Anti-angiogenic therapy increases intratumoral adenovirus distribution by inducing collagen degradation

Abstract

Conditionally replicating adenoviruses (CRAd) are a promising class of gene therapy agents that can overcome already known glioblastoma (GBM) resistance mechanisms but have limited distribution upon direct intratumoral (i.t.) injection. Collagen bundles in the extracellular matrix (ECM) have an important role in inhibiting virus distribution. In fact, ECM pre-treatment with collagenases improves virus distributions to tumor cells. Matrix metalloproteinases (MMPs) are an endogenous class of collagenases secreted by tumor cells whose function can be altered by different drugs including anti-angiogenic agents, such as bevacizumab. In this study we hypothesized that upregulation of MMP activity during anti-angiogenic therapy can improve CRAd-S-pk7 distribution in GBM. We find that MMP-2 activity in human U251 GBM xenografts increases (*P=0.03) and collagen IV content decreases (*P=0.01) during vascular endothelial growth factor (VEGF-A) antibody neutralization. After proving that collagen IV inhibits CRAd-S-pk7 distribution in U251 xenografts (Spearman rho=-0.38; **P=0.003), we show that VEGF-blocking antibody treatment followed by CRAd-S-pk7 i.t. injection reduces U251 tumor growth more than each individual agent alone (***P<0.0001). Our data propose a novel approach to improve virus distribution in tumors by relying on the early effects of anti-angiogenic therapy.

Figure 1

In vitro effects of VEGF neutralization on adenovirus replication. (A) Flow cytometry analysis of U251 glioma cell line for expression of surface receptor CAR, αvβ3, αvβ5 and CD138. The percentages of positive cells for the respective receptors are shown in bar diagrams, below the flow cytometry histograms. (B and C) CRAd-S-pk7 replication in U251 glioma treated with VEGF-Ab was quantified via quantitative real-time PCR for E1A (B) or adenovirus progeny titer (C). (D) Toxicity of CRAd-S-pk7 in combination with VEGF-Ab in U251 glioma cells five days after infection (ns: not significant; *p<0.05; *** p<0.001).

Figure 2

VEGF trapping up-regulates MMP-2 expression in glioma cell lines. (A) Western blot of U87, U118, A172 and U251 glioma cells after treatment with VEGF-Ab for 5 days. Before collection, cell secretion was blocked for 6 hours with Golgi-Plug. Human β-Actin was used as a protein loading control. (B) Quantitative real-time PCR for MMP-2 mRNA levels in glioma cell lines was evaluated using the ΔC_T method. Before plotted in the graph, the MMP-2 expression for each cell line was normalized to their Ig-Control treated condition, to which was given an arbitrary value of 1 (dotted, thin black line: Control). The relative expression levels of MMP-2 for all four cell lines upon VEGF-Ab treatment were then presented as fold of their Control treated cells at the indicated time points. (C) Immunocytochemistry (ICC) of glioma cell lines for MMP-2 and collagen IV after treatment with VEGF-Ab. Cells treated in vitro with VEGF-Ab for 5 days were blocked with Golgi-Plug for 6 hours before fixation for ICC. Nuclei are stained blue with DAPI; collagen IV is presented in yellow and MMP-2 in red. Bars equal 50μm.

Figure 3

Anti-angiogenic treatment alters the extracellular matrix (ECM) architecture of human glioma xenograft in nude mice. (A) Immunohistochemistry staining for MMP-2, Collagen IV, CD31 and Laminin. (B–E) Quantification of staining intensity was done through a computer based scoring for each of the corresponding IHC slides (n = 5 animals for each group) and mean values ± standard error of measurement (SEM) are presented in bar diagrams. Bars equal 50μm; *p < 0.05; ns, not significant.

Figure 4

Adenovirus infection does not increase MMP-2 expression. U251 xenografts sections (A) were stained for MMP-2 with HRP and counterstained with hematoxylin; then scanned for intensity of HRP staining, presented as percent of areas with similar intensity (B). Bars represent mean intensity values of all xenografts. ns, not significant; ***p<0.001. (C, D) Sections from U87 xenografts were stained for collagen IV content and scanned for HRP-positivity. (C) Representative images from each group; the insert is the ScanSoft rendered intensity-image of the same slide. (D) Bar graph representation of the staining intensities for each group (n=4). **p=0.008.

Figure 5

Collagen inhibits adenovirus distribution in intracranial glioma xenografts. (A) Collagen IV immunostaining of normal mouse brains (i), mock-treated glioma (iv) and VEGF-Ab treated IC glioma (vii). Representative areas are presented enlarged in (ii), (v) and (viii), respectively; and counterstained with DAPI (iii, vi and ix). (B) Representative area within glioma that depicts the distribution patterns of adenovirus in proximity to collagen bundles. This area was divided in 60 small quadrants and for each of them the percentage of area covered by collagen or adenoviral hexon staining was determined. The threshold was set at 5% and a heat map was constructed for each antigen based on the legend shown in the figure. The quantified distribution patterns were statistically analyzed for possible correlation. Bars equal 150μm.

Figure 6

VEGF trapping increases adenovirus distribution and replication in glioma xenografts. We assessed CRAd-S-pk7 intracranial distribution and replication in flank tumors with and without concomitant VEGF-Ab therapy. (A) Immunohistochemistry (IHC) staining for detection of adenoviral hexon protein in intracranial xenografts. For each animal we depicted 8 quadrants around the injection tract and quantified hexon distribution (B) within the tumor. Each dot represents a different animal. (C and D) Quantification of CRAd-S-pk7 replication in glioma xenografts. U251 flank tumors were established in nude mice. VEGFAb therapy was started after the tumors reached a diameter of 5mm. Animals received two i.p. injections of VEGF-Ab within 5 days and then injected with 10⁸ IU of CRAd-S-pk7. Adenoviral replication in tumor tissue was quantified at the indicated time points via qRTPCR for E1A (C) and adenoviral progeny titer (D); Bars equal 400μm (H&E) and 200μm (IHC); *p<0.05;.

Figure 7

Combining VEGF trapping with CRAd-S-pk7 resulted in more reduced tumor growth rates than each therapy alone. Nude mice with U251 glioma flank xenografts were treated for 5 days with VEGF-Ab; followed by 10⁸ IU of oncolytic adenovirus CRAd-S-pk7. Tumor size was measured twice weekly with caliper. Growth curves represent the mean tumor growth rate, for each group, as compared to the day 0, when the therapy was started. To compare tumor growth curves the log rank test was used; ***p < 0.001