Spatially resolved multi-omics to decipher molecular pathology for idiopathic pulmonary fibrosis

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by progressive scarring of lung tissue, resulting in respiratory failure. Despite extensive research, the underlying molecular mechanisms driving IPF progression remain elusive. Here, we highlight the application of multi-omics spatial mass spectrometry tissue imaging coupled with laser capture microdissection (LCM)-LC-MS/MS technologies to identify key drivers of fibrosis progression, enabling target identification and therapeutic invention strategies for IPF.

Methods: Sequential MALDI-MSI analysis was conducted on fresh-frozen IPF lung tissue sections for metabolite/lipid analysis and intact protein analysis using HiPlex-IHC (Ambergen) on a timsTOF fleX MALDI-2 (Bruker) instrument. SCiLS lab software (Bruker) was used for data visualization and analysis, while image segmentation algorithms embedded within QuPath and SCiLS lab were subsequently applied to enable the selection of regions of interest (ROI) for downstream analysis. LCM was performed using the LMD7 system (Leica) to isolate these ROIs for in-depth proteomic characterization via LC-MS/MS (timsTOF fleX MALDI-2 coupled with EvoSep).

Preliminary data: Existing spatial omics studies in IPF have analyzed single omics layers, limiting their ability to capture the complex interplay between biomolecules. Our innovative approach utilizes sequential multi-omics MALDI imaging, which enables us to concurrently analyze and integrate information from untargeted metabolites and lipids alongside targeted proteins within a single tissue section. Our multi-omics analysis found dysregulation of inflammatory mediators, including increased expression of inflammatory metabolites like lysophosphatidic acid and arachidonic acid. These elevated levels coincide with the spatial distribution of CD68 macrophages, indicating a possible connection between these mediators and macrophage activity in the progression of IPF.

Furthermore, our methodology incorporates laser capture microdissection (LCM) for targeted isolation of fibrotic regions, guided by a multi-modal approach. First, we utilize MALDI-IHC targeting α-SMA, an established protein marker for fibrosis, to identify regions of interest. The reliability of MALDI-IHC with α-SMA probes in determining fibrotic regions is then verified using pentachrome staining, a gold standard for characterizing fibrotic foci, ensuring that the most relevant areas for in-depth proteomic characterization via LC-MS/MS were isolated. Preliminary data from this approach indicates dysregulation in IPF-related pathways, including upregulated TGF-β signaling in fibrotic tissue, aligning with its role in ECM synthesis and remodeling. This localized analysis offers a deeper understanding of the molecular composition in fibrotic regions, overcoming limitations of bulk tissue analysis.