Selective anti-tumor activity of the novel fluoropyrimidine polymer F10 towards G48a orthotopic GBM tumors

Abstract

F10 is a novel anti-tumor agent with minimal systemic toxicity in vivo and which displays strong cytotoxicity towards glioblastoma (GBM) cells in vitro. Here we investigate the cytotoxicity of F10 towards GBM cells and evaluate the anti-tumor activity of locally-administered F10 towards an orthotopic xenograft model of GBM. The effects of F10 on thymidylate synthase (TS) inhibition and Topoisomerase 1 (Top1) cleavage complex formation were evaluated using TS activity assays and in vivo complex of enzyme bioassays. Cytotoxicity of F10 towards normal brain was evaluated using cortices from embryonic (day 18) mice. F10 displays minimal penetrance of the blood-brain barrier and was delivered by intra-cerebral (i.c.) administration and prospective anti-tumor response towards luciferase-expressing G48a human GBM tumors in nude mice was evaluated using IVIS imaging. Histological examination of tumor and normal brain tissue was used to assess the selectivity of anti-tumor activity. F10 is cytotoxic towards G48a, SNB-19, and U-251 MG GBM cells through dual targeting of TS and Top1. F10 is not toxic to murine primary neuronal cultures. F10 is well-tolerated upon i.c. administration and induces significant regression of G48a tumors that is dose-dependent. Histological analysis from F10-treated mice revealed tumors were essentially completely eradicated in F10-treated mice while vehicle-treated mice displayed substantial infiltration into normal tissue. F10 displays strong efficacy for GBM treatment with minimal toxicity upon i.c. administration establishing F10 as a promising drug-candidate for treating GBM in human patients.

Figures

Fig. 1

F10 is cytotoxic to GBM cells through induction of thymineless death. a Chemical structure of F10; b Metabolic conversion of F10 produces FdUMP and initiates Topoisomerase 1-mediated DNA double strand breaks; c Cytotoxicity of F10 to G48 cells; d TS inhibitory activity of F10 towards SNB-19 (top), G48a (middle), and U-251 (bottom) cells at 10−6 (asterisk) and 10−5 M (double asterisk) and for raltitrexed at 10−6 M. e Western blots (for three independent samples) evaluating TS expression in G48a, U-251, and SNB-19 cells relative to HL60 cells that are sensitive to F10 at nM concentrations. Overall sensitivity to F10 inversely correlates with TS expression

Fig. 2

F10 is not toxic to primary neuronal cultures and does not damage normal brain upon i.c. administration. a Viability of primary neuronal cells grown in tissue culture following treatment with F10 or 5-FU at the indicated doses. 5-FU, but not F10, significantly decreased neuronal survival at 1 μM relative to control (p < 0.05). ANOVA followed by Newman–Keuls multiple comparison test: control versus 1 μM 5-FU; p ≤ 0.05; 1 μm 5-FU versus 1 μM F10; p ≤ 0.05. b H&E stained section from the brain of mice treated with F10 at 120 mg/kg

Fig. 3

F10 treatment results in significant and dose-responsive regression of G48a orthotopic xenografts. a Luciferase signal from F10- and vehicle-treated nude mice at 80 and 120 mg kg doses. Treatment with F10 results in marked decrease in luciferase-signal for treated mice. b Mean tumor luminescence calculated from the luciferase images. F10 treatment results in regression of G48a xenografts that is highly significant (p < 0.01) relative to vehicle for the 120 mg/kg treatment

Fig. 4

F10 treatment results in selective eradication of G48a orthotopic brain tumors in nude mice. H&E staining from brain sections obtained from nude mice bearing G48a xenografts following treatment with vehicle (a, d, g) or F10 at 80 (b, e, h) or 120 mg/kg (c, f, i). F10, or vehicle, was administered i.c. over 7 days using an osmotic pump. Brain tissue from mice treated with vehicle-only revealed extensive infiltration of GBM cells into non-malignant tissue and a significant tumor mass (arrows point to tumor borders). Treatment with F10 at either 80 or 120 mg/kg resulted in extensive necrosis selectively for malignant cells with no apparent damage for non-malignant cells in the contralateral side at the level of the hippocampus. Invasive island of cells were observed in contralateral hippocampus of vehicle-treated animals (arrows in g) (Scale bar 100 μm in c and i, 50 μm in f)

Fig. 5

F10 treatment results in selective eradication of EphA2-stained G48a cells. EphA2 staining from brain sections obtained from nude mice bearing G48a xenografts following treatment with vehicle only (a, d) or F10 at 80 (b, e) or 120 mg/kg (c, f). Strong EphA2 staining is observed for tumor tissue in vehicle-only treated mice with no EphA2 staining in adjacent non-malignant tissue except in isolated invasive cells (arrows indicate tumor borders and point to invasive cells away from tumor core). EphA2 staining is greatly diminished in region of the residual tumor (indicated by arrows) from mice treated with F10 at 80 mg/kg and is absent in mice treated with F10 at 120 mg/kg (Scale bar 50 μm)