Industrialized, Artificial Intelligence-guided Laser Microdissection for Microscaled Proteomic Analysis of the Tumor Microenvironment

Abstract

The tumor microenvironment (TME) represents a complex ecosystem comprised of dozens of distinct cell types, including tumor, stroma, and immune cell populations.To characterize proteome-level variation and tumor heterogeneity at scale, highthroughput methods are needed to selectively isolate discrete cellular populations in solid tumor malignancies. This protocol describes a high-throughput workflow, enabled by artificial intelligence (AI), that segments images of hematoxylin and eosin (H&E)-stained, thin tissue sections into pathology-confirmed regions of interest for selective harvest of histology-resolved cell populations using laser microdissection (LMD). This strategy includes a novel algorithm enabling the transfer of regions denoting cell populations of interest, annotated using digital image software, directly to laser microscopes, thus enabling more facile collections. Successful implementation of this workflow was performed, demonstrating the utility of this harmonized method to selectively harvest tumor cell populations from the TME for quantitative, multiplexed proteomic analysis by high-resolution mass spectrometry. This strategy fully integrates with routine histopathology review, leveraging digital image analysis to support enrichment of cellular populations of interest and is fully generalizable, enabling harmonized harvests of cell populations from the TME for multiomic analyses.

ProteoMixture: A Cell Type Deconvolution Tool for Bulk Tissue Proteomics Data

“…After staining with aqueous hematoxylin and eosin, cell type annotation and counting were performed using the HALO image analysis software (Indica Labs, Albuquerque, NM, USA). Regions of tissue annotated using HALO were exported for LMD (LMD7, Leica Microsystems) as previously described (30). LMD harvested tissue for protein digestion was collected in MircoTubes (Pressure Biosciences, Inc., South Easton, MA, USA) containing 20 µL of 100 mM triethylammonium bicarbonate (TEAB, pH 8.0)/10% acetonitrile, capped and stored at -80 ºC until digestion.…”

Integration of Multi-omic Data in a Molecular Tumor Board Reveals EGFR-Associated ALK-Inhibitor Resistance in a Patient With Inflammatory Myofibroblastic Cancer

“…Under this research protocol, research use only (RUO)-based analysis of the expression and/or phosphorylation (“activation”) levels of 32 proteins that are the known direct drug targets of FDA-approved and experimental therapeutics was performed using reverse phase protein microarray (RPPA) by Theralink Technologies, Inc. (Golden, CO). 23 The patient’s TKI-naïve primary (pre-­alectinib, pre-lorlatinib) and recurrent (post-alectinib, on-lorlatinib) biopsy tissue specimens were processed via laser microdissection (LMD) to isolate tumor epithelial cells from the tumor microenvironment, 24 which were analyzed by RPPA. Of note, while the recurrent biopsy specimen was received only 6 days after beginning lorlatinib, precedent exists to suggest this interval represents sufficient time to see a visible clinical effect from a TKI targeting a clear driver mutation.…”