Positron emission tomography imaging for herpes virus infection: Implications for oncolytic viral treatments of cancer.

Molecular therapy using viruses would benefit greatly from a non-invasive modality for assessing dissemination of viruses. Here we investigated whether positron emission tomography (PET) scanning using [(124)I]-5-iodo-2'-fluoro-1-beta-d-arabinofuranosyl-uracil (FIAU) could image cells infected with herpes simplex viruses (HSV). Using replication-competent HSV-1 oncolytic viruses with thymidine kinase (TK) under control of different promoters, we demonstrate that viral infection, proliferation and promoter characteristics all interact to influence FIAU accumulation and imaging. In vivo, as few as 1 x 107 viral particles injected into a 0.5-cm human colorectal tumor can be detected by [(124)I]FIAU PET imaging. PET signal intensity is significantly greater at 48 hours compared with that at 8 hours after viral injection, demonstrating that PET scanning can detect changes in TK activity resulting from local viral proliferation. We also show the ability of FIAU-PET scanning to detect differences in viral infectivity at 0.5 log increments. Non-invasive imaging might be useful in assessing biologically relevant distribution of virus in therapies using replication-competent HSV.

Imaging the expression of transfected genes in vivo.

Imaging the expression of successful gene transduction has been demonstrated in vivo for the first time by using an appropriate combination of "marker gene" and "marker substrate" in an experimental animal model. The herpes simplex virus 1 thymidine kinase (HSV1-tk) gene was selected as an example of a marker gene, and the recombinant STK retrovirus containing HSV1-tk was used to transduce RG2 glioma cells in vitro and in vivo. RG2TK+ cell lines expressing the HSV1-tk gene and three potential marker substrates for the HSV1-TK enzyme were evaluated. Radiolabeled 5-iodo-2'-fluoro-2'deoxy-1-beta-D-arabinofuranosyluracil (FIAU) was shown to be a substantially better marker substrate for the HSV1-TK enzyme than 5-iodo-2'-deoxyuridine or ganciclovir. The magnitude of FIAU accumulation in different RG2TK+ clones corresponded to their sensitivity to ganciclovir and to the level of HSV1-tk mRNA expression. Imaging the expression of HSV1-tk in transduced RG2 tumor cells was demonstrated in animals using quantitative autoradiography; 2-[14C]FIAU accumulation was shown to be high in RG2TK+ brain tumors growing in one hemisphere and very low in nontransduced RG2 tumors in the contralateral hemisphere. Transduction of RG2 tumor cells with the HSV-tk gene in vivo resulted in tumors which accumulated FIAU to high levels and produced clearly defined images. Given the level of FIAU accumulation in the transduced tumors, it is likely that a clinically applicable method for imaging HSV1-tk gene expression can be implemented using existing clinical imaging techniques.

Tumor growth modulation by sense and antisense vascular endothelial growth factor gene expression: effects on angiogenesis, vascular permeability, blood volume, blood flow, fluorodeoxyglucose uptake, and proliferation of human melanoma intracerebral xenografts.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been investigated as a potent mediator of brain tumor angiogenesis and tumor growth. We evaluated the effect of VEGF expression on the pathophysiology of tumor growth in the brain. Human SK-MEL-2 melanoma cells, with minimal VEGF expression, were stably transfected with either sense or antisense mouse VEGF cDNA and used to produce intracerebral xenografts. Vascular permeability, blood volume, blood flow, and tumor fluorodeoxyglucose metabolism were assessed using tissue sampling and quantitative autoradiography. Tumor proliferation was assessed by measuring bromodeoxyuridine labeling indices. Tumor vascular density and morphological status of the blood-brain barrier were evaluated by immunohistochemistry. SK-MEL-2 cells transfected with sense VEGF (V+) expressed large amounts of mouse and human VEGF protein; V+ cells formed well-vascularized, rapidly growing tumors with minimal tumor necrosis. V+ tumors had substantial and significant increases in blood volume, blood flow, vascular permeability, and fluorodeoxyglucose metabolism compared to wild-type and/or V- (antisense VEGF) tumors. VEGF antisense transfected V- expressed no detectable VEGF protein and formed minimally vascularized tumors. V- tumors had a very low initial growth rate with central necrosis; blood volume, blood flow, vascular permeability, and glucose metabolism levels were low compared to wild-type and V+ tumors. A substantial inhibition of intracerebral tumor growth, as well as a decrease in tumor vascularity, blood flow, and vascular permeability may be achieved by down-regulation of endogenous VEGF expression in tumor tissue. VEGF-targeted antiangiogenic gene therapy could be an effective component of a combined strategy to treat VEGF-producing brain tumors.

Noninvasive imaging of herpes virus thymidine kinase gene transfer and expression: a potential method for monitoring clinical gene therapy.

Noninvasive imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression is possible with a clinical gamma camera and by single-photon emission tomography (SPECT) using 131I-labeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodo-uracil (FIAU). Studies were performed in rats bearing s.c. tumors. Tumors were produced by injection of wild-type RG2 glioma or W256 mammary carcinoma cells into one flank and RG2TK+ glioma or W256TK+ mammary carcinoma cells (that had been transduced in vitro with the HSV1-tk gene) into the opposite flank. In some animals, HSV1-tk gene transduction of the pre-established wild-type tumors was accomplished in vivo by direct intratumoral injection of retroviral vector-producer cells. Imaging studies were performed 2 weeks after tumor transduction to allow time for production and spread of the retroviruses through the tumor and for sufficient growth and increase in size of the tumors to facilitate imaging. The gamma camera and SPECT images revealed highly specific localization of [131I]FIAU-derived radioactivity to areas of HSV1-tk gene expression at 24, 36, and 48 h after i.v. administration of 1.6-2.8 mCi of [131I]FIAU. Comparative analysis of quantitative autoradiographic images obtained from the same tumors confirmed that the high levels of [131I]FIAU-derived radioactivity (> 1% dose) were localized to areas of HSV1-tk gene expression demonstrated by immunohistochemical staining for HSV1-tk protein. In contrast, significantly lower levels of [131I]FIAU-derived radioactivity (< 0.01%) were observed in the surrounding nontransduced tumor tissue, contralateral wild-type tumors, and other tissues that showed no immunohistochemical staining for the HSV1-tk protein. The magnitude of FIAU accumulation in RG2TK+, W256TK+, and wild-type tumors corresponded to the in vitro ganciclovir sensitivity of the cell lines used to produce these tumors, which indicates that the magnitude of FIAU accumulation reflects the level of HSV1-tk gene expression. We suggest that "clinically relevant" levels of HSV1-tk gene expression in transfected tissue can be imaged with [131I]FIAU and a gamma camera or SPECT, and that a significant improvement in imaging sensitivity and resolution is expected with [124I]FIAU and PET.

RG-2 glioma growth attenuation and severe brain edema caused by local production of interleukin-2 and interferon-gamma.

Two aspects of cytokine therapy of intracerebral tumors are considered in this study: modulation of tumor growth in vivo and central nervous system toxicity. Coimplantation of RG-2 glioma cells and retroviral vector producer cell lines was performed to provide a local source of interleukin-2 (IL-2) or IFN-gamma within the tumor and coinitiate an antitumor immune response. We demonstrated that local intratumoral production of IL-2 and IFN-gamma generates a cell-mediated antitumor response in vivo. This response was manifest as a diffuse infiltration of monocytes/macrophages, CD4+ and CD8+ T cells, and activation of microglial OX42+ cells in intracerebral RG2 tumors. The cell-mediated antitumor immune response resulted in the early suppression of intracranial and subcutaneous tumor growth, but the effect was not sustained and there were no tumor regressions. The absence of increased survival of animals with intracranial tumors is explained in part by the severe central nervous system toxicity caused by local production of IL-2 and IFN-gamma. Central nervous system toxicity induced blood-brain barrier disruption, vasogenic brain edema, and dislocation of the brain midline structures, as observed by dynamic magnetic resonance imaging and direct measurements of tissue water content. The clinical application of IL-2 and IFN-gamma gene transfer therapy for intracerebral tumors must consider the potential for severe vasogenic brain edema associated with intracerebral production of these cytokines.

Corticotropin-releasing factor decreases vasogenic brain edema.

We report the first series of studies comparing the anti-edematous effects of human corticotropin-releasing factor (hCRF) and dexamethasone in an experimental model of vasogenic peritumoral brain edema. Both hCRF and dexamethasone effectively decreased blood-brain barrier (BBB) permeability of intracerebral RG2 gliomas in rats as observed by contrast-enhanced T(1)-weighted magnetic resonance imaging. A decrease in the water content of tumor and peritumoral brain tissue was observed with proton-density magnetic resonance imaging and confirmed by direct wet/dry tissue measurements. The calculated ED(50) for hCRF was 59 micrograms/kg s.c. twice a day, and that for dexamethasone was 0.61 mg/kg i.m. twice a day; the hCRF:dexamethasone dose-potency ratio was 120:1 on a molar basis. The anti-edematous action of hCRF is not mediated by the release of adrenal corticosteroids. A direct action of hCRF on the tumor microvasculature results in restoration of BBB integrity and up-regulation of BBB-specific protein expression. The average survival time with chronic treatment was prolonged significantly in the hCRF-treated group (35 days) compared with the dexamethasone-treated group (28 days; P < 0.05) and the saline-treated control group (22 days; P < 0.0001). hCRF, as an alternative to corticosteroid therapy, may provide substantial benefits with respect to reducing the major side effects encountered with long-term, high-dose corticosteroid treatment.

Imaging adenoviral-mediated herpes virus thymidine kinase gene transfer and expression in vivo.

The feasibility of noninvasive imaging of adenoviral-mediated herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression was assessed in a well-studied animal model of metastatic colon carcinoma of the liver. Tumors were produced in syngeneic BALB/c mice by intrahepatic injection of colon carcinoma cells (MCA-26). Seven days later, three different doses (3 x 10(8), 1 x 10(8), and 3 x 10(7) plaque-forming units (pfu) of the recombinant adenoviral vector ADV. Rous sarcoma virus (RSV)-tk bearing the HSV1-tk gene were administered by intratumoral injection in separate groups of mice. Two control groups of tumor-bearing mice received intratumoral injections of the control adenoviral vector dl-312 or buffer alone, respectively. T2-weighted magnetic resonance (MR) images of mice were obtained before administering the virus and provided an anatomical reference of hepatic tumor localization. Eighteen h after the virus injection, one group of animals was given i.v. injections of 300 microCi of no-carrier-added 5-[131I]-2'-fluoro-1-beta-D-arabinofuranosyluracil (FIAU) and imaged 24 h later with a gamma camera. In some animals, the tumors were sampled and processed for histology and quantitative autoradiography (QAR). The gamma camera images demonstrated highly specific localization of [131I]FIAU-derived radioactivity to the area of ADV.RSV-tk-injected tumors in the liver, which was confirmed by coregistering the gamma camera and T2-weighted MR images. There was no accumulation of [131I]FIAU-derived radioactivity in tumors that were injected with the control vector or injection solution alone. A more precise distribution of radioactivity in the area of transfected tumor was obtained by histological and QAR comparisons. A heterogeneous pattern of radioactivity distribution in transfected tumors was observed. A punctate pattern of radioactivity distribution was observed in peritumoral liver tissue in animals given injections of 3 x 10(8) and 1 x 10(8) pfu of ADV.RSV-tk but not in animals given injections of 3 x 10(7) pfu nor in control animals. A QAR-microscopic comparison showed that the punctate areas of radioactivity colocalized with cholangial ducts. The level of [131I]FIAU-derived radioactivity accumulation (HSV1-tk expression) in the transfected tumors was viral dose-dependent. The viral dose-dependency of radioactivity accumulation was more pronounced in peritumoral liver, which was confirmed by reverse transcription-PCR analysis. A separate group of tumor-bearing animals received different doses of ADV.RSV-tk vector followed by treatment with ganciclovir (GCV), 10 mg/kg i.p. b.i.d. for 6 days. The ADV.RSV-tk transfected tumors significantly regressed with GCV treatment; the control tumors continued to grow. During the GCV treatment, the levels of liver transaminases (ALT and AST) were significantly increased in animals that received injections of 3 x 10(8) and 1 x 10(8) pfu of ADV.RSV-tk but not in animals that received injections of 3 x 10(7) pfu and in control animals. The observed liver toxicity confirms the results of gamma camera and QAR imaging, which demonstrated an unwanted spread of ADV.RSV-tk vector and HSV1-tk expression in peritumoral and remote liver tissue at higher doses. These and our previous results indicate that noninvasive imaging of adenoviral-mediated HSV1-tk gene expression is feasible for monitoring cancer gene therapy in patients.

Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography.

We report a series of studies that assess the feasibility and sensitivity of imaging of herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression with [124I]-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil ([124I]-FIAU) and positron emission tomography (PET) and the ability of [124I]-FIAU-PET imaging to discriminate different levels of HSV1-tk gene expression. Studies were performed in rats bearing multiple s.c. tumors derived from W256 rat carcinoma and RG2 rat glioma cells. In the first set, we tested the sensitivity of [124I]-FIAU-PET imaging to detect low levels of HSV1-tk gene expression after retroviral-mediated gene transfer. HSV1-tk gene transduction of one of preestablished wild-type W256 tumor in each animal was accomplished by direct intratumoral injection of retroviral vector-producer cells (W256-->W256TK* tumors). Tumors produced from W256 and W256TK+ cells served as the negative and positive control in each animal. Highly specific images of [124I]-FIAU-derived radioactivity were obtained in W256TK* tumors (that were transduced in vivo) and in W256TK+ tumors but not in nontransduced wild-type W256 tumors. The level of "specific" incorporated radioactivity in transduced portions of both W256TK* and W256TK+ tumors was relatively constant between 4 and 50 h. In the second set, we tested whether [124I]-FIAU and PET imaging can measure and discriminate between different levels of HSV1-tk gene expression. Multiple s.c. tumors were produced from wild-type RG2 cells and stably transduced RG2TK cell lines that express different levels of HSV1-tk. A highly significant relationship between the level of [124I]-FIAU accumulation [% injected dose/g and incorporation constant (Ki)] and an independent measure of HSV1-tk expression (sensitivity of the transduced tumor cells to ganciclovir, IC50) was demonstrated, and the slope of this relationship was defined as a sensitivity index. We have demonstrated for the first time that highly specific noninvasive images of HSV1-tk expression in experimental animal tumors can be obtained using radiolabeled 2'-fluoro-nucleoside [124I]-FIAU and a clinical PET system. The ability to image the location (distribution) of gene expression and the level of expression over time provides new and useful information for monitoring clinical gene therapy protocols in the future.

Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo.

To evaluate the efficiency of gene delivery in gene therapy strategies for malignant brain tumors, it is important to determine the distribution and magnitude of transgene expression in target tumor cells over time. Here, we assess the time- and vector dose-dependent kinetics of recombinant herpes simplex virus (HSV)-1 vector-mediated gene expression and vector replication in culture and in vivo by a recently developed radiotracer method for noninvasive imaging of gene expression (J. G. Tjuvajev et al., Cancer Res., 55: 6126-6132, 1995). The kinetics of viral infection of rat 9L gliosarcoma cells by the replication-conditional HSV-1 vector, hrR3, was studied by measuring the accumulation rate of 2-[14C]-fluoro-5-iodo-1-beta-D-arabinofuranosyl-uracil (FIAU), a selective substrate for viral thymidine kinase (TK). The level of viral TK activity in 9L cells was monitored by the radiotracer assay to assess various vector doses and infection times, allowing vector replication and spread. In parallel, viral yields and levels of Escherichia coli beta-galactosidase activity were assessed quantitatively. To study vector replication, spread and HSV-1-tk and lacZ gene coexpression in vivo, first- or second-generation recombinant HSV-1 vectors (hrR3 or MGH-1) were injected into s.c. growing rat 9L or human U87 deltaEGFR gliomas in nude rats at various times (8 h to 8 days) and at various vector doses [1 x 10(6) to 2 x 10(9) plaque-forming units (PFUs)] prior to imaging. For noninvasive assessment of HSV-1-tk gene expression (124I-labeled FIAU % dose/g), 0.15 mCi of 124I-labeled FIAU was injected i.v. 8 h after the last vector administration, and FIAU positron emission tomography (PET) was performed 48 h later. For the assessment of HSV-1-tk and lacZ gene coexpression, 0.2 mCi of 131I-labeled FIAU was injected i.v. 24 h after the last vector administration. Forty-eight h later, animals were killed, and tumors were dissected for quantitative autoradiographical and histochemical assessment of regional distribution of radioactivity (TK expression measured as 131I-labeled FIAU % dose/g) and coexpressed lacZ gene activity. The rates of FIAU accumulation (Ki) in hrR3-infected 9L cells in culture, which reflect the levels of HSV-1-tk gene expression, ranged between 0.12 and 3.4 ml/g/min. They increased in a vector dose- and infection time-dependent manner and correlated with the virus yield (PFUs/ml), where the PFUs:Ki ratios remained relatively constant over time. Moreover, a linear relationship was observed between lacZ gene expression and FIAU accumulation 5-40 h after infection of 9L cells with a multiplicity of infection of 1.5. At later times (> 52 h postinjection), high vector doses (multiplicity of infection, 1.5) led to a decrease of FIAU accumulation rates, viral yield, and cell pellet weights, indicating vector-mediated cell toxicity. Various levels of HSV-1-tk gene expression could be assessed by FIAU-PET after in vivo infection of s.c. tumors. The levels of FIAU accumulation were comparatively low (approximately ranging from 0.00013 to 0.003% injected dose/g) and were spatially localized; this may reflect viral-induced cytolysis of infected tumor cells and limited lateral spread of the virus. Image coregistration of tumor histology, HSV-1-tk related radioactivity (assessed by autoradiography), and lacZ gene expression (assessed by beta-galactosidase staining) demonstrated a characteristic pattern of gene expression around the injection sites. A rim of lacZ gene expression immediately adjacent to necrotic tumor areas was observed, and this zone was surrounded by a narrow band of HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue. These results demonstrate that recombinant HSV-1 vector-mediated HSV-1-tk gene expression can be monitored noninvasively by PET, where the areas of FIAU-derived radioactivity identify the viable portion of infected tumor tissue that retains FIAU accumulation ability, and that the accumulation rate of FIAU in culture, Ki, reflects the number of HSV-1 viral particles in the infected tumor cell population [4.1 +/- 0.6 x 10(6) PFUs/Ki unit (PFUs divided by ml/min/g)]. Moreover, time-dependent and spatial relationships of HSV-1-tk and lacZ gene coexpression in culture and in vivo indicate the potential for indirect in vivo imaging of therapeutic gene expression in tumor tissue infected with any recombinant HSV-1 vector where a therapeutic gene is substituted for the lacZ gene.

Imaging TCR-dependent NFAT-mediated T-cell activation with positron emission tomography in vivo.

A noninvasive method for molecular imaging of T-cell activity in vivo would be of considerable value. It would aid in understanding the role of specific genes and signal transduction pathways in the course of normal and pathologic immune responses, and could elucidate temporal dynamics and immune regulation at different stages of disease and following therapy. We developed and assessed a novel method for monitoring the T-cell receptor (TCR)-dependent nuclear factor of activated T cells (NFAT)-mediated activation of T cells by optical fluorescence imaging (OFI) and positron emission tomography (PET). The herpes simplex virus type 1 thymidine kinase/green fluorescent protein [HSV1-tk/GFP (TKGFP)] dual reporter gene was used to monitor NFAT-mediated transcriptional activation in human Jurkat cells. A recombinant retrovirus bearing the NFAT-TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting NFAT-specific enhancer. Transduced Jurkat cells were used to establish subcutaneous infiltrates in nude rats. We demonstrated that noninvasive OFI and nuclear imaging of T-cell activation is feasible using the NFAT-TKGFP reporter system. PET imaging with [(124)I]FIAU using the NFAT-TKGFP reporter system is sufficiently sensitive to detect T-cell activation in vivo. PET images were confirmed by independent measurements of T-cell activation (e.g., CD69) and induction of GFP fluorescence. PET imaging of TCR-induced NFAT-dependent transcriptional activity may be useful in the assessment of T cell responses, T-cell-based adoptive therapies, vaccination strategies and immunosuppressive drugs.

Functional coexpression of HSV-1 thymidine kinase and green fluorescent protein: implications for noninvasive imaging of transgene expression.

Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk genes was generated (tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2'-fluoro-2'-deoxy-1beta-D-arabinofuranosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.

A general approach to the non-invasive imaging of transgenes using cis-linked herpes simplex virus thymidine kinase.

Non-invasive imaging of gene expression opens new prospects for the study of transgenic animals and the implementation of genetically based therapies in patients. We have sought to establish a general paradigm to enable whole body non-invasive imaging of any transgene. We show that the expression and imaging of HSV1-tk (a marker gene) can be used to monitor the expression of the LacZ gene (a second gene) under the transcriptional control of a single promoter within a bicistronic unit that includes a type II internal ribosomal entry site. In cells bearing a single copy of the vector, the expression of the two genes is proportional and constant, both in vitro and in vivo. We demonstrate that non-invasive imaging of HSV1-tk gene accurately reflects the topology and activity of the other cis-linked transgene.

Imaging transgene expression with radionuclide imaging technologies.

A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review "marker/reporter gene" imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.

Imaging experimental brain tumors with 1-aminocyclopentane carboxylic acid and alpha-aminoisobutyric acid: comparison to fluorodeoxyglucose and diethylenetriaminepentaacetic acid in morphologically defined tumor regions.

The goal of this study was to evaluate the differences and define the advantages of imaging experimental brain tumors in rats with two nonmetabolized amino acids, 1-aminocyclopentane carboxylic (ACPC) acid and alpha-aminoisobutyric (AIB) acid compared with imaging with fluorodeoxyglucose (FDG) or the gallium-diethylenetriaminepentaacetic acid chelate (Ga-DTPA). 1-aminocyclopentane carboxylic acid, AIB, and FDG autoradiograms were obtained 60 minutes after intravenous injection to simulate positron emission tomography (PET) imaging, whereas the Ga-DTPA autoradiograms were obtained 5 or 10 minutes after injection to simulate gadolinium (Gd)-DTPA-enhanced magnetic resonance (MR) images. Three experimental tumors were studied (C6, RG2, and Walker 256) to provide a range of tumor types. Triple-label quantitative autoradiography was performed, and parametric images of the apparent distribution volume (Va, mL/g) for ACPC or AIB, relative glucose metabolism (R, micromol/100 g/min), vascular permeability to Ga-DTPA (K1, microL/min/g), and histology were obtained from the same tissue section. The four images were registered in an image array processor, and regions of interest in tumor and contralateral brain were defined on morphologic criteria (histology) and were transferred to the autoradiographic images. A comparative analysis of all measured values was performed. The location and morphologic characteristics of the tumor had an effect on the images and measurements of Va, R, and K1. Meningeal extensions of all three tumors consistently had the highest amino acid uptake (Va) and vascular permeability (K1) values, and subcortical portions of the tumors usually had the lowest values. Va and R (FDG) values generally were higher in tumor regions with high-cell density and lower in regions with low-cell density. Tumor areas identified as "impending" necrosis on morphologic criteria consistently had high R values, but little or no change in Va or K1. Tumor necrosis was seen consistently only in the larger Walker 256 tumors; low values of R and Va for AIB (less for ACPC) were measured in the necrotic-appearing regions, whereas K1 was not different from the mean tumor value. The highest correlations were observed between vascular permeability (K1 for Ga-DTPA) and Va for AIB in all three tumors; little or no correlation between vascular permeability and R was observed. The advantages of ACPC and AIB imaging were most convincingly demonstrated in C6 gliomas and in Walker 256 tumors. 1-aminocyclopentane was substantially better than FDG or Ga-DTPA for identifying tumor infiltration of adjacent brain tissue beyond the macroscopic border of the tumor; ACPC also may be useful for identifying low-grade tumors with an intact blood-brain barrier. Contrast-enhancing regions of the tumors were visualized more clearly with AIB than with FDG or Ga-DTPA; viable and necrotic-appearing tumor regions could be distinguished more readily with AIB than with FDG. [11C]-labeled ACPC and AIB are likely to have similar advantages for imaging human brain tumors with PET.

"Facilitated" amino acid transport is upregulated in brain tumors.

The goal of this study was to determine the magnitude of "facilitated" amino acid transport across tumor and brain capillaries and to evaluate whether amino acid transporter expression is "upregulated" in tumor vessels compared to capillaries in contralateral brain tissue. Aminocyclopentane carboxylic acid (ACPC), a non-metabolized [14C]-labeled amino acid, and a reference molecule for passive vascular permeability, [67Ga]-gallium-diethylenetriaminepentaacetic acid (Ga-DTPA), were used in these studies. Two experimental rat gliomas were studied (C6 and RG2). Brain tissue was rapidly processed for double label quantitative autoradiography 10 minutes after intravenous injection of ACPC and Ga-DTPA. Parametric images of blood-to-brain transport (K1ACPC and K1Ga-DTPA, microL/min/g) produced from the autoradiograms and the histology were obtained from the same tissue section. These three images were registered in an image array processor; regions of interest in tumor and contralateral brain were defined on morphologic criteria (histology) and were transferred to the autoradiographic images to obtain mean values. The facilitated component of ACPC transport (deltaK1ACPC) was calculated from the K1ACPC and K1Ga-DTPA data, and paired comparisons between tumor and contralateral brain were performed. ACPC flux, K1ACPC, across normal brain capillaries (22.6 +/- 8.1 microL/g/min) was >200-fold greater than that of Ga-DTPA (0.09 +/- 0.04 microL/g/min), and this difference was largely (approximately 90%) due to facilitated ACPC transport. Substantially higher K1ACPC values compared to corresponding K1DTPA values were also measured in C6 and RG2 gliomas. The deltaK1ACPC values for C6 glioma were more than twice that of contralateral brain cortex. K1ACPC and deltaK1ACPC values for RG2 gliomas was not significantly higher than that of contralateral cortex, although a approximately 2-fold difference in facilitated transport is obtained after normalization for differences in capillary surface area between RG2 tumors and contralateral cortex. K1ACPC, deltaK1ACPC, and K DTPA were directly related to tumor cell density, were higher in regions of "impending" necrosis, and the tumor/contralateral brain ACPC radio-activity ratios (0 to 10 minutes) were very similar to that obtained with 0 to 60 minutes experiments. These results indicate that facilitated transport of ACPC is upregulated across C6 and RG2 glioma capillaries, and that tumors can induce upregulation of amino acid transporter expression in their supporting vasculature. They also suggest that early imaging (e.g., 0 to 20 minutes) with radiolabeled amino acids in a clinical setting may be optimal for defining brain tumors.

Imaging of brain tumor proliferative activity with iodine-131-iododeoxyuridine.

METHODS: Iodine-131-iododeoxyuridine (IUdR) uptake and retention was imaged with SPECT at 2 and 24 hr after administering a 10-mCi dose to six patients with primary brain tumors. The SPECT images were directly compared to gadolinium contrast-enhanced MR images as well as to [18F]fluorodeoxyglucose (FDG) PET scans and 201Tl SPECT scans. RESULTS: Localized uptake and retention of IUdR-derived radioactivity was observed in five of six patients. The plasma half-life of [131I]IUdR was short (1.6 min) in comparison to the half-life of total plasma radioactivity (6.4 hr). The pattern of [131I]IUdR-derived radioactivity was markedly different in the 2-hr compared to 24-hr images. Radioactivity was localized along the periphery of the tumor and extended beyond the margin of tumor identified by contrast enhancement on MRI. The estimated levels of tumor radioactivity at 24 hr, based on semiquantitative phantom studies, ranged between < 0.1 and 0.2 microCi/cc (< 0.001% and 0.002% dose/cc); brain levels were not measurable. CONCLUSIONS: Iodine-131-IUdR SPECT imaging of brain tumor proliferation has low (marginal) sensitivity due to low count rates and can detect only the most active regions of tumor growth. Imaging at 24 hr represents a washout strategy to reduce 131I-labeled metabolites contributing to background activity in the tumors, and is more likely to show the pattern of [131I]IUdR-DNA incorporation and thereby increase image specificity. Iodine-123-IUdR SPECT imaging at 12 hr and the use of [124I]IUdR and PET will improve count acquisition and image quality.

Iododeoxyuridine uptake and retention as a measure of tumor growth.

Iodine-131-iododeoxyuridine (IUdR) uptake and retention was measured in two C6 glioma cell lines (C6m and C6a) with different growth characteristics. Animals with intracerebral (i.c.) C6a tumors had a mean survival of 16 days, whereas only 1 of 20 animals with i.c. C6m tumors died during an 8-wk period of observation. The growth of i.c. C6m tumors could be described by the Gompertz equation; tumor doubling time increased from 1.9 to 5.2 days between Days 8 and 16 after tumor inoculation. Corresponding measurements of 131I-IUdR uptake and retention (24 hr after IUdR administration) by i.c. C6m tumors were also time-dependent and decreased from 0.075 to 0.027 to 0.011 %dose/g in 8-, 10- and 16-day-old tumors, respectively. Iodine-131-IUdR uptake in "rapidly growing" i.c. C6a tumors was substantially higher (0.30 %dose/g at 24 hr) than that in "slowly growing" i.c. C6m tumors and corresponded with differences in the survival data. Subcutaneous C6a tumors had comparable high uptake values (0.49 %dose/g at 24 hr), and 93% of total tumor radioactivity was recovered in DNA 24 hr after IUdR administration. Clearance of radioactivity was rapid in nonproliferative tissues; more than 80% of plasma radioactivity was cleared in 24 hr. Tumor-to-cortex radioactivity ratios ranged from 100/1 to 120/1 and 150/1 between 24, 48 and 96 hr after IUdR injection respectively. A "washout strategy," which reduces tissue background activity and increases specificity for PET and SPECT imaging of IUdR-DNA incorporation, is possible with longer-lived radioisotopes of iodine.

Regression of C6 rat brain tumors by cells expressing an antisense insulin-like growth factor I receptor RNA.

We have previously shown that C6 cells expressing an antisense insulin-like growth factor I receptor (IGF-IR) RNA are no longer tumorigenic in syngeneic rats, protecting them from subsequent subcutaneous tumor challenge and causing regression of established subcutaneous tumors. In the present study, we have investigated the efficacy of this strategy on intracerebrally implanted C6 rat glioblastoma cells. We demonstrate that C6 cells expressing an antisense IGF-IR RNA implanted for 24 h in the subcutaneous tissue of the rats are able to elicit an anti-tumor response in the brain, leading to complete brain tumor regression and long-term survival of the rats. These findings suggest the possibility of therapeutic intervention in human gliomas.

Salmonella-based tumor-targeted cancer therapy: tumor amplified protein expression therapy (TAPET) for diagnostic imaging.

In preclinical studies, genetically engineered Salmonella have the ability to localize, selectively accumulate, and persist within transplantable murine tumors, spontaneous murine tumors and human tumor xenographs, and can express therapeutic proteins at high levels. These strains of engineered non-virulent Salmonella typhimurium display the capacity to accumulate and grow selectively in a variety of tumor types and to inhibit the growth of primary and metastatic tumors following intravenous injection into tumor-bearing mice. One strain of the bacteria (VNP20009) which has endogenous antitumor activity is currently in Phase I clinical trials. The bacteria are highly attenuated and genetically stable. The combination of the lipid mutation and the purine auxotrophy attenuate the virulence of the bacteria by greater than 10000-fold and enhance the specificity of the bacteria for tumor tissue. These bacteria have been found to be safe in mice, pigs and monkeys when administered intravenously. Second-generation Salmonella vectors will be developed to include transgenes that will express therapeutic agents and reporter transgenes for non-invasive imaging. We have performed a preliminary study to demonstrate localization of [(14)C]FIAU in tumored mice pretreated with Salmonella expressing HSV1-TK. The [(14)C]FIAU radioactivity and bacterial count data strongly support a Salmonella(TK)-dependent [(14)C]FIAU accumulation of at least 30-fold higher in tumor tissue compared to muscle tissue. These data warrant further investigation on the use of genetically engineered Salmonella as a systemically administered tumor-specific agents for tumor therapy and delivery of diagnostic imaging markers.

Anti-neoplastic properties of human corticotropin releasing factor: involvement of the nitric oxide pathway.

We report a series of the in vivo and in vitro studies that evaluate the anti-neoplastic potential of hCRF in W256 rat mammary carcinoma. Using magnetic resonance imaging (MRI) and direct measurements of tumor and peritumoral brain water content we found that hCRF treatment (100 micrograms/kg subcutaneously twice a day for 3 days) caused significant inhibition of growth and vascular permeability of the i.c. W256 tumors. hCRF also exhibited antiproliferative and differentiation-inducing effects in W256 cells in vitro. The calculated IC50 values were 70 nM and 100 nM of hCRF, as measured by digital videomicroscopic quantitation of tumor cell population growth rate and by [3H]-thymidine incorporation assay, respectively. The observed effects in W256 cells were CRF receptor mediated. This was shown in two ways: by the presence of relatively high levels of CRF1 receptor mRNA in W256 cells, and by the fact that the tumor growth inhibitory and differentiation inducing effects of hCRF in vitro were abolished by the CRF receptor antagonist a-helical CRF (9-41). Antiproliferative and differentiation inducing effects of hCRF in W256 cells involve activation of nitric oxide synthase (NOS) and L-arginine-NO pathway. This was shown by using the inhibitor of NOS, the L-nitro-arginine methyl esther (L-NAME), which prevented the antiproliferative and differentiation inducing effects of hCRF in vitro. The cytotoxicity of NO in W256 cells was assessed by the addition of sodium nitroprusside (SNP) to the media. SPN exhibited dose-dependent cytotoxicity in W256 cells with IC50 of 100 muM SNP as measured by [3H]-thymidine incorporation assay. We conclude, that hCRF has substantial anti-neoplastic effects which include inhibition of proliferation and induction of differentiation of the tumor cells in vitro, and a decrease in tumor vascular permeability (and possibly neo-angiogenesis) in vivo.