MicroRNAs for the treatment of breast cancer

McCarthy, Elan
MicroRNA-379 (miR-379) has been reported as a potent tumour suppressor miRNA with data supporting a potential therapeutic function in breast cancer. Despite this, the exact mechanism of action of miR-379 is still unknown. Moreover, successful delivery of this miRNA to the tumour site is required. Mesenchymal Stromal Cell Extracellular Vesicles (MSC-EVs) may act as therapeutic carriers due to their potential tumour homing and immune evasion properties. However, reproducible scale up of these vesicles is a necessity for clinical application. This study aimed to investigate the mechanism of action of miR-379 in an immune competent model of metastatic breast cancer. Scalable production of MSC-EVs was also established in a dynamic 3D serum free bioreactor to support longitudinal EV harvest. 4T1 luciferase expressing cells (4T1) were stably transduced with miR-379 (4T1-379). Balb/c immunocompetent mice received an orthotopic injection of 4T1 or 4T1-379 cells. IVIS imaging was employed to monitor disease progression. At study endpoint, tumour, lung, and bone tissues were harvested and fixed in 4% paraformaldehyde or RNAlater for immunohistochemical (IHC) or proteomic analysis respectively. Tissue sections were probed for selected hallmarks of cancer including proliferation (Ki67), angiogenesis (CD31, LYVE1) and immune cell infiltration (CD45, CD3, CD4, CD8). Mass spectrometry was performed to determine proteins differentially regulated by miR-379. 1 x 108 MSCs were inoculated into a 20kDa Fibercell bioreactor for 3D culture. Glucose consumption was monitored daily and when culture was stabilised, serum-free media was introduced. Cell conditioned media (CCM) was harvested every two days for seven weeks with cell samples withdrawn periodically. CCM EVs were isolated by size exclusion chromatography and characterised in terms of size distribution, concentration, and protein quantity. IVIS imaging reflected tumour progression with a leading edge and subsequent metastasis to distant organs. This was reinforced by Haematoxylin and Eosin staining revealing infiltration of tumour cells into lung tissue and significant remodelling of bone evident in tumour bearing animals compared to healthy controls. Tumour volume was significantly lower (P=0.05) in the miR-379 enriched vs 4T1 group. A significant reduction in Ki67 was found in the 4T1-379 tumours compared to 4T1 samples (P=0.01). However, no significant change in expression of CD31, LYVE1 or immune cell markers was observed between groups. Proteomic analysis revealed dysregulation of proteins associated with epithelial cell migration, immune response, and wound healing in tumours with elevated miR-379 expression. MSCs were successfully cultured in a 3D serum free bioreactor for 67 days with sampling at a variety of timepoints revealing viable cells with normal morphology and proliferation when transferred to culture flasks. Glucose readings revealed limited MSC proliferation in 3D culture. The average size of isolated EVs ranged from 133.3 ± 3.5 to 215.0 ± 3.2 nm in diameter with the majority of EVs < 200 nm representing small EVs. A cumulative yield of 3.89E+11 MSC-EVs was generated from the bioreactor culture over a timeframe of 48 days in serum free conditions, with limited protein contamination detected. The promising data presented highlights a role for miR-379 in regulation of tumour cell proliferation, with proteomic analysis revealing other potentially important novel targets. If miR-379 has the capacity to target these oncogenes individually or in concert, this could have important implications for therapeutic potency in breast cancer. In addition, successful scale up of reproducible high yields of MSC-EVs in a dynamic, serum free 3D system was demonstrated. Development of standardised large scale bioreactor production of tumour targeted MSC-EVs coupled with delivery of a potent tumour suppressor miRNA has exciting potential for future development of targeted therapeutics for metastatic breast cancer.
NUI Galway
Publisher DOI
Attribution-NonCommercial-NoDerivs 3.0 Ireland