Next-generation biomarkers based on 100-parameter functional super-resolution microscopy TIS

Abstract

Functional super-resolution (fSR) microscopy is based on the automated toponome imaging system (TIS). fSR-TIS provides insight into the myriad of different cellular functionalities by direct imaging of large subcellular protein networks in morphologically intact cells and tissues, referred to as the toponome. By cyclical fluorescence imaging of at least 100 molecular cell components, fSR-TIS overcomes the spectral limitations of fluorescence microscopy, which is the essential condition for the detection of protein network structures in situ/in vivo. The resulting data sets precisely discriminate between cell types, subcellular structures, cell states and diseases (fSR). With up to 16 bits per protein, the power of combinatorial molecular discrimination (PCMD) is at least 2(100) per subcellular data point. It provides the dimensionality necessary to uncover thousands of distinct protein clusters including their subcellular hierarchies controlling protein network topology and function in the one cell or tissue section. Here we review the technology and findings showing that functional protein networks of the cell surface in different cancers encompass the same hierarchical and spatial coding principle, but express cancer-specific toponome codes within that scheme (referred to as TIS codes). Findings suggest that TIS codes, extracted from large-scale toponome data, have the potential to be next-generation biomarkers because of their cell type and disease specificity. This is functionally substantiated by the observation that blocking toponome-specific lead proteins results in disassembly of molecular networks and loss of function.

Toponome imaging system: multiplex biomarkers in oncology

“…This limited ability to assess the true complexity of cancer accounts for their low prognostic value [1]. Toponome imaging systems (TIS; see Glossary) can analyze up to 100 proteins within a single cell [2]; therefore, this novel technology holds promise for developing a new generation of multiplex biomarkers. In this article, we review recent insights gained into cancer complexity and heterogeneity and describe the need for TIS in furthering our knowledge.…”

“…For example, 00001000111001000100 could be a sequence representation for 20 protein tags [42,44]. Functional super resolution (fSR) microscopy: a TIS technique that enables the subcellular colocalization of !100 proteins by quantification of signal intensity [2]. Hub protein: a protein that interacts with many others in a PPI network, with central importance in cellular functioning due to this aspect.…”

“…A high-dimensional intensity vector is constructed and a pseudocolor image is painted whereby the color of two pixels is similar if their corresponding high-dimensional intensity vector is located close by, and vice versa [55,56]. Multiplex biomarker: TIS technology can measure up to 100 biomarkers simultaneously within a single cell [2]. Combinatorial analysis of this information (such as CMPs, LAW codes, or MCEPs) is a 'multiplex' biomarker assay that yields highly ordered complex signatures of cells, as compared with conventional multiplex biomarkers that examine only approximately five substances.…”

“…When protein expression data is binarized (i.e., marked as either present or absent), the PCMD of TIS with n different proteins is 2 n . In fSR TIS examining co-expression of at least 100 proteins there is a PCMD of 2 100 [2]. In approaches where protein expression data are scaled rather than binarized (e.g., the MCEP approach), the PCMD increases manifoldly.…”

“…TIS subset surprisology similarity mapping (TIS-SIM): 'subset surprisology' refers to the observation that the number of pixels, subcellular locations, or cells whose intensities for various proteins/molecules are simultaneously high with respect to corresponding thresholds is surprisingly large. In the TIS-SIM approach [2] (which omits a binarization step), 8 bits, or potentially 16 bits, are used to describe pixel intensity per protein instead of 1 bit (present/absent) to generate the pixel protein profiles (PPPs). 'Lasagne' software enables an investigator to highlight a pixel location and reveal all other pixels with similar PPPs [57].…”