# Antigen-Specific Ganglioside Serological Profile of Pancreatic and Gastric Cancer Patients by Multiple TLC Overlay Assay and IR-MALDI Mass Spectrometry

## TL;DR

This study identifies unique glycolipid profiles in pancreatic and gastric cancer patients using advanced analytical techniques, revealing insights into immune evasion mechanisms.

## Contribution

A novel method combining micro-extraction, TLC overlay, and IR-MALDI MS enables first-time profiling of long-chain sialylated glycolipids in cancer sera.

## Key findings

- CD75s, CD15s, and iso-CD75s antigens are preferentially expressed on long-chain neolacto-species in both cancer types.
- A tumor-specific glycolipid profile is revealed, with reduced complex fucosylated neolacto-gangliosides during tumor progression.
- The method detected 80 sialylated GSLs containing specific antigens in human sera.

## Abstract

The serological CD75s, CD15s, and iso-CD75s antigens in glycolipids play a significant role in pancreatic and gastric cancer and also differentiate the two cancer types. We evidenced that in both cancers, these antigens are preferentially and significantly expressed on long-chain neolacto-species, pointing toward specific glycosylation processing in immune evasion in these cancer types. The new findings of this study are enabled by the technology we developed here for serological antigen glycolipid profiling, which combines efficient micro-extraction of long-chain sialylated glycolipids with overlay antibody/lectin thin-layer chromatography staining and on-plate matrix-assisted laser desorption mass spectrometry analysis of specific antigen-containing glycolipids. This technology enabled us to explore, for the first time in cancer sera, the long-chain sialylated glycolipids of neolacto-series, which are primarily involved in peripheral immunity, along with ganglio-series, while also robustly exploring and differentiating pancreatic cancer and gastric cancer, including the different developmental stages, in a cohort of patient samples.

Background: Altered glycosphingolipidome in cancerous tissues and cells reflects the circulatory glycosphingolipid (GSL) profiles, which is advantageous for establishing cancer biomarkers and/or unravelling GSL-associated mechanisms of immunity in cancer. Methods: Here, we combined a microscale extraction of GSLs with multiple overlay TLC assays and IR-MALDI-o-TOF MS and implemented it for the first time in serum analysis of CD75s-, CD15s-, and iso-CD75s-containing sialylated GSLs of ganglio- and neolacto-series. Results: This sensitive antigen-specific targeted GSL workflow enabled the identification of 80 sialylated GSLs containing the specific antigens in human sera and was applied for the investigation of clinical serum samples from gastric/stomach cancer patients (n = 40), pancreatic cancer patients (n = 40), and a cancer-free control group (n = 20). The CD75s-, CD15s-, and iso-CD75s-containing GSL series encompassing complex monosialylated and fucosylated GSLs of neolacto-series, with up to pentadecasaccharide chains, were detected in both cancer types, while differential semi-quantitative analysis indicates a tumor type-specific associated GSL profile. Both cancer types share a drop in the complex fucosylated neolacto-gangliosides during tumor progression, implying a decreased synthesis of long-chain neolacto-series. Conclusions: This drop suggesting a role of these highly polar complex ganglioside species in evading humoral tumor immune response in the early tumor stages.