Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy
Abstract
:1. Introduction
2. Fluorescent Immunohistochemistry
3. Multiplex Fluorescent Immunohistochemistry
3.1. Stain Removal Technologies
3.1.1. Multiepitope-Ligand Cartography
3.1.2. Sequential Immuno-Peroxidase Labelling and Erasing
3.1.3. Iterative Bleaching Extends Multiplexity
3.2. Fluorophore Inactivation Technologies
3.2.1. Multiplexed Fluorescence Microscopy
3.2.2. Cyclic Immunofluorescence
3.2.3. ChipCytometry
3.3. Multiplexed Signal Amplification
3.3.1. Multiplex Modified Hapten-Based
3.3.2. Tyramide Signal Amplification (TSA)
3.3.3. Nanocrystal Quantum Dots
3.4. DNA Barcoding Technologies
3.4.1. DNA Exchange Imaging
3.4.2. Codetection by Indexing
3.4.3. Signal Amplification by Exchange Reaction
3.4.4. Digital Spatial Profiling (DSP)
3.4.5. InSituPlex®
3.5. Mass Cytometry
3.5.1. Imaging Mass Cytometry
3.5.2. Multiplexed Ion Beam Imaging
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Method Name | Vendor Name | Sample | Maximal Labeling | Direct/ Indirect Detection | Antibody Conjugation | Stain Removal Method | Time Consuming (Per Cycle) * | Resolution |
---|---|---|---|---|---|---|---|---|
Stain removal technologies | ||||||||
MELC (Toponome imaging systems) | ToposNomos GmbH | FFPE | 100 | Direct | Fluorescent based | Bleaching | two proteins per hour (one tag/one dye per cycle) | <40 nm |
SIMPLE | NA | FFPE | 12 | Indirect | Fluorescent based | Alcohol-soluble red peroxidase substrate AEC method | 3 h | 15–20 μm |
IBEX | NA | Frozen/FFPE | >65 | Direct/indirect | Fluorescent based | LiBH4-based bleaching | Manual ~3.5 h/automated ~1.5 h | 160 nm |
Fluorophore inactivation technologies | ||||||||
MxIF | Cell IDx | FFPE | 60 | Indirect | Fluorescent based | Alkaline oxidation chemistry inactivation | 1 h 15 min | 1 μm |
CycIF | NA | FFPE | 60 | Direct | Fluorescent based | Hydrogen peroxide and light inactivation | ~24 h | 5 μm |
Chip Cytometry | Zellsafe™ | Cell suspensions /frozen/FFPE | 60 | Direct | Fluorescent based | Chemical bleaching or light photobleaching | ~1 h | 5 μm |
Multiplexed signal amplification | ||||||||
Multiplex modified hapten-based | UltraPlex™ | FFPE | 4 | Indirect | Fluorescent based | Antibody stripping | 2 h | NA |
TSA | Roche and Akoya Biosciences | Cell suspensions /FFPE | 9 | Indirect | Fluorescent based | Antibody stripping | 1 h | 0.25–0.9 μm |
QDs | NA | FFPE | 5 | Direct/Indirect | Fluorescent based | Chemical bleaching | 6 h | Super resolution |
DNA barcoding technologies | ||||||||
DEI | NA | FFPE | 8 | Indirect | DNA-barcoding based | NA | 2–3 h | 20 nm |
CODEX | Akoya Biosciences | Cell suspensions/frozen/FFPE | 60 | Indirect | DNA-barcoding based | NA | <1 day (Whole slide imaging) | 260 nm |
Immuno-SABER | NA | Cell suspensions/frozen/FFPE | 10 | Indirect | DNA-barcoding based | NA | 1 h | Super resolution |
DSP | NanoString | Frozen/FFPE | 96 | Indirect | DNA-barcoding based | NA | 1–2 h | 10 μm |
InSituPlex® | Ultivue | FFPE | 15 | Indirect | DNA-barcoding based | NA | 5.5 h | NA |
Mass cytometry | ||||||||
IMC | Hyperion | Cell suspensions /frozen/FFPE | >40 | Direct | Metal-based | NA | 2 weeks (0.5 mm × 0.5 mm ROI takes ~3.5 h with a slide scanner) | 1 μm |
MIBI | Ionpath | Cell suspensions /FFPE | 40–100 | Direct | Metal-based | NA | 2 weeks (Whole slide imaging) | 260 nm |
Method Name | Cancer Type | Biomarkers Studies | Refs. |
---|---|---|---|
Stain removal technologies | |||
MELC (Toponome imaging systems) | Colorectal cancer | CD3, CD4, CD25, CD29, CD44, human lymphocyte antigen (HLA)-DR | [22] |
SIMPLE | HNSCC PDAC | CD3, CD4, CD8, CD46, CD68, PD-1, Ki67, Eomes-odermin, GrzB, IDO, Tbet | [24] |
IBEX | NA | NA | NA |
Fluorophore inactivation technologies | |||
MxIF | Colon cancer | ER, androgen receptor (AR), p53, Her2, PLAC8 | [30,31] |
CycIF | Breast cancer | Her2, ER, PR | [35] |
ChipCytometry | Breast cancer | PD-L1, PD-L2 | [38] |
Multiplexed signal amplification | |||
Multiplex modified hapten-based | NSCLC | CD8, PD-L1, and panCK | [40] |
TSA | Metastatic gastric cancer (GC) | PD-L1 | [44] |
QDs | Gastric cancer/ breast cancer | type IV collagen, macrophages, matrix metalloproteinase 9 (MMP9), CD105 | [50] |
Breast cancer | type IV collagen, Her2 | [51] | |
DNA barcoding technologies | |||
DEI | N | NA | NA |
CODEX | Cutaneous T cell lymphoma (CTCL) | ICOS, IDO-1, LAG-3, PD-1, PD-L1, OX40, Tim-3, VISTA | [57] |
Immuno-SABER | NA | NA | NA |
DSP | NSCLC | CD3, CD4, CD8, CD20, PD-L1 | [64] |
InSituPlex® | Breast cancer | Kaiso | [66] |
Mass cytometry | |||
IMC | Melanoma | MHC-I, HMB45, S100, IFNGR1, IRF1, CD45RO, PD-L1, CD163, B7-H3, LAG3, TIM3, FOXP3, CD4, B7-H4, CD68, PD-1, CD20, CD8, PD-1H, Ki67, B2M, CD3a, CSF1R, PD-L2, Granzyme B, MHC-II, CXCL9, CXCL10, CXCL13 | [72,73] |
SUC | PD-1, PD-L1 | [74] | |
MIBI | Breast cancer | double-stranded DNA (dsDNA), ERα, PR, E-cadherin, Ki-67, vimentin, actin, keratin, HER2, PD-1, PD-L1 | [14,77] |
Method Name | Advantage | Disadvantage |
---|---|---|
Stain removal technologies | ||
MELC (Toponome imaging systems) | Detects hundreds of proteins and high resolution | The multiprobe image is limited to a single microscopic medium-to-high power field and high cost |
SIMPLE | Easy to perform by whole-slide scanner and can be labeled primary antibodies from same species | Up to 12 biomarkers |
IBEX | Allows over 65 biomarkers to detect and compatible with over 250 commercial antibodies | Not commercialized and few studies |
Fluorophore inactivation technologies | ||
MxIF | Up to 60 biomarkers | Time-consuming and relatively expensive |
CycIF | Use commonly reagents and instruments | Before the next staining, coverslip should be removed and time-consuming |
ChipCytometry | Detects unlimited number of biomarkers, long-storage samples, removes autofluorescence and instrument automaticity | Damage the tissue adherence and photobleachable dyes may generate weak signals during imaging processing |
Multiplexed signal amplification | ||
Multiplex modified hapten-based | Two-hour fast staining and cocktail antibodies are used in a single slide | Maximal four biomarkers can be labeled per slide and not applied widely |
TSA | Avoids antibody cross-reactivity and may realize an automated protocol | Nine biomarkers can be labeled per slide |
QDs | Removes autofluorescence and has much stronger signals | Big size relatively, has toxicity and limited nanocrystals |
DNA barcoding technologies | ||
DEI | Short-time staining and applies for most microscopy platforms | Lack of signal amplification, few studies |
CODEX | Allows 60 biomarkers labeled and can be imaged by conventional fluorescence microscopy, also keeps the morphology of normal and diseased tissues | Longer scanning and lack of signal amplification |
Immuno-SABER | High multiplexing, sensitivity and 5–180-fold signal amplification | Up to 10-plex and few publications |
DSP | No-damage staining protocol and performs high multiplexing image on FFPE samples | Chooses ROI manually and is not able to reconstruct images |
InSituPlex® | Good signal in low-expression antigen, 5.5 h workflow and relatively cheap | Few studies |
Mass cytometry | ||
IMC | Removes autofluorescence, reveals the quantity of proteins in subcellular level | Lack of signal amplification, the rate of image acquisition is slow and relatively low resolution in subcellular level |
MIBI | A large number of metal-antibodies can be labeled spectral overlap and high resolution | Time-consuming, instrument and metal-antibodies are expensive |
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Sheng, W.; Zhang, C.; Mohiuddin, T.M.; Al-Rawe, M.; Zeppernick, F.; Falcone, F.H.; Meinhold-Heerlein, I.; Hussain, A.F. Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy. Int. J. Mol. Sci. 2023, 24, 3086. https://doi.org/10.3390/ijms24043086
Sheng W, Zhang C, Mohiuddin TM, Al-Rawe M, Zeppernick F, Falcone FH, Meinhold-Heerlein I, Hussain AF. Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy. International Journal of Molecular Sciences. 2023; 24(4):3086. https://doi.org/10.3390/ijms24043086
Chicago/Turabian StyleSheng, Wenjie, Chaoyu Zhang, T. M. Mohiuddin, Marwah Al-Rawe, Felix Zeppernick, Franco H. Falcone, Ivo Meinhold-Heerlein, and Ahmad Fawzi Hussain. 2023. "Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy" International Journal of Molecular Sciences 24, no. 4: 3086. https://doi.org/10.3390/ijms24043086