Institute of Hygiene, University of Münster, Robert-Koch-Str. 41, 48149 Münster, Germany.
Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Domagkstr. 3, 48149 Münster, Germany.
Anal Chem. 2020 May 19;92(10):7096-7105. doi: 10.1021/acs.analchem.0c00480. Epub 2020 May 4.
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.
志贺毒素（Stx）的主要细胞受体是中性糖脂（GSLs），包括神经节苷脂三己糖苷（Gb3Cer/CD77）和神经节苷脂四己糖苷（Gb4Cer）。这些受体在大约一半的人类结直肠癌（CRC）和其他癌症中显著上调。因此，利用 Stx 的 Gb3Cer/Gb4Cer 结合 B 亚基（StxB）的缀合物引起了人们对诊断和辅助治疗干预的极大兴趣。此外，糖基化 GSL 被认为是潜在的肿瘤相关靶点。这些受体/配体系统更广泛应用的一个障碍是，特定 GSL 对肿瘤发生的贡献，特别是在改变的脂质代谢背景下，知之甚少。另一个障碍是，即使是非疾病器官（如肾脏）和血管也可以高水平表达某些 GSL，尤其是 Gb3Cer/Gb4Cer。在这里，我们在一项概念验证研究中使用基质辅助激光解吸/电离质谱成像结合激光诱导后电离（MALDI-2-MSI），同时在来自 3 名 CRC 患者的组织活检中可视化几种 Gb3Cer/Gb4Cer 脂类和相关 GSL（例如，Gb3Cer/Gb4Cer 前体和糖基化 GSL）的分布。使用 MALDI-2 和基于 StxB 的免疫荧光显微镜，Gb3Cer 和 Gb4Cer 主要存在于去分化肿瘤细胞区域、肿瘤基质和肿瘤浸润血管中。值得注意的是，糖基化 GSL，如 Fuc-(n)Lc4Cer，通常在发育不良的腺体和浸润脂肪组织的单行细胞中表现出高度局部化的表达。我们的“分子组织学”方法可以支持对肿瘤内 GSL 表达进行分层，以确定最佳治疗策略。MALDI-2 可提高化学覆盖度，从而有助于提高我们对肿瘤发展和 GSL 代谢的分子基础的理解。
