Mapping the in vivo distribution of herpes simplex virions.

We describe a method for labeling enveloped viral particles with a radiotracer, indium-111, allowing labeled viruses to be traced in vivo by nuclear imaging. After initial optimization experiments, a labeling efficiency of 83% (incorporation yield) was achieved for herpes simplex virus (HSV), resulting in a specific activity of 30 microCi/10(9) PFU. The labeling procedure did not significantly reduce the infectivity of the labeled virus and the virus did not release any significant amounts of the radionuclide within 12 hr after labeling. Sequential imaging of animals after intravenous administration of the labeled virus showed fast accumulation in the liver and redistribution from the blood pool (immediately after injection) to liver and spleen (12-24 hr after injection). At 12 hr after injection 7% of the virus-associated (111)In had been eliminated from the body and the remaining organ distribution of the virus was as follows: spleen 2.87 +/- 0.54% ID/g; liver, 2.60 +/- 0.51% ID/g; kidney, 0.98 +/- 0.31% ID/g; lung, 0.57 +/- 0.10% ID/g; [corrected] and lower amounts in other organs. Our results indicate that the described method allows qualitative and quantitative assessment of viral biodistribution in vivo by nuclear imaging.

Treatment of experimental brain tumors with trombospondin-1 derived peptides: an in vivo imaging study.

Antiangiogenic and antiproliferative effects of synthetic D-reverse peptides derived from the type 1 repeats of thrombospondin (TSP1) were studied in rodent C6 glioma and 9L gliosarcomas. To directly measure tumor size and vascular parameters, we employed in vivo magnetic resonance (MR) imaging and corroborated results by traditional morphometric tissue analysis. Rats bearing either C6 or 9L tumors were treated with TSP1-derived peptide (D-reverse amKRFKQDGGWSHWSPWSSac, n=13) or a control peptide (D-reverse amKRAKQAGGASHASPASSac, n=12) at 10 mg/kg, administered either intravenously or through subcutaneous miniosmotic pumps starting 10 days after tumor implantation. Eleven days later, the effect of peptide treatment was evaluated. TSP1 peptide-treated 9L tumors (50.7+/-44.2 mm3, n=7) and C6 tumors (41.3+/-34.2 mm3, n=6) were significantly smaller than tumors treated with control peptide (9L: 215.7+/-67.8 mm3, n=6; C6: 184.2+/-105.2 mm3, n=6). In contrast, the in vivo vascular volume fraction, the mean vascular area (determined by microscopy), and the microvascular density of tumors were not significantly different in any of the experimental groups. In cell culture, TSP1, and the amKRFKQDGGWSHWSPWSSac peptide showed antiproliferative effects against C6 with an IC of 45 nM for TSP1. These results indicate that TSP1-derived peptides retard brain tumor growth presumably as a result of slower de novo blood vessel formation and synergistic direct antiproliferative effects on tumor cells. We also show that in vivo MR imaging can be used to assess treatment efficacy of novel antiangiogenic drugs non-invasively, which has obvious implications for clinical trials.

Imaging of tumour neovasculature by targeting the TGF-beta 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.

Non-invasive in vivo mapping of tumour vascular and interstitial volume fractions.

Non-invasive measurement of haemodynamic parameters and imaging of neovasculature architecture is of importance in determining tumour prognosis, in directing tissue sampling and in assessing treatment efficacy. In the current research we investigated a dual tracer nuclear magnetic resonance (NMR) technique to map the tumour vascular (VVF) and interstitial volume fraction (IVF) non-invasively in vivo. We hypothesised that a NMR signal emanating after intravenous administrations of a vascular paramagnetic probe (MPEG-PL-GdDTPA) can be maximised so that additional signal after administration of a second interstitial probe (GdDTPA) would only reflect the IVF but not the VVF. The method and its assumptions were verified and experimental conditions optimised both in phantoms and in C6 glioma bearing rats. Data derived from in vivo studies show tumoral VVF and IVF values that are consistent with histology data and literature values; the relative ranking order of values was tumour > muscle > brain. Image maps showed intratumoral and intertumoral heterogeneity of both parameters at submillimetre pixel resolution. The method is applicable to a wide variety of tumour models and can theoretically be performed repeatedly to study tumour growth or involution during therapy.

Intracellular magnetic labeling of lymphocytes for in vivo trafficking studies.

Lymphocyte adhesion and trafficking is difficult to observe in vivo over time. We used magnetic resonance imaging (MRI) to identify magnetically labeled lymphocytes in phantom experiments and in tissue. A method of lymphocyte labeling was developed that is based on fluid-phase endocytosis of nanometer-sized biocompatible superparamagnetic particles. The maximum cell uptake in culture was 0.11 ng Fe/cell corresponding to 5 x 10(6) particles/lymphocyte. Cells stably retained the label and were fully viable for at least 3 days. Labeled lymphocytes showed adhesion to human endothelial cells similar to unlabeled cells, indicating no effect of labeling on cell surface expression of adhesion proteins. No particle-mediated cytotoxicity could be observed. The detection threshold of MRI for detecting labeled lymphocytes in the current study was 2.5 x 10(6) cells/30 microL sampling volume. Following intravenous injection of labeled lymphocytes into rats, cells accumulated in spleen, lymph nodes and liver with a similar bio-distribution as unlabeled cells. Lymphocyte accumulation in the spleen resulted in MRI signal intensity changes readily detectable by MRI. These findings suggest that intracellular lymphocyte labeling with superparamagnetic particles is feasible, does not alter the viability or tissue distribution of labeled cells and allows the detection of labeled lymphocytes by MRI.

In vivo localization of diglycylcysteine-bearing synthetic peptides by nuclear imaging of oxotechnetate transchelation.

A phenomenon of in vivo transchelation of oxotechnetate from a complex with glucoheptonic acid to synthetic peptides bearing oxotechnetate-binding motifs and a technique for in vivo visualization of these peptides are described. Using two model peptides bearing two tandem diglycylcysteine (GGC) motifs (P1) or three GGC motifs (P2), we demonstrated that: (i) these peptides efficiently transchelated oxo-[99mTc]technetate from a complex with glucoheptonic acid in vitro (a complex with peptides was stable at least 24 h; radiochemical purity exceeded 95% by high performance liquid chromatography); (ii) injection of peptides into the rectus femoris muscle (at 0.5-1 micromol of SH groups) followed by an intravenous injection of 99mTc-glucoheptonate (0.25-0.5 mCi per animal) yielded visualization of the injected muscle by nuclear imaging within 1 h after injection; (iii) the experimental/control (contralateral) thigh muscle ratio was 1.80 +/- 0.05 for peptide P1 and 3.0 +/- 0.1 for P2; (iv) the injection of a control peptide P2 with SH groups covalently modified with N-ethylmaleimide resulted in a ratio of 1.4 +/- 0.2. These findings argue for specific association of oxo-[99mTc]technetate with free thiols within the binding motif of injected peptides in vivo. In vivo transchelation of oxo-[99mTc]technetate may be useful for the purpose of noninvasive imaging of gene expression, i.e., when the expression product bears GGC motifs.

A long-circulating co-polymer in "passive targeting" to solid tumors.

A co-polymer of O-methyl polyethylene(glycol)-O'-succinate (MPEGs, m.w. 5100) and poly-l-lysine (PL, median m.w. 32700, degree of polymerization 256) has been synthesized by covalent grafting. The resultant MPEGs-PL (30% modification degree of epsilon-amino groups) had a hydrodynamic diameter corresponding to a 690 kD protein. Free amino groups (180/mol of the co-polymer) were used for conjugation of diethylene pentaacetic or succinic acid residues to MPEGs-PL. The potential of the resultant compound as a carrier of therapeutic and diagnostic drugs was studied using a rodent carcinoma models. The co-polymer had a blood pool half-life of 36h in adenocarcinoma-bearing rats. Radioactively labeled preparations were resistant to trans-chelation with apotransferrin and stable in blood for 24 h. The co-polymer accumulated in solid tumors at the level of 1.5-2% injected dose/g of tumor in 24 h. At that time, 34-40% of the accumulated polymer was associated with tumor cell fraction. The co-polymer non-covalently associated with cis-diamminedichloroplatinum(II), showed a cytostatic effect against mouse F9 carcinoma, and induced a reversal in tumor growth after intravenous administration.

Antibody-mediated versus nontargeted delivery in a human small cell lung carcinoma model.

The uptake of macromolecular agents in tumor cells (LX-1, human small cell lung carcinoma) and in corresponding tumor xenografts was compared in a parallel study utilizing a long-circulating biocompatible graft copolymer, MPEGs-PL-DTPA [Bogdanov, A., Jr., et al. (1995) Adv. Drug Delivery Rev. 16, 335-348; Bogdanov, A., Jr., et al. (1996) Bioconjugate Chem. 7, 144-149] and a tumor-specific chimeric monoclonal antibody, BR96 [Hellstrom, I., et al. (1990) Cancer Res. 50, 2183-2190; Garrigues, J., et al. (1993) Am. J. Pathol. 142, 607-622]. Covalent grafted conjugates of methoxy-(polyethylene glycol)succinate and polylysine and BR96 were modified with DTPA, biotinyl, or rhodamine-X-residues. Using radionuclide and fluorescent labeled derivatives of the copolymer and the antibody, we established that (1) the copolymer does not associate with the plasma membrane in N-ethylmaleimide-treated cells and is slowly internalized by live cells at 37 degrees C; (2) the antibody binds rapidly to the surface of LX-1 cells and shows active internalization in vesicles with a subsequent slow decrease in the cell-associated antibody concentration; (3) LX-1 cells bear more than 1 million BR96 binding sites/cell (with an apparent Kd of 4.5 x 10[-7] M); and (4) intravesicular fluorescence intensity in LX-1 cells was linearly dependent on copolymer concentration, suggesting fluid phase endocytosis. Tumor localization by nuclear imaging, biodistribution, microdistribution by histology, and determination of tumor cell fraction uptake was performed in LX-1 tumor xenografts. In vivo study showed that MPEGs-PL-DTPA progressively accumulates in the tumor, yielding from 2.8+/-1.5% injected dose per gram of tissue (ID/g) at 24 h to 5.2+/-1.7% ID/g of tissue at 48 h. The antibody accumulation peaked at 24 h (6.0+/-3.2% ID/g) and decreased thereafter. We determined that at 24 h 43.9+/-11.29% of the polymer accumulated in the tumor was associated with tumor cell fraction with the remainder of the accumulated dose localized in the interstitium. Accumulation of the biotinylated graft copolymer and the antibody in LX-1 xenografts and their uptake in cells were confirmed by histology using avidin-peroxidase staining. Our study demonstrates that, although BR96 is highly specific in vitro, tumoral drug delivery in vivo can be equally high with long-circulating graft copolymers because of slow extravasation at the tumor site.

Tumoral distribution of long-circulating dextran-coated iron oxide nanoparticles in a rodent model.

PURPOSE: To investigate the accumulation and cellular uptake of long-circulating dextran-coated iron oxide (LCDIO) particles in malignant neoplasms in vivo. MATERIALS AND METHODS: A gliosarcoma rodent model was established to determine the distribution of a model LCDIO preparation in tumors. LCDIO accumulation in tissue sections was evaluated with multichannel fluorescence microscopy with rhodaminated LCDIO, green fluorescent protein as a tumor marker, and Hoechst 33258 dye as an intravital endothelial stain. Uptake into tumor cells was corroborated with results of immunohistochemical and cell culture uptake experiments. The effect of intratumoral LCDIO uptake on magnetic resonance (MR) imaging signal intensity was evaluated with a 1.5-T superconducting magnet. RESULTS: Tumoral accumulation of LCDIO was 0.11% +/- 0.06 of the injected dose per gram of tissue in brain tumors and was sufficient for detection at MR imaging. In tumor sections, LCDIO was preferentially localized in tumor cells (49.0% +/- 4.6) but was also taken up by macrophages in tumors (21.0% +/- 3.1) and by endothelial cells in the areas of active angiogenesis (6.5% +/- 1.4). In cell culture, LCDIO uptake was strongly correlated with growth rate of tumor cell lines. CONCLUSION: Tumoral LCDIO accumulation was not negligible and helped explain MR imaging signal intensity changes observed in clinical trials. Microscopically, LCDIO accumulated predominantly in tumor cells and tumor-associated macrophages. Uptake into tumor cells appeared to be directly proportional to cellular proliferation rates.

Novel gliosarcoma cell line expressing green fluorescent protein: A model for quantitative assessment of angiogenesis.

Angiogenesis is essential for tumor proliferation and metastasis. The extent of angiogenesis is measured by microvessel density (MVD) which has been identified as an independent prognostic factor for relapse-free survival in cancer patients. Existing methods of MVD assessment measure the microvessel count in the most active area of neovascularization ("hot spots") using antibodies against vascular endothelial antigens. This may produce unreliable results because of tissue volume loss and misshapening during the fixation and dehydration procedures. We report here a genetically engineered 9L cell line constitutively expressing green fluorescent protein that can be visualized using fluorescence microscopy without additional histological staining. The model developed in this study allows for the performance of simple and easy MVD counting, assuming that nonfluorescent "black spots" visible by fluorescence microscopy within the borders of the tumor tissue represent blood vessels. This assumption was confirmed by a comparative study utilizing conventional histological methods, anti-CD31 immunohistology, and Hoechst 33258 dye exclusion. This model is also useful for delineation of the true borders between tumor and normal brain tissue, including microscopic tumor extensions, without multiple histological staining. The suggested model allows quantification of tumor angiogenesis in tissue specimens, thus providing independent prognostic information about tumor growth and regression. It is expected to be most valuable in evaluating the efficacy of anti-angiogenic therapy.

Mechanism of gadophrin-2 accumulation in tumor necrosis.

The molecular mechanism by which gadophrin-2 targets necrotic tumor tissue was investigated. Biodistribution studies and magnetic resonance imaging (MRI) and histologic/autoradiographic correlation were performed in xenograft mouse models bearing human tumors (HT 29, WiDr, LX 1). Binding of gadophrin-2 to DNA, lipids, or proteins was determined by fluorescence spectrophotometry. Protein binding was determined by dialysis and gel electrophoresis. Accumulation of gadophrin-2 was low (<0.7% injected dose/g tissue at 24 hours after injection) in viable tumor but higher in necrotic tumor regions and was readily detectable by MRI. Within a given tumor, the agent preferentially localized in the periphery of necrotic areas. Within these regions gadophrin-2 was bound to interstitial albumin and not other proteins, lipids, or DNA. Tumoral accumulation of gadophrin-2 most likely occurs through its binding to plasma albumin and subsequent slow extravasation into the tumor interstitium.

MR imaging of gene delivery to the central nervous system with an artificial vector.

PURPOSE: To determine whether gene delivery by means of a synthetic no viral DNA delivery system that is capable of gene transfer can be mapped with magnetic resonance (MR) imaging. MATERIALS AND METHODS: The DNA delivery system consisted of aminated (poly-L-lysine-conjugated) dextran chains anchored together with a central superparamagnetic core. Three different types of constructs were synthesized that differed in their amino content and, thus, DNA-loading capacity. The model plasmid consisted of complementary DNA encoding for humanized green fluorescent protein. Constructs were tested in cell culture and in vivo in a rat model. RESULTS: All three constructs were capable of transfecting human cells in culture with transfection efficiencies ranging from 0.3% to 4.1%, which is similar to that of diethylaminoethyl-dextran. MR imaging experiments showed that DNA constructs induced signal intensity changes that co-localized with phosphorus-33-labeled plasmid distribution at autoradiography. After injection of the constructs into the corpus callosum of rats, weak green fluorescent protein expression of neuronal and glial cells could be detected at immunohistologic examination. CONCLUSION: Dextran-based nonviral DNA delivery systems are capable of transfecting cells and can be visualized with MR imaging.

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.