Spatial and molecular resolution of diffuse malignant mesothelioma heterogeneity by integrating label-free FTIR imaging, laser capture microdissection and proteomics

Abstract

Diffuse malignant mesothelioma (DMM) is a heterogeneous malignant neoplasia manifesting with three subtypes: epithelioid, sarcomatoid and biphasic. DMM exhibit a high degree of spatial heterogeneity that complicates a thorough understanding of the underlying different molecular processes in each subtype. We present a novel approach to spatially resolve the heterogeneity of a tumour in a label-free manner by integrating FTIR imaging and laser capture microdissection (LCM). Subsequent proteome analysis of the dissected homogenous samples provides in addition molecular resolution. FTIR imaging resolves tumour subtypes within tissue thin-sections in an automated and label-free manner with accuracy of about 85% for DMM subtypes. Even in highly heterogeneous tissue structures, our label-free approach can identify small regions of interest, which can be dissected as homogeneous samples using LCM. Subsequent proteome analysis provides a location specific molecular characterization. Applied to DMM subtypes, we identify 142 differentially expressed proteins, including five protein biomarkers commonly used in DMM immunohistochemistry panels. Thus, FTIR imaging resolves not only morphological alteration within tissue but it resolves even alterations at the level of single proteins in tumour subtypes. Our fully automated workflow FTIR-guided LCM opens new avenues collecting homogeneous samples for precise and predictive biomarkers from omics studies.

Integrated Fourier Transform Infrared Imaging and Proteomics for Identification of a Candidate Histochemical Biomarker in Bladder Cancer

“…Multiplex FTIR imaging thus replaces not only a single biomarker but also a multi-IHC panel. 26 FTIRguided LCM allows label-free, observer-independent, and precise harvesting of homogeneous tissue for subsequent LC-MS/MS analysis.…”

“…The resultant pixel size of the focal plane array detector was determined to be 5.65 Â 5.75 mm, and the rotation between the visible and IR image microscope was approximately 0.06 degrees. 26…”

“…The spatially resolved FTIR spectra of labelfree, freshly frozen thin tissue sections were used as a molecular fingerprint of the proteome, genome, transcriptome, lipidome, and metabolome of the underlying tissue. 26 Index colors were assigned to the spectra to classify the tissue by digital labeling on the basis of an RF algorithm. Previously developed bioinformatics workflow was used.…”

“…Homogeneous ROIs in both specimens (cystitis and invasive high grade) were selected, transferred to the LCM, and then microdissected for subsequent proteome analysis, according to a previously described workflow. 26 The label-free tissue classification is exemplarily shown in Figure 1. The first RF classification differentiated between healthy and cancerous tissue.…”

“…This approach was recently implemented in a validation study of biomarkers in immunohistochemical panels to differentiate between pleural mesothelioma subtypes. 26 Tissue classification by FTIR imaging allows label-free annotation of thin sections with high accuracy (>90% sensitivity and specificity) and without interobserver variability as opposed to pathologic annotation. 27e31 Differential analyses can also be performed by label-free FTIR imaging, as demonstrated for lung, colon, and breast cancer.…”