Inhibition of laminin-8 in vivo using a novel poly(malic acid)-based carrier reduces glioma angiogenesis

Abstract

We have previously shown that laminin-8, a vascular basement membrane component, was overexpressed in human glioblastomas multiforme and their adjacent tissues compared to normal brain. Increased laminin-8 correlated with shorter glioblastoma recurrence time and poor patient survival making it a potential marker for glioblastoma diagnostics and prediction of disease outcome. However, laminin-8 therapeutic potential was unknown because the technology of blocking the expression of multi-chain complex proteins was not yet developed. To inhibit the expression of laminin-8 constituents in glioblastoma in vitro and in vivo, we used Polycefin, a bioconjugate drug delivery system based on slime-mold Physarum polycephalum-derived poly(malic acid). It carries an attached transferrin receptor antibody to target tumor cells and to deliver two conjugated morpholino antisense oligonucleotides against laminin-8 alpha4 and beta1 chains. Polycefin efficiently inhibited the expression of both laminin-8 chains by cultured glioblastoma cells. Intracranial Polycefin treatment of human U87MG glioblastoma-bearing nude rats reduced incorporation of both tumor-derived laminin-8 chains into vascular basement membranes. Polycefin was thus able to simultaneously inhibit the expression of two different chains of a complex protein. The treatment also significantly reduced tumor microvessel density (p < 0.001) and area (p < 0.001) and increased animal survival (p < 0.0004). These data suggest that laminin-8 may be important for glioblastoma angiogenesis. Polycefin, a versatile nanoscale drug delivery system, was suitable for in vivo delivery of two antisense oligonucleotides to brain tumor cells causing a reduction of glioblastoma angiogenesis and an increase of animal survival. This system may hold promise for future clinical applications.

Fig. 1

Schematic drawing of Polycefin. This delivery device was used for inhibiting the expression of laminin α4 and β1 chains by morpholino AONs in vitro and in vivo to prevent angiogenesis of glial tumors. The modules are (1) morpholino AON 1a (to α4 chain) and 1b (to β1 chain) conjugated to the scaffold by disulfide bonds, which are cleaved in the cytoplasm by glutathi-one to release the free drugs, (2) mAb to transferrin receptor for cancer cell targeting and receptor-mediated endocytosis, (3) PEG for protection, (4) stretches of conjugated L-valine to provide pH-dependent lipophilicity for the disruption of endosomal membranes, and (5) optional fluorescent reporter dye (Fluorescein or Alexa Fluor 680) for detection

Fig. 2

Dot blot analysis of species cross-reactivity of transferrin receptor antibody OX-26. Lysate of the rat glioma cell line RG2 (positive control) reacts well with OX-26. Mouse glioma GL26 cell lysate shows background reactivity (negative control). Lysates of human glioma U87MG and of human brain microvascular endothelial cells (HBMVEC) also react well with the antibody. Omission of primary antibody (secondary antibody; middle panel) only produced background signal. A blot for β-actin as positive loading control is shown on the right panel. Results are shown for 8 μl lysates in triplicates

Fig. 3

In vitro targeting and inhibition of laminin chain synthesis by Polycefin. Immunofluorescent confocal microscopy of U87MG glioma cultures stained for laminin chains. Left, untreated control cells expressing α4 and β1 laminin-8 chains. Right, Polycefin that bears AONs to α4 and β1 chains markedly inhibits the expression of both laminin chains. Nuclei are counterstained with DAPI

Fig. 4

In vivo target inhibition by Polycefin and animal survival. (a) Survival of Polycefin-treated (0.5 mg/kg body weight) and control animals. After intracranial administration of four doses of Polycefin, the animal survival time was significantly increased (p < 0.0004) compared to saline treated or Polycefin(-mAb) treated rats. (b) Immunofluorescent analysis of xenotransplanted brain tumors with anti-human mAbs to laminin α4 or β1 chains. After Polycefin treatment, the number of tumor vessels positive for either laminin chain was markedly diminished. Therefore, Polycefin inhibited the expression of both its targets and their incorporation into basement membrane by human tumor cells. Asterisks denote tumor-adjacent (normal) brain area. This area has significantly decreased cellularity (revealed by blue nuclear staining with DAPI) compared to highly cellular tumor at the left. No vascular staining is observed in tumor-adjacent area with both mAbs, because the antibodies only recognized human laminin chains. Left lower panel, a hematoxylin-eosin (H&E) stained tumor showing a sharp boundary between highly cellular tumor and surrounding brain parenchyma with significantly fewer cells. Right lower panel, staining of a serial section with a pAb to laminin α4 chain recognizing human and rat protein that reveals all vessels. Note increased vascularity and cellularity of the tumor as opposed to hypocellular surrounding tissue (asterisk) that has only scattered vessels (arrows)

Fig. 5

Decreased vascular density and area in tumors treated with Polycefin. (a) Double immunohistochemical staining of rat brain vessels using antibodies to two endothelial markers, von Willebrand factor (vWF, green) and CD31 (red). The two markers were used to optimize the screening accuracy. In most vessels both markers co-distribute (yellow color). The vessel number is higher in the xenotransplanted U87MG tumor than in normal brain, and this number is decreased after four intracranial Polycefin treatments. (b) Quantitative assessment of vascular density in treated and untreated tumors compared to normal brain. Vessels were revealed by either marker (Fig. 5a) and their number was quantitated at × 200 direct magnification. Images were analyzed using NIH ImageJ software. Statistical significance was determined by ANOVA. Microvascular density in xenotransplanted U87MG human glioma mock-treated with saline is significantly increased compared to normal brain (p < 0.001). After four intracranial treatments with Polycefin, tumor vessel density was significantly decreased (p < 0.001) and became similar to normal brain tissue (NS, not significant with p > 0.05). (c) Quantitative assessment of vascular area in treated and untreated tumors compared to normal brain. Vessels were revealed by either marker (Fig. 5a) and their relative area quantitated as for vessel density. Vessel area in xenotransplanted U87MG human glioma mock-treated with saline is significantly increased compared to normal brain (p < 0.001). After four intracranial treatments with Polycefin, tumor vascular area significantly decreased (p < 0.001) but remained somewhat higher than in normal brain (p < 0.05)