We employed laser microdissection to selectively harvest histology-resolved tumors and stroma from formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma (HNSCC) tissues. Peptide digests from the LMD-enriched HNSCC tissue were analyzed by quantitative mass-spectrometry-based proteomics using a data independent analysis approach. In paired samples, excellent proteome coverage was achieved, having quantified 6668 proteins with a median quantitative coefficient of variation under 10%. Significant differences in relevant functional pathways between the tumor and the stroma regions were observed. Extracellular matrix (ECM) was identified as a major component enriched in the stroma, including many cancer-associated fibroblast signature proteins in this compartment. We demonstrate the potential for comparative deep proteome analysis from a very low starting input in a scalable format. Correlating such results with clinical features or disease progression will likely enable the identification of novel targets for disease classification and interventions.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). In spite of its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the secretome of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP has functional relevance, and may regulate physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Local invasiveness of head and neck squamous cell carcinoma (HNSCC) is a complex phenomenon supported by interaction of the cancer cells with the tumor microenvironment (TME). We and others have shown that cancer-associated fibroblasts (CAFs) are a component of the TME that can promote local invasion in HNSCC and other cancers. Here we report that the secretory enzyme heparan-6-O-endosulfatase 2 (Sulf-2) directly affects the CAF-supported invasion of the HNSCC cell lines SCC35 and Cal33 into Matrigel. The Sulf-2 knockout (KO) cells differ from their wild type counterparts in their spheroid growth and formation, and the Sulf-2-KO leads to decreased invasion in a spheroid co-culture model with the CAF. Next, we investigated whether a fucosylated chondroitin sulfate isolated from the sea cucumber Holothuria floridana (HfFucCS) affects the activity of the Sulf-2 enzyme. Our results show that HfFucCS not only efficiently inhibits the Sulf-2 enzymatic activity but, like the Sulf-2 knockout, inhibits Matrigel invasion of SCC35 and Cal33 cells co-cultured with primary HNSCC CAF. These findings suggest that the heparan-6-O-endosulfatases regulate local invasion and could be therapeutically targeted with the inhibitory activity of a marine glycosaminoglycan.

Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the postsynthetic editing of heparan sulfate (HS), which regulates many important biological processes. The activity of the Sulf-2 and its substrate specificity remain insufficiently characterized in spite of more than two decades of studies of this enzyme. This is due, in part, to the difficulties in the production and isolation of this highly modified protein and due to the lack of well-characterized synthetic substrates for the probing of its catalytic activity. We introduce synthetic HS oligosaccharides to fill this gap, and we use our recombinant Sulf-2 protein to show that a paranitrophenol (pNP)-labeled synthetic oligosaccharide allows a reliable quantification of its enzymatic activity. The substrate and products of the desulfation reaction are separated by ion exchange high-pressure liquid chromatography and quantified by UV absorbance. This simple assay allows the detection of the Sulf-2 activity at high sensitivity (nanograms of the enzyme) and specificity. The method also allowed us to measure the heparan 6-O-endosulfatase activity in biological samples as complex as the secretome of cancer cell lines. Our in vitro measurements show that the N-glycosylation of the Sulf-2 enzyme affects the activity of the enzyme and that phosphate ions substantially decrease the Sulf-2 enzymatic activity. This assay offers an efficient, sensitive, and specific measurement of the heparan 6-O-endosulfatase activity that could open avenues to in vivo activity measurements and improve our understanding of the enzymatic editing of the sulfation of heparan.

Targeted quantification of glycoproteins has not reached its full potential because of limitations of the existing analytical workflows. In this study, we introduce a targeted microflow LC-MS/MS-PRM method for the quantification of multiple glycopeptides in unfractionated serum samples. The entire preparation of 16 samples in a batch is completed within 3 h, and the LC-MS quantification of all the glycoforms in a sample is completed in 15 min in triplicate, including online capture and desalting. We demonstrate applicability of the workflow on a multiplexed quantification of eight N-glycoforms of immunoglobulin G (IgG) together with two O-glycoforms of hemopexin (HPX). We applied the assay to a serologic study of fibrotic liver disease in patients of HCV etiology. The results document that specific IgG- and HPX-glycoforms detect efficiently fibrotic disease of different degree, and suggest that the LC-MS/MS-PRM assays may provide rapid and reproducible biomarker assay targeting simultaneously the N- and O-glycoforms of the peptides. We propose that such high throughput multiplexed methods may advance the clinical use of the LC-MS/MS assays.

Development of high throughput robust methods is a prerequisite for a successful clinical use of LC-MS/MS assays. In earlier studies, we reported that nLC-MS/MS measurement of the O-glycoforms of HPX is an indicator of liver fibrosis. In this study, we show that a microflow LC-MS/MS method using a single column setup for capture of the analytes, desalting, fast gradient elution, and on-line mass spectrometry measurements, is robust, substantially faster, and even more sensitive than our nLC setup. We demonstrate applicability of the workflow on the quantification of the O-HPX glycoforms in unfractionated serum samples of control and liver disease patients. The assay requires microliter volumes of serum samples, and the platform is amenable to one hundred sample injections per day, providing a valuable tool for biomarker validation and screening studies.

In humans, mutations in the transcription factor encoding gene, FOXP2, are associated with language and Autism Spectrum Disorders (ASD), the latter characterized by deficits in social interactions. However, little is known regarding the function of Foxp2 in male or female social behavior. Our previous studies in mice revealed high expression of Foxp2 within the medial subnucleus of the amygdala (MeA), a limbic brain region highly implicated in innate social behaviors such as mating, aggression, and parental care. Here, using a comprehensive panel of behavioral tests in male and female Foxp2+/– heterozygous mice, we investigated the role Foxp2 plays in MeA-linked innate social behaviors. We reveal significant deficits in olfactory processing, social interaction, mating, aggressive, and parental behaviors. Interestingly, some of these deficits are displayed in a sex-specific manner. To examine the consequences of Foxp2 loss of function specifically in the MeA, we conducted a proteomic analysis of microdissected MeA tissue. This analyses revealed putative sex differences expression of a host of proteins implicated in neuronal communication, connectivity, and dopamine signaling. Consistent with this, we discovered that MeA Foxp2-lineage cells were responsive to dopamine with differences between males and females. Thus, our findings reveal a central and sex-specific role for Foxp2 in social behavior and MeA function.

Neoantigen discovery in pediatric brain tumors is hampered by their low mutational burden and scant tissue availability. Here we develop a proteogenomic approach combining tumor DNA/RNA sequencing and mass spectrometry proteomics to identify tumor-restricted (neoantigen) peptides arising from multiple genomic aberrations to generate a highly target-specific, autologous, personalized T cell immunotherapy. Our data indicate that aberrant splice junctions are the primary source of neoantigens in medulloblastoma, a common pediatric brain tumor. Proteogenomically identified tumor-specific peptides are immunogenic and generate MHC II-based T cell responses. Moreover, polyclonal and polyfunctional T cells specific for tumor-specific peptides effectively eliminate tumor cells in vitro. Targeting tumor-specific antigens obviates the issue of central immune tolerance while potentially providing a safety margin favoring combination with other immune-activating therapies. These findings demonstrate the proteogenomic discovery of immunogenic tumor-specific peptides and lay the groundwork for personalized targeted T cell therapies for children with brain tumors.

The development of therapeutics for muscle diseases such as facioscapulohumeral dystrophy (FSHD) is impeded by a lack of objective, minimally invasive biomarkers. Here we identify circulating miRNAs and proteins that are dysregulated in early-onset FSHD patients to develop blood-based molecular biomarkers. Plasma samples from clinically characterized individuals with early-onset FSHD provide a discovery group and are compared to healthy control volunteers. Low-density quantitative polymerase chain reaction (PCR)-based arrays identify 19 candidate miRNAs, while mass spectrometry proteomic analysis identifies 13 candidate proteins. Bioinformatic analysis of chromatin immunoprecipitation (ChIP)-seq data shows that the FSHD-dysregulated DUX4 transcription factor binds to regulatory regions of several candidate miRNAs. This panel of miRNAs also shows ChIP signatures consistent with regulation by additional transcription factors which are up-regulated in FSHD (FOS, EGR1, MYC, and YY1). Validation studies in a separate group of patients with FSHD show consistent up-regulation of miR-100, miR-103, miR-146b, miR-29b, miR-34a, miR-454, miR-505, and miR-576. An increase in the expression of S100A8 protein, an inflammatory regulatory factor and subunit of calprotectin, is validated by Enzyme-Linked Immunosorbent Assay (ELISA). Bioinformatic analyses of proteomics and miRNA data further support a model of calprotectin and toll-like receptor 4 (TLR4) pathway dysregulation in FSHD. Moving forward, this panel of miRNAs, along with S100A8 and calprotectin, merit further investigation as monitoring and pharmacodynamic biomarkers for FSHD.

Abstract T cell immunotherapies are promising new tools to combat high-risk subgroups of medulloblastoma without increasing the late effects burden. The ideal target antigen of an effective antitumor T-cell response is abundantly expressed by tumor cells but not by normal tissues, in order to limit off-target effects. Tumors translate a host of highly novel transcripts that are the result of aberrations in tumor DNA and the unmasking of alternative or novel exons. We developed a novel proteogenomic approach to identify tumor-restricted peptides and used them to select and expand T cells capable of mounting a tumor-specific cytotoxic immune response. Using RNA-seq and WGS data, we created personalized custom searchable databases containing predicted novel proteins from somatic mutations, novel junctions and fusion transcripts from 56 medulloblastoma tumors. By searching these databases with raw mass spectrometry data from paired medulloblastoma tumors, we identified tens of neoantigen peptides arising from the translation of tumor-specific transcripts; novel isoforms being the predominant source. We tested these peptides for their ability to select and expand autologous polyclonal populations of T cells and tested the immunogenicity of each individual peptide. Flow cytometry revealed populations of CD4+ and CD8+ cells with an activation profile marked by IFN-γ and TNF-α. Immunosuppressive marker profiles were also characterized. Using cytotoxicity assays, we demonstrated that tumor specific T cells can eliminate neoantigen bearing tumor cells. Thus, proteogenomics can identify immunogenic tumor specific peptides that can be used to create a personalized, targeted T cell therapy for children with high risk medulloblastoma.

Medulloblastoma is a common childhood brain tumor for which current therapies fail to cure high-risk subgroups, including metastatic and recurrent disease. Toxicity limits further intensification of conventional chemo-radiation therapy, and most survivors endure significant lifelong sequelae from treatment. There is an urgent need to develop new tools to combat this cancer without increasing the late effects burden. Neoantigens, which are exclusively expressed on tumor cells, are one of the main targets of an effective antitumor T-cell response. The ideal target antigen is abundantly expressed by tumor cells but not by normal tissues, in order to limit off-target effects. Tumors translate a host of highly novel transcripts that are the result of aberrations in tumor DNA and the unmasking of alternative or novel exons. We used a novel proteogenomic approach to identify tumor-restricted peptides and used them to select and expand T cells capable of mounting a tumor-specific cytotoxic immune response, with no off-target effects for patients with MB. Using RNA-seq and WGS data, we created personalized custom searchable databases containing predicted novel proteins from somatic mutations, novel isoforms, and fusion proteins from 56 medulloblastoma tumors. By searching this database with raw mass spectrometry data from the paired medulloblastoma tumor, we have identified tens of peptides arising from the translation of tumor-specific transcripts; these peptides are potential neoantigens. Our data indicate that in cases of tumors with low mutation rates, such as pediatric brain tumors, novel isoforms are the main source of neoantigens. In a pilot study, we have tested these peptides for their ability to select and expand polyclonal populations of T cells from the same patient whose tumor was the source of the peptides. The immunogenicity of each individual peptide was then determined. Flow cytometry cellular characterization reveals populations of both CD4+ and CD8+ cells with an activation profile marked by IFN-γ and TNF-α production. Immunosuppressive marker profiles have also been characterized for these cells. Using cytotoxicity assays, we demonstrated that tumor-specific T cells can eliminate neoantigen-bearing tumor cells. These findings demonstrate an initial proof of principle that proteogenomics can be used to identify immunogenic tumor specific peptides and lay the groundwork for a personalized, targeted T-cell therapy for children with high-risk medulloblastoma. Citation Format: Samuel Rivero-Hinojosa, Melanie Grant, Aswini Panigrahi, Huizhen Zhang, Veronika Caisova, Catherine Bollard, Brian Rood. Proteogenomic discovery of novel tumor proteins as neoantigens for personalized T-cell immunotherapy in pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A23.

Cardiac disease in Duchenne muscular dystrophy (DMD) is now the leading cause of death. Increasing cardiac fibrosis demonstrated by late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging (CMR) is associated with worse outcomes in DMD. We sought to identify novel serum biomarkers in

Objectives Otitis media (OM) is a ubiquitous pediatric disease leading to a significant health care burden. There is no medication beneficial to resolving COM fluid, highlighting the need for research in the field. Crucially, current human middle ear epithelial cell models are transformed cells not recapitulating physiological functions. Herein, we describe a new method to proliferate and differentiate pediatric primary middle ear epithelial cells (pMEEC) from patients as a physiological model for the study of OM. Methods We adapted a cell reprogramming protocol using irradiated fibroblast feeder medium in addition to Rho kinase inhibitor to proliferate pMEEC collected during cochlear implant surgery. Cells were plated on transwell membranes, proliferated with conditionally reprogrammed culture medium, and transferred to air–liquid interface (ALI). Cultures were maintained for 4 weeks at ALI, photos were taken and cell lysates and secretions were collected over time for characterization analysis using quantitative polymerase chain reaction, Western bolt, and proteomics. Keratins, MUC5B and MUC5AC mucins, and beta tubulin (TUBB) were analyzed at the mRNA and protein level. Results Cultures took a mean of 2 weeks to proliferate before transwell plating and forming a tight epithelium at ALI from 2 to 4 weeks. Although mRNA expression of MUC5B, MUC5AC, TUBB, and keratin 5 (KRT5) were variable depending on the differentiation stage and the patient, both TUBB and KRT5 proteins were detected until week 2. Conclusion We demonstrate a novel method to proliferate and differentiate pMEECs that express epithelial markers and that are able to secrete mucins for the study of OM. Level of Evidence NA

BackgroundPaediatric ulcerative colitis (UC) patients would be well-served by a non-steroidal drug to control mucosal inflammation, without long-term and costly commitment to biologics. Vamorolone is a first-in-class alternative to glucocorticoids (GCs), under development for children with Duchenne muscular dystrophy (DMD); preliminary findings demonstrate improved safety compared with GCs1,2. We sought to define NFkB-regulated, GC-responsive serum biomarkers for use in a proof-of-concept pilot trial of vamorolone in UC, focusing on TFF3 (produced by intestinal epithelia, GC-responsive in UC), and CCL22 (produced by macrophages, GC-responsive in UC and other inflammatory diseases)3,4.MethodsSera from 10 children with IBD (7 UC, 3 CD) were tested pre and post prednisone/prednisolone (1 mg/kg/day, max 40 mg, 1–4 weeks); 210 proteins responsive to GCs in UC 3 were analyzed using SOMAscan. Proteins that showed significant change over time were correlated with change in Paediatric Ulcerative Colitis Activity Index (PUCAI) (p < 0.05 significance). Percent change in circulating CCL22 was compared with percent change in DMD patients treated with vamorolone (2 and 6 mg/kg/day, 2 weeks)2. Immunoassays were utilized to validate SOMAscan data.ResultsCCL22 and TFF3 validated as decreased by GCs in IBD (p=0.005, p < 0.001). Decrease in TFF3 correlated with decrease in PUCAI (r=0.741, p=0.022); decrease in CCL22 did not correlate with change in PUCAI. Magnitude of CCL22 decrease in GC-treated UC patients was comparable to that seen in DMD patients treated with 6 mg/kg of vamorolone (47% vs. 33%). SOMAscan findings in UC validated by immunoassays.ConclusionDecreases in serum CCL22 likely reflect effect on innate immune response, while decreases in serum TFF3 may reflect intestinal-specific effects of GCs in UC. CCL22 and TFF3, measured by immunoassays, may be useful as objective secondary outcomes reflective of NFkB inhibition and anti-inflammatory activity in a proof-of-concept trial of vamorolone in paediatric UC.ReferencesHoffman EP, et al. Phase 1 trial of vamorolone, a first-in-class steroid, shows improvements in side effects via biomarkers bridged to clinical outcomes. Steroids. 2018 Jun;134:43–52.Conklin LS, et al. Phase IIa trial in Duchenne muscular dystrophy shows vamorolone is a first-in-class dissociative steroidal anti-inflammatory drug. Pharmacol Res. 2018 Oct;136:140–150.Heier CM, et al. Identification of pathway-specific serum biomarkers of response to glucocorticoid and infliximab treatment in children with inflammatory bowel disease. Clin Transl Gastroenterol. 2016 Sep 15;7(9): e192.Conklin LS, et al. Serum biomarkers of glucocorticoid response and safety in anti-neutrophil cytoplasmic antibody-associated vasculitis and juvenile dermatomyositis. Steroids. 2018 Dec; 140:159–166.Disclosure(s)LSC, JNvdA, and EPH are employees of ReveraGen BioPharma. LSC and JNvdA own stock options of ReveraGen. EPH is a co-founder of ReveraGen and owns founder shares.

The generation of high-quality pilot data that leads to “go/no-go decisions” regarding new or repurposed drugs is often problematic, particularly with a high placebo response rate. In short duration trials, pharmacodynamic (PD) biomarkers can demonstrate proof-of-concept of drug action that may or may not be bridged to clinical outcomes. We sought to identify early-changing PD biomarkers in patients with IBD. Clinical scores and serum were obtained at baseline and after 1–2 weeks of corticosteroids in 5 IBD patients. Using the SOMAscan assay, 45 published chronic steroid and infliximab-responsive proteins were evaluated. All were log-transformed and normality verified using the Shapiro-Wilk test. Pre- to post- treatment comparisons were performed using a paired t-test (p<0.01). Label-free proteome profiling using mass spectrometry was performed. Thirteen proteins responded consistently to 1–2 weeks of corticosteroids. These included biomarkers previously shown to be responsive in pediatric IBD patients after 2 doses of infliximab (CCL23, carnosinase, p-cadherin, CCL25, testican-2, AGT, and FAM3B) and after longer courses of corticosteroid therapy (AGT, afamin, GHBP, CCL22, MMP12, PROT C, Ly9). Four were detected by label-free mass spectrometry (AGT, afamin, PROT C, and carnosinase). Most are involved in regulation of inflammation, immune homeostasis, and extracellular matrix binding. In this small sample, change in some biomarkers correlated with short-term clinical response (carnosinase, testican-2, FAM3B). We have identified treatment-responsive proteins in IBD patients after several weeks of steroids, after 2 doses of infliximab, and within 1–2 weeks of treatment with steroids; these biomarkers may be useful for screening trials of anti-inflammatory drugs. For selection of candidates to study in larger, controlled trials, short-duration biomarker-based trials would be beneficial for patients.

Nontypeable Haemophilus influenzae (NTHi), one of the most common acute otitis media (OM) pathogens, is postulated to promote middle-ear epithelial remodeling in the progression of OM from acute to chronic. The goal of this study was to examine early quantitative proteomic secretome effects of NTHi lysate exposure in a human middle-ear epithelial cell (HMEEC) line. NTHi lysates were used to stimulate HMEEC, and conditional quantitative stable isotope labeling with amino acids in cell culture of cell secretions was performed. Mass spectrometry analysis identified 766 proteins across samples. Of interest, several heterogeneous nuclear ribonucleoproteins (hnRNPs) were regulated by NTHi lysate treatment, especially hnRNP A2B1 and hnRNP Q, known to be implicated in microRNA (miRNA) packaging in exosomes. After purification, the presence of exosomes in HMEEC secretions was characterized by dynamic light scattering (<100 nm), transmission electron microscopy, and CD63/heat shock protein 70 positivity. hnRNP A2B1 and hnRNP Q were confirmed to be found in exosomes by Western blot and proteomic analysis. Finally, exosomal miRNA content comprised 110 unique miRNAs, with 5 found to be statistically induced by NTHi lysate (miR-378a-3p + miR-378i, miR-200a-3p, miR-378g, miR30d-5p, and miR-222-3p), all known to target innate immunity genes. This study demonstrates that NTHi lysates promote release of miRNA-laden exosomes from middle-ear epithelium in vitro.

Although Bacillus Calmette-Guérin (BCG) vaccines against tuberculosis have been available for more than 90 years, their effectiveness has been hindered by variable protective efficacy and a lack of lasting memory responses. One factor contributing to this variability may be the diversity of the BCG strains that are used around the world, in part from genomic changes accumulated during vaccine production and their resulting differences in gene expression. We have compared the genomes and transcriptomes of a global collection of fourteen of the most widely used BCG strains at single base-pair resolution. We have also used quantitative proteomics to identify key differences in expression of proteins across five representative BCG strains of the four tandem duplication (DU) groups. We provide a comprehensive map of single nucleotide polymorphisms (SNPs), copy number variation and insertions and deletions (indels) across fourteen BCG strains. Genome-wide SNP characterization allowed the construction of a new and robust phylogenic genealogy of BCG strains. Transcriptional and proteomic profiling revealed a metabolic remodeling in BCG strains that may be reflected by altered immunogenicity and possibly vaccine efficacy. Together, these integrated-omic data represent the most comprehensive catalogue of genetic variation across a global collection of BCG strains.