Supplementary MaterialsSupp FigS1: Figure S1. were between 80 and 120% (Gapdh: 104%, Wsb2: 90%, and Ptpn11: 118%). The standard deviation in values suggests that a difference by a factor of two in template concentration could be detected with 99% confidence using a sample size of 3. (C) values estimate the abundance of mRNA obtained from whole cell lysates (red circles) versus exosomes (black squares) for each cell line (B16F0, S91, and Melan-A) and target tested (Wsb2, Ptpn11, Gapdh, Eif4abp2, Kpnb1, and Rnd2). ** indicates that the difference in between whole cell lysates and exosome samples was significant (p-value 0.0002). NIHMS837419-supplement-Supp_FigS2.tiff (1.2M) GUID:?BCF314AA-EBB9-4453-8A20-42E21F280AE1 Supp TableS1: Table S1. Enriched pathways associated with mRNA from B16F0 exosomes P-values were computed using the Fisher exact test. The Z-score is a statistical ranking metric derived from running the Fisher exact test for many random gene sets in order to compute a mean rank and standard deviation from the expected rank for each term in the gene-set library and finally calculating a z-score to assess the deviation from the expected rank. Combined score is calculated from p-value and z-score. NIHMS837419-supplement-Supp_TableS1.docx (16K) GUID:?B3C76BC6-3364-4F88-9013-5899724B28AB Summary As exosomes are emerging as a new mode of intercellular communication, we hypothesized that the payload contained within exosomes is shaped by somatic evolution. To test this, we assayed the impact on primary CD8+ T cell function, a key mechanism for anti-tumor immunity, of exosomes derived from three melanoma-related cell lines. While morphologically similar, exosomes from each cell line were CP 31398 dihydrochloride functionally different, as B16F0 exosomes dose-dependently suppressed T cell proliferation. In contrast, Cloudman S91 exosomes promoted T cell proliferation and Melan-A exosomes had a negligible effect on primary CD8+ T cells. Mechanistically, transcript profiling suggested that exosomal mRNA is enriched for full-length mRNAs that target immune-related pathways. Interestingly, B16F0 exosomes were unique in that they contained both protein and mRNA IL13 antibody for and and and and and were enriched in the B16F0 exosome samples. Collectively, CP 31398 dihydrochloride the gene expression results suggest that mRNAs are selectively packaged into exosomes and that the mRNAs are intact ORFs. Open in a separate window Figure 4 Relative mRNA abundance between B16F0 exosomes and cells were consistent between qRT-PCR and microarray analyses(a) The abundance of 10 genes (Kpnb1, Rnf14, Rnd2, Ptp4a3, Ptpn11, Eif2c2, Hipk2, Eif4ebp2, Dnmt3a, and Wsb2) in B16F0 exosomes versus B16F0 cells were quantified by quantitative RT-PCR (mean s.d., N = 3). The qRT-PCR results were normalized to the average differential abundance of three control genes: Kpnb1, Rnf14, and Rnd2. (b) The relative abundances of mRNAs assayed by qRT-PCR were compared against the relative abundances of mRNAs assayed by cDNA microarray. The dotted collection indicates that the two different assays provide the same results for relative large quantity. (c) Full-length coding sequences (ORFs) were amplified by semi-quantitative RT-PCR. Equal concentrations of RNA were reverse-transcribed into cDNA and amplified by PCR. After 25 cycles, full-length open-reading framework amplicons were monitored every three cycles and resolved on agarose gel before the amplification was saturated. B16F0 exosomes deliver a biological payload to T lymphocytes Like a subset of mRNAs were selectively enriched in exosomes, we used the Enrichr pathway enrichment algorithm to identify biological pathways that are associated with mRNAs that are enriched in exosomes. Using 145 enriched mRNAs in B16F0 exosomes, we recognized 18 signaling CP 31398 dihydrochloride pathways that experienced positive combined scores (observe Supplemental Table S1). Interestingly, several of the pathways are closely connected to the anti-tumor immunity, with the Type I Interferon signaling pathway having the least expensive p-value and the IL-2, the T cell receptor, and Type II Interferon signaling pathways all possessing a positive combined score. One of the difficulties with pathway enrichment results is definitely that genes associated with a specific pathway can either promote or inhibit transmission transduction. The gene that was common to 12 out of the 18 enriched pathways was.