Vyjayanthi Kinhal

Tumour-derived exosomes as a signature of pancreatic cancer - liquid biopsies as indicators of tumour progression

Pancreatic cancer is the fourth most common cause of death due to cancer in the world. It is known to have a poor prognosis, mostly because early stages of the disease are generally asymptomatic. Progress in pancreatic cancer research has been slow, leaving several fundamental questions pertaining to diagnosis and treatment unanswered. Recent studies highlight the putative utility of tissue-specific vesicles (i.e. extracellular vesicles) in the diagnosis of disease onset and treatment monitoring in pancreatic cancer. Extracellular vesicles are membrane-limited structures derived from the cell membrane. They contain specific molecules including proteins, mRNA, microRNAs and non-coding RNAs that are secreted in the extracellular space. Extracellular vesicles can be classified according to their size and/or origin into microvesicles (∼150-1000 nm) and exosomes (∼40-120 nm). Microvesicles are released by budding from the plasmatic membrane, whereas exosomes are released via the endocytic pathway by fusion of multivesicular bodies with the plasmatic membrane. This endosomal origin means that exosomes contain an abundance of cell-specific biomolecules which may act as a fingerprint of the cell of origin. In this review, we discuss our current knowledge in the diagnosis and treatment of pancreatic cancer, particularly the potential role of EVs in these facets of disease management. In particular, we suggest that as exosomes contain cellular protein and RNA molecules in a cell type-specific manner, they may provide extensive information about the signature of the tumour and pancreatic cancer progression.

Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells-liquid biopsies for monitoring complications of pregnancy

Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.

Proteomics method to identification of protein profiles in exosomes

Exosomes are membrane-bound nanovesicles that transport molecular signals (e.g., proteins) between cells and are released from a wide range of cells, including the human placenta. Interestingly, the levels of exosomes present in maternal circulation are higher in preeclamptic pregnancies and their protein content profile change in response to the microenvironment milieu. Through the discovery of candidate biomarkers, mass spectrometry (MS)-based proteomics may provide a better understanding of the pathophysiology underlying pregnancy-associated disorders. With advances in sample preparation techniques, computational methodologies, and bioinformatics, MS-based proteomics have addressed the challenge of identifying and quantifying thousands of proteins and peptides from a variety of complex biological samples. Despite increasing interest in biomarker diagnostics, the complex nature of biological matrices (e.g., plasma) poses a challenge for candidate biomarker discovery. Here we describe a workflow to prepare exosomes for proteomic analysis.

OP01.02: Placenta‐derived exosomes in pregnancies complicated with fetal growth restriction at term

Objectives: Evaluation of the fetus at risk for uteroplacental insufficiency and growth restriction applies spectral Doppler measurements of the fetal circulation. Our objective is to determine if fetuses with abdominal circumference (AC) below the 5th or weight (EFW) below the 10th percentile will have lower values for the cerebroplacental ratio or the cerebrorenal ratio. Methods: We evaluated 2900 unselected women with multiple associated fetal and maternal co morbidities in whom we measured both the CPR and the CRR using previously standardised methodology. Results: No discernible differences were found between fetuses with low AC <5 or 10 percentile and fetuses with EFW <10% and the CRR or CPR of the appropriately grown fetuses. We plotted the values of each on previously created reference curves (figure). Conclusions: The measurement of the CPR or the CRR among fetuses with AC <10th percentile was not better than EFW <10th percentile to identify fetuses that would ultimately have a lower value consistent with centralisation of fetal blood flow.

Pregnancy-Associated Exosomes Changes in Pregnancies Complicated by Small-for-Gestational-Age (SGA) Neonates and Intrauterine Growth Restriction (IUGR)

Figures will be available only online Underline represents presenting author; Asterisk represents senior author; Dagger represents an in-training author.

Extracellular vesicles (ev) size in shallow trophoblast invasion in old world non-human primates

Materials and Methods Introduction Marcel Chuecos, BS1, Edward Dick2, Gene Hubbard3, Cathy Perez2, Soumyalekshmi Nair4, Carlos Palma4,Vyjayanthi Kinhal4, Carlos Salomon4 and Natalia Shlabritz-Loutsevitch, MD, PhD1. 1OB/GYN Research, TTUHSC-Permian Basin, Odessa, Texas, United States. 2Southwest National Primate Center, Texas Biomedical Research Institute, San Antonio, Texas, USA . 3University of Texas Health Sciences Center at San Antonio, San Antonio, Texas, USA. 4Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Womens Hospital, The University of Queensland, Brisbane, QLD, Australia. 5Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Ochsner Clinic Foundation, New Orleans, Louisiana USA; Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, University of Concepción, Concepción, Chile