Ronald G. Blasberg

Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts.

Adoptive transfer of large numbers of tumor-reactive CD8+ cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer. However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8+ CTLs, with much less emphasis on the role and contribution of CD4+ T cells. Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4+ T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation. Surprisingly, CD4+ T cells developed cytotoxic activity, and tumor rejection was dependent on class II–restricted recognition of tumors by tumor-reactive CD4+ T cells. Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4+ T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma. These findings suggest a novel potential therapeutic role for cytotoxic CD4+ T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4+ cells in vivo over current protocols favoring in vitro expansion and differentiation.

Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo

A noninvasive method for molecular imaging of the activity of different signal transduction pathways and the expression of different genes in vivo would be of considerable value. It would aid in understanding the role specific genes and signal transduction pathways have in various diseases, and could elucidate temporal dynamics and regulation at different stages of disease and during various therapeutic interventions. We developed and assessed a method for monitoring the transcriptional activation of endogenous genes by positron-emission tomography (PET) imaging. The HSV1-tk/GFP (TKGFP) dual reporter gene was used to monitor transcriptional activation of p53-dependent genes. A retrovirus bearing the Cis-p53/TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting p53-specific enhancer. U87 glioma and SaOS-2 osteosarcoma cells were transduced with this retrovirus and used to establish xenografts in rats. We demonstrated that DNA damage-induced up-regulation of p53 transcriptional activity correlated with the expression of p53-dependent downstream genes, such as p21, in U87 (wild-type p53), but not in SaOS-2 osteosarcoma (p53 −/−) cells. We showed that PET, with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-[124I]iodouracil) and the Cis-p53TKGFP reporter system, is sufficiently sensitive to image the transcriptional regulation of genes in the p53 signal transduction pathway. These imaging results were confirmed by independent measurements of p53 activity and the expression levels of downstream genes (e.g., p21) by using conventional molecular-biological assays. PET imaging of p53 transcriptional activity in tumor xenografts by using the Cis-p53TKGFP reporter system may be useful in assessing novel therapeutic approaches.

Molecular-genetic imaging: current and future perspectives

Medical imaging has undergone a revolution in the past decade. This is largely due to improved technology involving all the major imaging modalities: MRI, computed tomography (CT), positron emission tomography (PET), ultrasound, and optical imaging. These advances and improvements in technology are being rapidly translated into the clinic and have established new standards of medical practice. Cancer imaging was identified as one of six “extraordinary scientific opportunities” by the National Cancer Institute in 1997–1998, and the institute’s subsequent funding initiatives have provided a major stimulus for further developments. A major target of these initiatives has been development of and support for molecular imaging. Molecular-genetic imaging provides visualization in space and time of normal as well as abnormal cellular processes at a molecular or genetic level. Needless to say, current gamma camera, PET, MRI, and optical technologies do not visualize individual cells, much less molecules. Perhaps the most exciting aspect of this emerging new field are the novel imaging paradigms being developed — paradigms that image molecular-genetic processes rather than anatomy. These paradigms can be successful within the inherent spatial-resolution limits of existing imaging systems, provided that the volume element, or voxel, of the tissue (cells) is relatively homogenous. This review will focus primarily on radionuclide imaging, although many of the principles described are directly applicable to optical and MRI technology as well. A more extensive discussion of these issues was recently published (1). Molecular imaging has its roots in molecular biology and cell biology as well as in imaging technology. Three different noninvasive, in vivo imaging technologies have developed more or less in parallel: (a) MRI (2–6); (b) nuclear imaging (quantitative autoradiography, gamma camera, and PET) (7–11); and (c) optical imaging of small animals (12–14). The convergence of these disciplines is at the heart of the molecular imaging success story and constitutes the wellspring for further advances in the field. The development of versatile and sensitive assays that do not require tissue samples will be of considerable value for monitoring of molecular-genetic and cellular processes in animal models of human disease, as well as for studies in human subjects. Noninvasive imaging of molecular-genetic and cellular processes will complement established ex vivo molecular-biological assays that require tissue sampling; it also provides a spatial as well as a temporal dimension to our understanding of various diseases.

Molecular Imaging of Temporal Dynamics and Spatial Heterogeneity of Hypoxia-Inducible Factor-1 Signal Transduction Activity in Tumors in Living Mice

Tumor hypoxia is a spatially and temporally heterogeneous phenomenon, which results from several tumor and host tissue-specific processes. To study the dynamics and spatial heterogeneity of hypoxia-inducible factor-1 (HIF-1)-specific transcriptional activity in tumors, we used repetitive noninvasive positron emission tomography (PET) imaging of hypoxia-induced HIF-1 transcriptional activity in tumors in living mice. This approach uses a novel retroviral vector bearing a HIF-1–inducible “sensor” reporter gene (HSV1-tk/GFP fusion) and a constitutively expressed “beacon” reporter gene (DsRed2/XPRT). C6 glioma cells transduced with this multireporter system revealed dose-dependent patterns in temporal dynamics of HIF-1 transcriptional activity induced by either CoCl2 or decreased atmospheric oxygen concentration. Multicellular spheroids of C6 reporter cells developed a hypoxic core when >350 μm in diameter. 18F-2′-fluoro-2′deoxy-1β-D-arabionofuranosyl-5-ethyl-uracil (FEAU) PET revealed spatial heterogeneity of HIF-1 transcriptional activity in reporter xenografts in mice as a function of size or ischemia-reperfusion injury. With increasing tumor diameter (>3 mm), a marked increase in HIF-1 transcriptional activity was observed in the core regions of tumors. Even a moderate ischemia-reperfusion injury in small C6 tumors caused a rapid induction of HIF-1 transcriptional activity, which persisted for a long time because of the inability of C6 tumors to rapidly compensate acute changes in tumor microcirculation.

midazolam Changes Cerebral Blood Flow in Discrete Brain Regions : an H2-15o Positron Emission Tomography Study

Background: Changes in regional cerebral blood flow (rCBF) determined with H215 O positron emission tomographic imaging can identify neural circuits affected by centrally acting drugs. Methods: Fourteen volunteers received one of two midazolam infusions adjusted according to electroencephalographic response. Low or high midazolam effects were identified using post‐hoc spectral analysis of the electroencephalographic response obtained during positron emission tomographic imaging based on the absence or presence of 14‐Hz spindle activity. The absolute change in global CBF was calculated, and relative changes in rCBF were determined using statistical parametric mapping with localization to standard stereotactic coordinates. Results: The low‐effect group received 7.5 +/‐ 1.7 mg midazolam (serum concentrations, 74 +/‐ 24 ng/ml), and the high‐effect group received 9.7 +/‐ 1.3 mg midazolam (serum concentrations, 129 +/‐ 48 ng/ml). Midazolam decreased global CBF by 12% from 39.2 +/‐ 4.1 to 34.4 +/‐ 6.1 ml [center dot] 100 g sup ‐1 [center dot] min sup ‐1 (P < 0.02 at a partial pressure of carbon dioxide of 40 mmHg). The rCBF changes in the low‐effect group were a subset of the high‐effect group. Decreased rCBF (P < 0.001) occurred in the insula, the cingulate gyrus, multiple areas in the prefrontal cortex, the thalamus, and parietal and temporal association areas. Asymmetric changes occurred, particularly in the low‐effect group, and were more significant in the left frontal cortex and thalamus and the right insula. Relative rCBF was increased in the occipital areas. Conclusion: Midazolam causes dose‐related changes in rCBF in brain regions associated with the normal functioning of arousal, attention, and memory.

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.

“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, μL/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 (∂KlACPC) 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 μL/g/min) was >200-fold greater than that of Ga-DTPA (0.09 ± 0.04 μL/g/min), and this difference was largely (~90%) due to facilitated ACPC transport. Substantially higher K1ACPC values compared to corresponding K1DTPA values were also measured in C6 and RG2 gliomas. The ∂K1ACPC values for C6 glioma were more than twice that of contralateral brain cortex. K1ACPC and ∂K1ACPC values for RG2 gliomas was not significantly higher than that of contralateral cortex, although a ~2-fold difference in facilitated transport is obtained after normalization for differences in capillary surface area between RG2 tumors and contralateral cortex. K1ACPC, ∂K1ACPC, and K1DTPA 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.

Surface-Enhanced Resonance Raman Scattering Nanostars for High Precision Cancer Imaging

Surface-enhanced resonance Raman scattering gold nanostars allow detection of macro- and microscopic foci of premalignant and cancerous lesions in vivo. Seeing Nanostars Microscopic tumors may be difficult for the naked eye to see, but they are no match for nanosized imaging agents, which home in on cancerous tissues to signal the presence of disease. Harmsen and colleagues created a new generation of cancer imaging agents, called “surface-enhanced resonance Raman scattering (SERRS) nanostars” −75-nm star-shaped gold cores wrapped in Raman reporter molecule-containing silica. When hit by a near-infrared laser, these nanostars emit a unique photonic signature (Raman “fingerprint”). The authors used a new silica encapsulation method and a reporter molecule that was “in resonance” with the laser, which meant that they shone nearly 400 times brighter than their “nonresonant” counterparts during Raman imaging. The SERRS nanostars were used to image macro- and microscopic malignant lesions in animal models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma with high precision. As endoscopic and handheld Raman imaging devices are further developed for the clinic, the SERRS nanostars are sure to find a place in human tumor detection. The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread, and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the true extent of tumors is desired clinically and surgically. We show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal locoregional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed SERRS nanostars. The SERRS nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, and in one human sarcoma xenograft model, SERRS nanostars enabled accurate detection of macroscopic malignant lesions, as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 fM limit of detection) of SERRS nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS nanostars a promising imaging agent for more precise cancer imaging and resection.

In vivo molecular-genetic imaging: multi-modality nuclear and optical combinations

Multi-modality, noninvasive in vivo imaging is increasingly being used in molecular-genetic studies and will soon become the standard approach for reporter gene imaging studies in small animals. The coupling of nuclear and optical reporter genes, as described here, represents only the beginning of a far wider application of this technology in the future. Optical imaging and optical reporter systems are cost-effective and time-efficient; they require less resources and space than PET or MRI, and are particularly well suited for imaging small animals, such as mice. Optical reporter systems are also very useful for the quantification and selection of transduced cells using FACS, and for performing in vitro assays to validate the function and sensitivity of constitutive and specific-inducible reporter systems. However, optical imaging techniques are limited by depth of light penetration and do not yet provide optimal quantitative or tomographic information. These issues are not limiting for PET- or MRI-based reporter systems, and PET- and MRI-based animal studies are more easily generalized to human applications. Many of the shortcomings of each modality alone can be overcome by the use of dual- or triple-modality reporter constructs that incorporate the opportunity for PET, fluorescence and bioluminescence imaging.

Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats.

SummaryHerpes simplex virus (HSV) mutants kill dividing tumor cells but spare non-proliferating, healthy brain tissue and may be useful in developing new treatment strategies for malignant brain tumors. Two HSV mutants, a thymidine kinase deficient virus (TK-) and a ribonucleotide reductase mutant (RR-), killed 7/7 human tumor cell lines in tissue culture. The TK-HSV killed Rat RG2 glioma and W256 carcinoma lines but not the rat C6 glioma in culture. TK-HSV replication (12 pfu/cell) was similar to wild-type HSV (10 pfu/cell) in rapidly dividing W256 cells in tissue culture, but was minimal (<1 pfu/cell) in serum-starved cells, suggesting that the proliferative activity of tumor cells at the site and time of TK-HSV injection may influence efficacyin vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or virus free inoculum. A significant effect of TK-HSV therapy on W256 tumor growth was demonstrated compared to controls (p=0.002). Complete regression was observed in 4/9 experimental tumors, with no recurrence over 6 months. Tumor growth in the remaining 5/9 animals was attenuated during the first 3 to 5 days after treatment, but not beyond 5 days compared to 9 matched control animals; no tumor regression was observed in any of the control animals. These results suggest that HSV mutants are potentially useful as novel therapeutic agents in the treatment of tumors in immunocompetent subjects.