Bis(4-(1,3-benzoxazol-2-yl)) phenyl analogues as novel matrices for Fourier transform ion cyclotron resonance mass spectrometric imaging of lipids in dual ion mode in vulvar squamous cell carcinomas

Abstract

Vulvar squamous cell carcinoma (VSCC) is a rare malignancy, which accounts for only 2-5% of cancers related to the female reproductive system. Current histopathological diagnoses rely on the subjective interpretation of immunohistochemical staining, highlighting the need for more objective and comprehensive analytical techniques. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometric imaging (MSI) using dual-polarity matrices has emerged as a powerful tool for identification and spatial mapping of diverse metabolites in tissue because it can broaden the molecular coverage and acquire two ion images, but most are restricted in brain or kidney tissue. MSI-based research on VSCC remains scarce and research on the distribution patterns of the metabolites is necessary in order to investigate the molecular mechanisms in their physiological and pathological functions. In this study, bis(4-(1,3-benzoxazol-2-yl)) phenyl analogues including N,N-bis(4-(1,3-benzoxazole-2-yl) phenyl)aniline (BPA), N,N-bis(4-(1,3-benzoxazole-2-yl)phenyl)-1-naphthylamine (BPNA), N,N-bis(4-(1,3-benzoxazole-2-yl)phenyl)-9-aminoanthracene (BPAA), and N,N-bis(4-(1,3-benzoxazol-2-yl)phenyl)-6-(phenanthren-9-yl) naphthalen-2-amine (BPYA) were synthesized and evaluated as matrices for MALDI-Fourier transform ion cyclotron resonance (FT ICR)-MSI of lipid metabolites in VSCCs in both positive and negative ion modes. Among the four candidate matrices, BPYA exhibited superior performance in dual-polarity MALDI-MSI, including higher detection sensitivity, lower laser power, and lower background noise, when compared with commonly used MALDI matrices. The use of BPYA enabled the detection of 402 and 107 lipids in VSCC tissue in positive and negative ion modes, respectively. Various VSCCs were analyzed using MALDI-MSI and co-registered with haematoxylin and eosin (H&E) stained images; 140 lipids were found to be different. The difference analysis results of MSI were also validated using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis and 18 lipids were consistent in our MALDI-MSI and UPLC-MS results. This approach allowed the investigation of lipid distribution in these gynecological tissues and may broaden the molecular coverage. The technology may have potential to provide faster and user-independent tissue diagnoses using the tissue's spatial lipidomic distributions. The characteristic distribution patterns of different lipids provide useful insights into investigating the molecular mechanisms of the physiological and pathological functions of VSCC. This method can also be extended to the other widely used benchmark tissues, such as mouse brain.

Keywords: Bis(4-(1,3-benzoxazol-2-yl)) phenyl analogues; Lipids; Mass spectrometric imaging; Matrix-assisted laser desorption/ionization; Vulvar squamous cell carcinomas.