Employing mesenchymal stem cells to support tumor-targeted delivery of extracellular vesicle (EV)-encapsulated microRNA-379.

TLDR: The data presented support miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2.

Abstract: 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.

Figures

Figure 1. Analysis of miR-379 expression in matched primary tumors and lymph node metastases from the same patients (t-test, P<0.005).

Figure 2. Functional Impact of miR-379 in Breast Cancer. A (i) Sample image of transduced RFPpositive T47D cells following puromycin selection; (ii) Equivalent DAPI stained nuclei confirming all cells express RFP; (iii) Confirmation of elevated miR-379 expression following transduction of T47D and HCC cells. B Human angiogenesis proteome profile array of (i) HCC-NTC lysate compared to (ii) HCC-379 lysate to determine the effect of miR-379 on pro-angiogenic proteins; (iii) Impact of miR379 on COX-2 protein expression determined using western blot in HCC cells.

Figure 6. Schematic showing timeline of In Vivo study tracking HCC-luciferase(luc) tumor progression following IV injection of MSC-379, MSC-NTC, Exo-NTC or Exo-379

Figure 5. In Vivo analysis of therapeutic impact of IV administered engineered MSCs or EVs. A RFPlabeled MSCs engrafted within tumor following IV administration B (i) Quantitative bioluminescence following IP administration of D-Luciferin (15mg/ml) weekly following IV injection of 1 x 106 cells/50µl PBS of MSC-NTC or MSC-379; (ii) Sample IVIS images taken over the course of the study of mice that received MSC-NTC or MSC-379 cells. C (i) Imaging performed on mice following 4 repeat IV injections of 2.6 x 10 7 EVs/50 µl PBS EV-NTC or EV-379. (ii) Sample IVIS images over the course of the study of mice that received EV-NTC or EV-379.