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Knowledge of lectin and glycosidase specificities is fundamental to the study of glycobiology. The primary specificities of such molecules can be uncovered using well-established tools, but the complex details of their specificities are difficult to determine and describe. Here we present a language and algorithm for the analysis and description of glycan motifs with high complexity. The language uses human-readable notation and wildcards, modifiers, and logical operators to define motifs of nearly any complexity. By applying the syntax to the analysis of glycan-array data, we found that the lectin AAL had higher binding where fucose groups are displayed on separate branches. The lectin SNA showed gradations in binding based on the length of the extension displaying sialic acid and on characteristics of the opposing branches. A new algorithm to evaluate changes in lectin binding upon treatment with exoglycosidases identified the primary specificities and potential fine specificities of an α1-2-fucosidase and an α2-3,6,8-neuraminidase. The fucosidase had significantly lower action where sialic acid neighbors the fucose, and the neuraminidase showed statistically lower action where α1-2 fucose neighbors the sialic acid or is on the opposing branch. The complex features identified here would have been inaccessible to analysis using previous methods. The new language and algorithms promise to facilitate the precise determination and description of lectin and glycosidase specificities.

Outcomes following tumor resection vary dramatically among patients with pancreatic cancer. A challenge in defining predictive biomarkers is to discern within the complex tumor tissue the specific subpopulations and relationships that drive recurrence. Multiplexed immunofluorescence is valuable for such studies when supplied with markers of relevant subpopulations and analysis methods to sort out the intra-tumor relationships that are informative of tumor behavior. We hypothesized that the glycan biomarkers CA19-9 and STRA, which detect separate subpopulations of cancer cells, define intra-tumoral features associated with recurrence. We probed this question using automated signal thresholding and spatial cluster analysis applied to the immunofluorescence images of the STRA and CA19-9 glycan biomarkers in whole-block tumor sections. The tumors (N = 22) displayed extreme diversity between them in the amounts of the glycans and in the levels of spatial clustering, but neither the amounts nor the clusters of the individual and combined glycans associated with recurrence. The combined glycans, however, marked divergent types of spatial clusters, alternatively only STRA, only CA19-9, or both. The co-occurrence of more than one cluster type within a tumor associated significantly with disease recurrence, in contrast to the independent occurrence of each type of cluster. In addition, intra-tumoral regions with heterogeneity in biomarker clusters spatially aligned with pathology-confirmed cancer cells, whereas regions with homogeneous biomarker clusters aligned with various non-cancer cells. Thus, the STRA and CA19-9 glycans are markers of distinct and co-occurring subpopulations of cancer cells that in combination are associated with recurrence. Furthermore, automated signal thresholding and spatial clustering provides a tool for quantifying intra-tumoral subpopulations that are informative of outcome.