An end-to-end workflow for multiplexed image processing and analysis

Windhager, Jonas; Bodenmiller, Bernd; Eling, Nils

doi:10.1101/2021.11.12.468357

Cited by 58 publications

(78 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All cells located within this expanded hull obtain a milieu ID that is identical to the patch ID. We have meanwhile implemented the patch detection algorithm in the imcRtools R package available via Bioconductor ( 69 ).…”

Section: Methodsmentioning

confidence: 99%

“…To quantify additional exhaustion markers on CXCL13 + /CD8 + T cells from another consecutive cut of the TMA, we acquired data from 17 patients with high T cell infiltration. For segmentation, we used an implementation of DeepCell ( 68 ) within the dockerized steinbock pipeline to generate single-cell data in a csv format ( 69 ). Steinbock version 0.7.3 was applied with standard parameters as described in the steinbock documentation.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Multiplexed imaging mass cytometry of the chemokine milieus in melanoma characterizes features of the response to immunotherapy

et al. 2022

Self Cite

View full text Add to dashboard Cite

Intratumoral immune cells are crucial for tumor control and antitumor responses during immunotherapy. Immune cell trafficking into tumors is mediated by binding of specific immune cell receptors to chemokines, a class of secreted chemotactic cytokines. To broadly characterize chemokine expression and function in melanoma, we used multiplexed mass cytometry–based imaging of protein markers and RNA transcripts to analyze the chemokine landscape and immune infiltration in metastatic melanoma samples. Tumors that lacked immune infiltration were devoid of most of the profiled chemokines and exhibited low levels of antigen presentation and markers of inflammation. Infiltrated tumors were characterized by expression of multiple chemokines. CXCL9 and CXCL10 were often localized in patches associated with dysfunctional T cells expressing the B lymphocyte chemoattractant CXCL13. In tumors with B cells but no B cell follicles, T cells were the sole source of CXCL13 , suggesting that T cells play a role in B cell recruitment and potentially in B cell follicle formation. B cell patches and follicles were also enriched with TCF7 + naïve-like T cells, a cell type that is predictive of response to immune checkpoint blockade. Our data highlight the strength of targeted RNA and protein codetection to analyze tumor immune microenvironments based on chemokine expression and suggest that the formation of tertiary lymphoid structures may be accompanied by naïve and naïve-like T cell recruitment, which may contribute to antitumor activity.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Multiplexed imaging mass cytometry of the chemokine milieus in melanoma characterizes features of the response to immunotherapy

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…It does not allow spatial visualization of cells in tissues and does not scale well as the image volume increases (26, 47). This could be circumvented by different software utilities implemented in one of the prevalent programing languages in bioinformatics, such as Python or R (48). Here, we developed and share with open-source license, a basic IMC data analysis workflow that can be used in subsequent studies.…”

Section: Discussionmentioning

confidence: 99%

Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues

Tornaas

Kleftogiannis

Fromreide

et al. 2022

Preprint

View full text Add to dashboard Cite

IntroductionCancer associated fibroblast (CAF) mediated crosstalk with other tumor compartments is shown to modulate cancer development and progression. Conventionally, CAFs are known to promote tumorigenesis either by secreting a wide range of growth factors, cytokines, and chemokines or by remodeling the underlying stroma. However, recent studies aimed at depleting CAFs have shown progression to more aggressive tumors leading to poorer outcomes. This may be attributed to the heterogeneity and plasticity of CAFs. Understanding CAF heterogeneity has been limited due to the lack of specific markers. Imaging mass cytometry (IMC) can analyze up to 40 parameters at subcellular resolution simultaneously and hence it can be an ideal tool to unravel the heterogeneity of CAFs and decipher the role of various CAF subsets in cancer development and progression. The aim of this study was to develop a panel of antibodies (ABs) for IMC to identify different CAF phenotypes and their spatial distribution in tissues and other tumor components in formalin-fixed paraffin-embedded (FFPE) archival tissues.MethodsA comprehensive panel of ABs were chosen based on known literature on CAFs and other tumor and microenvironment-related markers. ABs were conjugated to rare earth metals using the protocol provided by the supplier (Fluidigm, CA, USA). FFPE tissue samples from different organs/lesions known to express certain markers were used for making control tissue microarrays (cTMAs) for verification of conjugated ABs. Immunohistochemistry (IHC) with metal-conjugated ABs was used as the “gold standard” for optimization of IMC. Tissue images were segmented using ImcSegmentationPipeline (Ilastik and CellProfiler). Downstream analysis of single-cell data was performed using a newly developed bioinformatics pipeline as well as ImaCytE, an existing software solution.ResultsThe final panel of ABs was decided based on a comparable performance of conjugated ABs validated using IHC and IMC. Different cell types were identified including various subsets of CAFs and immune cells using cell segmentation and unsupervised clustering.ConclusionThis study establishes an extensive panel of IMC ABs on archival FFPE tissue material for unravelling CAF heterogeneity. In addition, the panel described here, the downstream analysis and the newly developed pipeline can be readily applied to different tissue types in context of cancer research or other fibrotic disease.

show abstract

“…The raw mcd files were processed using the Steinbock pipeline, version 0.70 (Windhager et al 2021). In brief, the raw files were converted into tiff files, and the cells were segmented using a pre-trained neural network (Greenwald et al 2021) using the H3K9ac channel as the nuclear channel and CD45RA/RO and Vimentin as the membrane channels.…”

Section: Methodsmentioning

confidence: 99%

Optimizing multiplexed imaging experimental design through tissue spatial segregation estimation

Bost

Schulz

Engler

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Recent advances in multiplexed imaging methods allow simultaneous detection of dozens of proteins or RNAs enabling deep spatial characterization of both healthy and tumor tissues. Parameters for design of optimal sequencing-based experiments have been established, but such parameters are lacking for multiplex imaging studies. Here, using a spatial transcriptomic atlas of healthy and tumor human tissues, we developed a new statistical framework that determines the number of fields of view necessary to accurately identify all cell types that are part of the tissue. Using this strategy on imaging mass cytometry data, we identified a measurement of tissue spatial segregation that enables optimal experimental design and that is technology invariant. This strategy will enable significantly improved design of multiplexed imaging studies.

show abstract

An end-to-end workflow for multiplexed image processing and analysis

Cited by 58 publications

References 44 publications

Multiplexed imaging mass cytometry of the chemokine milieus in melanoma characterizes features of the response to immunotherapy

Multiplexed imaging mass cytometry of the chemokine milieus in melanoma characterizes features of the response to immunotherapy

Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues

Optimizing multiplexed imaging experimental design through tissue spatial segregation estimation

Contact Info

Product

Resources

About