Abstract
The paradigm of molecular histopathology is shifting from a single-marker immunohistochemistry towards multiplexed detection of markers to better understand the complex pathological processes. However, there are no systems allowing multiplexed IHC (mIHC) with high-resolution whole-slide tissue imaging and analysis, yet providing feasible throughput for routine use. We present an mIHC platform combining fluorescent and chromogenic staining with automated whole-slide imaging and integrated whole-slide image analysis, enabling simultaneous detection of six protein markers and nuclei, and automatic quantification and classification of hundreds of thousands of cells in situ in formalin-fixed paraffin-embedded tissues. In the first proof-of-concept, we detected immune cells at cell-level resolution (n = 128,894 cells) in human prostate cancer, and analysed T cell subpopulations in different tumour compartments (epithelium vs. stroma). In the second proof-of-concept, we demonstrated an automatic classification of epithelial cell populations (n = 83,558) and glands (benign vs. cancer) in prostate cancer with simultaneous analysis of androgen receptor (AR) and alpha-methylacyl-CoA (AMACR) expression at cell-level resolution. We conclude that the open-source combination of 8-plex mIHC detection, whole-slide image acquisition and analysis provides a robust tool allowing quantitative, spatially resolved whole-slide tissue cytometry directly in formalin-fixed human tumour tissues for improved characterization of histology and the tumour microenvironment.
Highlights
It is important to understand the spatial cellular composition and heterogeneity of tissues, especially in cancer where cell subpopulations and the tumour microenvironment provide insights about the biology and clinical progression of the disease
The method utilizes a combination of fluorescent and chromogenic tissue staining for the simultaneous detection of six proteins and nuclei in formalin-fixed tissue section (Supplementary Fig. S1 and Supplementary Fig. S2)
The other panel was designed for the characterisation of prostate epithelium and consisted of antibodies for p63, Pan-CK, CK5, CK8, CK18, alpha-methylacyl-CoA racemase (AMACR), and androgen receptor (AR) (Epithelial cell panel)
Summary
It is important to understand the spatial cellular composition and heterogeneity of tissues, especially in cancer where cell subpopulations and the tumour microenvironment provide insights about the biology and clinical progression of the disease. One solution to overcome this limitation is to utilize a “hotspot” imaging where a low-resolution scan of whole tissue is performed first followed by a subsequent “hotspot” analysis at higher resolution[1,16] This assay design does not allow true whole-slide analytics. As a proof-of-concept, we demonstrate automated classification of epithelial and immune cells in human prostate cancer and a simultaneous marker analysis at single cell level To our knowledge, this is the first open-source 8-plex mIHC assay design that enables whole-slide imaging with true quantitative whole-slide analysis in FFPE samples at high resolution across the whole tissue
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