Fusion-negative rhabdomyosarcoma orthotopic tongue xenografts for study of invasion, intravasation and metastasis in live animals.

PAX3/7 Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Towards achieving a more complete understanding of advanced FN-RMS, we developed an orthotopic tongue xenograft model for studies of molecular basis of FN-RMS invasion and metastasis. The behavior of FN-RMS cells injected into murine tongue was examined using in vivo bioluminescence imaging, non-invasive intravital microscopy (IVM), and histopathology and compared to the prevailing hindlimb intramuscular and subcutaneous xenografts. FN-RMS cells were retained in the tongue and invaded locally into muscle mysial spaces and vascular lumen. While evidence of hematogenous dissemination to the lungs occurred in tongue and intramuscular xenografts, evidence of local invasion and lymphatic dissemination to lymph nodes only occurred in tongue xenografts. IVM and RNA-seq of tongue xenografts reveal shifts in cellular phenotype and differentiation state in tongue xenografts. IVM also shows homing to blood and lymphatic vessels, lymphatic intravasation, and dynamic membrane protrusions. Based on these findings, the tongue orthotopic xenograft of FN-RMS is a valuable model for tumor progression studies at the tissue, cellular and subcellular levels providing insight into kinetics and molecular bases of tumor invasion and metastasis and, hence, new therapeutic avenues for advanced FN-RMS.

Identification of global inhibitors of cellular glycosylation.

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.

An atlas of O-linked glycosylation on peptide hormones reveals diverse biological roles.

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.

A strategy for generating cancer-specific monoclonal antibodies to aberrant O-glycoproteins: identification of a novel dysadherin-Tn antibody.

Successful application of potent antibody-based T-cell engaging immunotherapeutic strategies is currently limited mainly to hematological cancers. One major reason is the lack of well-characterized antigens on solid tumors with sufficient cancer specific expression. Aberrantly O-glycosylated proteins contain promising cancer-specific O-glycopeptide epitopes suitable for immunotherapeutic applications, but currently only few examples of such antibody epitopes have been identified. We previously showed that chimeric antigen receptor T-cells directed towards aberrantly O-glycosylated MUC1 can control malignant growth in a mouse model. Here, we present a discovery platform for the generation of cancer-specific monoclonal antibodies targeting aberrant O-glycoproteins. The strategy is based on cancer cell lines engineered to homogeneously express the truncated Tn O-glycoform, the so-called SimpleCells. We used SimpleCells of different cancer origin to elicit monoclonal antibodies with selectivity for aberrant O-glycoproteins. For validation we selected and characterized one monoclonal antibody (6C5) directed to a Tn-glycopeptide in dysadherin (FXYD5), known to be upregulated in cancer and promote metastasis. While dysadherin is widely expressed also in normal cells, we demonstrated that the 6C5 epitope is specifically expressed in cancer.

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells.

The hemostatic system comprises platelet aggregation, coagulation, and fibrinolysis, and is critical to the maintenance of vascular integrity. Multiple studies indicate that glycans play important roles in the hemostatic system; however, most investigations have focused on N-glycans because of the complexity of O-glycan analysis. Here we performed the first systematic analysis of native-O-glycosylation using lectin affinity chromatography coupled to liquid chromatography mass spectrometry (LC-MS)/MS to determine the precise location of O-glycans in human plasma, platelets, and endothelial cells, which coordinately regulate hemostasis. We identified the hitherto largest O-glycoproteome from native tissue with a total of 649 glycoproteins and 1123 nonambiguous O-glycosites, demonstrating that O-glycosylation is a ubiquitous modification of extracellular proteins. Investigation of the general properties of O-glycosylation established that it is a heterogeneous modification, frequently occurring at low density within disordered regions in a cell-dependent manner. Using an unbiased screen to identify associations between O-glycosites and protein annotations we found that O-glycans were over-represented close (± 15 amino acids) to tandem repeat regions, protease cleavage sites, within propeptides, and located on a select group of protein domains. The importance of O-glycosites in proximity to proteolytic cleavage sites was further supported by in vitro peptide assays demonstrating that proteolysis of key hemostatic proteins can be inhibited by the presence of O-glycans. Collectively, these data illustrate the global properties of native O-glycosylation and provide the requisite roadmap for future biomarker and structure-function studies.

Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma.

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.