A 40-Marker Panel for High Dimensional Characterization of Cancer Immune Microenvironments by Imaging Mass Cytometry

Ijsselsteijn, Marieke E.; Breggen, Ruud van der; Sarasqueta, Arantza Fariña; Koning, Frits; Miranda, Noel F C C de

doi:10.3389/fimmu.2019.02534

Cited by 119 publications

(120 citation statements)

References 19 publications

Supporting

Mentioning

113

Contrasting

Order By: Relevance

“…As staining quality is strongly influenced by duration of and temperature during antibody incubation ( 15 ), we tested two different incubation conditions for the individual antibodies in the 34-marker panel: 5 h at RT or overnight at 4°C, after which the signal intensity and specificity were assessed by IMC for each antibody. We also determined the maximum signal threshold for all antibodies within several ROIs to compare the staining intensity between the two conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, IMC offers a major advantage over classical immunohistochemistry techniques which are limited by the numbers of markers that can be included simultaneously. Together with other studies that have developed antibody panels for FFPE tissue (15,21,22) this sets the stage for detailed studies to determine immune heterogeneity and cell-cell interactions in situ, providing a novel layer of understanding of functioning of the immune system on tissues. We anticipate that our study will guide other researchers that wish to use IMC for analysis of tissue of choice.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue

et al. 2020

Self Cite

View full text Add to dashboard Cite

Imaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass cytometry. As such, it allows us to gain spatial information and antigen quantification in situ, and can be applied to both snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue sections. Herein, we have developed and optimized the immunodetection conditions for a 34-antibody panel for use on human snap-frozen tissue sections. For this, we tested the performance of 80 antibodies. Moreover, we compared tissue drying times, fixation procedures and antibody incubation conditions. We observed that variations in the drying times of tissue sections had little impact on the quality of the images. Fixation with methanol for 5 min at −20 • C or 1% paraformaldehyde (PFA) for 5 min at room temperature followed by methanol for 5 min at −20 • C were superior to fixation with acetone or PFA only. Finally, we observed that antibody incubation overnight at 4 • C yielded more consistent results as compared to staining at room temperature for 5 h. Finally, we used the optimized method for staining of human fetal and adult intestinal tissue samples. We present the tissue architecture and spatial distribution of the stromal cells and immune cells in these samples visualizing blood vessels, the epithelium and lamina propria based on the expression of α-smooth muscle actin (α-SMA), E-Cadherin and Vimentin, while simultaneously revealing the colocalization of T cells, innate lymphoid cells (ILCs), and various myeloid cell subsets in the lamina propria of the human fetal intestine. We expect that this work can aid the scientific community who wish to improve IMC data quality.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…The mass cytometry quantitative strategy can also be extended to intact tissue sections like MSI. Imaging mass cytometry was used to visualise the tumor microenvironment in FFPE CRC tissues with a focus on immune cell infiltration [128] or more recently to evaluate the singlecell pathology landscape of a large cohort of 352 breast cancer tissues [129].…”

Section: Spatial Resolution In Discovery Proteomicsmentioning

confidence: 99%

Recent advances in mass spectrometry based clinical proteomics: applications to cancer research

2020

View full text Add to dashboard Cite

Cancer biomarkers have transformed current practices in the oncology clinic. Continued discovery and validation are crucial for improving early diagnosis, risk stratification, and monitoring patient response to treatment. Profiling of the tumour genome and transcriptome are now established tools for the discovery of novel biomarkers, but alterations in proteome expression are more likely to reflect changes in tumour pathophysiology. In the past, clinical diagnostics have strongly relied on antibody-based detection strategies, but these methods carry certain limitations. Mass spectrometry (MS) is a powerful method that enables increasingly comprehensive insights into changes of the proteome to advance personalized medicine. In this review, recent improvements in MS-based clinical proteomics are highlighted with a focus on oncology. We will provide a detailed overview of clinically relevant samples types, as well as, consideration for sample preparation methods, protein quantitation strategies, MS configurations, and data analysis pipelines currently available to researchers. Critical consideration of each step is necessary to address the pressing clinical questions that advance cancer patient diagnosis and prognosis. While the majority of studies focus on the discovery of clinically-relevant biomarkers, there is a growing demand for rigorous biomarker validation. These studies focus on high-throughput targeted MS assays and multi-centre studies with standardized protocols. Additionally, improvements in MS sensitivity are opening the door to new classes of tumour-specific proteoforms including posttranslational modifications and variants originating from genomic aberrations. Overlaying proteomic data to complement genomic and transcriptomic datasets forges the growing field of proteogenomics, which shows great potential to improve our understanding of cancer biology. Overall, these advancements not only solidify MS-based clinical proteomics' integral position in cancer research, but also accelerate the shift towards becoming a regular component of routine analysis and clinical practice.

show abstract

“…Both techniques are, however, low-throughput due to the relatively long imaging time of 2 h per field of 1 mm 2 in IMC and 1 h 12 min per field of 1 mm 2 in MIBI-TOF ( 129 ). IMC has been applied to study tumor heterogeneity in several types of cancers, such as pancreatic cancer ( 130 ), biliary tract cancer ( 131 ), breast cancer ( 126 , 132 , 133 ), and colorectal cancer ( 108 , 134 ). MIBI-TOF has been used to study the tumor-immune microenvironment of breast cancer ( 127 , 128 , 135 , 136 ) and the metabolic state of T cells in colorectal cancer ( 109 ).…”

Section: Multidimensional Single-cell Technologies and Their Strengthmentioning

confidence: 99%

Unraveling the Complexity of the Cancer Microenvironment With Multidimensional Genomic and Cytometric Technologies

et al. 2020

Self Cite

View full text Add to dashboard Cite

Cancers are characterized by extensive heterogeneity that occurs intratumorally, between lesions, and across patients. To study cancer as a complex biological system, multidimensional analyses of the tumor microenvironment are paramount. Single-cell technologies such as flow cytometry, mass cytometry, or single-cell RNA-sequencing have revolutionized our ability to characterize individual cells in great detail and, with that, shed light on the complexity of cancer microenvironments. However, a key limitation of these single-cell technologies is the lack of information on spatial context and multicellular interactions. Investigating spatial contexts of cells requires the incorporation of tissue-based techniques such as multiparameter immunofluorescence, imaging mass cytometry, or in situ detection of transcripts. In this Review, we describe the rise of multidimensional single-cell technologies and provide an overview of their strengths and weaknesses. In addition, we discuss the integration of transcriptomic, genomic, epigenomic, proteomic, and spatially-resolved data in the context of human cancers. Lastly, we will deliberate on how the integration of multi-omics data will help to shed light on the complex role of cell types present within the human tumor microenvironment, and how such system-wide approaches may pave the way toward more effective therapies for the treatment of cancer.

show abstract

A 40-Marker Panel for High Dimensional Characterization of Cancer Immune Microenvironments by Imaging Mass Cytometry

Cited by 119 publications

References 19 publications

A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue

A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue

Recent advances in mass spectrometry based clinical proteomics: applications to cancer research

Unraveling the Complexity of the Cancer Microenvironment With Multidimensional Genomic and Cytometric Technologies

Contact Info

Product

Resources

About