Imaging Mass Spectrometry and Lectin Analysis of N-Linked Glycans in Carbohydrate Antigen–Defined Pancreatic Cancer Tissues

Abstract

The early detection of pancreatic ductal adenocarcinoma is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers CA19-9 and sTRA are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and non-cancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry approach was utilized to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of PDAC patients represented by tissue microarrays and whole tissue sections. Orthogonally, these same tissues were characterized by multi-round immunofluorescence which defined expression of CA19-9 and sTRA as well as other lectins towards carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated bi-antennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9 expressing tissues tended to be bi-, tri- and tetra-antennary structures with both core and terminal fucose residues and bisecting N-acetylglucosamines. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored tri- and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-IHC and IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

Complete spatial characterisation of N-glycosylation upon striatal neuroinflammation in the rodent brain

“…Released N- glycan ions were detected using a MALDI timsTOF fleX trapped ion mobility separated QTOF mass spectrometer (Bruker Daltonics, Germany) operating in a positive mode as described by McDowdell et al [ 47 ]. This had a SmartBeam 3D laser operating at 10 kHz and a laser spot size of 20 μm.…”

Direct N-Glycosylation Profiling of Urine and Prostatic Fluid Glycoproteins and Extracellular Vesicles

“…A dual source timsTOF fleX MALDI-QTOF mass spectrometer (Bruker) was used to image the slides as previously described ( McDowell et al, 2021 ). Images were collected with a SmartBeam 3D laser operating at 10,000 Hz with a 20 µm laser spot size at a 150 µm raster with 300 laser shots per pixel.…”

Oligomannosylation and MAN1A1 expression associate strongly with a subset of human cancer types

“…Importantly, the cancer-associated elevation of oligomannose was supported by multiple independent studies for several cancer types. Notably, we also found papers describing unchanged or reduced levels of oligomannosylation in five other cancer types including gastric (GC) [57], hepatocellular carcinoma (HCC) [58], kidney (KC) [59], pancreas (PanCa) [60], and thyroid cancer (TC) [61]. Thus, our initial literature survey pointed to a strong association between oligomannosidic N -glycans and a subset of human cancers.…”

“…Cell surface glycoepitopes are known to play important roles in cellcell and cell-extracellular matrix communication [1,24,73]. To this end, we explored the cell surface expression of oligomannosidic epitopes of the promyelocytic HL- 60…”