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Adult Mesenchymal Stem Cells (MSCs) have a well-established tumor-homing capacity, highlighting potential as tumor-targeted delivery vehicles. MSCs secrete extracellular vesicle (EV)-encapsulated microRNAs, which play a role in intercellular communication. The aim of this study was to characterize a potential tumor suppressor microRNA, miR-379, and engineer MSCs to secrete EVs enriched with miR-379 for in vivo therapy of breast cancer. miR-379 expression was significantly reduced in lymph node metastases compared to primary tumor tissue from the same patients. A significant reduction in the rate of tumor formation and growth in vivo was observed in T47D breast cancer cells stably expressing miR-379. In more aggressive HER2-amplified HCC-1954 cells, HCC-379 and HCC-NTC tumor growth rate in vivo was similar, but increased tumor necrosis was observed in HCC-379 tumors. In response to elevated miR-379, COX-2 mRNA and protein was also significantly reduced in vitro and in vivo. MSCs were successfully engineered to secrete EVs enriched with miR-379, with the majority found to be of the appropriate size and morphology of exosomal EVs. Administration of MSC-379 or MSC-NTC cells, or EVs derived from either cell population, resulted in no adverse effects in vivo. While MSC-379 cells did not impact tumor growth, systemic administration of cell-free EVs enriched with miR-379 was demonstrated to have a therapeutic effect. The data presented support miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Exploiting the tumor-homing capacity of MSCs while engineering the cells to secrete EVs enriched with miR-379 holds exciting potential as an innovative therapy for metastatic breast cancer.

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Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer.

It is believed that microRNAs have potential as circulating biomarkers of disease; however, successful clinical implementation remains a challenge. This chapter highlights broad variations in approaches to microRNA analysis where whole blood, serum and plasma have each been employed as viable sources. Further discrepancies in approaches are seen in endogenous controls and extraction methods utilized. This has resulted in contradictory publications, even when the same microRNA is targeted in the same disease setting.Analysis of blood samples highlighted the impact of both collection method and storage, on the microRNA profile. Analysis of a panel of microRNAs across whole blood, serum, and plasma originating from the same individual emphasized the impact of starting material on microRNA profile. This is a highly topical field of research with immense potential for translation into the clinical setting. Standardization of sample harvesting, processing and analysis will be key to this translation. Methods of sample harvesting, preservation, and analysis are outlined, with important mitigating factors highlighted.

Mesenchymal Stem Cells (MSCs) are multipotent stromal cells known to migrate specifically to the sites of tumors and metastases, raising potential as tumor-targeted delivery vehicles for therapeutic agents. MSCs secrete exosomes containing genetic material including microRNAs (miRs), that are effectively taken up by cells. Previous work has shown miR-379 to be significantly reduced in breast tumor tissue, highlighting a potential tumor suppressor role. This study aimed to establish a mechanism of action for miR-379, and to engineer MSCs to secrete exosomes enriched with the miR for tumor-targeted delivery. Methods: RNA was isolated from breast tumors and matching lymph node metastases from the same patients, and miR-379 expression quantified by RQ-PCR. HCC1954 breast cancer cells were transduced with lentivirus to express elevated miR-379 (HCC-379) or a control sequence (HCC-NTC). Cells were implanted into the mammary fat pad (MFP) of mice and tumor progression monitored. Subsequently, MSCs were transduced to generate MSC-379 and MSC-NTC. MSC-secreted exosomes were isolated by differential centrifugation, microfiltration and ultracentrifugation. The morphology, size and number of isolated exosomes was characterized using Transmission Electron Microscopy, Western Blot and Nanoparticle Tracking Analysis respectively. Exosomal miRs were analysed by RQ-PCR. MFP tumors were established using HCC1954-luciferase cells, followed by IV injection of MSC-379 or MSC-NTC cells, or sequential doses of exosomes derived from either cell population. Results: miR-379 expression was significantly reduced in lymph node metastases compared to primary tumors from the same patients (p=0.009), supporting a tumor suppressor role. Analysis of HCC-379 and HCC-NTC tumor growth In Vivo showed no difference in tumor size, however an increase in tumour necrosis (5-50%) and decrease in lymph node invasion was observed in HCC-379 tumors. Investigation of a potential role for miR-379 in regulating COX-2, revealed an inverse relationship (r = -0.48, p=0.02) at a mRNA level, further confirmed at a protein level. MSC-379 cells showed no significant change in migratory or proliferative capacity In Vitro. MSC-secreted exosomes were confirmed to be the correct morphology and size (30-120nm), and to express the exosome-associated protein CD63. An increase in miR-379 (>5 fold) was observed in exosomes secreted by MSC-379 compared to MSC-NTC cells. Administration of MSC-379 or MSC-NTC cells, or exosomes derived from either cell population, was well tolerated In Vivo with no adverse effects observed. Monitoring tumor response to therapy using IVIS is ongoing. Conclusion: The data presented supports miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Engineering tumor-targeted MSCs to secrete exosomes enriched with miR-379 holds exciting potential as a novel therapy for breast cancer. Citation Format: Killian P. O'Brien, Katie Gilligan, Sonja Khan, Brian Moloney, Kerry Thompson, Pierce Lalor, Peter Dockery, Helen Ingoldsby, Michael J. Kerin, Roisin M. Dwyer. Engineering Mesenchymal Stem Cells (MSCs) to support tumor-targeted delivery of exosome-encapsulated microRNA-379 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3045. doi:10.1158/1538-7445.AM2017-3045

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