This protocol outlines a comprehensive mass cytometry (cytometry by time-of-flight [CyTOF]) analysis method for evaluating both systemic and local immune responses in hepatocellular carcinoma (HCC). The approach aims to provide insights into the immune landscape of HCC, offering a deeper understanding of the tumor microenvironment and the associated immune mechanisms.
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Method Article
Mass Cytometry Analysis of Systemic and Local Immune Responses in Hepatocellular Carcinoma
* These authors contributed equally
In This Article
Summary
Abstract
Hepatocellular carcinoma (HCC) is one of the most common and deadliest forms of liver cancer worldwide. Despite advances in treatment, the prognosis for HCC patients remains poor due to the complex interplay of genetic, environmental, and immunological factors driving its progression. Understanding the immune landscape of HCC is crucial for developing effective therapies, particularly in the field of immunotherapy, which holds great promises for improving patient outcomes. This study employs mass cytometry (cytometry by time-of-flight [CyTOF]) technology to investigate both systemic and local immune responses in patients with HCC. By analyzing peripheral blood and tumor samples, the research aims to identify unique immune cell populations, and their functional states associated with HCC progression. The findings provide a comprehensive overview of the immune landscape in HCC, highlighting potential biomarkers and therapeutic targets. This approach offers valuable insights into the immune mechanisms underlying HCC and paves the way for the development of more effective immunotherapies for this malignancy.
Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver cancer and a significant global health issue due to its high incidence and mortality rates1. According to the World Health Organization, HCC ranks as the fifth most common cancer and the second leading cause of cancer-related deaths worldwide2. It is particularly prevalent in regions with high rates of chronic hepatitis B and C infections, such as East Asia and sub-Saharan Africa3. Major risk factors include viral hepatitis, cirrhosis, and metabolic syndrome4. HCC requires long-term treatment, imposing substant....
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Protocol
The steps for blood and HCC sample collection, peripheral blood mononuclear cells (PBMCs) isolation, single-cell dissociation, and staining are outlined in the following plan. The experimental reagents and materials are all listed in the Table of Materials. All experiments were carried out with the approval of the Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University, ensuring that the collection of tumor samples did not interfere with pathological diagnosis. Written informed consent was obtained from all human subjects.
1. Isolation of PBMCs
- Draw a blood sample from the vein....
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Results
To elucidate the immunological characteristics associated with HCC, a comprehensive analysis of immune cell populations was conducted. Paired PBMCs and HCC tissue samples were collected from 4 patients with HCC. Mass cytometry profiling was performed to examine immune cell populations at the single-cell proteomic level, using two antibody panels for both PBMCs and HCC tissue samples.
After quality control, 45,326 cells were incl.......
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Discussion
This study leverages mass cytometry technology to provide an in-depth analysis of both systemic and local immune responses in HCC. The application of mass cytometry in this context enables the simultaneous detection of multiple markers at a single-cell level, offering a detailed immunophenotypic characterization that is crucial for understanding the complex immune landscape of HCC. Mass cytometry has revolutionized immunological studies by facilitating high-dimensional single-cell analysis. This technique employs rare me.......
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Disclosures
The authors declare that they have no conflicts of interest.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (grant 2019YFA0803000 to J.S.), the Excellent Youth Foundation of Zhejiang Scientific (grant R22H1610037 to J.S.), the National Natural Science Foundation of China (grant 82173078 to J.S.), the Natural Science Foundation of Zhejiang Province (grant 2022C03037 to J.S.).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 1×PBS | HyClone | SH30256.01 | |
| 10×PBS | HyClone | SH30256.01 | |
| 100 mm×20 mm tissue-culture-treated culture dish | Corning | 430167 | |
| 1000 mL pipette tips | Rainin | 30389218 | |
| 15 mL centrifuge tube | NEST | 601052 | |
| 200 mL pipette tips | Rainin | 30389241 | |
| 40 mm nylon cell strainer/70-mm nylon cell strainer | Falcon | 352340 | |
| 50 mL centrifuge tube | NEST | 602052 | |
| 70 μm syringe fifilter | Sangon Biotech | F613462-9001 | |
| Anti-Human CCR2 Antibody (clone: K036C2) | BioLegend | 357224 | |
| Anti-Human CCR3 Antibody (clone: 5E8) | BioLegend | 310724 | |
| Anti-Human CCR7 Antibody (clone: G043H7) | BioLegend | 353240 | |
| Anti-Human CD103 Antibody (clone: Ber-ACT8) | BioLegend | 350202 | |
| Anti-Human CD115 Antibody (clone: 9-4D2-1E4) | BioLegend | 347314 | |
| Anti-Human CD117 Antibody (clone: 104D2) | BioLegend | 313201 | |
| Anti-human CD11b Antibody (clone: 1CRF44) | BD | 562721 | |
| Anti-human CD11c Antibody (clone: B-ly6) | BD | 563026 | |
| Anti-Human CD123 Antibody (clone: 6H6) | BioLegend | 306002 | |
| Anti-Human CD127 Antibody (clone: A019D5) | BioLegend | 351337 | |
| Anti-Human CD13 Antibody (clone: WM19) | BioLegend | 301701 | |
| Anti-Human CD138 Antibody (clone: MI15) | BioLegend | 356535 | |
| Anti-human CD14 Antibody (clone: HCD14) | BioLegend | 325604 | |
| Anti-Human CD141 Antibody (clone: M80) | BioLegend | 344102 | |
| Anti-Human CD15 Antibody (clone: QA19A61) | BioLegend | 376302 | |
| Anti-human CD16 Antibody (clone: B7311) | BD | 561313 | |
| Anti-Human CD161 Antibody (clone: HP-3G10) | BioLegend | 339902 | |
| Anti-Human CD163 Antibody (clone: GHI/61) | BioLegend | 333603 | |
| Anti-Human CD169 Antibody (clone: 7-239) | BioLegend | 346002 | |
| Anti-human CD19 Antibody (clone: HIB19) | BioLegend | 302226 | |
| Anti-Human CD1c Antibody (clone: L161) | BioLegend | 331501 | |
| Anti-Human CD20 Antibody (clone: 2H7) | BioLegend | 302301 | |
| Anti-Human CD206 Antibody (clone: 15-2) | BioLegend | 321151 | |
| Anti-Human CD24 Antibody (clone: ML5) | BioLegend | 311129 | |
| Anti-Human CD25 Antibody (clone: BC96) | BioLegend | 302624 | |
| Anti-human CD3 Antibody (clone: UCHT1) | BD | 555916 | |
| Anti-Human CD31 Antibody (clone: W18200D) | BioLegend | 375902 | |
| Anti-Human CD32 Antibody (clone: FUN-2) | BioLegend | 303232 | |
| Anti-Human CD326 Antibody (clone: CO17-1A) | BioLegend | 369812 | |
| Anti-Human CD33 Antibody (clone: WM53) | BioLegend | 303402 | |
| Anti-human CD4 Antibody (clone: L200) | BD | 563094 | |
| Anti-Human CD45 Antibody (clone: HI30) | BD | 563716 | |
| Anti-Human CD45RO Antibody (clone: UCHL1) | BioLegend | 304220 | |
| Anti-human CD56 Antibody (clone: 5.1H11) | BioLegend | 362510 | |
| Anti-Human CD64 Antibody (clone: S18012C) | BioLegend | 399502 | |
| Anti-Human CD66b Antibody (clone: 6/40c) | BioLegend | 392917 | |
| Anti-human CD68 Antibody (clone: Y1/82A) | BioLegend | 333808 | |
| Anti-Human CD69 Antibody (clone: FN50) | BioLegend | 310902 | |
| Anti-Human CD7 Antibody (clone: 4H9/CD7) | BioLegend | 395602 | |
| Anti-human CD8 Antibody (clone: RPA-T8) | BD | 557750 | |
| Anti-Human CD80 Antibody (clone: W17149D) | BioLegend | 375402 | |
| Anti-Human CD86 Antibody (clone: BU63) | BioLegend | 374202 | |
| Anti-Human FOXP3 Antibody (clone: 206D) | BioLegend | 320101 | |
| Anti-Human HLA_ABC Antibody (clone: W6/32) | BioLegend | 311426 | |
| Anti-human HLA-DR Antibody (clone: L243) | BioLegend | 307650 | |
| Anti-Human IgD Antibody (clone: IA6-2) | BioLegend | 348211 | |
| Anti-Human Ki67 Antibody (clone: Ki-67) | BioLegend | 350501 | |
| Anti-Human PD_L2 Antibody (clone: MH22B2) | BD | 567783 | |
| Anti-Human PD1 Antibody (clone: EH12.2H7) | BioLegend | 329951 | |
| Anti-Human PDL1 Antibody (clone: MIH2) | BioLegend | 393602 | |
| Anti-human TCR-γδ Antibody (clone: B1) | BD | 740415 | |
| Cell cryopreservation solution | Thermo Fisher | A2644601 | |
| Cell-lD Cisplatin | Standard BioTools | 201064 | |
| Cell-lD Intercalator-lr | Standard BioTools | 201192A | |
| Collagenase, Type IV | Gibco | 17104019 | |
| Constant-temperature shake | FAITHFUL | FS-50B | |
| CyTOF System | Fluidigm Corporation | Helios | |
| Cytosplore | Cytosplore Consortium | 2.3.1 | |
| Dispase II | Gibco | 17105041 | |
| DNase I | Merck | DN25 | |
| Eppendorf centrifuge | Eppendorf | 5702 | |
| EQ Four Element Calibration Beads | Standard BioTools | 201078 | |
| FBS | Gibco | 16000-044 | |
| Ficoll-paque | Cytiva | 17-1440-02 | |
| Finnpipette | Thermo Scientific | 4700870 | |
| Fixation buffer | Thermo Scientific | FB001 | |
| FlowJo | BD Life Sciences | 10.1 | |
| Formaldehyde solution | Thermo Scientific | 28906 | |
| Granzyme B Antibody, anti-human/mouse (clone: QA16A02) | BioLegend | 396413 | |
| Heparin Tubes | BD | 367874 | |
| Human BD Fc Block 2.5 mg/mL | BD | 564220 | |
| MACS Tissue Storage Solution | Miltenyi | 130-100-008 | |
| Maxpar Fix and Perm Buffer | Standard BioTools | 201067 | |
| Maxpar metal-coniugated antibodies | Standard BioTools | Various | |
| Maxpar PBS | Standard BioTools | 201058 | |
| Maxpar Water | Standard BioTools | 201069 | |
| Maxpare Cell Staining Buffer | Standard BioTools | 201068 | |
| Metal-conjugated Anti-Human α-SMA Antibody (clone: 1A4) | Miltenyi Biotec | 130-098-145 | |
| Percoll | Merck | P4937-500ML | |
| Permeabilization buffer | Thermo Scientific | 00833356 | |
| RBC lysis buffer | BD | 555899 | |
| Refrigerated centrifuge | Eppendorf | 5910ri | |
| RPMI 1640 medium | GE HealthCare | SH30027.0 | |
| Scalpel | APPLYGEN | TB6298-1 | |
| Sterile Pasteur pipette | ZDAN | ZD-H03 | |
| Tissue digestion solution | Yeasen Biotech | 41423ES30 | |
| Tuning Solution | Standard BioTools | 201072 | |
| Vortex Mixer | Thermo Scientific | 88882012 |
References
- Zhang, C. H., Cheng, Y., Zhang, S., Fan, J., Gao, Q. Changing epidemiology of hepatocellular carcinoma in Asia. Liver Int. 42 (9), 2029-2041 (2022).
- Renne, S. L., et al. Hepatocellular carcinoma: A clinical and pathological overview. Pathologica. 113 (3), 203-2....
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