Comparative genome analysis of exosomal mRNA derived from Tspan8-overexpressing and Tspan8-negative rat pancreas tumors

We recently described that tumor-derived exosomes containing the tetraspanin Tspan8 induce angiogenesis. However, not much is known about exosome-endothelial cell interactions and there are no clues on the role of tetraspanins, which are a constitutive component of exosomes. We used the stably Tspan8 expressing rat adenocarcinoma cell line AS-Tspan8, to explore how exosomal Tspan8 supports angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes. A genome-wide microarray analysis revealed that both, AS- and AS-Tspan8-exosomes contain about 1500 transcripts, from which 285 are over 3 fold enriched in AS-Tspan8-exosomes. We confirmed high levels mRNA of transcription factors MycN and Spi-C, elongation factor B(SIII), TCEB-1, fibroblast growth factor binding protein 1 (FGFBP1) and growth and differentiation factor-3 (GDF3) in AS-Tspan8-exosomes compared to AS-exosomes and to the parental cells by RT-PCR. VEGF mRNA was not enriched in AS-Tspan8 cells or exosomes. Expression of CCL20, prothrombin, bone morphogenetic protein-2, several Ras and MAPK family members and a phosphatase2A subunit was below the detection level in exosomes. The array consists of 12 samples. Samples 1-6 represent total RNA from non-treated endothelial cells (1,2) cells co-cultured with Tspan8-negative exosomes (3,4) and cells co-cultured with Tspan8-positive exosomes (5,6). Samples 7 and 8 contain total RNA isolated from cells, from which exosomes are derived. Total RNA from Tspan8-negative exosomes represets samples 9,10; and total RNA from Tspan8-positive exosomes represents samples 11,12.