Viable Hypoxic Cells Research Articles

Development of Novel 18F-PET Agents for Tumor Hypoxia Imaging

Tumor hypoxia is a major factor responsible for tumor progression, metastasis, invasion, and treatment resistance, leading to low local tumor control and recurrence after radiotherapy in cancers. Here,18F-positron emission tomography (PET) probes are developed for visualizing viable hypoxic cells in biopsies. Pimonidazole derivatives and nitroimidazole-based agents bearing sulfonyl linkers were evaluated. A small-animal PET study showed that the tumor uptake of [18F]-23 [poly(ethylene glycols) (PEG)-sulfonyl linker] of 3.36 ± 0.29%ID/g was significantly higher (P < 0.01) than that of [18F]-20 (piperazine-linker tracer, 2.55 ± 0.49%ID/g) at 2 h postinjection in UPPL tumors. The tumor-to-muscle uptake ratio of [18F]-23 (2.46 ± 0.48 at 2 h pi) was well improved compared with that of [18F]-FMISO (1.25 ± 0.14 at 2 h pi). A comparable distribution pattern was observed between ex vivo autoradiography of [18F]-23 and pimonidazole staining of the neighboring slice, indicating that [18F]-23 is a promising PET agent for hypoxia imaging.

Synthesis of 99mTc-labeled 2-Mercaptobenzimidazole as a novel radiotracer to diagnose tumor hypoxia.

Discovery of 99mTc-labeled imidazole derivatives as a potential radiotracer for hypoxic tumor imaging is considered to be of great interest because of non-invasive detection capabilities. 2-Mercaptobenzimidazole (2-MBI) was successfully synthesized, characterized and radiolabeled with [99mTc (CO)3(H2O)3]+ intermediate to form 99mTc-2-MBI complex with radiochemical purity of ≥95% yield as observed by instant-thin layer chromatography (ITLC) and radio-high performance liquid chromatography (radio-HPLC). The 99mTc-2-MBI complex was observed to be stable in saline and serum with no noticeable decomposition at room temperature and 37 °C, respectively, over a time period of 24 h. Biodistribution results in Balb/c mice bearing S180 tumor show that 99mTc-2-MBI highly internalized in tumor tissue, also possess preferably high tumor/muscle and tumor/blood ratios 4.14 ± 0.77 and 3.91 ± 0.63, respectively at 24 h incubation. Scintigraphic imaging study shows 99mTc-2-MBI is visibly accumulated in hypoxic tumor tissue, suggesting it would be a promising radiotracer for early stage diagnosis of tumor hypoxia.

Ruthenium Complexes with Hydrophobic Ligands That Are Key Factors for the Optical Imaging of Physiological Hypoxia

The phosphorescence emission of ruthenium complexes was applied to the optical imaging of physiological hypoxia. We prepared three complexes with hydrophobic substituents on the phenanthroline ligand and characterized their emission, which was quenched by molecular oxygen. Among the complexes synthesized in this study, a pyrene chromophore-linked ruthenium complex, Ru-Py, exhibited optimal properties for the imaging of hypoxia; the prolonged lifetime of the triplet excited state of the ruthenium chromophore, which was induced by efficient energy distribution and transfer from the pyrene unit, provided the highest sensitivity towards molecular oxygen. The introduction of hydrophobic pyrene increased the lipophilicity of the complex, leading to enhanced cellular uptake. Consequently, the bright phosphorescence of Ru-Py was seen in the cytoplasm of viable hypoxic cells, whereas the signal from aerobic cells was markedly weaker. Thus, we could clearly discriminate between hypoxic and aerobic cells by monitoring the phosphorescence emission. Furthermore, Ru-Py was applied to optical imaging in live mice. An intramuscular injection of Ru-Py successfully visualized ischemia-based hypoxia, which was constructed by leg banding.

Hypoxia and Metastasis

In this issue of Clinical Cancer Research , Rofstad et al. ([1][1]) report on the significance of chronic (diffusion-limited) and acute (fluctuating or perfusion-limited) hypoxia in D-12 and R-18 human melanoma xenografts on the development of metastatic disease. That tumors may contain different

Dynamics of Tumor Hypoxia Measured with Bioreductive Hypoxic Cell Markers

Hypoxic cells are common in tumors and contribute to malignant progression, distant metastasis and resistance to radiotherapy. It is well known that tumors are heterogeneous with respect to the levels and duration of hypoxia. Several strategies, including high-oxygen-content gas breathing, radiosensitizers and hypoxic cytotoxins, have been developed to overcome hypoxia-mediated radioresistance. However, with these strategies, an increased tumor control rate is often accompanied by more severe side effects. Consequently, development of assays for prediction of tumor response and early monitoring of treatment responses could reduce both over- and undertreatment, thereby avoiding unnecessary side effects. The purpose of this review is to discuss different assays for measurement of hypoxia that can be used to detect changes in oxygen tension. The main focus is on exogenous bioreductive hypoxia markers (2-nitroimidazoles) such as pimonidazole, CCI-103F, EF5 and F-misonidazole. These are specifically reduced and bind to macromolecules in viable hypoxic cells. A number of these bioreductive drugs are approved for clinical use and can be detected with methods ranging from noninvasive PET imaging (low resolution) to microscopic imaging of tumor sections (high resolution). If the latter are stained for multiple markers, hypoxia can be analyzed in relation to different microenvironmental parameters such as vasculature, proliferation and endogenous hypoxia-related markers, for instance HIF1alpha and CA-IX. In addition, temporal and spatial changes in hypoxia can be analyzed by consecutive injection of two different hypoxia markers. Therefore, bioreductive exogenous hypoxia markers are promising as tools for development of predictive assays or as tools for early treatment monitoring and validation of potential endogenous hypoxia markers.

A kinetic model for dynamic [18F]-Fmiso PET data to analyse tumour hypoxia

A method is presented to identify and quantify hypoxia in human head-and-neck tumours based on dynamic [18F]-Fmiso PET patient data, using a model for the tracer transport. A compartmental model was developed, inspired by recent immunohistochemical investigations with the tracer pimonidazole. In order to take the trapping of the tracer and the diffusion in interstitial space into account, the kinetic model consists of two compartments and a specific input function. This voxel-based data analysis allows us to decompose the time-activity curves (TACs) into their perfusion, diffusion and hypoxia-induced retention components. This characterization ranges from well perfused tumours over diffusion limited hypoxia to strong hypoxia and necrosis. The overall shape of the TAC and the model parameters may point at the structural architecture of the tissue sample. The model addresses the two main problems associated with hypoxia imaging with PET. Firstly, the hypoxic areas are spatially separated from well perfused vessels, causing long diffusion times of the tracer. Secondly, tracer uptake occurs only in viable hypoxic cells, which constitute only a small subpopulation in the presence of necrosis. The resulting parameters such as the concentration of hypoxic cells and the perfusion are displayed in parameter plots (‘hypoxia map’). Quantification of hypoxia performed with the presented kinetic model is more reliable than a criterion based on static standardized uptake values (SUV) at an early timepoint, because severely hypoxic/necrotic tissues show low uptake and are thus overlooked by SUV threshold identification. The derived independent measures for perfusion and hypoxia may provide a basis for individually adapted treatment planning.

HER-2/neuOverexpression Increases the Viable Hypoxic Cell Population within Solid Tumors without Causing Changes in Tumor Vascularization

AbstractThe effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6HER-2) into SCID-Rag2M mice. LCC6HER-2 tumors were more viable (H&amp;E-stained tumor sections) than isogenic vector control tumors (LCC6Vector). Correspondingly, a 2.7-fold increase in trypan blue–excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6HER-2 versus LCC6Vector tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6HER-2 tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content ≥ 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6HER-2 than in LCC6Vector tumors (16.41 ± 8.1% and 5.96 ± 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser536) nuclear factor-κB p65 were significantly elevated in LCC6HER-2 tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1α protein levels was observed in LCC6HER-2 compared with LCC6Vector tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6HER-2 tumors, no significant differences were found (P &amp;gt; 0.05) between LCC6HER-2 and LCC6Vector vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.

Treatment of HER-2/neu overexpressing breast cancer xenograft models with trastuzumab (Herceptin) and gefitinib (ZD1839): drug combination effects on tumor growth, HER-2/neu and epidermal growth factor receptor expression, and viable hypoxic cell fraction.

The purpose of this research was to assess the effects of single agent and combination treatment with trastuzumab and gefitinib on tumor growth and tumor microenvironment in two HER-2/neu overexpressing breast xenograft models, MDA-MB-435/LCC6(HER-2) (LCC6(HER-2); estrogen receptor negative) and MCF-7(HER-2) (estrogen receptor positive). LCC6(HER-2) and MCF-7(HER-2) cells, both in tissue culture and xenografts grown in SCID-Rag 2M mice, were treated with trastuzumab and gefitinib, alone or in combination. The rate of tumor growth was determined. In addition, tumor HER-2/neu and epidermal growth factor receptor expression, cell viability, cell cycle distribution, and proportion of viable hypoxic cells were determined by flow cytometric analyses of single tumor cell suspensions. Both tumor models were very sensitive to trastuzumab and moderately sensitive to gefitinib in vivo. The combination resulted in therapeutic effects, as judged by inhibition of tumor growth, which was greater (albeit not statistically significant) than that observed with trastuzumab administered as a single agent. Trastuzumab was effective in down-regulating HER-2/neu, and gefitinib mediated a reduction in epidermal growth factor receptor expression on tumor cells. In LCC6(HER-2) tumors, trastuzumab significantly reduced tumor cell viability, which was not improved by the addition of gefitinib. Gefitinib dramatically reduced the proportion of viable hypoxic cells in LCC6(HER-2) and MCF-7(HER-2) tumors. This effect was abrogated by the addition of trastuzumab. Although in vivo efficacy studies in two HER-2/neu overexpressing breast xenograft models showed that the combination of trastuzumab and gefitinib was effective, analyses of various cellular parameters failed to reveal beneficial effects and argue that this drug combination may not be favorable.

Modulation of cell death in the tumor microenvironment

The microenvironment of solid human tumors is characterized by heterogeneity in oxygenation. Hypoxia arises early in the process of tumor development because rapidly proliferating tumor cells outgrow the capacity of the host vasculature. Formation of solid tumors thus requires coordination of angiogenesis with continued tumor cell proliferation. However, despite such neovascularization, hypoxia is persistent and frequently found in tumors at the time of diagnosis. Tumors with low oxygenation have a poor prognosis, and strong evidence suggests this is because of the effects of hypoxia on malignant progression, angiogenesis, metastasis, and therapy resistance. The presence of viable hypoxic cells is likely a reflection of the development of hypoxia tolerance resulting from modulation of cell death in the microenvironment. This acquired feature has been explained on the basis of clonal selection—the hypoxic microenvironment selects cells capable of surviving in the absence of normal oxygen availability. However, the persistence and frequency of hypoxia in solid tumors raises a second potential explanation. We suggest that stable microregions of hypoxia may play a positive role in tumor growth. Although hypoxia inhibits cell proliferation and in tumor cells will eventually induce cell death, hypoxia also provides angiogenic and metastatic signals. The development of hypoxia tolerance will thus allow prolonged survival in the absence of oxygen and generation of a persistent angiogenic signal. We will discuss the concept of hypoxia tolerance and review mechanisms used by cancer cells to acquire this phenotype. The concept of hypoxia tolerance has important implications for current and future therapeutic approaches. Most therapeutic efforts to combat hypoxia have focused on targeting the presence of hypoxia itself. Our hypothesis predicts that targeting the biological responses to hypoxia and the pathways leading to hypoxia tolerance may also be attractive therapeutic strategies. Copyright 2003, Elsevier Science (USA). All rights reserved.

Measuring hypoxia and predicting tumor radioresistance with nuclear medicine assays

Tumor cells at low oxygen tension are relatively radioresistant. The hypoxic fraction of individual tumors before, during and after radiotherapy is likely to have prognostic value but its diagnosis still awaits an accurate and acceptable assay. The recent indications that hypoxia can also induce the expression of specific genes and promote a more aggressive tumor phenotype makes its diagnosis even more important. Over 15 years ago, misonidazole, an azomycin-based hypoxic cell radiosensitizer, was found to link covalently to cellular molecules at rates inversely proportional to intracellular oxygen concentration. The use of bioreducible markers to positively label zones of viable hypoxic cells within solid tumors and to predict for tumor radioresistance was proposed. Several hypoxic markers have now been identified and their selective binding within tumors has been measured by both invasive and non-invasive assays. Research from our laboratory has emphasized both mechanistic and preclinical studies associated with nuclear medicine procedures for measuring tumor hypoxia and predicting tumor radioresistance. This report updates radiation oncologists about the status of nuclear medicine hypoxic marker research and development as of mid-1997. While several potential imaging agents have been identified, their testing and validation in appropriate human tumors will require focused research efforts by individual academic departments and, possibly, by clinical trials performed through cooperative groups. Since the prediction of hypoxia in individual tumors could strongly impact radiotherapy treatment planning, the radiation oncology research community is best positioned to execute the validation studies associated with these markers.

Anomalous patterns of nitroimidazole binding adjacent to necrosis in human glioma xenografts: possible role of decreased oxygen consumption.

In contrast to reports of extensive hypoxia in human gliomas in situ measured by pO2 histography, non-invasive methods of assessing glioma oxygenation, including nitroimidazole binding, have yielded surprisingly contradictory results. In order to investigate the relationship of necrosis, hypoxia, nitroreductase activity and cellular respiration in human gliomas, subcutaneous models using the human glioma cell lines M059K, M006 and M010b were developed in the murine SCID host. Intracranial growth of the M006 line was achieved in nude rats. The nitroreductive capacity of glioma cell lines was assessed and found to be similar to transplanted tumours previously reported in the literature. This suggests that if substantial numbers of viable hypoxic cells were present in situ in gliomas, then nitroimidazole-binding techniques should be capable of identifying them. Inter-tumour variability in the amount of necrosis was seen. M006 xenografts growing in subcutaneous and intracranial sites revealed extensive necrotic regions histologically, some of which were surrounded by cells labelled heavily for [3H]misonidazole, while other areas were lightly labelled. Similar binding patterns were seen for subcutaneous M059K tumours, while subcutaneous M010b tumours display necroses of which almost all were surrounded by heavily labelled cells. The oxygen consumption rates of tumour cell lines grown in vivo, in which venous pO2 concentrations are of the order of 2-5%, were two to sevenfold less than those of the same lines grown as monolayers in vitro under oxygen concentrations of 18%. We postulate that glioma cell lines behave as 'oxygen conformers', in that their rate of oxygen consumption appears to vary with the availability of oxygen. Together with the misonidazole-binding data, the results in this glioma tumour model are consistent with coordinate inhibition or down-regulation of respiration under moderate hypoxia.

Fluoromisonidazole. A metabolic marker of myocyte hypoxia.

Fluoromisonidazole is metabolically trapped in viable hypoxic cells in vitro. This property is the basis for the hypothesis that [18F]fluoromisonidazole can be used to detect hypoxic tissues noninvasively using positron emission tomography. To assess the potential usefulness of this compound as a marker for hypoxic myocardium, we measured the accumulation of [3H]fluoromisonidazole in isolated adult rat myocytes under normoxic, hypoxic (5,000 ppm O2), and anoxic conditions. Both anoxia and hypoxia caused a marked increase in [3H]fluoromisonidazole accumulation. Relative to uptake during normoxia, uptake during anoxia was increased by 8.4-fold at 60 minutes and 26.5-fold at 180 minutes (p less than 0.001). During hypoxia, uptake was increased by 4.4-fold at 60 minutes and by 15.3-fold at 180 minutes (p less than 0.0001) and occurred in the absence of significant cell injury as measured by release of creatine kinase and changes in cell morphology. Additional studies demonstrated a slow oxygen-insensitive efflux of fluoromisonidazole or labeled metabolite(s) from myocytes reincubated in drug-free medium. We conclude that fluoromisonidazole is avidly retained in hypoxic myocytes and thus may be suitable for noninvasive detection of hypoxic myocardium using positron emission tomography.

Heterogeneity in tumor microvascular response to radiation

Viable hypoxic cells have reduced radiosensitivity and could be a potential cause for treatment failure with radiotherapy. The process of reoxygenation, which may occur after radiation exposure, could increase the probability for control. However, incomplete or insufficient reoxygenation may still be a potential cause for local treatment failure. One mechanism that has been thought to be responsible for reoxygenation is an increase in vascular prominence after radiation. However, the effect is known to be heterogeneous. In this study, tumor microvascular hemodynamics and morphologies were studied using the R3230 Ac mammary adenocarcinoma transplanted in a dorsal flap window chamber of the Fisher-344 rat. Measurements were made before and after (at 24 and 72 hr) 5-Gy radiation exposure to assess microvascular changes and to explore possible explanations for the heterogeneity of the effect. There was considerable heterogeneity between tumors prior to radiation. Vascular densities ranged from 67 to 3000 vessels/mm 3 and median vessel diameters from 22 to 85 μm. Pretreatment perfusion values varied by a factor of six. In irradiated tumors, conjoint increases in both vascular density and perfusion occurred in most tumors, although the degree of change was variable from one individual to the next. The degree of change in density was inversely related to median pretreatment diameter. Relative change in flow, as predicted by morphometric measurements, overestimated observed changes in flow measured hemodynamically. These results support that heterogeneity in tumor vascular effects from radiation are somewhat dependent on pretreatment morphology as well as relative change in morphology. Since changes in flow could not be completely explained by morphometric measurements, however, it is likely that radiation induced changes in pressure and/or viscosity contribute to the overall effect. Further work in this laboratory will investigate these hypotheses.

Chemical modifiers of radiosensitivity-theory and reality: A review

In this review the poor clinical gains from hyperbaric oxygen (HBO) and misonidazole (MISO) are discussed critically. The biggest factor reducing clinical gains is almost certainly reoxygenation. Other possible reasons include vasoconstrictive self-limitation of HBO and neurotoxicity of MISO, so that the radiosensitization of any hypoxic cells in human tumors was not adequate. Nevertheless, there have been some positive clinical results, so that hypoxic cells can sometimes be a problem in some tumors, especially those of the head and neck, even after multiple fraction radiotherapy. While hypoxic cell radioresistance is obviously only one form of radioresistance it is a large factor of resistance when hypoxic cells are present. Current developments are briefly reviewed: the ‘new’ clinical sensitizers Ro-03-8799 and SR-2508 which should be 3 to 10 times more efficient than MISO if viable hypoxic cells are present; and methods of measuring which human tumors might have significant numbers of hypoxic viable cells.

Further Characterization of 4-Bromomisonidazole as a Potential Detector of Hypoxic Cells

[14C]Bromomisonidazole was prepared by direct bromination of [ring-2] [14C]misonidazole in dioxane. The uptake and binding of the two labeled sensitizers were compared in vitro in 1-mm EMT-6 spheroids which contain a necrotic core. Using liquid scintillation counting it was shown that spheroids incubated with 50 microM [14C]bromomisonidazole concentrated drug above levels in the medium by 1 1/2 hr and achieved maximum concentration by 10 hr with no further increase at 23 hr. Spheroids incubated with 50 microM [14C]misonidazole may concentrate the sensitizer more slowly but ultimately reached the same fivefold increase over levels in the medium by 23 hr as was observed for bromomisonidazole. Autoradiographs prepared from spheroids after incubation with [14C]misonidazole or [14C]bromomisonidazole showed silver grains preferentially located over viable hypoxic cells in the inner half of the spheroid rim adjacent to the necrotic center, with lower grain density over nonviable necrotic areas and many fewer grains over oxic cells at the periphery of the spheroid. The results indicate that both severely and moderately hypoxic cells may preferentially bind [14C]bromomisondiazole. The data support the potential of radiolabeled bromomisonidazole for in vivo imaging pending additional studies of the metabolism of this agent.

The detection and measurement of hypoxic cells in solid tumors.

The presence of viable hypoxic cells in human cancers has concerned oncologists for years. Cells in tissues that are deficient in oxygen are relatively resistant to radiation inactivation and may not be accessible to some systemic chemotherapy. The premise that hypoxic tumor cells do, indeed, control the radiocurability of some cancers is supported by some clinical evidence. The presence of hypoxic regions within tumors can be directly and indirectly inferred from invasive procedures such as oxygen electrode techniques and histologic study, respectively, but such information does not significantly contribute to current prescriptions given by oncologists for tumor treatment. Novel procedures (some of which are noninvasive) for detecting hypoxic regions within solid tumors have been proposed and are based upon two recent developments: (1) the discovery that some radiosensitizing drugs become selectively bound by metabolism to the molecules of viable hypoxic cells, and (2) the growing availability of new imaging procedures based upon positron-emission tomography, single-photon emission tomography, and nuclear magnetic resonance spectroscopy. Preliminary research results from these novel procedures are reviewed, and the potential clinical impact of each is discussed.

Evidence for acutely hypoxic cells in mouse tumours, and a possible mechanism of reoxygenation.

Abstract The proportion of viable hypoxic cells in EMT6/St/lu tumours assayed 18 and 24 hours after a dose of misonidazole (1.2 mg/g), which killed 80–90% of the tumour cells, was reduced from 20% in the controls to 7% in the misonidazole treated mice. In other experiments, the radiation cell-survival curve of the RIF-1 tumour was assayed in both intradermal and intramuscular sites under both air-breathing and acutely hypoxic conditions. The slope of the survival curve of the intramuscular tumours irradiated under air-breathing conditions was significantly steeper than that of the same tumours growing intradermally (D0 values of 187 rad vs. 357 rad), or when the tumours were irradiated in either site under acutely hypoxic conditions (D0 = 375 rad). Both of these experimental observations are examined in the light of data from other sources, notably from the radiation response of hypoxic cells in spheroids and from observations made from tumours implanted between close, parallel mica sheets (“sandwich” tum...