BackgroundTumor irradiation blocks local angiogenesis, forcing any recurrent tumor to form new vessels from circulating cells. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas in the brains of nude mice can be delayed or prevented by inhibiting circulating blood vessel–forming cells by blocking the interaction of CXCR4 with its ligand stromal cell-derived factor (SDF)–1 (CXCL12). In the present study we test this strategy by directly neutralizing SDF-1 in a clinically relevant model using autochthonous brain tumors in immune competent hosts.MethodsWe used NOX-A12, an l-enantiomeric RNA oligonucleotide that binds and inhibits SDF-1 with high affinity. We tested the effect of this inhibitor on the response to irradiation of brain tumors in rat induced by n-ethyl-N-nitrosourea.ResultsRats treated in utero with N-ethyl-N-nitrosourea began to die of brain tumors from approximately 120 days of age. We delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age, began treatment with NOX-A12 immediately following irradiation, and continued with either 5 or 20 mg/kg for 4 or 8 weeks, doses and times equivalent to well-tolerated human exposures. We found a marked prolongation of rat life span that was dependent on both drug dose and duration of treatment. In addition we treated tumors only when they were visible by MRI and demonstrated complete regression of the tumors that was not achieved by irradiation alone or with the addition of temozolomide.ConclusionsInhibition of SDF-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme.

Despite the approval of antiangiogenic therapy for glioblastoma multiforme (GBM) patients, survival benefits are still limited. One of the resistance mechanisms for antiangiogenic therapy is the induction of hypoxia and subsequent recruitment of macrophages by stromal-derived factor (SDF)-1α (CXCL-12). In this study, we tested whether olaptesed pegol (OLA-PEG, NOX-A12), a novel SDF-1α inhibitor, could reverse the recruitment of macrophages and potentiate the antitumor effect of anti–vascular endothelial growth factor (VEGF) therapy. We also tested whether magnetic resonance imaging (MRI) with ferumoxytol as a contrast agent could provide early information on macrophage blockade. Orthotopic human G12 glioblastomas in nude mice and rat C6 glioblastomas were employed as the animal models. These were treated with bevacizumab or B-20, both anti-VEGF antibodies. Rats were MR imaged with ferumoxytol for macrophage detection. Tumor hypoxia and SDF-1α expression were elevated by VEGF blockade. Adding OLA-PEG to bevacizumab or B-20 significantly prolonged the survival of rodents bearing intracranial GBM compared with anti-VEGF therapy alone. Intratumoral CD68+ tumor associated macrophages (TAMs) were increased by VEGF blockade, but the combination of OLA-PEG + VEGF blockade markedly lowered TAM levels compared with VEGF blockade alone. MRI with ferumoxytol as a contrast agent noninvasively demonstrated macrophage reduction in OLA-PEG + anti-VEGF–treated rats compared with VEGF blockade alone. In conclusion, inhibition of SDF-1 with OLA-PEG inhibited the recruitment of TAMs by VEGF blockage and potentiated its antitumor efficacy in GBM. Noninvasive MRI with ferumoxytol as a contrast agent provides early information on the effect of OLA-PEG in reducing TAMs.

We have previously shown that spores of the nonpathogenic clostridial strain C. sporogenes genetically engineered to express the E. coli-derived cytosine deaminase gene are effective in converting systemically injected nontoxic 5-fluorocytosine into the toxic anticancer drug 5-fluorouracil, thereby producing tumor-specific antitumor activity. To improve the expression of E. coli-derived genes with this system, we first replaced the original fdP promoter in the vector with one of two powerful endogenous clostridial promoters: that of the thiolase gene (thlP) and that for the clostridial transcription factor abrB310 (abrBP). These substitutions improved protein expression levels of the prodrugactivating genes by 2-to 3-fold in comparison with fdP-driven expression. However, despite these strong promoters, we found much higher expression of the nitroreductase (NTR) protein in the E. coli host compared with the clostridial host, which we hypothesized could be the result of different codon use between the two organisms.