CRISPR-screen identifies ZIP9 and dysregulated Zn2+ homeostasis as a cause of cancer-associated changes in glycosylation.

INTRODUCTION: In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are upregulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear. METHODS: To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole-genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn. RESULTS AND CONCLUSIONS: We show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in upregulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as downregulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.

Mapping of truncated O-glycans in cancers of epithelial and non-epithelial origin.

BACKGROUND: Novel immunotherapies targeting cancer-associated truncated O-glycans Tn (GalNAcα-Ser/Thr) and STn (Neu5Acα2-6GalNacα-Ser/Thr) are promising strategies for cancer treatment. However, no comprehensive, antibody-based mapping of truncated O-glycans in tumours exist to guide drug development. METHODS: We used monoclonal antibodies to map the expression of truncated O-glycans in >700 tissue cores representing healthy and tumour tissues originating from breast, colon, lung, pancreas, skin, CNS and mesenchymal tissue. Patient-derived xenografts were used to evaluate Tn expression upon tumour engraftment. RESULTS: The Tn-antigen was highly expressed in breast (57%, n = 64), colorectal (51%, n = 140) and pancreatic (53%, n = 108) tumours, while STn was mainly observed in colorectal (80%, n = 140) and pancreatic (56%, n = 108) tumours. We observed no truncated O-glycans in mesenchymal tumours (n = 32) and low expression of Tn (5%, n = 87) and STn (1%, n = 75) in CNS tumours. No Tn-antigen was found in normal tissue (n = 124) while STn was occasionally observed in healthy gastrointestinal tissue. Surface expression of Tn-antigen was identified across several cancers. Tn and STn expression decreased with tumour grade, but not with cancer stage. Numerous xenografts maintained Tn expression. CONCLUSIONS: Surface expression of truncated O-glycans is limited to cancers of epithelial origin, making Tn and STn attractive immunological targets in the treatment of human carcinomas.

Hip-fracture osteosynthesis training: exploring learning curves and setting proficiency standards.

Background and purpose - Orthopedic surgeons must be able to perform internal fixation of proximal femoral fractures early in their career, but inexperienced trainees prolong surgery and cause increased reoperation rates. Simulation-based virtual reality (VR) training has been proposed to overcome the initial steep part of the learning curve but it is unknown how much simulation training is necessary before trainees can progress to supervised surgery on patients. We determined characteristics of learning curves for novices and experts and a pass/fail mastery-learning standard for junior trainees was established. Methods - 38 first-year residents and 8 consultants specialized in orthopedic trauma surgery performed cannulated screws, Hansson pins, and sliding hip screw on the Swemac TraumaVision VR simulator. A previously validated test was used. The participants repeated the procedures until they reached their learning plateau. Results - The novices and the experts reached their learning plateau after an average of 169 minutes (95% CI 152-87) and 143 minutes (CI 109-177), respectively. Highest achieved scores were 92% (CI 91-93) for novices and 96% (CI 94-97) for experts. Plateau score, defined as the average of the 4 last scores, was 85% (CI 82-87) and 92% (CI 89-96) for the novices and the experts, respectively. Interpretation - Training time to reach plateau varied widely and it is paramount that simulation-based training continues to a predefined standard instead of ending after a fixed number of attempts or amount of time. A score of 92% comparable to the experts' plateau score could be used as a mastery learning pass/fail standard.