High content, quantitative AFM analysis of the scalable biomechanical properties of extracellular vesicles.

Extracellular vesicles (EVs) are studied extensively as natural biomolecular shuttles and for their diagnostic and therapeutic potential. This exponential rise in interest has highlighted the need for highly robust and reproducible approaches for EV characterisation. Here we optimise quantitative nanomechanical tools and demonstrate the advantages of EV population screening by atomic force microscopy (AFM). Our high-content informatics analytical tools are made available for use by the EV community for widespread, standardised determination of structural stability. Ultracentrifugation (UC) and sonication, the common mechanical techniques used for EV isolation and loading respectively, are used to demonstrate the utility of optimised PeakForce-Quantitative Nano Mechanics (PF-QNM) analysis. EVs produced at an industrial scale exhibited biochemical and biomechanical alterations after exposure to these common techniques. UC resulted in slight increases in physical dimensions, and decreased EV adhesion concurrent with a decrease in CD63 content. Sonicated EVs exhibited significantly reduced levels of CD81, a decrease in size, increased Young's modulus and decreased adhesive force. These biomechanical and biochemical changes highlight the effect of EV sample preparation techniques on critical properties linked to EV cellular uptake and biological function. PF-QNM offers significant additional information about the structural information of EVs following their purification and downstream processing, and the analytical tools will ensure consistency of analysis of AFM data by the EV community, as this technique continues to become more widely implemented.

Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11.

Polycystic ovary syndrome (PCOS) is a common gynaecological disorder, with a prevalence of up to 12% of women of reproductive age, and is in part characterised by elevated circulating androgens and aberrant expression of androgen receptor (AR) in the endometrium. A high percentage of PCOS patients suffer from infertility, a condition that appears to be linked to mistimed and incomplete decidualisation critically affecting events surrounding embryo implantation. The aim of this study was to examine the involvement of MAGEA11, and the genome-wide role of AR in PCOS. We determined that elevated androgen levels on PCOS cells had an impact on the delayed and incomplete decidual transformation of endometrial cells. The AR co-regulator MAGEA11, a known enhancer of AR function, was constitutively overexpressed throughout the menstrual cycle of PCOS patients, co-localised in the nucleus of PCOS stromal tissue and cells and formed a molecular complex with AR. Genome-wide AR analysis in PCOS stromal cells revealed that AR targets included genes involved in cell death and apoptosis, as well as genes commonly dysregulated in endometrial cancer. Enhanced MAGEA11 and AR-mediated transcriptional regulation may impact on a correct endometrial decidualisation response, subsequently affecting endometrial receptivity in these infertile women. KEY MESSAGES: MAGEA11 and AR are overexpressed in hyperandrogenic PCOS patients. MAGEA11-AR overexpression in PCOS correlates with delayed decidualisation. AR and MAGEA11 associate in a molecular complex. AR directly regulates a unique set of genes controlling gene differentiation.

Antibody-drug conjugates and other nanomedicines: the frontier of gynaecological cancer treatment.

Gynaecological cancers: malignancies of the cervix, uterus, ovaries, vagina and vulva, are responsible for over 1.1 million new cancer cases and almost half a million deaths annually. Ovarian cancer in particular is difficult to treat due to often being diagnosed at a late stage, and the incidence of uterine and vulvar malignancies are both on the rise. The field of nanomedicine is beginning to introduce drugs into the clinic for oncological applications exemplified by the liposomal drugs, Doxil and Myocet, the nanoparticle, Abraxane and antibody-drug conjugates (ADCs), Kadcyla and Adcetris. With many more agents currently undergoing clinical trials, the field of nanomedicine promises to have a significant impact on cancer therapy. This review considers the state of the art for nanomedicines currently on the market and those being clinically evaluated for the treatment of gynaecological cancers. In particular, it focuses on ADCs and presents a methodology for their rational design and evaluation.

Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity in HER2 overexpressing breast cancer.

AIM: Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. METHODS: We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. RESULTS: In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. CONCLUSION: Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development.