Method Article

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

DOI:

10.3791/67425

⸱

April 25th, 2025

 ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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

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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

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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

  1. Draw a blood sample from the vein....

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Results

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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

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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

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The authors declare that they have no conflicts of interest.

Acknowledgements

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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

List of materials used in this article
NameCompanyCatalog NumberComments
1×PBSHyCloneSH30256.01
10×PBSHyCloneSH30256.01
100 mm×20 mm tissue-culture-treated culture dishCorning430167
1000 mL pipette tipsRainin30389218
15 mL centrifuge tubeNEST601052
200 mL pipette tipsRainin30389241
40 mm nylon cell strainer/70-mm nylon cell strainerFalcon352340
50 mL centrifuge tubeNEST602052
70 μm syringe fifilterSangon BiotechF613462-9001
Anti-Human CCR2 Antibody (clone: K036C2)BioLegend357224
Anti-Human CCR3 Antibody (clone: 5E8)BioLegend310724
Anti-Human CCR7 Antibody (clone: G043H7)BioLegend353240
Anti-Human CD103 Antibody (clone: Ber-ACT8)BioLegend350202
Anti-Human CD115 Antibody (clone: 9-4D2-1E4)BioLegend347314
Anti-Human CD117 Antibody (clone: 104D2)BioLegend313201
Anti-human CD11b Antibody (clone: 1CRF44) BD562721
Anti-human CD11c Antibody (clone: B-ly6) BD563026
Anti-Human CD123 Antibody (clone: 6H6)BioLegend306002
Anti-Human CD127 Antibody (clone: A019D5)BioLegend351337
Anti-Human CD13 Antibody (clone: WM19)BioLegend301701
Anti-Human CD138 Antibody (clone: MI15)BioLegend356535
Anti-human CD14 Antibody (clone: HCD14)BioLegend325604
Anti-Human CD141 Antibody (clone: M80)BioLegend344102
Anti-Human CD15 Antibody (clone: QA19A61)BioLegend376302
Anti-human CD16 Antibody (clone: B7311) BD561313
Anti-Human CD161 Antibody (clone: HP-3G10)BioLegend339902
Anti-Human CD163 Antibody (clone: GHI/61)BioLegend333603
Anti-Human CD169 Antibody (clone: 7-239)BioLegend346002
Anti-human CD19 Antibody (clone: HIB19)BioLegend302226
Anti-Human CD1c Antibody (clone: L161)BioLegend331501
Anti-Human CD20 Antibody (clone: 2H7)BioLegend302301
Anti-Human CD206 Antibody (clone: 15-2)BioLegend321151
Anti-Human CD24 Antibody (clone: ML5)BioLegend311129
Anti-Human CD25 Antibody (clone: BC96)BioLegend302624
Anti-human CD3 Antibody (clone: UCHT1) BD555916
Anti-Human CD31 Antibody (clone: W18200D)BioLegend375902
Anti-Human CD32 Antibody (clone: FUN-2)BioLegend303232
Anti-Human CD326 Antibody (clone: CO17-1A)BioLegend369812
Anti-Human CD33 Antibody (clone: WM53)BioLegend303402
Anti-human CD4 Antibody (clone: L200) BD563094
Anti-Human CD45 Antibody (clone: HI30) BD563716
Anti-Human CD45RO Antibody (clone: UCHL1)BioLegend304220
Anti-human CD56 Antibody (clone: 5.1H11)BioLegend362510
Anti-Human CD64 Antibody (clone: S18012C)BioLegend399502
Anti-Human CD66b Antibody (clone: 6/40c)BioLegend392917
Anti-human CD68 Antibody (clone: Y1/82A)BioLegend333808
Anti-Human CD69 Antibody (clone: FN50)BioLegend310902
Anti-Human CD7 Antibody (clone: 4H9/CD7)BioLegend395602
Anti-human CD8 Antibody (clone: RPA-T8) BD557750
Anti-Human CD80 Antibody (clone: W17149D)BioLegend375402
Anti-Human CD86 Antibody (clone: BU63)BioLegend374202
Anti-Human FOXP3 Antibody (clone: 206D)BioLegend320101
Anti-Human HLA_ABC Antibody (clone: W6/32)BioLegend311426
Anti-human HLA-DR Antibody (clone: L243)BioLegend307650
Anti-Human IgD Antibody (clone: IA6-2)BioLegend348211
Anti-Human Ki67 Antibody (clone: Ki-67)BioLegend350501
Anti-Human PD_L2 Antibody (clone: MH22B2)BD567783
Anti-Human PD1 Antibody (clone: EH12.2H7)BioLegend329951
Anti-Human PDL1 Antibody (clone: MIH2)BioLegend393602
Anti-human TCR-γδ Antibody (clone: B1) BD740415
Cell cryopreservation solutionThermo FisherA2644601
Cell-lD CisplatinStandard BioTools201064
Cell-lD Intercalator-lrStandard BioTools201192A
Collagenase, Type IVGibco17104019
Constant-temperature shakeFAITHFULFS-50B
CyTOF SystemFluidigm CorporationHelios
CytosploreCytosplore Consortium2.3.1
Dispase IIGibco17105041
DNase IMerckDN25
Eppendorf centrifugeEppendorf5702
EQ Four Element Calibration BeadsStandard BioTools201078
FBSGibco16000-044
Ficoll-paqueCytiva17-1440-02
FinnpipetteThermo Scientific4700870
Fixation bufferThermo ScientificFB001
FlowJoBD Life Sciences10.1
Formaldehyde solutionThermo Scientific28906
Granzyme B Antibody, anti-human/mouse (clone: QA16A02)BioLegend396413
Heparin TubesBD367874
Human BD Fc Block 2.5 mg/mLBD564220
MACS Tissue Storage SolutionMiltenyi130-100-008
Maxpar Fix and Perm BufferStandard BioTools201067
Maxpar metal-coniugated antibodiesStandard BioToolsVarious
Maxpar PBSStandard BioTools201058
Maxpar WaterStandard BioTools201069
Maxpare Cell Staining BufferStandard BioTools201068
Metal-conjugated Anti-Human α-SMA Antibody (clone: 1A4)Miltenyi Biotec130-098-145
PercollMerckP4937-500ML
Permeabilization bufferThermo Scientific00833356
RBC lysis bufferBD555899
Refrigerated centrifugeEppendorf5910ri
RPMI 1640 medium GE HealthCareSH30027.0
ScalpelAPPLYGENTB6298-1
Sterile Pasteur pipetteZDANZD-H03
Tissue digestion solutionYeasen Biotech41423ES30
Tuning SolutionStandard BioTools201072
Vortex MixerThermo Scientific88882012

References

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  1. 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).
  2. Renne, S. L., et al. Hepatocellular carcinoma: A clinical and pathological overview. Pathologica. 113 (3), 203-2....

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Tags

Mass CytometryHepatocellular CarcinomaImmune ProfilingCyTOF AnalysisPeripheral BloodTumor Immune ResponseSingle Cell AnalysisImmune Cell SubsetsBiomarker DiscoverySPADE Clustering

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