Automatic single cell segmentation on highly multiplexed tissue images

Schüffler, Peter J.; Schapiro, Denis; Giesen, Charlotte; Wang, Hao A. O.; Bodenmiller, Bernd; Buhmann, Joachim M.

doi:10.1002/cyto.a.22702

Cited by 60 publications

(52 citation statements)

References 19 publications

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“…Previous MxIF studies focused entirely on cancer, where more uniform cell shape and loss of polarity allow for relatively straightforward cell segmentation (1, 2). This issue was solved by the addition of multiple membrane markers to provide coverage on all sides of the cell (Figure 1), similar to those described by Schüffler et al, using imaging mass cytometry (34). In addition, cloud processing facilitated rapid image processing and analysis of the large data sets collected for these studies (>1 terabyte raw image files).…”

Section: Discussionmentioning

confidence: 91%

Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity

McKinley¹,

Sui²,

Al-Kofahi³

et al. 2017

JCI Insight

120

128

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Section: Discussionmentioning

confidence: 91%

Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity

McKinley¹,

Sui²,

Al-Kofahi³

et al. 2017

JCI Insight

120

128

View full text Add to dashboard Cite

“…Approaches that enable multiplexing protein measurements include iterative rounds of fluorescence imaging (23–26) or metal-based detection (27, 28). To achieve single-cell resolution, single or multiple cell border markers are used in conjunction with sophisticated image processing algorithms to extract single cell objects from images (29). Oblique sectioning and imperfect segmentation of partial cells can lead to inaccurate quantification, making these approaches semi-quantitative at best.…”

Section: Introductionmentioning

confidence: 99%

Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

et al. 2016

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Cellular heterogeneity poses a significant challenge to understanding tissue level phenotypes and confounds conventional bulk analyses. To facilitate the analysis of signaling at the single-cell level in human tissues, we applied mass cytometry using CyTOF (Cytometry Time-of-Flight) to formalin-fixed paraffin-embedded (FFPE) normal and diseased intestinal specimens. We developed and validated a technique called FFPE-DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue), a single-cell approach for characterizing native signaling states from embedded solid tissue samples. We applied FFPE-DISSECT coupled to mass cytometry and found differential signaling by tumor necrosis factor α (TNF-α) in intestinal enterocytes, goblet cells and enteroendocrine cells, implicating the role of the downstream RAS-RAF-MEK-ERK signaling pathway in dictating goblet cell identity. In addition, application of FFPE-DISSECT, mass cytometry, and data-driven computational analyses to human colon specimens confirmed reduced differentiation in colorectal cancer (CRC) compared to normal colon, and revealed quantitative increases in inter- and intra-tissue heterogeneity in CRC with regards to the modular regulation of signaling pathways. Specifically, modular co-regulation of the kinases P38 and ERK, the translation regulator 4EBP1, and the transcription factor CREB in the proliferative compartment of the normal colon was loss in CRC, as evidenced by their impaired coordination over samplings of single cells in tissue. Our data suggest that this single-cell approach, applied in conjunction with genomic annotation, such as microsatellite instability and mutations in KRAS and BRAF, allows rapid and detailed characterization of cellular heterogeneity from clinical repositories of embedded human tissues. FFPE-DISSECT coupled of mass cytometry can be used for deriving cellular landscapes from archived patient samples, beyond CRC, and as a high resolution tool for disease characterization and subtyping.

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“…Another impressive example is the use of labeled antibodies to detect biomarkers in breast cancer tissue; see Figure . Here, multiplexing by means of lanthanoide labeling demonstrate its preponderance convincingly.…”

Section: Imaging Mass Spectrometrymentioning

confidence: 82%

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side‐by‐side

Linscheid

2018

Mass Spectrometry Reviews

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To understand biological processes, not only reliable identification, but quantification of constituents in biological processes play a pivotal role. This is especially true for the proteome: protein quantification must follow protein identification, since sometimes minute changes in abundance tell the real tale. To obtain quantitative data, many sophisticated strategies using electrospray and MALDI mass spectrometry (MS) have been developed in recent years. All of them have advantages and limitations. Several years ago, we started to work on strategies, which are principally capable to overcome some of these limits. The fundamental idea is to use elemental signals as a measure for quantities. We began by replacing the radioactive P with the "cold" natural P to quantify modified nucleotides and phosphorylated peptides and proteins and later used tagging strategies for quantification of proteins more generally. To do this, we introduced Inductively Coupled Plasma Mass Spectrometry (ICP-MS) into the bioanalytical workflows, allowing not only reliable and sensitive detection but also quantification based on isotope dilution absolute measurements using poly-isotopic elements. The detection capability of ICP-MS becomes particularly attractive with heavy metals. The covalently bound proteins tags developed in our group are based on the well-known DOTA chelate complex (1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid) carrying ions of lanthanoides as metal core. In this review, I will outline the development of this mutual assistance between molecular and elemental mass spectrometry and discuss the scope and limitations particularly of peptide and protein quantification. The lanthanoide tags provide low detection limits, but offer multiplexing capabilities due to the number of very similar lanthanoides and their isotopes. With isotope dilution comes previously unknown accuracy. Separation techniques such as electrophoresis and HPLC were used and just slightly adapted workflows, already in use for quantification in bioanalysis. Imaging mass spectrometry (MSI) with MALDI and laser ablation ICP-MS complemented the range of application in recent years.

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Automatic single cell segmentation on highly multiplexed tissue images

Cited by 60 publications

References 19 publications

Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity

Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity

Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side‐by‐side

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