Hypoxic Cell Marker Research Articles

Comparison of two techniques for detecting tumour hypoxia: a fluorescent immunochemical method and an in vitro colony assay

The aim of this study was to compare the percentage of hypoxic cells obtained with two methods: an in vitro colony assay and a new method based on immunodetection of a marker for hypoxic cells (NITP) which could be used in patients. These studies have been carried out using one rodent tumour EMT6 (a mammary carcinoma) and one human tumour HRT18 (a rectal adenocarcinoma). The hypoxic cell fraction was assessed in control mice and in mice receiving two treatments: 250 mg/kg nicotinamide + carbogen, and 250 mg/kg nicotinamide + carbogen + 4 ml/kg perflubron emulsion. The two treatments increased the radiosensitivity of the two cell lines, nicotinamide plus carbogen plus perflubron emulsion having the greatest radiosensitising effect. For untreated and treated tumours, the percentage of hypoxic cells obtained with the in vitro colony assay were comparable to those obtained with immunodetection using NITP. Whatever the treatment, NITP detection was a convenient test to detect the hypoxic cell fraction in the two solid tumours we have studied.

Bioreductive markers for hypoxic cells: 2-nitroimidazoles with biotinylated 1-substituents.

The interference by oxygen with the bioreductive metabolism and binding within cells of 2-nitroimidazoles has been used to identify hypoxic cells. Three novel compounds were synthesized with a 1-substituent containing a biotin moiety. Bound adducts of these compounds could be identified in hypoxic cells in vitro by the biotin binding proteins, avidin or streptavidin, labeled with fluorescein. The metabolism and discrimination of these compounds between well-oxygenated and hypoxic cells was evaluated by flow cytometry. Ester or amide links between the 2-nitroimidazole and the biotin were degraded in the presence of mouse serum, but a compound with a C5 hydrocarbon link was stable, and this compound was suitable for evaluation in an in vivo tumor model.

Bioreductive fluorescent markers for hypoxic cells: a study of 2-nitroimidazoles with 1-substituents containing fluorescent, bridgehead-nitrogen, bicyclic systems.

The oxygen-sensitive bioreductive binding of 2-nitroimidazoles labeled with fluorescent side chains has been used to stain hypoxic mammalian cells selectively. Several novel compounds were synthesized with a 1-substituent containing a fluorescent, bicyclic system having a bridgehead-nitrogen atom. Additional amine and secondary alcohol substituents were also included in the link between the fluorophor and the nitroimidazole to improve water solubility. Their ability to discriminate between hypoxic and oxic cells was compared by flow cytometric analysis. A wide range of cellular fluorescence and hypoxic-oxic differentials in fluorescence was observed when compounds with indolizine fluorophors were incubated with cells, and one such compound was considered suitable for further evaluation in vivo. Two compounds with bimane fluorophors gave very little cellular fluorescence when incubated with hypoxic cells.

Abstract #7 — Retention of the hypoxic cell marker, misonidazole, in dental pulp

Abstract #7 — Retention of the hypoxic cell marker, misonidazole, in dental pulp

Non-invasive assessment of human tumour hypoxia with 123I-iodoazomycin arabinoside: preliminary report of a clinical study.

Non-invasive predictive assays which can confirm the presence or absence of hypoxic cells in human tumours show promise for understanding the natural history of tumour oxygenation, and improving the selection of patient subsets for novel radiotherapeutic strategies. Sensitiser adducts have been proposed as markers for hypoxic cells. Misonidazole analogues radiolabelled with iodine-123 have been developed for the detection of tumour hypoxia using conventional nuclear medicine techniques. In this pilot study, we have investigated one such potential marker, 123I-iodoazomycin arabinoside (123I-IAZA). Patients with advanced malignancies have undergone planar and single-photon emission computed tomographic (SPECT) imaging after intravenous administration of 123I-IAZA. We have observed radiotracer avidity in three out of ten tumours studied to date. Normal tissue activity of variable extent was also seen in the thyroid and salivary glands, upper aerodigestive tract, liver, intestine, and urinary bladder. Quantitative analysis of those images showing radiotracer avidity revealed tumour/normal tissue (T/N) ratios of 2.3 (primary small cell lung carcinoma), 1.9 (primary malignant fibrous histiocytoma) and 3.2 (brain metastasis from small cell lung carcinoma) at 18-24 h post injection. These preliminary data suggest that the use of gamma-emitter labelled 2-nitroimidazoles as diagnostic radiopharmaceuticals is feasible and safe, and that metabolic binding of 123I-IAZA is observed in some, but not all tumours. The inference that tumour 123I-IAZA avidity could be a non-invasive measure of tumour hypoxia deserves independent confirmation with needle oximetry.

Fluorescent markers for hypoxic cells: a study of nitroaromatic compounds, with fluorescent heterocyclic side chains, that undergo bioreductive binding

Several novel compounds having both a 2-nitroimidazole nucleus and a fluorescent ring system in their molecular structure were prepared and evaluated as potential fluorescent probes for hypoxia. Bioreduction of nitroimidazoles, which is inhibited by oxygen, is known to lead to binding of bioreductive metabolites to cellular macromolecules and this provides a mechanism for binding the fluorescent moiety to hypoxic cells. These compounds can incorporate a wide range of fluorophors and can therefore be designed to suit the laser-line wavelengths available for excitation of fluorescence in the flow cytometer. Several nitroimidazoles with naphthalimide side chains were rapidly taken up into cells and became concentrated in the cells, thus reducing their concentration in the extracellular medium. This suggests a potential microscopic bioavailability problem with probes of this type when used in vivo as they would become progressively depleted in the extracellular fluid as they diffused from blood vessels, through layers of packed cells in tumors, to the hypoxic cells where they could undergo hypoxia-specific metabolism. Synthesis of nitroimidazoles with coumarin fluorophors led to several potentially useful probes for hypoxia; substituents on the coumarin fluorophor had a marked effect on the cellular fluorescence of these compounds.

Synthesis of an (iodovinyl)misonidazole derivative for hypoxia imaging

Nitroimidazoles undergo a bioreduction in viable hypoxic tissue, resulting in trapping within these tissues, as demonstrated by misonidazole. A radioiodinated analogue of misonidazole (IVM, (E)-5-(2-Nitroimidazolyl)-4-hydroxy-1-iodopent-1-ene, 3) has been synthesized by halodestannylation, for evaluation as an imaging agent for hypoxia. A key step in the synthetic sequence involves the use of the Lewis acid BF3.Et2O to promote the nucleophilic ring opening of glycidyl tosylate with (E)-1-lithio-2-(tributylstannyl)ethylene. Direct comparison of IVM versus F-MISO (2) another misonidazole type hypoxic cell marker, in several in vitro cell culture studies, indicates that IVM behaves in analogous fashion to F-MISO and has promise as a hypoxia imaging agent for SPECT.

Fluorescent markers for hypoxic cells: A study of novel heterocyclic compounds that undergo bio-reductive binding

The bioreductive metabolism and binding of nitroaromatic compounds has been suggested as a method for the identification of hypoxic tumour cells. Bound metabolites of suitable nitroaryl compounds (and some other reducible aromatic compounds) may fluoresce, offering an alternative to radiolabelling or NMR etc. as a diagnostic method. In this paper, the synthesis of some heteroaromatic nitro-compounds is given together with the results obtained from testing of these and other mainly nitroaromatic compounds in vitro as potential bioreductive fluorescent probes for hypoxic cells in tumours. Compounds were incubated with oxygenated or hypoxic mammalian cell suspensions for various times before evaluation of the cellular fluorescence from bioreductive metabolites by fluorescence microscopy and flow cytometry. Among those compounds yielding fluorescent metabolites in cells, considerable variation in hypoxic: oxic differential fluorescence was observed. The in vitro mammalian cell test system showed several of the compounds to be sufficiently promising to merit further investigation in vivo.

Radiosensitisation and radioprotection by BSO and WR-2721: the role of oxygenation

Endogenous and exogenous thiols are thought to influence cellular radiosensitivity directly by radical scavenging and/or hydrogen donation processes, and indirectly, by regulating the amount of oxygen (or other electron affinic radiosensitiser) able to reach the radiosensitive targets of the cell. The relative importance of these two mechanisms was evaluated in multicell spheroids treated with two agents currently undergoing clinical testing, the thiophosphate WR-2721 and the glutathione synthesis inhibitor BSO. Fluorescence-activated cell sorting techniques were used to recover cells selectively from different depths (different oxygenation status) within the spheroids. The radiosensitivity of cell populations recovered from different regions suggested that both agents acted primarily by affecting the oxygenation status of the spheroid. Similarly, the binding of a fluorescent marker for hypoxic cells, the nitrofuran AF-2, was markedly enhanced by WR-2721 addition, and decreased by BSO-induced thiol depletion. We conclude that the major radiobiological consequence of thiol manipulation in multicell systems is to increase or decrease the availability of oxygen.

Keynote address: Cellular reduction of nitroimidazole drugs: Potential for selective chemotherapy and diagnosis of hypoxic cells

Nitroimidazole drugs were initially developed as selective radiosensitizers of hypoxic cells and, consequently, as adjuvants to improve the local control probabilities of current radiotherapies. Misonidazole (MISO), the prototype radiosensitizing drug, was found in Phase I clinical studies to cause dose-limiting neurotoxicities (mainly peripheral neuropathies). MISO was also found to be cytotoxic in the absence of radiation and to covalently bind to cellular molecules, both processes demonstrating rates much higher in hypoxic compared with oxygenated cells. It is likely that neurotoxicity, cellular cytotoxicity and adduct formation results from reactions between reduction intermediates of MISO and cellular target molecules. Spin-offs from radiosensitizer research include the synthesis and characterization of more potent hypoxic cytotoxins and the exploitation of sensitizer-adducts as probes for measuring cellular and tissue oxygen levels. Current developments in hypoxic cell cytotoxin and hypoxic cell marker research are reviewed with specific examples from studies which characterize the cellular reduction of TF-MISO, (1-(2-nitro-l-imidazolyl)-3[2,2,2-trifluoroethoxy]-2-propanol).

3H-Misonidazole labeling and viability of hypoxic cells in the sandwich system, an in vitro tumor analogue

3H-misonidazole was used as a marker of hypoxic cells in an in vitro tumor analogue, the sandwich system. MISO binding was assessed in situ, using autoradiography. Binding profiles indicate that there are regions of radiobiological hypoxia surrounding the necrotic center in sandwiches of the V79 cell line and in sandwiches of the 9L cell line. Grains per cell were counted and detailed statistics on the variation of intrinsic binding among cells in the same microenvironment are presented. There is a systematic decrease in the standard deviation of grains per cell as one examines populations of cells further and further from the nutrient and oxygen source. Kinetic studies show that the growth fraction of the cell population also decreases with distance from the nutrient source. These findings taken together suggest that MISO binding is proportional to cell size and cells in the inner noncycling portion of the sandwich are more nearly uniform in size. Sandwich cells which exhibit heavy MISO binding, and are presumably radiobiologically hypoxic, were shown to be still viable if restored to good nutrient and oxygen conditions.

Iodoazomycin Riboside (1-(5′-Iodo-5′-Deoxyribofuranosyl)-2-Nitroimidazole), a Hypoxic Cell Marker: I. Synthesis and in Vitro Characterization

Misonidazole (MISO), a selective radiosensitizer of hypoxic cells, forms adducts with cellular biomolecules with rates which are 30-50 X higher under hypoxic as compared to aerobic conditions of incubation. This technique of sensitizer adduct formation was proposed as a possible means of measuring the hypoxic fraction of solid tumors by noninvasive procedures. Iodoazomycin riboside (5'-IAZR) and 5'-[125I]AZR were synthesized and chemically characterized. Measurements of in vitro cytotoxicity and radiosensitizing ability with EMT-6 tumor cells in vitro indicated that 5'-IAZR is approximately 3 X more toxic and effective than is azomycin riboside (AZR) and approximately 10 X more toxic and effective than is MISO. 5'-[125I]AZR was shown to selectively bind to hypoxic EMT-6 cells at rates which were 2.5-3 X faster than those of MISO. The absolute rates of binding of 5'-IAZR to hypoxic cells at concentrations of 10-100 microM are the highest observed in this laboratory for any hypoxic cell radiosensitizer tested to date. These data suggest that 5'-IAZR, when labeled with an appropriate radioisotope (e.g., 131I), might be a useful marker for hypoxic cells in solid tumors amenable to noninvasive detection. Additional studies with animal tumor models appear to be warranted.

Synthesis and biodistribution of 18F-labeled fluoronitroimidazoles: Potential in vivo markers of hypoxic tissue

Three 18F labeled fluoronitroimidazoles have been prepared as potential in vivo markers of hypoxic cells in tumors, and ischemic areas of the heart and brain. 1-(2-Nitroimidazolyl)-3-[ 18F]fluoro-2-hydroxypropanol ( 18F]fluoro-normethoxymisonidazole) 4, 1-(2-[ 18F]fluoroethyl)-2-nitroimidazole 7, and 1-(2-[ 18F]fluoroethyl)-2-methyl-5-nitroimidazole ([ 18F]fluoro-norhydroxymetronidazole) 10 were prepared in average radiochemical yields of <1%, 23% and 15–43% (8% at the no carrier-added level) respectively at end-of-synthesis. The in vivo biodistribution in rats was determined for each of the 18F labeled fluoronitroimidazoles. At 1 and 3 h after administration, the tissue distribution of each of the 18F labeled nitroimidzaoles was quite uniform and consistent with that of nitroimidazoles previously studied. These results suggest the need for a suitable animal model to evaluate their potential as in vivo markers of hypoxic tissue in the brain.

Hypoxic Fraction and Binding of Misonidazole in EMT6/Ed Multicellular Tumor Spheroids

Misonidazole has been shown to bind selectively to hypoxic cells in tissue culture and to cells which are presumed to be chronically hypoxic in EMT6 spheroids and tumors. Thus it has considerable potential as a marker of hypoxic cells in vivo. To further evaluate this potential EMT6/Ed spheroids were used to quantitate misonidazole binding under conditions which resulted in hypoxic fractions between 0 and 1. Hypoxic fractions were quantitated using radiation survival curves. A doubling of the oxygen in the gas phase to 40% was required to fully oxygenate all chronically hypoxic cells. The patterns of binding of 14C-labeled misonidazole determined by autoradiography were consistent with the regions of radiobiological hypoxia as predicted by oxygen diffusion theory. The overall uptake of 3H-labeled misonidazole by spheroids correlated well with the hypoxic fraction, although binding to aerobic cells and necrotic tissue contributed appreciably to the total label in the spheroids. It is concluded that misonidazole is an excellent marker of hypoxia in EMT6/Ed spheroids at the microscopic level, and the total amount bound per spheroid provides a potentially useful measure of the hypoxic fraction.

The Radiation Sensitivities of R3327-H and R3327-AT Rat Prostate Adenocarcinomas

Dunning R3327-H and R3327-AT tumors growing subcutaneously in the flanks of Fischer X Copenhagen rats were irradiated with 137Cs γ-rays at volumes of approximately 300mm.3 The effects of various doses of radiation were estimated by measurements of subsequent tumor growth as well as by histological evaluation. The well-differentiated, hormonally-responsive R3327-H tumor was more radiosensitive than the anaplastic R3327-AT tumor. The reasons for this increased radiation sensitivity of the R3327-H tumor include a greater “apparent” radiation sensitivity of tumor cells and the absence of tumor hypoxia. The presence of hypoxic tumor stem cells was inferred from significant radiosensitization of tumor growth delay by 0.5mg./gm. misonidazole and by a technique which utilizes radioactively-labelled misonidazole as a marker for hypoxic cells. The persistence of a mass of R3327-H tumor tissue after aggressive radiotherapy was not indicative of tumor cells. Furthermore, the rapid increase in volume of R3327-AT cells after aggressive radiotherapy was attributed to limited proliferation of tumor cells which were destined to ultimately die. Possible implications of these findings for the management of human prostatic adenocarcinoma are discussed.

Binding of 14C-misonidazole to hypoxic cells in V79 spheroids.

The metabolism-induced binding of 14C-labelled misonidazole (MISO) to hypoxic V79 cells in multicell spheroids has been quantitated using autoradiography. Hypoxia was shown to be the major determinant of the rate of binding. Maximally hypoxic cells bound MISO several times more rapidly than necrotic material in the centre of the spheroids, and up to 50 times more rapidly than well oxygenated cells. The rate of binding to chronically hypoxic cells at the edge of the necrotic centre was 20 times less than to similar cells in other spheroids made maximally hypoxic with N2. This difference is consistent with the greater radio-sensitivity of the chronically hypoxic cells, which is a consequence of their intermediate level of oxygenation. The results indicated that the ability to bind MISO might have considerable potential as a marker for hypoxic cells in tumours. However, some binding patterns cannot be explained by the simplest model of O2 diffusion. It may be necessary to invoke more complex models of O2 diffusion or metabolic gradients within the spheroid which affect the rate of binding.

A marker for hypoxic cells in tumours with potential clinical applicability.

A marker for hypoxic cells in tumours with potential clinical applicability.