Abstract 3599: Long-distance intercellular transport of microRNAs via tunneling nanotubes: Role in tumor-stroma interactions and malignant potential

Abstract

Abstract Introduction: Intercellular communication is vital to cancer cell organization and invasion. Identification of key cellular cargo and their preferred modes of transport is important, and yet it remains underdeveloped. Gap junctions and exosomes are known to play a key role as conduits in mediating intercellular transport. Tunneling nanotubes (TnTs) - long narrow actin-based cytoplasmic extensions - are unique structures that facilitate direct long distance cell-to-cell transport of cargo, potentially including microRNAs (miRNAs). These small non-coding RNAs play a significant role in pathogenesis, primarily in post-transcriptional gene regulation. We hypothesized that miRNA transport via TnTs occurs between malignant and stromal cells and can lead to changes in gene regulation that propagate the cancer phenotype. In this study, we used murine osteosarcoma and human ovarian cancer as model systems to investigate tumor-tumor and tumor-stroma interactions via TnTs. Methods: miR-19a was labelled with the fluorophore Alexa-488 by in vitro transcription, and visualized using confocal fluorescent video microscopy. K7M2 (murine osteosarcoma) cells were transfected with Alexa-488 labeled miR-19a prior to co-culture with K7M2 or MC3T3 murine osteoblast cells. Parallel studies were performed using miR-199a in human ovarian cancer (SKOV3 and C200) and normal human ovarian epithelial cells (IOSE). Movement of miRNAs was imaged by time-lapse microscopy every 5 minutes up to 48 hours. Intercellular transport of miRNAs was further studied by co-culture of cells separated by a membrane filter with pores 0.4 µm in size in a transwell chamber, which is restrictive of cell migration and exosome transfer but permissive of TnT formation. Cells were collected at 48 hours and relative miR-19a levels were assessed in recipient cells using qPCR. Results: TnT-mediated cell-to-cell transport of miR-19a was demonstrated among K7M2 cells themselves and between K7M2 and MC3T3 cells using fluorescence time-lapse imaging. miRNA transport was detected in TnTs spanning 40-200 µm in length, occurring over 25 minutes to several hours. Relative endogenous levels of miR-19a were elevated up to 2.4-fold in MC3T3 osteoblast cells after 48 hours of co-culture with osteosarcoma cells. Similar experiments performed in human ovarian cancer and ovarian epithelial cells also confirmed transport of miR-199a between malignant and stromal cells. Conclusions: To our knowledge, we provide the first demonstration of intercellular transport of miRNAs between cancer and stromal cells via TnTs. This discovery establishes a novel mechanism by which miRNAs mediate altered gene regulation in the complex and heterogeneous tumor microenvironment. We are further investigating the effects of TnT-mediated miRNA transfer on recipient stromal cells and its effects on the invasive phenotype of solid tumors. Citation Format: Venugopal Thayanithy, Elizabeth L. Dickson, Subbaya Subramanian, Clifford J. Steer, Emil Lou. Long-distance intercellular transport of microRNAs via tunneling nanotubes: Role in tumor-stroma interactions and malignant potential. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3599. doi:10.1158/1538-7445.AM2014-3599