Glioblastoma stem cells (GSCs) are invasive, treatment-resistant brain cancer cells that express downregulated in renal cell carcinoma (DRR), also called FAM107A, a genetic driver of GSC invasion. We developed antibody-antisense oligonucleotide (AON) conjugates to target and reduce DRR/FAM107A expression. Specifically, we used antibodies against antigens expressed on the GSCs, such as CD44 and EphA2, conjugated to chemically modified AONs against DRR/FAM107A, which were designed as chimeras of DNA and 2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid (FANA) for increased nuclease stability and mRNA affinity. We demonstrate that these therapeutic conjugates successfully internalize, accumulate, and reduce DRR/FAM107A expression in patient-derived GSCs. This is the first example of an antibody-antisense strategy against cancer stem cells.

No abstract

Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 (ITGB1)—a gene that promotes invasion and metastasis—and siRNA against eukaryotic translation initiation factor 3 subunit b (eIF-3b)—a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of ITGB1 led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of eIF-3b resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.

In this study, we explore the effect of a library of 2¢-, 4¢-, and 2¢,4¢-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2¢-F-ribose, 2¢-F-arabinose, 2¢-OMe-ribose, 2¢-F,4¢-OMe-ribose, 2¢-F,4¢-OMe-arabinose, and 2¢-OMe,4¢-F-ribose. We found that 2¢,4¢-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting. The impact of the dual (2¢,4¢) modification was assessed by comparing gene silencing ability to 2¢-or 4¢-(singly) modified siRNA counterparts. siRNAs with (2¢,4¢)-modified overhangs generally outperformed the native siRNA as well as siRNAs with a 2¢-or 4¢-modified overhang, suggesting that 2¢,4¢-modified nucleotides interact favorably with Argonaute protein's PAZ domain. Among the most active siRNAs were those with 2¢-F,4¢-OMe-ribose or 2¢-F,4¢-OMe-arabinose at the overhangs. When modifications were placed at both overhangs and internal positions, a duplex with the 2¢-F (internal) and 2¢-F,4¢-OMe (overhang) combination was found to be the most potent, followed by the duplex with 2¢-OMe (internal) and 2¢,4¢-diOMe (overhang) modifications. Given the nuclease resistance exhibited by 2¢,4¢-modified siRNAs, particularly when the modification is placed at or near the overhangs, these findings may allow the creation of superior siRNAs for therapy.

Glioblastoma stem cells (GSCs) play an important role in the invasive nature of glioblastoma (GBM); yet, the mechanisms driving this behavior are poorly understood. To recapitulate tumor invasion in vitro, we developed a GBM tumor-mimetic hydrogel using extracellular matrix components upregulated in patients. We show that our hydrogel facilitates the infiltration of a subset of patient-derived GSCs, differentiating samples based on phenotypic invasion. Invasive GSCs are enriched for injury-responsive pathways while noninvasive GSCs are enriched for developmental pathways, reflecting established GSC stratifications. Using small molecule inhibitors, we demonstrate that the suppression of matrix metalloprotease and rho-associated protein kinase processes results in a significant reduction of cell invasion into the hydrogel, reflecting mesenchymal- and amoeboid-dependent mechanisms. Similar reduction in cell invasion was observed by siRNA knockdown of ITGB1 and FAK focal adhesion pathways. We elucidate the transcriptomic profile of cells invading in the hydrogel by performing bulk RNA sequencing of cells cultured in the hydrogel and compare these to cells cultured in conventional tissue culture polystyrene (TCP). In our 3D hydrogel cultures, invasion-related molecular signatures along with proliferation and injury response pathways are upregulated while development processes are downregulated compared to culture on 2D TCP. With this validated in vitro model, we establish a valuable tool to find therapeutic intervention strategies against cellular invasion in glioblastoma.