Abstract
We describe here a method, termed immunoFISH, for simultaneous in situ analysis of the composition and distribution of proteins and individual RNA transcripts in single cells. Individual RNA molecules are labeled by hybridization and target proteins are concurrently stained using immunofluorescence. Multicolor fluorescence images are acquired and analyzed to determine the abundance, composition, and distribution of hybridized probes and immunofluorescence. We assessed the ability of immunoFISH to simultaneous quantify protein and transcript levels and distribution in cultured HER2 positive breast cancer cells and human breast tumor samples. We demonstrated the utility of this assay in several applications including demonstration of the existence of a layer of normal myoepithelial KRT14 expressing cells that separate HER2+ cancer cells from the stromal and immune microenvironment in HER2+ invasive breast cancer. Our studies show that immunoFISH provides quantitative information about the spatial heterogeneity in transcriptional and proteomic features that exist between and within cells.
Highlights
The abundance, composition and spatial distribution of diverse RNA transcripts and proteins are fundamental determinants of the behavior of normal and cancer cells and numerous technologies have been developed to measure these cellular features
We show in this paper that simultaneous in situ measurement of transcripts location and composition and protein levels is possible in most circumstances by combining aspects of RNA fluorescence in situ hybridization (FISH) and immunofluorescence staining
We demonstrate the utility of immunoFISH by assessing the location and abundance of HER2 introns and exons relative to the nuclear boundary defined by immunostaining for nuclear lamins and HER2 protein in
Summary
The abundance, composition and spatial distribution of diverse RNA transcripts and proteins are fundamental determinants of the behavior of normal and cancer cells and numerous technologies have been developed to measure these cellular features. Work has focused on the development of ‘omic methods such as mass spectrometry and massively parallel sequencing that enable comprehensive and quantitative measures of protein and transcript profiles[1,2] These methods have enabled discovery of important functional components of normal cells and have revealed changes in specific transcripts and proteins from normal that suggest mechanisms by which diseases arise, progress and respond to treatment. Signals from fluorescently labeled proteins and individual transcripts are quantified by analysis of images acquired using high-resolution fluorescence microscopy This approach builds on previous reports showing that coding and noncoding RNA transcripts can be quantified and spatially localized[15,16] and even sequenced in situ[17]. We used immunoFISH to assess KRT14 and HER2 protein levels and HER2 mRNA expression in formal-fixed, paraffin embedded tissues and showed the remarkable spatial heterogeneity that exists within individual HER2+ cancers
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