Sebastian Bredow

Imaging of tumour neovasculature by targeting the TGF-β binding receptor endoglin

In vivo imaging of endothelial markers in intact tumour neovasculature would have applications in assessing the efficacy of anti-angiogenic agents in clinical trials. Although a variety of different endothelial markers have been described, few have been evaluated as imaging markers. The transforming growth factor-beta (TGF-beta) binding receptor endoglin is a proliferation-associated endothelial marker. We hypothesised that endoglin would be an ideal target for imaging since it is strongly upregulated in proliferating endothelial cells of the tumour neovasculature. We used a radiolabelled monoclonal anti-endoglin antibody and compared its neovascular binding, accumulation and in vivo behaviour to an isotype-matched control IgG(2a). Our data show that the probe binds specifically and rapidly within minutes in vivo and that correlative autoradiography and immunohistology support the in vivo imaging findings. Imaging of abundantly expressed endothelial targets circumvents delivery barriers normally associated with other tumour targeting strategies, and can potentially be used to quantitate molecular angiogenic markers.

In vivo assessment of vascular endothelial growth factor-induced angiogenesis.

To determine whether vascular endothelial growth factor (VEGF)‐induced tumor microvascularity is detectable by in vivo NMR imaging, an experimental study was conducted in nude mice. Human breast cancer cells (MCF‐7) and MCF‐7 cells stably transfected with the cDNA for the VEGF165 isoform (MV165) were grown in nude mice and models were characterized by RT‐PCR, Western blotting, ELISA, immunohistochemistry and NMR imaging using a novel synthetic protected graft copolymer (PGC) as a vascular probe. MV165 tumors showed a 1.6‐fold higher microvascular density by histology. Both tumors showed identical MR signal intensities on non‐contrast and Gd‐DTPA enhanced images. PGC enhanced MR imaging of tumoral vascular volume fraction (VVF), however, revealed significant differences between the 2 tumor types (MV165: 8.9 ± 2.1; MCF‐7: 1.7 ± 0.5; p < 0.003), as expected from histology. VVF changes were more heterogeneous in the MV165 model both among tumors as well as within tumors as determined 3‐dimensionally at submillimeter resolutions. Our results have potential applications for non‐invasive assessment of angiogenesis by in vivo imaging and for clinical monitoring during angiogenic therapies. Int. J. Cancer 83:798–802, 1999.

Spontaneous release of stable viral double-stranded RNA into the extracellular medium by influenza virus-infected MDCK epithelial cells: implications for the viral acute phase response

SummaryThe viral factor responsible for triggering the acute phase response, or ‘flu’ syndrome, associated with many acute viral infections is not defined. One candidate viral factor is double-stranded RNA (dsRNA) generated during viral replication. In this report we demonstrate by reverse-transcriptase polymerase-chain reaction that nuclease-stable viral RNA was released from influenza-infected MDCK epithelial cells at the time of cell lysis. Removal of virion-associated RNA by ultracentrifugation left equal amounts of positive-and negative-strand viral RNA in the medium that resisted degradation by endogenous RNase in the medium and by exogenous RNase added prior to phenol extraction. These data are the first demonstration that viral RNA with characteristics of dsRNA is spontaneously released from dying influenza virus-infected cells, and thus is available to amplify cytokine induction and contribute to systemic disease.

A Model System to Quantitate Tumor Burden in Locoregional Lymph Nodes during Cancer Spread

In order to quantitate the metastatic burden of secondary tumor deposits in locoregional lymph nodes, we produced a green fluorescent protein (GFP)-expressing murine cell line (B16F1) metastatic to lymph nodes in immunocompetent mice. When implanted into the hindleg of mice, all animals developed paraaortic lymph node metastases. Fluorescence microscopy, immunohistochemistry, and RT-PCR confirmed the presence of metastatic cells in lymph nodes. Tumoral deposits occurred preferentially in marginal sinuses and spread superficially to the subcortical area. Western blot analysis showed that the local tumor burden could be quantitated in individual lymph nodes. This model system should be useful for quantitating metastatic invasion particularly of micrometastases and aid in the development of lymphotropic drugs to detect and/or treat lymph node metastases in advanced cancers.

DNA binding chelates for nonviral gene delivery imaging.

Noninvasive in vivo monitoring of gene delivery would provide a critically important information regarding the spatial distribution, local concentration, kinetics of removal and/or biodegradation of the expression vector. We developed a novel approach to noninvasive gene delivery imaging using heterobifunctional peptide-based chelates (PBC) bearing double-stranded DNA-binding groups and a technetium-binding amino acid motif. One of such chelates: Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Lys4-Lys-(N-ε-[4-(psoralen-8-yloxy)]butyrate)-NH2 has been characterized and labeled with reduced 99mtc pertechnetate (oxotechnetate). the psoralen moiety (a dna binding group of pbc) allowed linking to double-stranded dna upon short-term irradiation with the near uv range light (>320 nm). Approximately 30–40% of added 99mTc-labeled PBC was nonextractable and co-eluted with a model pCMV-GFP vector during the gel-permeation chromatography. Nuclear imaging of ‘naked’ DNA and DNA complexes with lipid-based transfection reagents (‘lipoplexes’) has been performed after systemic or local administration of 99mTc-PBC-labeled DNA in mice. Imaging results were corroborated with the biodistribution using 99mTc-PBC and 32P-labeled DNA and lipoplexes. A markedly different biodistribution of 99mTc PBC-labeled DNA and lipoplexes was observed with the latter being rapidly trapped in the liver, spleen and lung. 99mTc PBC-DNA was used as an imaging tracer during in vivo transfection of B16 melanoma by local injection of ‘naked’ 99mTc PBC-DNA and corresponding lipoplexes. As demonstrated by nuclear imaging, 99mTc PBC-DNA lipoplexes showed a slower elimination from the site of injection than 99mTc PBC-DNA alone. This result correlated with a higher expression of marker mRNA and green fluorescent protein as determined using RT-PCR and immunohistochemistry, respectively.

Synthetic influenza viral double‐stranded RNA induces an acute‐phase response in rabbits

Numerous studies have characterized the physiological effects of synthetic, high‐molecular‐weight, homopolymeric, double‐stranded RNA (dsRNA), particularly polyriboinosinic·polyribo‐ cytidylic acid [Carter and De Clercq (1974): Science 186:1172–1178], but limited information exists regarding the physiological effects of dsRNA of viral composition and size. In this report, we determined sleep and fever responses of rabbits to intracerebroventricular injection of different doses of synthetic viral dsRNA (either 108 base pairs or 661 base pairs) derived from the N‐terminal sequence of gene segment 3 of the A/PR/8/34‐H1N1 (PR8) influenza virus. Both the108‐mer and the 661‐mer dsRNAs increased nonrapid eye movement sleep, suppressed rapid eye movement sleep, and induced fever. The 661‐mer dsRNA had more potent somnogenic and pyrogenic effects than the 108‐mer dsRNA on the basis of weight. Neither single‐stranded RNA from the corresponding sequences had significant effects on sleep or brain temperature. These results demonstrate for the first time that low‐molecular‐weight, viral dsRNA has the stability in vivo that is required to induce the fever and sleep changes found in natural viral infections, and the hypothesis is supported that virus‐associated dsRNA may be responsible for initiating the acute‐phase response during viral infections. J. Med. Virol. 57:198–203, 1999.