Additional Supporting Information may be found in the online version of this article.

sc-12-0785_sm_SupplFigure1.pdf2724KSupplemental Figure 1: Cell and stimulatory RNA portfolio. (A) Pie chart of the 9 cell populations investigated in this study. We used patient-specific, previously described tumor cells (17), i.e. stem cell-enriched primary GBM cultures (pGBM CSC), primary GBM cultures without stem cell properties (pGBM w/o CSC) and Residual GBM cultures (pGBM Residual). The commercially available U87 glioma cell line was applied as a reference. To assess toxicity and tumor-specificity, a variety of human non-tumor cells were analyzed as control. Specifically, we investigated embryonic stem cell-derived neural stem cells (ESCd NSCs; (19)), iPSC-derived neural stem cells (iPSCd NSC, n=2; (20)), iPSCd microglia (iPSCd MC; (21)) and astrocytes (iPSCd Astro) as well as primary adult human neural progenitor cells (pAHNPs, 18). (B) Phase contrast micrograph of pGBM #046 at in vitro passage 7. (C-E) Multistep procedure as standard evaluation platform for portfolio tumor cells (all display items from case #046). (C) SNPbased genotyping for discrimination of GBM-typical alterations (40). Evident in this case are a gain of chromosome 7, a deletion of the CDKN2A/ELAVL2/LINGO2 locus, and loss of chromosome 10, 14, and 22. (D) Neurosphere assay (23). Bright-field appearance of neurospheres (left). Immature (middle) and mature (right) neurons (green, beta III tubulin) and astroglial progeny (red, GFAP) early after plating of individual secondary neurospheres. (E) Xenograft studies. After engraftment of pGBMs into the right frontal lobe, manifestation of a large donor-derived tumor is observed. The arrowhead depicts the central fissure (left) and the presence of tumor-neovascularization, beginning vascular proliferation and concomitant thrombosis (middle). The upper panel right shows GFAPexpressing heteromorphous glial tumor cells (brown DAB reaction); the lower panel a high proliferative tumor cell activity (KI67, brown DAB reaction). (F) Table outlining the various stimulatory RNA molecules and the respective transfection methods used in this study. Scale bars (μm): B=50; D/left=150; D/middle, D/right, E/right=20, E/left=300; E/middle=100.
sc-12-0785_sm_SupplFigure2.tif2173KSupplemental Figure 2: Immunostimulation data with controls. Graphs extend on data presented in Figure 1B and Figure 1C, demonstrating the effects induced by stimulatory RNA molecules compared to their respective controls (Ctrl). Medium, media control. pCA, untransfected pCA. pCA/m, pCA used with Mirus transfection reagent. pCA/R, pCA used with RNAiMAX. (A) CXCL10 secretion, (B) type I IFN secretion.
sc-12-0785_sm_SupplFigure3.pdf131KSupplemental Figure 3: Immunostimulatory impact on NK cell activity. (A) Co-culture experiment for the revealing of increased NK cell activity following immune stimulation of GBM cells. After plating and stimulation of GBM CSCs with untransfected p(I:C), transfected p(I:C), untransfected p(I:C)-LC, or transfected 3pRNA supernatants were collected and added to freshly sorted primary CD56+/CD3− cells (NK cells). pGBM CSCs were additionally provided on day 4, and FACS analysis was conducted 4 hours later (right). Note: CD107a is a functional marker used in this degranulation-assay for the identification of natural killer cell activity. Non-stimulatory 20mer pCA-RNA was used as control reagent for these experiments. MFI, mean fluorescence intensity. (*p<0.05). (B) Using the experimental setting of (A), NK cell activity was evaluated following stimulation with p(I:C), 3pRNA, or type I IFN in the presence of blocking antibodies against IFNα/β chain receptor 2 (black bars) or IgG2A as a control (gray bars). (*p<0.05, **p<0.01). (C) Lysis assay. Based on the experimental paradigm presented in (A), DiO pre-labeled pGBM CSCs were added to activated NK cells (left). NK mediated pGBM CSC lysis was determined by FACS after 24 h of coculture (non-stimulatory 20mer pCA-RNA was used as a control for the experiments). Similar experiments (right) were conducted in the presence of blocking antibodies against IFNα/β chain receptor 2 (black bars) or IgG2A as a control (gray bars). Data presented relative to the effect of type I IFN. (*p<0.05, **p<0.01).
sc-12-0785_sm_SupplFigure4.pdf104KSupplemental Figure 4: Analysis of cancer stem-like cell properties. Data extend on Figure 2E. (A) Experimental layout for generating the cells used for analysis. Maximum of cell death was determined at 5±1 days following RNA molecule stimulation (see B, refer to Figure 2E). Less than 10% of pGBM CSCs survived the stimulation paradigm. However, these cells recovered from the cytotoxic stimulus proliferating to confluent cultures at day 28±14 post RNA molecule stimulation (see C and D). (C) The graphs demonstrate quantitative data from the neurosphere assay (NSA, left; inset: neurosphere (NS)-size calculation) and qRT-PCR analysis on stem cell-typic mRNA expression levels of pGBM CSCs. Note: Results are calculated as percent of the non-stimulatory pCA (RNA)-control. (D) The table summarizes findings from NS-differentiation and xenotransplantation experiments. Cells that recovered from antineoplastic RNA molecule activity in vitro retained their capacity to generate neurons and glia, and their ability to induce tumors upon orthotopic transplantation. Together, these data imply that the frequency of cancerous stem-like GBM cells remains unchanged. Thus, the immunostimulatory RNA molecules p(I:C) and 3pRNA seem to effectively target GBM cells with and without stem-like properties.
sc-12-0785_sm_SupplFigure5.tif2806KSupplemental Figure 5: Tumor cell specificity-related experiments. Western blot analysis of antiapoptotic proteins and Noxa. (A) Representative Bcl-xL and Noxa expression levels of human non-malignant control cells (n=5; ESCd NSC-1 data following stimulation with transfected p(I:C) and 3pRNA are shown). (B) Incongruent Bcl-xL post-stimulation levels in three pGBM CSC cultures. Ctrl = pCA (RNA) control. (C) Representative data for tumor and non-tumor control cells: Baseline levels of the antiapoptotic proteins Bcl-xL, Bcl-w, and Mcl-1 in pGBM CSCs (#'s 035, 046, 078, 106) cases and iPSCd NSC cell populations (right lanes) are presented.
sc-12-0785_sm_SupplFigure6.pdf49KSupplemental Figure 6: RNA transfection efficacy. Cy3-labeled control RNA was transfected (see Methods) and cells were analyzed by flow cytometry 24h later. Comparable transfection efficacies were observed in tumor (pGBM CSCs #'s046, 078, 106) vs. non-malignant control cells (pAHNP-1; iPSCd NSC-2; ESCd NSC-2) (mean data ± SEM). The inset depicts characteristic scatter plots.
sc-12-0785_sm_Suppltable1.tif1487KSupplementary Table 1

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