Gene expression profiling of acidic cell compartments from HCT116 spheroids.

We deciphered specific traits of acidic cancer cells by comparing gene expression profiles in acidic (negative K-pHLIP and positive pHLIP cells) and buffered (negative pHLIP cells) cell compartments from HCT116 colorectal cancer 3D spheroids. Overall design: HCT116 3D spheroids were labeled with AlexaFluor 568 pHLIP (pH low insertion peptide) and AlexaFluor 594 K-pHLIP (pH-independent control peptide) conjugated peptides, and sorted by FACS to isolate negative K-pHLIP and positive pHLIP cells (acidic) from pHLIP-negative cell population (buffered). We then subjected both cell populations to RNA-seq analysis to compare the transcriptomes acidic vs. buffered cancer cells.