Purified cell populations (>93% purity) were processed for mass spectrometry (MS) analysis using a 9 M urea-based lysis buffer, proteins were enzymatically digested and purified using a bead-based method. Then, 3 μg of each sample were labeled with tandem mass tags (TMT10plex+TMT131C) and measured on an Orbitrap Fusion™Lumos™Tribrid™ mass spectrometer. Functional enrichment analysis (FEA) was performed using DAVID v2021.

Flow cytometry analyses indicated varying population distributions related to the anatomical origin of colon samples. Most cells belonged to the other EC subgroup across all samples, with right and left colon samples containing the CD166bright EC population (<0.1% frequency) absent in sigmoid and rectum samples. MS identified 3,991 distinct proteins in all colon samples, with 3,435 common proteins, including EC-specific sets of 4 to 5 proteins. While no unique proteins were found for any subset, different expression levels were observed between populations, reflecting different functional profiles. Other ECs exhibited typical colon functions such as absorption, microorganism interaction, and villus formation, while the CD166^-/CD44⁺ population showed additional metabolism-related functions. ISCs displayed enhanced cell cycle and development functions, akin to the more active CD166bright population. Comparing normal and tumoral populations, the latter closely resembled the general EC profile, sharing 3,101 proteins without unique identifications. FEA showed functions highlighting metabolic, RNA, mutational processes, and protein transport functions in tumoral cells, indicative of increased activity and tumoral development.

This study presents the proteomic landscapes of ISCs and EC populations from normal and tumoral colon samples, revealing distinct functional patterns that could aid in identifying new tumor biomarkers for CRC diagnosis, treatment, and monitoring when validated in additional cohorts.