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Glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate, are an important component of the tumor microenvironment (TME). These carbohydrates are attached to proteoglycans at the surface of tumor cells and play a vital role in malignant transformation. Heparan sulfate (HS) glycosaminoglycans have been demonstrated to play a pleiotropic role in regulating the activity of important cellular receptors and downstream signaling pathways, and are implicated in cancer cell proliferation, angiogenesis, invasion, and metastasis. Heparinase, an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans is expressed in several sarcoma subtypes. We measured heparinase activity in several cell lines and found that A673, CHP100, CHLA9 and RD-ES Ewing’s sarcoma cell lines have significantly higher levels of heparinase activity compared to the heparinase-deficient CHO-K1 psgD-677 cell line. Interference with heparinase or heparin sulfate function(s) may represent a therapeutic approach in sarcoma. To this end, we tested these cell lines by measuring cell growth using a cell viability assay, in the presence or absence of GTC3300P, a small molecule inhibitor of protein interaction with glycosaminoglycan. We demonstrate that the Ewing’s sarcoma cell lines exhibit potent and significant inhibition in cell growth in vitro, in the presence of drug. Western blot analysis shows induction of cleaved Poly-ADP Ribose Polymerase (PARP), yH2AX, as well as cleaved caspase-3, known markers of apoptosis and down regulation of mTORC1 targets such as phospho-S6 (p-S6), in Ewing’s sarcoma. Apoptosis was confirmed by the induction of sub-G1 population in Ewing’s sarcoma by cell cycle analysis. Previous reports have indicated that heparinase activity is strongly implicated in cell migration and invasion and is associated with tumor metastasis, angiogenesis and inflammation. This was confirmed by LC-MS/MS proteomics and bioinformatics (gene ontology and gene set enrichment) analysis of these multiple cell lines, which identified alterations in numerous biomarkers in these pathways, including multiple ribosomal protein subunits, spliceosomes components, and complement coagulation cascade. We further conducted a cell migration assay and demonstrate that GTC3300 significantly inhibits cell migration in Ewing’s sarcoma cell lines, but not in heparinase-deficient psg677 cells. Taken together our data strongly suggests that targeting protein interaction with glycosaminoglycans including heparan sulfate in Ewing’s sarcoma represents a novel approach and merits further investigation Citation Format: Tahir N. Sheikh, Elgilda Musi, Alex J. Rai, Gary K. Schwartz. Development of small molecule inhibitors of protein interaction with glycosaminoglycans. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4977.

Uveal melanoma (UM) is a rare melanoma subtype with an estimated annual incidence of approximately 2000 in the United States. Despite excellent rates of local disease control with surgery or radiotherapy, nearly 50% of patients will develop metastatic disease, mostly to the liver, with a life expectancy of 12 months. The receptor tyrosine kinase MET is overexpressed in 60-86% of cases and, together with its ligand HGF, it plays a significant role in UM migration and metastasis. In recent years, it has been demonstrated that tumor cells communicate with the microenvironment at distant organs by secreting exosomes, extracellular small vesicles released into body fluids, to support cancer development, immune evasion and metastatic progression. Exosomes are able to stably transfer their unique cargos of proteins and RNA from the primary tumor to distant sites, such as the pre-metastatic “niche”. The functional role of UM exosomes in the tumor microenvironment cross-talk is largely unknown. To get deeper insight into UM exosome biology, we have isolated exosomes from UM cells before and after treatment with the MET inhibitor crizotinib and examined their content by proteomic analysis. Approximately 600 proteins were identified from each of these samples, with 444 proteins in common and 145 and 167 proteins uniquely detected in exosomes from untreated and treated cells, respectively. The expression of several exosome-associated proteins, including MET, were suppressed by crizotinib, suggesting that the exosomal cargo can be pharmacologically manipulated. Exposure of normal hepatocytes to UM-derived exosomes results in activation of MET in a dose dependent fashion, with induction of both the MAPK and AKT/PI3K pathways. Exosomes derived from serum of patients with advanced uveal melanoma also express MET and induced similar effects in hepatocytes. Furthermore, UM-derived exosomes activate the expression of pro-angiogenic and pro-inflammatory factors in normal hepatocytes, while exosomes isolated from crizotinib-treated UM cells reduced these effects. UM-derived exosomes also stimulated immune cells to release cytokines and chemokines, including IL-6, IL-8 CCL2, CCL3, CCL5 and CXCL1. Functionally, we demonstrated that conditioned media from exosome-treated hepatocyte highly stimulated UM cell migration, which could be repressed by the combination of MET and cytokine receptor inhibitors. These findings provide evidence that UM derived exosomes play a role in reprogramming the recipient cells to facilitate the establishment of metastasis, and the inhibition of exosome-mediated signaling may provide a potential therapeutic strategy in the early stages of metastasis. Citation Format: Grazia Ambrosini, Alex J. Rai, Blake Ebert, Gary K. Schwartz. Uveal melanoma exosomes express MET and re-program the recipient cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6082.

Early detection remains the most promising approach to improve long-term survival of patients with ovarian cancer. In a five-center case-control study, serum proteomic expressions were analyzed on 153 patients with invasive epithelial ovarian cancer, 42 with other ovarian cancers, 166 with benign pelvic masses, and 142 healthy women. Data from patients with early stage ovarian cancer and healthy women at two centers were analyzed independently and the results cross-validated to discover potential biomarkers. The results were validated using the samples from two of the remaining centers. After protein identification, biomarkers for which an immunoassay was available were tested on samples from the fifth center, which included 41 healthy women, 41 patients with ovarian cancer, and 20 each with breast, colon, and prostate cancers. Three biomarkers were identified as follows: (a) apolipoprotein A1 (down-regulated in cancer); (b) a truncated form of transthyretin (down-regulated); and (c) a cleavage fragment of inter-α-trypsin inhibitor heavy chain H4 (up-regulated). In independent validation to detect early stage invasive epithelial ovarian cancer from healthy controls, the sensitivity of a multivariate model combining the three biomarkers and CA125 [74% (95% CI, 52–90%)] was higher than that of CA125 alone [65% (95% CI, 43–84%)] at a matched specificity of 97% (95% CI, 89–100%). When compared at a fixed sensitivity of 83% (95% CI, 61–95%), the specificity of the model [94% (95% CI, 85–98%)] was significantly better than that of CA125 alone [52% (95% CI, 39–65%)]. These biomarkers demonstrated the potential to improve the detection of early stage ovarian cancer.

Biomarkers have become invaluable tools in the clinical space for cancer detection, diagnosis, patient prognosis and treatment selection. An ideal biomarker should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. Although many candidate biomarkers for various diseases have been proposed in the literature, very few have made their way to clinical use, and for certain types of cancer there are no reliable biomarker options. The use of prostate specific antigen (PSA) has led to a dramatic increase in the diagnosis of prostate cancer - over 230,000 new cases in 2014. However, most of these cases are of low risk disease that is unlikely to cause significant morbidity and mortality, leading to the accepted notion that prostate cancer is overdiagnosed. On the other hand, while it is a sensitive test, less than ¼ of patients undergoing a prostate biopsy based on PSA testing are actually found to have the disease. The identification of biomarkers that are more senstitive and specific predictors for the presence of prostate cancer, as well as for the aggressiveness of the disease, could decrease the complications and morbidity related to prostate biopsies and definitive treatment. The circulating miRNA in blood could potentially fill this void, providing biomarkers suitable for real-time detection and application to the management of prostate cancer patients. The main advantages of miRNAs versus longer molecules (mRNA, lncRNA) include: (i) smaller number of sequences The objective of this project was to develop a set of reagents and tools for efficient recovery and analysis of circulating miRNAs. Our team has built a complete workflow to provide: (1) high throughput isolation of total serum RNA using a new MagMaxTM mirVanaTM reagent kit for the KingFisherTM Flex and Duo instruments, and (2) downstream analysis of microRNA profiles by qRT-PCR using next generation TaqMan® microRNA assays. The effectiveness of this workflow is exemplified by analysis of a panel of miRNAs in samples derived from serum of patients with metastatic prostate cancer versus control groups. Following this fast and easy workflow suitable for serum and other body fluids, disease-specific RNA signatures can be identified and used as biomarkers. TaqMan® is a registered Trademark of Roche Molecular Systems, Inc. used under permission and license. Citation Format: Emily M. Zeringer, Alex J. Rai, Joel DeCastro, Luming Qu, Marie Gonzalez, Laura Chapman, Alexander V. Vlassov, Susan M. Magdaleno, Chunmei Liu, Fangqi Hu, Shoulian Dong, Linda Wong. A complete workflow for high throughput isolation of serum microRNAs and downstream analysis by qRT-PCR: application to cancer biomarker discovery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3387. doi:10.1158/1538-7445.AM2015-3387

Exosomes are small vesicles (30-150 nm) found in abundance in human body fluids which function as carriers of different species of RNA and protein between diverse locations in the body. The spectrum of current scientific interest in exosomes is wide and ranges from studying their functions and pathways to utilizing them in diagnostics and therapeutics development. As such, there is a growing need for quick and easy methods for both isolation of exosomes and analysis of their cargo. We present herein a workflow for exosome isolation and analysis which entails: (i) fast and efficient isolation of exosomes from serum, plasma, and urine of both healthy donors and patients with prostate cancer, using Total Exosome Isolation reagents; (ii) characterization of their size distribution and count with Nanosight LM10 instrument; (iii) extraction of exosome “cargo” with Total Exosome RNA and Protein Isolation kit; (iv) characterization of exosomal RNA content using the Ion Torrent PGM sequencing and qRT-PCR. The protocol described herein lays the groundwork for the development of a standardized operating procedure (SOP) for isolation of exosomes and downstream analysis of their constituents, using clinical samples. We demonstrate that cancer-specific RNA signatures residing within the exosomes can be delineated from different patient cohorts. This is the first step towards developing a method whereby performance characteristics can be measured and used to optimize a validated assay useful for routine testing of clinical samples. Citation Format: Robert A. Setterquist, Alex J. Rai, Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Susan Magdaleno, Alexander V. Vlassov. Development of a standard operating procedure for exosome isolation and analysis using clinical samples: Application to cancer biomarker discovery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1870. doi:10.1158/1538-7445.AM2014-1870