Figure 1.

Nanoparticle-enabled cytosolic delivery of 2’3’-cGAMP activates the STING pathway in neuroblastoma cells. (A) Integrated molecular analysis of mRNA expression of genes from the pediatric neuroblastoma TARGET dataset that have been distinguished by functional significance and clustered into evenly split tertiles based on high (upper tertile, n=47), intermediate (median tertile, n=47), and low (bottom tertile, n=47) TMEM173 mRNA expression. (B)TMEM173 mRNA expression in MYCN non-amplified and amplified samples profiled by microarray in the TARGET pediatric neuroblastoma (n=55) datasets. Data were accessed through the cBioPortal.69 Mann-Whitney U test (two-tailed) was used for statistical comparison.(C) Schematic representation of STING-NPs designed to enhance cytosolic delivery of cGAMP via endosomal escape, resulting in potent activation of STING signaling. (D) Neuroblastoma cell lines (Neuro-2a, 9464D, SK-N-SH, and LAN-1) were treated with vehicle (PBS), empty nanoparticles (NP), 200 nM (or 400 nM for 9464D) cGAMP, or STING-NPs for 48 hours; cells were collected for western blot analysis using anti-IRF3 and anti-phospho-IRF3 (p-IRF3) antibodies. Gel loading was normalized for equal actin; representative blots from one of two independent experiments. The relative density of bands is shown under each immunoblot, after normalization to the levels of actin. (E) qRT-PCR gene expression of IFNB1, TNF, CXCL10, and IL12 in neuroblastoma cell lines at 6, 24 and 48 hours after treatment with cGAMP or STING-NPs. (n=2, data shown as mean±SD; data was analyzed by two-way ANOVA followed by Dunnett’s posthoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate a statistically significant difference relative to vehicle (PBS). ANOVA, analysis of variance; cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; qRT-PCR, quantitative real-time PCR; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.