Effect of radiation on the expression of E-cadherin and alpha-catenin and invasive capacity in human lung cancer cell line in vitro

PURPOSE: To investigate whether electrode measurements of tumor oxygenation obtained under a range of different treatment conditions designed to alter the degree of tumor hypoxia could be correlated with estimates of radiobiological hypoxia measured under the same conditions. METHODS AND MATERIALS: Experiments were performed in restrained, nonanesthetized, female C3H/He mice, which had approximately 0.5 g KHT sarcomas growing intramuscularly in the hind limbs. The treatments used to modify tumor oxygenation status included breathing gas mixtures of varying oxygen content, altering tumor blood flow, and shifting the hemoglobin oxygen dissociation curve. Radiobiological hypoxic fraction was estimated using the paired survival curve assay, while electrode measurements of tumor oxygenation were obtained with an Eppendorf histograph. RESULTS: With the selected manipulations it was possible to vary the radiobiological hypoxic fraction in the tumors from approximately 1 to approximately 100% of the total viable cell population. Furthermore, these changes in radiation response were directly reflected in the changes in tumor oxygenation measurements made with the Eppendorf histograph. CONCLUSION: These findings suggest that in the KHT tumor model the Eppendorf electrode measurements could predict the response of the tumors to radiation as determined by the proportion of hypoxic cells.     

Gel electrophoresis of individual cells to quantify hypoxic fraction in human breast cancers

A new gel electrophoresis method has been used to quantify hypoxic fraction in human tumors. Radiation-induced DNA damage was measured in individual tumor cells, where the radiobiologically hypoxic cells were observed as a subpopulation showing a 3-fold reduction in DNA strand breaks. Patients receiving palliative radiotherapy for breast cancers were given a single dose of 5-10 Gy, and a fine needle aspiration biopsy was taken immediately after irradiation. Hypoxic cells were detected in seven of eight tumors. In four tumors, bivariate analyses of DNA content versus DNA damage to individual cells allowed distinction between the response of diploid normal cells and aneuploid tumor cells. These early results indicate that "comet assay" shows considerable promise for resolving the extent and significance of hypoxia in human tumors.     

Enhancement of tumor radiation response by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid

PURPOSE: 5,6-dimethylxanthenone-4-acetic acid (DMXAA) selectively damages tumor vasculature and is currently in clinical trial as an antitumor agent. Its ability to induce synthesis of tumor necrosis factor (TNF), and its apparent selectivity for poorly-perfused regions in tumors, suggests it possible use in combination with radiotherapy. This investigation examines activity of DMXAA as a radiation modifier using two murine tumors. METHODS AND MATERIALS: Tumor growth delay was evaluated using i.m. RIF-1 and MDAH-MCa-4 tumors irradiated in unanaesthetised, restrained mice (cobalt-60) using single dose or multiple fractions (8 x 2.5 Gy over 4 days) with DMXAA administered i.p. at various times in relation to irradiation. RESULTS: Administration of DMXAA (80 micromol/kg, i.p.) immediately after radiation resulted in a large increase in tumor growth delay, giving a radiation dose modifying factor of 2.3 for RIF-1 and 3.9 for MDAH-MCa-4. The combination was less active when radiation was given 1-4 h after DMXAA, but was highly active 12-48 h after DMXAA. At the latter times, clamping the tumor blood supply caused a large increase in radioresistance. These studies suggest that cells surviving DMXAA are hypoxic for only a short period. DMXAA increased overall growth delay when administered daily during fractionated irradiation, giving an approximately additive response. CONCLUSIONS: The marked synergy between DMXAA and single dose ionising radiation may reflect the complementarity of these agents at the microregional level, with DMXAA preferentially killing hypoxic cells in poorly perfused regions. Despite additional hypoxia shortly after DMXAA treatment, surviving cells appear to reoxygenate quickly which makes it feasible to use DMXAA before and during fractionated radiotherapy. The combination of fractionated radiation and DMXAA appears to be less effective than for single dose radiation (possibly because of the smaller contribution of hypoxia under these conditions), but may be therapeutically useful.     

RSR13, a synthetic allosteric modifier of hemoglobin, as an adjunct to radiotherapy: preliminary studies with EMT6 cells and tumors and normal tissues in mice

RSR13, 2[4-[[(3,5dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropion ic acid, a synthetic allosteric modifier of hemoglobin, reduces the affinity of hemoglobin for oxygen. The experiments reported here examined the effect of treatment with RSR13, combined with oxygen breathing, on the radiation response of EMT6 mammary tumors in BALB/c mice and of two normal tissues. RSR13 plus oxygen breathing increased the response of EMT6 tumors to irradiation. RSR13 had no discernible effects on tumors rendered maximally hypoxic by nitrogen asphyxiation, no discernible cytotoxic effects in EMT6 tumors, and no effect on the viability or radiation response of EMT6 cells in vitro under either aerobic or hypoxic conditions. The effects of RSR13 therefore reflect changes in tumor oxygenation, rather than a direct cytotoxic or radiosensitizing effect of the drug. RSR13 plus oxygen reduced the hypoxic fraction to 9% from the value of 24% found in both air-breathing and oxygen-breathing mice. Treatment with RSR13 plus oxygen did not alter the radiation response of the bone marrow progenitor cells (CFU-S) or acute radiation reactions in the skin. The improvement in tumor radiation response produced by treatment with RSR13 plus oxygen, combined with the absence of enhanced radiation reactions in the normal tissues, support further testing of RSR13 as an adjunct to radiotherapy.     

Direct evidence that apoptosis enhances tumor responses to fractionated radiotherapy

Currently, the contribution of cellular apoptotic sensitivity to tumor response after radiation therapy remains controversial. To address this issue, the survival of Rat-1 fibroblasts containing a 4-hydroxytamoxifen-regulated c-Myc allele, c-MycER (T. D. Littlewood et al., Nucleic Acids Res., 23: 1686-1690, 1995), after single and fractionated doses of radiation was investigated. This model system allows pharmacological regulation of apoptosis sensitivity in the same cells in vitro and as xenograft tumors derived from these cells in vivo (G. I. Evan et al., Cell, 69: 119-128, 1992; R. M. Alarcon et al., Cancer Res., 56: 4315-4319, 1996). Activating c-MycER in vitro resulted in marked sensitization of Rat-1 fibroblasts to the effects of both single-dose and fractionated irradiation as measured by the induction of apoptosis and clonogenic survival. Overexpression of the antiapoptosis protein Bcl-2 suppressed the induction of apoptosis and increased clonogenic survival in cells with activated c-Myc after single-dose and fractionated radiation. Systemic time-release implant delivery of 4-hydroxytamoxifen to severe combined immunodeficient mice bearing Rat-1-MycER tumors over the course of either single-dose (10 Gy) or fractionated (five fractions of 2 Gy) radiotherapy resulted in prolonged tumor growth delay relative to identical tumors from mice that received placebo implants. Furthermore, tumors derived from Rat-1-MycER cells that overexpressed Bcl-2 exhibited shorter tumor growth delays relative to similarly treated Rat-1-MycER tumors. The length of tumor growth delay after single-dose or fractionated radiotherapy strongly correlated with the extent of radiation-induced apoptosis in the xenograft tumors as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling. These in vivo results provide direct evidence that increasing the sensitivity of tumor cells to die by apoptosis increases the efficacy of fractionated radiotherapy by reducing tumor cell clonogenic survival.     

Neuralgic amyotrophy or an isolated lesion of the anterior interosseal nerve?]

OBJECTIVE: Considerable evidence exists to suggest that tumor hypoxia results in radioresistance. Historically, it has been difficult to assess tumor oxygen tension levels reliably. These levels can now be assessed in head and neck malignancies using the Eppendorf pO2 histograph, which uses a fine-needle electrode and a computerized micromanipulator. This technology was used to compare the pretreatment tumor oxygen tension level in lymph node metastases of patients with head and neck cancer to measurements taken during nonsurgical treatment after a partial response had been achieved. STUDY DESIGN: Prospective study. METHODS: Oxygen tension levels were measured in the cervical lymph nodes of 10 patients with biopsy-proven head and neck squamous cell carcinoma and cervical metastases who were being treated with nonsurgical management. These levels were obtained using the Eppendorf pO2 histograph system. Measurements were taken before the start of treatment and were repeated when the size of the cervical metastatic node had decreased by 50%. Normal subcutaneous tissue was measured during the same session. The mean and median pO2 levels, as well as the percentage of measurements with pO2 less than 5 mm Hg were determined. RESULTS: A mean of 72.6 measurements per session was taken from each lymph node. The median tumor pO2 measurement fell from a mean (+/-SD) of 13.9+/-8.0 mm Hg to 7.3+/-9.9 mm Hg. Even more dramatic, however, was the substantial increase in the percentage of values less than 5 mm Hg, a rise from 29% to 52%. CONCLUSIONS: While there is variability both in the pretreatment oxygenation of head and neck cervical metastases and in the change in tumor oxygen tension during treatment, there appears to be a decrease in the overall oxygenation of the tumors. The dramatic increase in very low oxygen measurements may reflect selective survival of radioresistant or chemoresistant hypoxic tumor cells. Cells at the very low level would be expected to be radiobiologically hypoxic (resistant to radiation-induced cell kill).     

Interstitial fluid pressure in cervical carcinoma: within tumor heterogeneity, and relation to oxygen tension

BACKGROUND: Interstitial fluid pressure (IFP) is elevated in many animal and human tumors. The authors assessed tumor IFP and its relation to tumor oxygenation in a prospective clinical study of patients with cervical carcinoma. METHODS: Measurements were made in 77 patients with cervical carcinoma prior to treatment. IFP was measured in normal paravaginal submucosal tissue and at one to five positions in the visible tumor with the patients anesthetized and in the lithotomy position. Tumor oxygen tension was measured immediately prior to IFP using a polarographic needle electrode. Patients were treated with radiotherapy only. Response was evaluated 3 months after the completion of radiotherapy. RESULTS: There was substantial variation in IFP from region to region in some tumors. The mean IFP in individual tumors ranged from 3 to 48 millimeters of mercury (mmHg). The overall mean and median values for the entire patient group were 19 mmHg and 17 mmHg, respectively. IFP was significantly higher in tumor tissue than in normal tissue (P < 0.0001). Tumors with high IFP were more likely to be hypoxic (P < 0.007) and less likely to regress completely with radiotherapy (P < 0.04). CONCLUSIONS: IFP in cervical carcinoma is elevated above normal tissue values. Multiple measurements are needed to evaluate IFP in these tumors. High IFP is associated with hypoxia and may provide information about the mechanism of hypoxia on which treatment can be based.     

Studies on the turnover of procarboxypeptidase B, its active enzyme and the activation peptide in the pig

Four human melanoma xenograft lines (A-07, D-12, R-18, U-25) grown orthotopically in Balb/c nu/nu mice were characterized with respect to the fraction of radiobiologically hypoxic cells. The purpose of the study was to establish a firm radiobiological basis for future use of the lines in the development and evaluation of non-invasive assays of tumour hypoxia. The hypoxic fractions were assessed using three different assays, the single cell survival assay, the tumour growth delay assay and the local tumour control assay, and the means +/- s.e. were found to be 6 +/- 3%, 3 +/- 1% and 5 +/- 2% respectively (A-07), 26 +/- 5%, 25 +/- 6% and 22 +/- 6% respectively (D-12), 55 +/- 9%, 65 +/- 8% and 48 +/- 7% respectively (R-18) and 52 +/- 8%, 59 +/- 7% and 47 +/- 7% respectively (U-25). The three assays gave numerical values for the hypoxic fraction that were not significantly different for any of the lines. The hypoxic fraction differed significantly among the lines; the R-18 and U-25 lines showed higher hypoxic fractions than the D-12 line (P < 0.05), which in turn showed a higher hypoxic fraction than the A-07 line (P < 0.05), regardless of the assay. The wide range of the hypoxic fractions and the significant differences among the lines suggest that A-07, D-12. R-18 and U-25 tumours should be useful models in future studies attempting to develop non-invasive assays of tumour hypoxia.     

Identification of nonproliferating but viable hypoxic tumor cells in vivo

We have used the combination of pimonidazole labeling of hypoxic cells, bromodeoxyuridine labeling of proliferating cells, and cell sorting based on Hoechst 33342 perfusion to directly study hypoxia and proliferation in human tumor xenografts and transplantable murine tumors in vivo. Hypoxia was largely confined to cells in regions with the least perfusion, although in tumors exhibiting transient blood flow, hypoxic cells were not as highly localized. Similarly, proliferation and hypoxia were mutually exclusive except in areas of a tumor subjected to transient changes in perfusion. By determining the clonogenic potential, pimonidazole labeling intensity, and radiosensitivity of sorted tumor cell subpopulations, we have provided direct evidence that pimonidazole identifies hypoxic tumor cells of therapeutic relevance in vivo. Given that pimonidazole exhibits few diffusion or delivery problems and no apparent cytotoxicity, it appears to be a versatile and useful label for hypoxic cells in solid tumors.     

Expression of manganese superoxide dismutase reduces tumor control radiation dose: gene-radiotherapy

This study investigated the in vitro and in vivo radiation response of tumor cells transfected with human manganese superoxide dismutase (MnSOD) cDNA. A major objective was to test the potential tumor suppressive effect of MnSOD in vivo. Tumor cells studied were an in vitro line derived from a murine spontaneous fibrosarcoma, FSa-II, which expressed an undetectable MnSOD activity. These cells were transfected with pSV2-NEO plasmid (NEO line) or cotransfected with MnSOD plasmid plus pSV2-NEO plasmid (SOD lines) as described previously. The cell lines used were SOD-L and SOD-H, which expressed, respectively, low and high MnSOD activities after transfection, and NEO and parental FSa-II controls. Both SOD-L and SOD-H cell lines were slightly more resistant to ionizing radiation than were the two control cell lines when irradiated in vitro in the presence of oxygen. The dose-modifying factors calculated at the survival level of 0.01 were 1.13 and 1.15 for the SOD-L and SOD-H cells, respectively. To investigate potential tumor suppressive effects, animal tumors of 4 mm diameter were irradiated in vivo under hypoxic conditions, and the radiation dose to control one-half of the irradiated tumors (TCD50) was determined for each tumor. The TCD50S obtained on the basis of the tumor control rate in 120 days after irradiation were substantially lower for the SOD-H and SOD-L tumors compared to the NEO tumors. They were 22.9, 28.6, and 47.5 Gy for SOD-H, SOD-L and NEO tumors, respectively. To analyze these data, survival curves were obtained for hypoxic cells by irradiating NEO and SOD-H tumors under hypoxic conditions in vivo and assaying in vitro. Analysis of these curves suggests that the decrease in the TCD50S of SOD tumors is attributable to the reduced tumorigenicity in these tumors. The hypoxic cell survival curves also showed that SOD did not protect cells from radiation in the absence of oxygen. Electron microscopy showed no morphological differences between these cells. These results suggest that the fraction of tumorigenic cells could be reduced by expression of MnSOD, resulting in a substantial decrease in the TCD50.     

Preclinical development and current status of the fluorinated 2-nitroimidazole hypoxia probe N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR 4554, CRC 94/17): a non-invasive diagnostic probe for the measurement of tumor hypoxia by magnetic resonance spectroscopy and imaging, and by positron emission tomography

Hypoxia occurs to a variable extent in a vast majority of rodent and human solid tumors. It results from an inadequate and disorganized tumor vasculature, and hence an impaired oxygen delivery. A probe for the non-invasive detection of tumor hypoxia could find important utility in the selection of patients for therapy with bioreductive agents, anti-angiogenic/anti-vascular therapies and hypoxia-targeted gene therapy. In addition, tumor hypoxia has been shown to predict for treatment outcome following radio- or chemotherapy in human cancers, the underlying mechanism for which may involve hypoxia driving genetic instability and resulting tumor progression. Beyond oncology, utility can also be envisaged in stroke, ischemic heart disease, peripheral vascular disease, arthritis and other disorders. Design, validation, preclinical development and current status of a fluorinated 2-nitroimidazole, N-(2-hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-l-imidazolyl) acetamide (SR 4554, CRC 94/17), which has been rationally designed for the measurement of tumor hypoxia by magnetic resonance spectroscopy (MRS) and imaging (MRI), are reviewed. Application in positron emission tomography (PET) detection is also proposed. Design goals were: (i) a nitro group with appropriate redox potential for selective reduction and binding in hypoxic tumor cells; (ii) hydrophilic/hydrogen bonding character in the side chain to limit nervous tissue penetration and prevent neurotoxicity; and (iii) three equivalent fluorine atoms to enhance MRS/MRI detection, located in a metabolically stable position. Reduction of SR 4554 by mouse liver microsomes was dependent on oxygen content, with a half-maximal inhibition at 0.48 +/- 0.06%. SR 4554 underwent nitroreduction by hypoxic but not oxic tumor cells in vitro and electron energy loss spectroscopic analysis showed selective retention in the hypoxic regions of multicellular tumor spheroids. Pharmacokinetic design goals were met. In particular, low brain tissue concentrations were seen in contrast to excellent tumor levels, as measured by high performance liquid chromatography. The extent of this restricted entry to brain tumor was surprising given the overall octanol/water partition coefficient and was attributed to the hydrophilic/hydrogen bonding character of the side chain. Quantitative MRS was used to assess the retention of 19F signal in murine tumors and human tumor xenografts. The 19F retention index (FRI; ratio of 19F signal levels at 6 h relative to that at 45 min) ranged from 0.5 to 1.0 and 0.2 to 0.9 for murine tumors and human xenografts respectively. The correlation between SR 4554 retention and pO2 was not a linear one, but when FRI was > 0.5, the % pO2 < or = 5 mmHg was always > 60%, indicating that high FRI was associated with low levels of oxygenation. Finally, whole body 19F-MRI in mice demonstrated that SR 4554 and related metabolites localized mainly in tumor, liver and bladder regions. A selective MRS signal was readily detectable in tumors at doses at least 7-fold lower than those likely to cause toxicity in mice. We conclude that proof of principle is established for the use of SR 4554 as a non-invasive MRS/MRI probe for the detection of tumor hypoxia. Based on these promising studies, SR 4554 has been selected for clinical development.     

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

In a previous study an apparent discrepancy was found between the radiobiological hypoxic fraction of tumours and the tumour oxygenation: the lowest percentage of low pO2 values was observed in the most hypoxic tumour, a heavily pigmented melanoma Na11+. This report describes a similar study with two other less pigmented melanomas. The influence of melanin on pO2 readings was also studied using synthetic melanin and L-tyrosine. Tumour oxygenation was measured using the KIMOC 6650 histograph, apparent pO2 was also measured in the calibration chamber in a buffer containing melanin or L-Tyr at three pHs (6.5, 7.0, 7.5) and bubbled with three different oxygen concentrations (0.2, 2.0, 20.9%). The proportion of hypoxic cells, measured by an in vivo/in vitro colony assay, was 58% for Na11+, 30% for Be11 and 51% for Ma11 tumours. The melanin content (microgram/10(6) cells) was 6.5 (Na11+), 2.0 (Be11), and 4.3 (Ma11). The percentages of radiobiologically hypoxic cells and low pO2 reading values (<2 mmHg) were inversely correlated, contrary to what was expected. In buffer, the pO2 values increased significantly with the melanin concentration: the lower the oxygen concentration, the greater was the increase in pO2. The pO2 readings values increased to a lesser extent with L-Tyr concentration. These results indicate that clinical determination of pO2 in melanoma tumours requires careful attention.     

Allelic loss on chromosome 22 in oral cancer: possibility of the existence of a tumor suppressor gene on 22q13

PURPOSE: To model the influence of hypoxic radioprotection in fractionated treatments over a range of fraction sizes. To determine whether there is a "therapeutic window" of dose per fraction where hypoxic radioresistance could be reduced, and if so, where it occurs in different cell lines. MATERIALS AND METHODS: A mathematical model has been used to simulate the response of cells to low doses of radiation, in the region of clinical interest. We have used the inducible repair variant of the linear quadratic (LQ) equation, with a hypersensitive region (alphaS) at low doses that gradually transforms to the accepted "resistance" in the shoulder region (alphaR). It contains two new parameters, the ratio alphaS/alphaR, and D(C). We have accepted that the "induction dose" D(C) is modified by anoxia to the same extent as the other parameters. We have initially modeled using theoretical parameters and then checked the conclusions with 14 sets of published experimental data for cell lines investigated for inducible repair. RESULTS: We have computed the clinical hypoxic protection (OER') as a function of dose per fraction in simulations of clinical fractionated schedules. We have identified a therapeutic window in terms of dose per fraction at about 0.5 Gy, where the OER' is minimized, regardless of the precise cell survival curve parameters. The minimum OER' varies from one cell line to another, falling to about 1.0 if alphaS/alphaR = 6-10 and even far below 1.0 if alphaS/alphaR > or = 20. DISCUSSION: Hyperfractionation using 0.5 Gy fractions may therefore be more effective than oxygen mimetic chemical sensitizers, since it could even make some tumor cells more sensitive than oxic normal tissues. The tumor lines that benefit most from this type of sensitization are those with the highest intrinsic oxic radioresistance, i.e. those with high SF2 values.     

Interleukin 2 expression by tumor cells alters both the immune response and the tumor microenvironment

Microenvironmental conditions within solid tumors can have marked effects on the growth of the tumors and their response to therapies. The disorganized growth of tumors and their attendant vascular systems tends to result in areas of the tumors that are deficient in oxygen (hypoxic). Cells within these hypoxic areas are more resistant to conventional therapies such as radiation and chemotherapy. Here, we examine the hypoxic state of EMT6 mouse mammary tumors and the location of host cells within the different areas of the tumors to determine whether such microenvironmental conditions might also affect their ability to be recognized by the immune system. Hypoxia within tumors was quantified by flow cytometry and visualized by immunohistochemistry using a monoclonal antibody (ELK3-51) against cellular adducts of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide (EF5), a nitroimidazole compound that binds selectively to hypoxic cells. Thy-1+ cells, quantified using a monoclonal antibody, were found only in the well-oxygenated areas. The location of these Thy-1+ cells was also examined in EMT6 tumors that had been transfected with the gene for interleukin-2 (IL-2) because these tumors contain greatly increased numbers of host cells. Surprisingly, we found that IL-2-transfected tumors had significantly decreased hypoxia compared to parental tumors. Furthermore, using the fluorescent dye Hoechst 33342, an in vivo marker of perfused vessels, combined with immunochemical staining of PECAM-1 (CD31) as a marker of tumor vasculature, we found increased vascularization in the IL-2-transfected tumors. Thus, expression of IL-2 at the site of tumor growth may enhance tumor immunity not only by inducing the generation of tumor-reactive CTLs but also by allowing increased infiltration of activated T cells into the tumors.     

Use of 2-nitroimidazoles as bioreductive markers for tumour hypoxia

Tumour hypoxia is thought to contribute to some failures of radiotherapy to achieve local control. Polarographic measurements of tumour oxygenation have been shown to predict clinical response to radiotherapy and patient survival. Hypoxia is also involved in many common types of normal tissue morbidity. However, at present there is no widely used method of measuring hypoxia in the clinic, or for individualizing therapy on the basis of tumour or tissue oxygenation. The bioreductive metabolism of 2-nitroimidazoles provides a way of labelling hypoxic cells in vivo and a variety of isotopic labels have been proposed for the non-invasive detection of bound metabolites of these markers. Several 2-nitroimidazoles with immunologically identifiable side-chains have been described and conventional immunostaining procedures can be used to locate their metabolites, bound to hypoxic cells in histological sections. Use of fluorescent immunoreagents allows flow cytometric assessment of hypoxia and multiple colour fluorescent staining allows hypoxia to be correlated with other markers on a cell by cell basis. 2-Nitroimidazole hypoxia markers show considerable promise for clinical use in diagnosing hypoxia and their use could allow rational application of hypoxia-related therapies to those patients most likely to benefit from them.     

Fibroblasts prevent apoptosis of IL-2-deprived T cells without inducing proliferation: a selective effect on Bcl-XL expression

Tumour hypoxia is well recognised as a major factor contributing to radioresistance. This article examines the role of hypoxia in influencing the treatment outcome following radiotherapy (RT), and reviews the rationale and results of clinical trials that utilise hypoxic sensitizers or cytotoxins in the treatment of head and neck carcinoma. Histologic evidence for tumour hypoxia in human neoplasms was first reported in 1955. Since then, direct measurement by microelectrodes has revealed heterogeneity in intratumoural oxygen concentrations, and low oxygen concentrations are associated with poor local-regional control by RT. These findings coupled with the result of nuclear imaging studies employing radiolabelled imidazoles, provide strong evidence for the existence of tumour hypoxia which influences RT treatment outcome. Hyperbaric oxygen (HBO) trials for head and neck cancer, conducted in the early 1970s, demonstrated that HBO improved local control and survival rates in patients with head and neck cancer receiving radiotherapy (RT). Since the mid-1970s, clinical research in overcoming tumour hypoxia was mainly centred on the use of nitro-imidazoles as hypoxic cell sensitizers. However, the results from several major clinical trials remain inconclusive. Specifically, the Radiation Therapy Oncology Group (RTOG) misonidazole head and neck trial (298 patients) showed no benefit. The Danish misonidazole trial (626 patients) showed no overall benefit, however positive results were observed in a subgroup (304 pharyngeal cancer patients). Although the European Organisation for Research and Teaching of Cancer (EORTC) misonidazole trial with hyperfractionated RT showed no benefit, the Danish nimorazole trial demonstrated an overall benefit in survival as well as local control. The European etanidazole (ETA) trial (374 patients) showed no advantage of adding the drug to RT. The RTOG ETA trial (504 patients) showed no global benefit. However, positive results were observed in a subset of patients with early nodal disease (197 patients). In addition, a recent meta-analysis by Overgaard, utilising pooled results in the literature demonstrated that modification of tumour hypoxia significantly improved local-regional control in head and neck cancers with an odds ratio of 1.23 (95% confidence limits 1.09 to 1.37). Hypoxic cytotoxins, such as tirapazamine, represent a novel approach in overcoming radioresistant hypoxic cells. Tirapazamine is a bioreductive agent which, by undergoing one electron reduction in hypoxic conditions, forms cytotoxic free radicals that produce DNA strand breaks causing cell death. In vitro and in vivo laboratory studies demonstrate that tirapazamine is 40 to 150 times more toxic to cells under hypoxic conditions as compared to oxygenated conditions and that tirapazamine is superior to ETA in enhancing fractionated irradiation in mouse SCCVII and other tumour types with an enhancement ratio of 1.5 to 3.0. Phase I studies demonstrated that therapeutic doses of tirapazamine can be given safely. A multi-institutional phase II trial using tirapazamine with concurrent RT for head and neck cancer is now in progress.     

Recurrence after different primary treatment for cancer of the supraglottic larynx

BACKGROUND: Hypoxic tumors are frequently resistant to radiation therapy. Polyethylene glycol conjugated bovine hemoglobin (PEG-Hb) was tested for its ability to increase oxygen tension in the hypoxic rat osteogenic sarcoma UMR-106, murine Lewis lung carcinoma LL2 and rat gliosarcoma 9L. In addition, PEG-Hb was tested as an adjunct for radiotherapy in UMR-106 and human prostate carcinoma PC-3 solid tumors. MATERIAL AND METHODS: Rodents bearing established subcutaneous tumors were intravenously administered PEG-Hb. Tumor surface tissue oxygen tension was measured by both OxySpot and OxyMap systems, which utilize the same phosphorescence quenching method. RESULTS: A time-dependent rise in oxygen tension was noted, and the maximum tissue oxygen tensions were observed two hours post PEG-Hb administration, and sustained for at least 2 hours. Following a single dose radiation of 4 Gray, osteogenic sarcoma tumors in the PEG-Hb treated group showed dramatic regression (complete remission occurred in 100% of the high dose PEG-Hb treated rats), as compared to control (Ringer's lactate) group tumors that showed continued aggressive growth. All PEG-Hb plus radiation treated animals bearing human prostate carcinoma (PC-3) showed significant tumor growth delay compared to both control (Ringer's lactate) and irradiation only treated animals. CONCLUSION: PEG-Hb increased tumor oxygen content and improved the effectiveness of radiotherapy in these rodent models.     

Coronary bypass surgery: what is changing? Analysis of 3834 patients undergoing primary isolated myocardial revascularization

We have previously demonstrated in primary cancer of the uterine cervix that tumor hypoxia, as determined polarographically, is strongly associated with clinical malignant progression of the disease. Having applied a similar methodological approach to investigate loco-regional relapses, we found a pronounced shift to more hypoxic oxygenation profiles in the recurrent tumors than in the primary tumors. Median pO2 values in 53 pelvic recurrences were significantly lower than the median pO2 values of 117 primary tumors of comparable sizes (7.1 +/- 1.1 mmHg vs. 12.1 +/- 1.0 mmHg, p = 0.0013). The differences in tumor oxygenation between primary and recurrent tumors mirrored the differences in the patients' 5-year survival probabilities. In the cohort of patients with pelvic relapses, median tumor pO2 < 4 mmHg indicated a significantly shorter median survival time as compared to median tumor pO2 > or = 4 mmHg. Our results further support our thesis that in cervical cancer, tumor hypoxia and clinical aggressiveness in terms of resistance to therapy and tumor dissemination, are interrelated.     

Induction thermochemotherapy increases therapeutic gain factor for the fractionated radiotherapy given to a mouse fibrosarcoma

PURPOSE: It has been shown that thermochemotherapy (TC) given prior to radiation reduces the number of clonogens, with a resultant decrease in the tumor control radiation dose. The purpose of this article was to investigate using an animal tumor model how this clonogen reduction affects subsequent fractionated radiotherapy, including repopulation of surviving clonogens, and whether the induction TC can increase the therapeutic gain factor (TGF). METHODS AND MATERIALS: The single-cell suspensions prepared from the fourth-generation isotransplants of a spontaneous fibrosarcoma, FSa-II, were transplanted into the C3Hf/Sed mouse foot. TC was given by heating tumors at 41.5 degrees C for 30 min immediately after an intraperitoneal injection of cyclophosphamide (200 mg/kg) when tumors reached an average diameter of 4 mm. Fractionated radiotherapy (R) with equally graded daily doses was initiated 24 h after TC either in air (A) or under hypoxic conditions (H). The 50% tumor control dose (TCD50) and the radiation dose to induce a score 2.0 reaction (complete epilation with fibrosis) in one-half of irradiated animals, RD50(2.0), were obtained, and the TGF was calculated. Our previous results on the fractionated radiotherapy using the same tumor system served as controls. RESULTS: The TCD50(A, single dose) and TCD50(H, single dose) following TC+R were 52.2 and 57.3 Gy, respectively, which were 14.0 and 20.4 Gy lower than those following radiation alone. The TCD50(A, TC+R) increased only slightly when the number of fractions was increased from one to 10 doses, and all TCD50s were significantly lower than the TCD50(A, R alone). Both TCD50(H, TC+R) and TCD50(H, R alone) increased consistently from a single dose to 20 doses, but all TCD50(H, TC+R) were significantly lower than the TCD50(H, R alone). Regarding the normal tissue reaction, the RD50 values both following TC+R and R alone increased consistently from a single dose to 20 daily doses. However, the RD50(TC+R) and RD50(R alone) for each corresponding number of fractions was not significantly different, resulting in the TGFs significantly > 1.0 for combined TC+R treatments, with the exception of 20 daily doses given in air. CONCLUSION: The induction TC decreased the TCD50 values substantially without altering the RD50 for a late reaction, resulting in an significant increase in the TGF. These results encourage the use of TC as an induction treatment prior to fractionated radiotherapy.     

Vanadium induces AP-1- and NFkappB-dependent transcription activity

PURPOSE: For hypoxic and anoxic cells in solid tumors to be a therapeutic problem, they must live long enough to be therapeutically relevant, or else be rapidly recruited into the proliferating compartment during therapy. We have, therefore, estimated lifetime and recruitment rate of hypoxic human tumor cells in multicell spheroids in vitro, or in xenografted tumors in SCID mice. MATERIALS AND METHODS: Cell turnover was followed by flow cytometry techniques, using antibodies directed at incorporated halogenated pyrimidines. The disappearance of labeled cells was quantified, and verified to be cell loss rather than label dilution. Repopulation was studied in SiHa tumor xenografts during twice-daily 2.5-Gy radiation exposures. RESULTS: The longevity of hypoxic human tumor cells in spheroids or xenografts exceeded that of rodent cell lines, and cell turnover was slower in xenografts than under static growth as spheroids. Human tumor cells remained viable in the hypoxic regions of xenografts for 4-10 days, compared to 3-5 days in spheroids, and 1-3 days for most rodent cells in spheroids. Repopulation was observed within the first few radiation treatments for the SiHa xenografts and, with accumulated doses of more than 10 Gy, virtually all recovered cells had progressed through at least one S-phase. CONCLUSION: Our results suggest an important difference in the ability of human vs. rodent tumor cells to withstand hypoxia, and raise questions concerning the increased longevity seen in vivo relative to the steady-state spheroid system.