A U87 non-stem U87 stem-like U251 non-stem U251 stem-like 0 200 400 600 800 MFI/FOV:TLR9 0 200 400 1,000 1,400 MFI/FOV:pSTAT3 600 800 1,200 * 0 200.

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A U87 non-stem U87 stem-like U251 non-stem U251 stem-like MFI/FOV:TLR9 ** ,000 1,400 MFI/FOV:pSTAT ,200 *** , ***** ,000 2,000 2,500 MFI/FOV:TLR9 1,500 pSTAT3 TLR9 Hoechst non-stem stem-like non-stem stem-like BC non-stem stem-like Tlr9 mRNA P = 0.06 GL261DBT - TLR9 (180 kDa) -  -Actin - TLR9 (115 kDa) M r [kDa] D - pStat3 (91 kDa) - Stat3 (91 kDa) - MSI-1 (39 kDa) - SOX2 (40 kDa) - Actin (42 kDa) GL261DBT - Nestin (177 kDa) non-stem stem-like non-stem stem-like non-stem stem-like non-stem stem-like Figure S1, Herrmann, A., Cherryholmes, G. et al. Figure S1. Increased TLR9 expression in induced glioma spheres. (A) TLR9 and activated STAT3 are significantly increased in glioma stem cell enriched culture using sphere formation assay. TLR9 and pSTAT3 expression in cells grown in non-cancer stem cell conditions, compared to sphere forming culture condition (left panels). Scale bar 20 μm. Quantification of mean fluorescent intensity (MFI) of TLR9 and pSTAT3, respectively (right panels). (B) Quantitative RT-PCR to analyze expression of Tlr9 mRNA in murine GL261 glioma cells, in sphere stem-cell culture condition compared to adherent non-stem cell culture condition. (C) Expression of TLR9 protein and (D) CSC-associated factors by Western blotting in murine glioma cells cultured in sphere stem-cell culture condition, compared to adherent non-stem cell culture condition. MFI/FOV:pSTAT3

F - TLR9 GL261 DBT Input IgG  Stat3 IgG  Stat3 stem-likenon-stem ChIP: TLR9 mRNASTAT3 mRNA scr-shRNA STAT3-shRNA G P = 0.01P = 0.03 A ** none, n = 4 CpG-ODN 1668, n = B No. of spheres No. of spheres, Ø1-2 mm none CpG P = Sox2 mRNA Nestin mRNA 10 2 none CpG P = 0.03 Days No. of spheres 0246 Days DBT nt-shRNA, n = 9 *** DBT Tlr9-shRNA (1), n = 9 DBT Tlr9-shRNA (2), n = 9 DC Tlr9-shRNA nt-shRNA Musashi-1 mRNA 1.5 P = Sox2 mRNA 1.5 P = Nestin mRNA 1.5 P = nt-shRNA, n = 24 TLR9shRNA 1, n = 24 TLR9shRNA 2, n = 24 – 4 – 3 – 2 – 1 0 U251 non-responding [log] number of cells E Figure S2, Herrmann, A., Cherryholmes, G. et al. BLANK 2 nd ab ctrl DBT-nt-RNA DBT-Tlr9shRNA#1 TLR9 DBT-Tlr9shRNA#2 % of Max.

Figure S2. TLR9 and STAT3 form a feed-forward loop in GSCs. (A) Sphere formation assay to phenocopy cancer stem cell-like cells upon TLR9 signaling activation in DBT glioma spheres triggered with CpG-ODN. SD and significance are shown: *P ≤ 0.05; **P ≤ Quantification of sphere diameter at day 8 of sphere formation is shown. SD shown and significance are indicated. (B) Quantitative RT-PCR of Sox2 and Nestin mRNA levels in DBT spheres with or without CpG-ODN treatments. SD and significance are shown. (C) Effect of TLR9 signaling on human GSC-like cells using limiting dilution assays. U251 glioma spheres stably transduced with indicated TLR9shRNAs were grown in stem cell-enriching (sphere-forming) culture conditions, followed by a limiting dilution assay to assess the frequency of GSCs. (D) Tlr9shRNA was introduced into DBT glioma cells and TLR9 protein knockdown efficacy was determined by flow cytometry (left panel). Sphere formation assay of murine DBT glioma cells expressing shRNA against Tlr9 in sphere stem-cell culture condition. SD and significance shown: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ (right panel). (E) Analysis of mRNA expression levels of stem cell-associated factors in glioma such as Msi-1, Sox2, and Nestin by RT-PCR upon introduction of Tlr9 shRNA; SD and significance are shown. (F) Chromatin immunoprecipitation (ChIP) of Stat3 binding to the Tlr9 promoter in GL261 and DBT murine glioma cells cultured in sphere stem-cell culture condition, compared to adherent non-stem cell culture condition. (G) Expression of STAT3 mRNA and TLR9 mRNA levels upon STAT3 knockdown in human glioma cells grown in sphere culture condition analyzed by quantitative RT-PCR. SD and significance are shown. Figure S2 cont’d, Herrmann, A., Cherryholmes, G. et al.

A Days DBT ,000 1,500 Tumor volume [mm 3 ] , * * * vehicle, n = 4 CpG-Stat3siRNA, n = 4 CpG-lucsiRNA, n = 4 Neovasculature ECM Annexin V noneCpG-lucsiRNACpG-Stat3siRNA C Figure S3, Herrmann, A., Cherryholmes, G. et al. noneCpG-Stat3siRNA FITC SSC-H FL-1A:: FITC noneCpG-Stat3siRNA FITC B D DBTGL261 none CpG-lucsiRNA CpG-Stat3siRNA Hoechst pStat3 Hoechst cl.Casp.3 CD31 Hoechst E 0 25 Percent Survival Days Vehicle, n = 8 CpG, n = 8 CpG-Stat3 siRNA, n = CpG-Stat3siRNA FITC Hoechst33342

Figure S3. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits tumor growth. (A) Internalization of CpG-Stat3siRNA-FITC into glioma tumors engrafted subcutaneously visualized by in vivo multi-photon imaging (upper panel, scale bar 50  m) and quantified by flow cytometry (lower panel). (B) Tumor growth of DBT murine glioma in immunocompromised mice treated locally as indicated. SD and significance shown (*P ≤ 0.05). (C) Tumor vasculature and induction of cell apoptosis in DBT murine glioma treated with CpG- Stat3siRNA, CpG-lucsiRNA, or left untreated was analyzed by in vivo multi-photon imaging. Scale bar 100  m. (D) Antitumor effects on glioma treated with CpG-Stat3siRNA, CpG- lucsiRNA, or vehicle control was analyzed in two rodent glioma models DBT and GL261, respectively, showing decreased Stat3 activation (upper panel), diminished tumor CD31 + vasculature (middle panel) and cleaved caspase 3 + induced tumor cell apoptosis (lower panel). Scale bar 100  m. (E) Improved survival rate as a result of locally administered CpG- Stat3siRNA was acquired in a syngenic model by treating mice bearing brain tumors twice a week for two weeks. CpG1668 and vehicle treatment were included as controls. Significance shown (**P ≤ 0.01). Figure S3 cont’d, Herrmann, A., Cherryholmes, G. et al.

A SSC-H ’30’60’120’ DBT sphere CpG-Stat3siRNA-FITC ,000 No. of spheres none, n = 6 02 Days CpG-lucsiRNA, n = 6 CpG-Stat3siRNA, n = 6 DBT 64 B ** Tlr9 mRNASox2 mRNASSEA1 mRNA none, n = 3 CpG-lucsiRNA, n = 3 CpG-Stat3siRNA, n = Stat3 mRNA C noneCpG-lucsiRNACpG-Stat3siRNA pStat3 Nestin Hoechst Nestin TLR9 Hoechst D Figure S4, Herrmann, A., Cherryholmes, G. et al. P=0.024P=0.008 P=0.001

Figure S4. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits GSCs. (A) Internalization of CpG-Stat3siRNA-FITC into DBT glioma sphere culture determined by flow cytometry. (B) Sphere formation capacity of DBT murine glioma cells upon CpG-Stat3siRNA treatment compared to controls as indicated. SD and significance shown (*P ≤ 0.05; **P ≤ 0.01). (C) Quantitative RT-PCR analyses showing mRNA levels of stem cell associated factors in glioma tumors grown subcutaneously and treated as indicated. SD and P values shown. (D) Confocal microscopic analyses showing protein levels of pStat3 and TLR9 in Nestin + tumor areas from tumors treated with indicated CpG-siRNAs. Scale bar 100  m. Figure S4 cont’d, Herrmann, A., Cherryholmes, G. et al.

noneCpGlucsiRNACpGscrRNA BLU 10 6 Figure S5, Herrmann, A., Cherryholmes, G. et al none CpGlucsiRNA CpGscrRNA P = 0.07 P = 0.25 Figure S5. Targeting Brain tumors systemically with CpG-siRNA reaches the tumor site. Xenogen imaging of GL261 luc+ brain tumors orthotopically engrafted in C57BL/6 mice showing decreasing bioluminescent signals upon treatment with CpG-lucsiRNA as well as indicated controls (left panel). Luciferase signals upon indicated treatments was acquired by non-invasive xenogen imgaging and quantified. P values shown (right panel).