Abstract
Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA’s multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA’s analysis is strongly correlated with sequencing data (Pearson’s r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.
Highlights
Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications
We investigated whether MOSAICA can provide multiplexed mRNA detection and phasor-based background correction and error detection to clinically relevant and challenging sample matrices
To validate these puncta counts and their relative expressions, we examined the relationship between the decodified puncta with matching bulk RNA-sequencing obtained from The Cancer Genome Atlas (TCGA) database
Summary
Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Fluorescence lifetime is a measure of the time a fluorophore spends in the excited state before returning to the ground state and is an inherent characteristic of the fluorophore and its surrounding environment[26,27] By utilizing both time and spectral domains for labeling and imaging, we were able to discriminate a repertoire of 10 different fluorescent signatures against autofluorescent moieties and nonspecific binding events within the same sample in this study and expect to scale up to at least 60plex in the future to enable increased multiplexing capabilities with standard optical systems. We showcased MOSAICA in analyzing a 10-plex gene expression panel in colorectal SW480 cells based on combinatorial spectral and lifetime barcoding of only five generic commercial fluorophores Using this model, we illustrated the multiplexing scalability and MOSAICA’s ability to correct for stochastic nonbinding artifacts present within the sample. MOSAICA is rapid, cost-effective, and easy-to-use and can fill a critical gap between conventional FISH and sequential- and sequencing-based techniques for targeted and multiplexed spatial transcriptomics
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