Blockade of SDF-1 after irradiation inhibits tumor recurrences of autochthonous brain tumors in rats

Abstract

Background: Tumor 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.

Methods: We 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.

Results: Rats 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.

Conclusions: Inhibition of SDF-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme.

Keywords: CXCL12; CXCR4; CXCR7; ENU-induced tumors; NOX-A12; SDF-1; angiogenesis; glioblastoma; irradiation; vasculogenesis.

Fig. 1.

(a) NOX-A12 inhibits SDF-1–mediated and CXCR4-dependent chemotaxis of THP-1 myelomonocytes. THP-1 cells are attracted by 5 nM of human SDF-1 (set to 100%). SDF-1 was preincubated with NOX-A12 at various concentrations. One representative dose-response curve (mean ± SD of triplicates) of 3 independent experiments is shown. NOX-A12 inhibits SDF-1–mediated chemotaxis of CXCR4-expressing THP-1 cells with an IC₅₀ of 3.9 ± 0.2 nM. (b) NOX-A12 inhibits SDF-1–mediated internalization of CXCR7. PathHunter eXpress CXCR7 activated GPCR internalization cells show SDF-1–mediated internalization of CXCR7. Internalization of CXCR7 by incubation with 10 nM SDF-1 was set to 100%. SDF-1 was preincubated with various concentrations of NOX-A12. One representative dose-response curve (mean ± SD of triplicates) of 4 independent experiments is shown. NOX-A12 inhibits SDF-1–mediated CXCR7 internalization with an IC₅₀ of 3.0 ± 0.9 nM. (c) NOX-A12 inhibits SDF-1–mediated migration of human ECs. HUVECs were stained with the fluorescent dye DiIC₁₂(3), and migration through the FluoroBlok membrane was quantified in a bottom reading plate reader; 1 µg/mL of human SDF-1 was preincubated either with or without an equimolar concentration of NOX-A12 and then added to the bottom of the transwell inserts, which were coated with fibronectin. SDF-1 immobilized on fibronectin increases migration of HUVECs, which is completely inhibited by NOX-A12. Each curve reflects the means of duplicates ± SD from a single experiment and is representative of 5 independent experiments.

Fig. 2.

SDF-1 inhibition after irradiation prolongs the survival of the brain tumor–bearing rats. Rats born to mothers treated with a single injection of the carcinogen ENU on day 18 of gestation were sham irradiated or given a dose of 20 Gy to the whole brain with shielding of the buccal cavity. The rats receiving NOX-A12 were injected subcutaneously every 2 days with either 5 or 20 mg/kg starting soon after irradiation and continued for either 4 or 8 weeks. Survival times are shown in Table 1.

Fig. 3.

MRI is effective in detecting the ENU-induced brain tumors. Rats born to mothers given a single injection of ENU on day 18 of gestation were imaged by MRI, and when 1 or more tumors per rat were detected, the rats were sacrificed and the brains serially sectioned and stained with hematoxylin and eosin to identify the same lesions as seen on the MRI scans. MRI also detected all but the smallest lesions detected from the histology. Shown are representative images of 3 rats from a total of 11 analyzed.

Fig. 4.

Addition of NOX-A12 following irradiation of the ENU-induced brain tumors produces complete responses by MRI. In utero ENU-treated rats were imaged by MR starting on day 130 of age, repeated every 2 weeks until death. Rats were distributed into the various treatment groups so as to have approximately equal total tumor volumes in each group at the start of treatment.

Fig. 5.

Model showing the vasculogenesis pathway that restores tumor vasculature after irradiation and the various inhibitors of this pathway that can improve the radiation response of the tumor. In addition to summarizing the data from the present study, the figure draws data from prior publications.^, Abbreviations: MAb, monoclonal antibody; HIF-, hypoxia-inducible factor 1; VEGF, vascular endothelial growth factor; IR, irradiation.