The Role of the Peritoneal Microenvironment in the Pathogenesis of Colorectal Peritoneal Carcinomatosis: Single-cell Transcriptomics

Research methodology

Before inclusion of patients, patients will be screened for the presence of hepatitis B, hepatitis C and HIV to ensure researcher safety. During cytoreductive surgery, a total of five samples of tumoral tissue will be acquired. Three of those samples will be utilized for ScRNASeq analysis, and two samples of a macrometastasis will be snap frozen and cryopreserved.

Single-cell RNA-Sequencing analysis We will comprehensively catalog the stromal cell types in the microenvironment of colorectal cancer peritoneal metastases utilizing single-cell RNA sequencing (scRNAseq) on three samples (one of which should preferably originate from the omentum majus, to encompass milky spots) acquired from surgical waste material of patients undergoing cytoreductive surgery. After sample prelevation, samples will be transported as soon as possible while cooled and submerged in 10% PBS to the VIB facilities, where single cell suspensions will be made according to standard protocols. After cell selection by FACS (CD45+ and live/dead stain, to ensure a 50/50 mix of immune and stromal cells) and library prep, ScRNASeq will be performed.

Single cell sequencing analysis will allow us to identify cell clusters that by usage of marker genes will be assigned to different known cell lineages. After annotation of data, a "landscape" of the composition of the tumor stroma will be acquired.