MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue

The main cellular receptors of Shiga toxins (Stxs), the neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer/CD77) and globotetraosylceramide (Gb4Cer), are significantly upregulated in about half of the human colorectal carcinomas (CRC) and in other cancers. Therefore, conjugates exploiting the Gb3Cer/Gb4Cer-binding B subunit of Stx (StxB) have attracted great interest for both diagnostic and adjuvant therapeutic interventions. Moreover, fucosylated GSLs were recognized as potential tumor-associated targets. One obstacle to a broader use of these receptor/ligand systems is that the contribution of specific GSLs to tumorigenesis, in particular, in the context of an altered lipid metabolism, is only poorly understood. A second is that also nondiseased organs (e.g., kidney) and blood vessels can express high levels of certain GSLs, not least Gb3Cer/Gb4Cer. Here, we used, in a proof-of-concept study, matrix-assisted laser desorption/ionization mass spectrometry imaging combined with laser-induced postionization (MALDI-2-MSI) to simultaneously visualize the distribution of several Gb3Cer/Gb4Cer lipoforms and those of related GSLs (e.g., Gb3Cer/Gb4Cer precursors and fucosylated GSLs) in tissue biopsies from three CRC patients. Using MALDI-2 and StxB-based immunofluorescence microscopy, Gb3Cer and Gb4Cer were mainly found in dedifferentiated tumor cell areas, tumor stroma, and tumor-infiltrating blood vessels. Notably, fucosylated GSL such as Fuc-(n)Lc4Cer generally showed a highly localized expression in dysplastic glands and indian file-like cells infiltrating adipose tissue. Our "molecular histology" approach could support stratifying patients for intratumoral GSL expression to identify an optimal therapeutic strategy. The improved chemical coverage by MALDI-2 can also help to improve our understanding of the molecular basis of tumor development and GSL metabolism.