Abstract 2136: Transcriptome analysis of osteoblasts fused with cancer-derived exosomes

Abstract

Abstract Breast cancer tumors have a high rate of metastasis and certain aggressive subtypes favor the bone environment. Clinically, it is estimated that metastatic breast cancer has a prevalence of up to 70% of bone tumors. Exosomes are important in cell-cell communication, and are increasingly being recognized as key contributors for priming targeted tissues for metastasis. Despite numerous advances in exosome detection and cargo characterization, transcriptional effects of cancer exosomes on targeted cells remain difficult to characterize at physiologically relevant levels. This is largely due to a combination of limited exosome secretion/uptake and an inability to segregate affected cells from the population. Conventional approaches to circumvent these limitations include spiking cell environments with exosome quantities far in excess of physiological concentrations. We sought to analyze the transcriptional effect of cancer exosomes on cells that have uptaken cancer-derived exosomes in a dynamic co-culture environment. In order to accomplish this, we first engineered a human breast cancer cell line (MDA-MB-231) to express GFP labeled exosomes. This novel cell line expresses three GFP fusion proteins for tetraspanin exosome markers (CD9, CD63, and CD81) known to be found on exosomes derived from MDA-MB-231 cells. Stable, intrinsic, fluorescent labeling of three exosome markers allows for an improved, more sensitive method for the analysis of exosome transfer. We can now examine transfer representing a more diverse pool of exosomes requiring no manipulation post exosome isolation prior to visualization. To investigate breast cancer exosome-mediated alterations on gene expression in bone cells to mimic breast cancer bone metastasis, this GFP exosome expressing line (MDA-MB-231 exo-GFP) was co-cultured with a mouse osteoblast cell line (MC3T3) then analyzed via flow cytometry to quantify the uptake of cancer exosomes in target cell population over time. MC3T3 cells that become GFP positive, indicative of exosome mediated transfer, increased from 0.91% of the population on day 3 post co-culture to 17.3% on day 14. Subsequent cell sorting and RNA-seq of GFP positive MC3T3 populations yielded novel insights into the progressive transcriptional effect of cancer exosomes on target cells. Future research will expand upon this approach to examine the effects on bone cells non-metastatic and metastatic cancer cell lines have on the metastatic niche through exosome-mediated signaling. This approach introduces a novel method for prolonged transcriptional analysis of cancer and target cell co-culture allowing the segregation of exosome affected and unaffected subpopulations of target cells. This study was funded by DOD grant BC151687. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas H. Hum, Kelly A. Martin, Gabriela G. Loots. Transcriptome analysis of osteoblasts fused with cancer-derived exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2136.