Investigating Metabolomic and Microbiota Shifts in Hepatocellular Carcinoma Patients During Immunotherapy. META GUT HCC study Metabolomics and Gut Microbiota in HCC

Blood samples for metabolomic analysis will be collected using a dried blood spot (DBS) card. Participants will clean their hands thoroughly, and the finger to be pricked will be sanitized with an alcohol swab. A lancet will be used to prick the finger, and each drop of blood will be placed on the DBS card, ensuring that at least 35% of each spot is covered. After drying, the DBS cards will be sealed in ziplock bags and either shipped at ambient temperature or stored at 4°C for later processing. In the laboratory, metabolites will be extracted by immersing the DBS card in a solution of methanol, acetonitrile, and formic acid. The samples will be vortexed, dried, and resuspended for analysis using Liquid Chromatography-Mass Spectrometry (LCMS), with each LCMS run lasting approximately 20 minutes per sample.

Fecal samples will be collected at baseline and after three cycles of treatment using a sterile collection kit. Samples will be stored at -80°C until processing. For microbiota analysis, fecal material will be mixed with phosphate-buffered saline (PBS) and centrifuged to isolate bacterial cells. DNA will be extracted from the bacterial cells and analyzed using 16S rRNA gene sequencing to assess microbial diversity and composition. The sequencing data will identify specific microbial taxa that may correlate with treatment response or disease progression.