Mass Cytometry Analysis of Systemic and Local Immune Responses in Hepatocellular Carcinoma

Mass cytometry allows us to analyze both systematic and local immune responses in hepatocellular carcinoma. By profiling immune cells at the single-cell level, we identify unique subsets and the functional states linked to tumor progression, offering insights into immune evasion mechanisms and the potential therapeutic targets for personalized treatments. Our protocol uses mass cytometry to analyze over 14 markers simultaneously at the single-cell level, avoiding spectral overlap seen in traditional flow cytometry. This involves precise identification of real cell populations, and provides high dimensional data for detailed immune profiling and the biomarker discovery.

[Narrator] To begin, thaw the frozen peripheral blood mononuclear cell and the hepatocarcinoma cell suspensions. After cell recovery, resuspend the cells in one milliliter of PBS without calcium and magnesium. Add cisplatin to a final concentration of 0.5 micromolar, mix well, and incubate at room temperature for two minutes. To stop the reaction, centrifuge the tubes at 500 G for five minutes at room temperature. Discard the supernatant, then add one milliliter of cell staining buffer to each tube. After centrifuging again, carefully discard the supernatant. To perform FC receptor blocking, first, prepare a 50 microliter block mix for each sample, combine 48 microliters of cell staining buffer with two microliters of FC blocking solution. Resuspend the cell pellet in the block mix and incubate. Next, add 1.1 microliters of each antibody per sample into a labeled tube to prepare the membrane protein antibody mix. Make up the volume to 55 microliters with cell staining buffer. Transfer 50 microliters of the prepared antibody mix into each sample tube, bringing the total volume to 100 microliters. Then gently swirl the samples to mix before incubating at room temperature for 15 minutes. Add one milliliters of cell staining buffer to each sample tube. Centrifuge at 500 G for five minutes at room temperature and discard the supernatant. After the final wash, briefly vortex the remaining liquid with the cell pellet to thoroughly resuspend the cells. For nucleus protein staining, add 500 microliters of the mixed fixation solution to the resuspended cells. Gently mix the samples before incubating at room temperature for 30 minutes. After incubation, centrifuge at 500 G for five minutes at room temperature and discard the supernatant. Next, pipette 1000 microliters of permeabilization buffer to each tube to wash the cells. Then centrifuge the tubes at 1000 G for five minutes at room temperature. Add 50 microliters of the antibody mixture into each tube after discarding the supernatant. Gently pipette the cells to mix thoroughly before incubating. Next, pipette 1000 microliters of permeabilization buffer to each tube, centrifuge again, then discard the supernatant. To fix the cells, add one milliliter of 1.6% formaldehyde prepared in PBS into each sample tube and vortex to mix the contents thoroughly. After incubating at room temperature for 10 minutes, centrifuge the samples and discard the supernatant. For nuclear intercalation staining, first dilute Cell-ID Intercalator Iridium with fix and permeabilization buffer. Pipette one milliliter of the intercalation solution into each sample. Gently mix and vortex. Place the samples at four degrees Celsius overnight, then centrifuge as before. When incubation is complete, pipette 1000 microliters of cell staining buffer into the tube. Centrifuge the tubes at 800 G for five minutes and discard the supernatant. Finally, resuspend the cells in 450 to 900 microliters of deionized water. Stain the cells with trypan blue for cell counting before further analysis. To begin, switch on the mass cytometer and launch the program. Analyze the samples in the CyTOF system and acquire the mass cytometric data of the peripheral blood mononuclear cells and the hepatocarcinoma cell samples. Launch the data processing software on the instrument and pre-process the CD45+ cell population in the FCS file. Then, eliminate dead cells using viability staining, such as cisplatin exclusion. Gate the CD45+ population to focus on immune cells, and export the gated population for further analysis. Transform the data with a co-factor of five. Next, launch the clustering software and identify the main clusters based on the SPADE algorithm, grouping cells with similar marker expression profiles into clusters. Apply hierarchical stochastic neighbor embedding, or HSNE, for dimensionality reduction and identification of distinct clusters. Then use the CyTOF kit package in our software to perform re-clustering of the major clusters. Use the PhenoGraph program to identify sub-clusters with default parameters. A total of 14 distinct cell types were identified in the peripheral blood mononuclear cell samples. Marker expression patterns for each of the 14 cell types were detailed in the heat map, showing unique expression profiles for distinct cell populations. The distribution of these cell types varied across samples with sample D having a higher proportion of CD4 T cells, samples A and B showing significant enrichment of B cells, and CD141+, conventional dendritic cells, predominantly present in sample C. Eight cell types were identified in the tissue samples including monocytes, T cells, neutrophils, natural killer cells, B cells, plasmacytoid dendritic cells, eosinophils, and myeloid dendritic cells. Marker expression profiles for the tissue cell types indicate distinct patterns for each cell population. Tissue samples showed a consistent proportion of cell types across all patients, reflecting shared immunological characteristics with hepatocellular carcinoma.