Utility of 19F MRS detection of the hypoxic cell marker EF5 to assess cellular hypoxia in solid tumors.

BACKGROUND AND PURPOSE: The present studies were undertaken to determine whether 19F MRS could be used to quantify the binding of the pentafluorinated derivative of etanidazole (EF5) in hypoxic cells of solid tumors. MATERIALS AND METHODS: A 4.7 T imaging magnet was used for the in situ and in vitro evaluation of EF5 signals. In order to develop a better understanding of these NMR measurements the characteristics of parent, reduced unbound, and reduced bound EF5 signals were examined in vitro using a 12 T spectrometer. RESULTS: In situ data acquired using a 4.7 T imaging magnet, showed retention of EF5 signals in KHT sarcomas that was absent in muscles for 6 h after EF5 injection. In vitro studies showed no difference in the NMR detectable signal of parent and reduced unbound EF5. T2 values determined using parent EF5 samples revealed a T2 time of 675 ms. In contrast, EF5 bound to KHT tumor cells gave rise to signals of low intensity, broad line widths, and T2 relaxation times of less than 30 ms. When the same samples were analyzed using the 4.7 T imaging magnet, the CF3 and CF2 fluorine peaks were readily identifiable in the parent EF5 sample but no fluorine signal could be detected from EF5 bound to KHT tumor cells. CONCLUSION: The inability to resolve bound EF5 metabolites even at high field strengths (12 T), coupled with the short T2 relaxation times of the bound EF5, and the limits of detection of the in situ applied imaging magnet (4.7 T), meant that hypoxic cells could not be quantified in tumors using the 19F MRS technique. In situ 19F MRS measurements of EF5 signals (parent/reduced unbound) may reflect conditions of tumor physiology and thus indicate the extent of tumor hypoxia but they are not capable of resolving the cellular oxygenation status of the tumor cells.     

Hypoxia-activated cytotoxicity of benznidazole against clonogenic tumor cells

Solid tumors contain numerous regions with insufficient oxygen concentrations, a condition termed hypoxia. Tumor hypoxia is significantly associated with metastasis, refractory to conventional cancer therapies, and poor patient survival. Therefore, eradication of hypoxic tumor cells will likely have significant impact on the overall progression-free patient survival. This article reports a new discovery that Benznidazole, a bioreductive drug currently used to treat Chagas disease caused by the parasitic protozoan Trypanosoma cruzi, is activated by hypoxia and can kill clonogenic tumor cells especially those under severe hypoxic conditions (≤0.1 % O₂). This type of hypoxia selectivity is important in that severely hypoxic tumor microenvironment is where tumor cells exhibit the strongest resistance to therapy. Mechanistically, activation of Benznidazole coincides with the stabilization of the Hypoxia-Inducible Factor 1α (HIF-1α), suggesting that Benznidazole is activated after tumor cells have entered into a fully hypoxic state. Under such hypoxic conditions, Benznidazole induces the formation of 53BP1 foci, a hallmark of DNA double-stranded breaks that can cause clonogenic inhibition or cell death. These results demonstrate that Benznidazole is a hypoxia-activated cytotoxin with the potential to specifically eliminate hypoxic tumor cells.     

The in vivo radiation response of an experimental tumor: the effect of exposing tumor-bearing mice to a reduced oxygen environment prior to but not during irradiation.

The effect of single or fractionated doses of radiaton on the transplantable KHT sarcoma has been studied in mice which were exposed to a reduced oxygen (O₂) environment prior to but not during irradiation. Tumor-bearing mice were kept in a 12% O₂ environment for a period of time; then their tumors were irradiated locally with a single dose of 2000 rad at a dose rate of 1140 rad/min while the mice breathed air, O₂ or carbogen (5% CO₂:95% O₂). Following irradiation, tumor cell survival was determined using an in vivo lung colony or an in vitro agar colony assay.The results indicate that overnight exposure of tumor-bearing mice (15 hr) to 12% O₂, prior to irradiation of their tumors under air-breathing conditions, leads to a reduction in tumor cell survival by a factor of 3–4 over that found for tumors of air-breathing mice not given a low O₂ pre-treatment. A similar reduction in tumor cell survival is observed for irradiation without prior exposure to the low O₂ environment when the mice are given O₂ or carbogen to breathe instead of air. If the tumors are irradiated while the animals breathe O₂ or carbogen after exposure to 12% O₂, tumor cell survival is reduced to a value ～10 times lower than is obtained in tumors of air-breathing mice not given a low O₂ pre-treatment.During a course of fractionated radiation (seven 500 rad fractions at 24 hr intervals) delivered while the tumor-bearing mice breathed air or carbogen, a 12% O₂ exposure prior to alternate dose fractions was found to lead to significant enhancement of tumor cell killing above that observed when no pre-treatment with 12% O₂ was employed. The reduction in tumor cell survival almost certainly results from a decrease in the number of hypoxic cells in the tumors but the mechanism by which this reduction occurs is not known at present. The decrease may be because of direct killing of hypoxic cells by the low O₂ environment or because of physiological adaptation to the low O₂ environment which persists when the animals are returned to an air atmosphere, leading to an improved O₂ delivery to the tumor. A number of adaptation mechanisms were studied but these shpwed little correlation with the radiobiological effect. Consequently, no clear explanation of the results can be given at present. Regardless of the mechanism, the findings indicate an approach to increasing the effectiveness of radiation treatment of tumors.     

Augmenting tumor sensitivity to topotecan by transient hypoxia

We examined how the effect of topotecan is modulated by transient hypoxia in three different tumor lines, Lewis lung carcinoma (LLC), U87 human glioblastoma and DMS273 human small cell lung cancer. Four groups of tumor bearing mice were treated with saline or a single dose of topotecan, immediately followed by 6-h or 72-h exposure to a hypoxic environment (10% O₂) or normal air. Topotecan + hypoxia resulted in significantly greater suppression of tumor growth than normoxic topotecan or hypoxia alone. Correspondingly, the sensitivity of LLC cells to topotecan in a clonogenic survival assay was significantly higher under hypoxia. This effect of hypoxia was not a general phenomenon, since the tumor growth inhibitory effect of the alkylating agent cisplatin was not changed by hypoxic environment. In a parallel series of in vitro experiments, cell cultures were exposed to hypoxia (0.1% or 0.7% O₂) in a hypoxic chamber or normoxia for 24 h. We found a dose-dependent downregulation of HIF-1 by topotecan (30–270 nM). The hypoxic upregulation of Glucose transporter-1 and VEGF secretion to the culture medium was inhibited by the addition of topotecan, while doses up to 270 nM had no effect on VEGF under normoxia. VEGF protein levels in tumors were also reduced by topotecan. These data show that the effect of topotecan is increased by transient hypoxia, and this may be a direct effect on the ability of cells to survive under hypoxia as well as an antiangiogenic effect, mediated through the HIF-1 inhibitory effect of topotecan.     

Intravascular HBO(2) saturations, perfusion and hypoxia in spontaneous and transplanted tumor models

Clinical trials utilizing strategies to manipulate tumor oxygenation, blood flow and angiogenesis are under way, although limited quantitative information exists regarding basic tumor pathophysiology. The current study utilized murine KHT fibrosarcomas, spontaneous mammary carcinomas and first-generation spontaneous transplants to examine heterogeneity in vascular structure and function, to relate these changes to the distribution of tumor hypoxia and to determine whether fundamental relationships among the different pathophysiological parameters exist. Three methods were included: (i) immunohistochemical staining of anatomical and perfused blood vessels, (ii) cryospectrophotometric measurement of intravascular oxyhemoglobin saturations and (iii) fluorescent detection of the EF5 hypoxic marker. While a distinct pattern of decreasing oxygenation with increasing distance from the tumor surface was observed for KHT tumors, striking intertumor variability was found in both spontaneous and first-generation transplants, with a reduced dependence on tumor volume. EF5 hypoxic marker uptake was also much more heterogeneous among individual spontaneous and first-generation tumors compared to KHT. Although mammary carcinomas demonstrated fewer anatomical blood vessels than fibrosarcomas, the proportion of perfused vessels was substantially reduced in KHT tumors, especially at larger tumor volumes. Vascular morphology, tissue histological appearance and pathophysiological parameters differed substantially between KHT tumors and both spontaneous and first-generation tumors. Such differences in vascular structure and function are also likely to correlate with altered response to therapies targeted to the vascular system. Finally, spontaneous differentiation status, tumor morphology, vascular configuration and function were well preserved in first-generation transplanted tumors, suggesting a close relationship between vascular development and function in early-generation transplants and spontaneous tumor models.     

Induction of severe tumor hypoxia by modifiers of the oxygen affinity of hemoglobin

Methods have been compared for inducing severe hypoxia in experimental tumors. Hypoxic fractions in the tumors were obtained from measurements of the displacement of cellular survival plots in vitro following tumor irradiation in vivo. Two compounds that displace to the left, oxygen-hemoglobin association curves greatly increase the hypoxic fractions in the tumors. The compound BW12C increases the hypoxic fractions in the KHT and Lewis-Lung tumors from about 10% to between 50-100%. The longer acting analogue BW589C increases hypoxic fraction in the KHT tumor to the same level achievable by treatment with the vaso-active drug hydralazine. The effect is also observed in the RIF-1 tumor even though the hypoxic fraction in this tumor is normally only about 1-3%. The kinetics for hypoxia induction by BW589C and its subsequent return to normal levels are comparable to those for the left-shifting of the oxy-hemoglobin association curve observable up to about 2 days post treatment.     

Radiobiological Hypoxia in the KHT Sarcoma: Predictions Using the Eppendorf Histograph

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 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 1 to 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.     

Acute (cyclic) hypoxia enhances spontaneous metastasis of KHT murine tumors.

Hypoxia exists in most human and rodent solid tumors and has been shown to correlate with poor survival in carcinoma of the cervix, carcinoma of the head and neck, and soft tissue sarcoma. It exists both chronically, due to the poorly organized vasculature of solid tumors, and acutely, due to fluctuations in blood flow. It has been found that tumors that are more hypoxic are more likely to metastasize in humans and in rodent models, and it has been demonstrated that exposure of tumor cells to hypoxia in vitro can transiently enhance their metastatic potential when they are reinjected i.v. into mice. The purpose of the present study was to determine whether experimentally imposed hypoxic stress in vivo, either chronic or acute, affects the process of spontaneous metastasis in tumor-bearing mice. We exposed mice bearing KHT tumors to low oxygen conditions (5-7% O(2) breathing) daily during tumor growth in an attempt to induce additional chronic (2 h/day) and acute (12 x 10 min/day) hypoxia in their tumors. By monitoring tumor pO(2) levels over the course of treatment, we demonstrated that these treatments produce acute and chronic hypoxia within the tumor tissue. The acute but not the chronic hypoxia treatment significantly increased the number of spontaneous microscopic lung metastases in the mice by a factor of about 2, and the results suggest that this effect was due to the changes induced in the primary tumor. This study describes a novel method for studying the effects of hypoxia in solid tumors and demonstrates that acute and chronic hypoxia can have different effects on tumor cell behavior in vivo.     

Changes in misonidazole binding with hypoxic fraction in mouse tumors

Binding of misonidazole (MISO) or a derivative to hypoxic cells in tumors has been proposed as a method for identifying tumors and measuring their level of hypoxia. We have recently shown that the hypoxic fraction of tumor cells can be altered over a wide range in vivo by acutely changing the hematocrit of the host animal by transfusion. The present study aimed to investigate the changes in binding by 14C MISO that accompanied this procedure. Tumor bearing mice were injected with 14C MISO, irradiated with a single dose of X rays (20 Gy) and their tumor excised and bisected. One half of each tumor was used to determine cell survival in vitro, the other was used for 14C scintillation counting. As previously described, tumor cell survival was dramatically increased in acutely anemic mice and this was accompanied by an increase in 14C MISO binding to the tumors. The relationship between clonogenic cell survival and binding was found to be linear on a log-log plot for each of the tumor lines studied, but the slopes of the lines were different tumor lines and generally steeper than the value of 1.0 expected for a 1:1 correspondence between cells binding radioactivity and radiobiological resistance. We attribute these differences to MISO binding to cells in the tumor which were not clonogenic.     

Overcoming hypoxia-induced tumor radioresistance in non-small cell lung cancer by targeting DNA-dependent protein kinase in combination with carbon ion irradiation

Background

Hypoxia-induced radioresistance constitutes a major obstacle for a curative treatment of cancer. The aim of this study was to investigate effects of photon and carbon ion irradiation in combination with inhibitors of DNA-Damage Response (DDR) on tumor cell radiosensitivity under hypoxic conditions.

Methods

Human non-small cell lung cancer (NSCLC) models, A549 and H1437, were irradiated with dose series of photon and carbon ions under hypoxia (1% O₂) vs. normoxic conditions (21% O₂). Clonogenic survival was studied after dual combinations of radiotherapy with inhibitors of DNA-dependent Protein Kinase (DNAPKi, M3814) and ATM serine/threonine kinase (ATMi).

Results

The OER at 30% survival for photon irradiation of A549 cells was 1.4. The maximal oxygen effect measured as survival ratio was 2.34 at 8 Gy photon irradiation of A549 cells. In contrast, no significant oxygen effect was found after carbon ion irradiation. Accordingly, the relative effect of 6 Gy carbon ions was determined as 3.8 under normoxia and. 4.11 under hypoxia. ATM and DNA-PK inhibitors dose dependently sensitized tumor cells for both radiation qualities. For 100 nM DNAPKi the survival ratio at 4 Gy more than doubled from 1.59 under normoxia to 3.3 under hypoxia revealing a strong radiosensitizing effect under hypoxic conditions. In contrast, this ratio only moderately increased after photon irradiation and ATMi under hypoxia. The most effective treatment was combined carbon ion irradiation and DNA damage repair inhibition.

Conclusions

Carbon ions efficiently eradicate hypoxic tumor cells. Both, ATMi and DNAPKi elicit radiosensitizing effects. DNAPKi preferentially sensitizes hypoxic cells to radiotherapy.

     

Characterization of radiation resistant hypoxic cell subpopulations in KHT sarcomas. (I). Centrifugal elutriation

Studies were performed to determine the location, with respect to cell cycle phase, of the radiobiologically hypoxic cells in KHT sarcomas. Cells dispersed from solid KHT tumours were separated into subpopulations at different stages of the cell cycle by centrifugal elutriation. Flow cytometric analysis of the DNA content of these subpopulations indicated that the degree of synchrony that could be achieved was greater than or equal to 95% for G1 cells, 70-75% for S cells and 70-75% for G2M cells. The approach to locate and characterize hypoxic cells was based on the premise that due to their lack of oxygen such cells would preferentially survive radiation treatment. Consequently KHT sarcomas were irradiated in situ either in dead animals or in animals breathing air. Following treatment, the tumours were dissociated, the cells elutriated into the various phases of the cell cycle and clonogenic cell survival was determined. Complete dose-response curves were determined for cells in the G1, S and G2M cell cycle phases. The various cell cycle subpopulations obtained from tumours irradiated while mice breathed air, all demonstrated survival curves with a break and final slope which paralleled that of the corresponding anoxic cell survival curve. From these curves the proportion of hypoxic tumour cells in the G1 phase was calculated to be 10.1 +/- 1.7%. Because the elutriated S and G2M enriched cell fractions were to some extent contaminated by cells from other phases of the cell cycle, the percentage of hypoxic S and G2M tumour cells was estimated to range from 0-7% and 0-5% respectively. However, since G1 cells comprised the majority of all the neoplastic cells in these tumours, the data suggest that hypoxic cells in KHT sarcomas are found primarily in this cell cycle stage.     

Comparison of in vivo Efficacy of Hypoxic Cytotoxin Tirapazamine and Hypoxic Cell Radiosensitizer KU-2285 in Combination with Single and Fractionated Irradiation

Development of strategies to eradicate radioresistant hypoxic cells would be of great benefit for clinical radiotherapy. In the present study, the in vivo effects of a promising hypoxic cytotoxin, tirapazamine (3-amino-1,2,4-benzotriazine 1,4-di-N-oxide), were examined in comparison with those of KU-2285, one of the best hypoxic cell radiosensitizers, in combination with both single and fractionated irradiation. The tumor response was assessed by the standard in vivo-in vitro clonogenic assay using SCCVII tumors in C3H mice and EMT-6/KU tumors in Balb/c mice with different characteristics of tumor hypoxia. With single-dose irradiation (18 Gy), both tirapazamine and KU-2285 showed significant enhancement of cell killing in a dose-dependent manner, but tirapazamine was more effective for SCCVII tumors with acutely hypoxic cells, while KU-2285 was more effective for EMT-6/KU tumors predominantly with chronically hypoxic cells. In fractionated irradiation regimens (4 fractions of 5 Gy at 12 h intervals), tirapazamine showed more marked combined effects at 10 and 20 mg/kg than KU2285 at 100–200 mg/kg in both SCCVII and EMT-6/KU tumors. We concluded that the effectiveness of KU-2285 and tirapazamine was correlated with the nature of tumor hypoxia with single-dose irradiation, whereas tirapazamine appeared more potent than KU-2285 with fractionated irradiation. These findings suggest the potential usefulness of tirapazamine in clinical fractionated radiotherapy.     

31P nuclear magnetic resonance spectroscopy studies of tumor energy metabolism and its relationship to intracapillary oxyhemoglobin saturation status and tumor hypoxia

Relationships between tumor bioenergetic status on the one hand and intracapillary oxyhemoglobin (HbO2) saturation status and fraction of radiobiologically hypoxic cells on the other were studied using two murine sarcoma lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI). Tumor energy metabolism was studied in vivo by 31P nuclear magnetic resonance (NMR) spectroscopy and the resonance area ratio (PCr + NTP beta)/Pi was used as parameter for bioenergetic status. Intracapillary HbO2 saturation status reflects the oxygen supply conditions in tumors and was measured in vitro using a cryospectrophotometric method. The KHT, RIF-1, and MLS lines showed decreasing bioenergetic status, i.e., decreasing PCr and NTP beta resonances and an increasing Pi resonance, with increasing tumor volume, whereas the OWI line showed no changes in these resonances during tumor growth. The volume-dependence of the HbO2 saturation status differed similarly among the tumor lines; HbO2 saturation status decreased with increasing tumor volume for the KHT, RIF-1, and MLS lines and was independent of tumor volume for the OWI line. Moreover, linear correlations were found between bioenergetic status and HbO2 saturation status for individual tumors of the KHT, RIF-1, and MLS lines. These observations together indicated a direct relationship between 31P-NMR spectral parameters and tumor oxygen supply conditions. However, this relationship was not identical for the different tumor lines, suggesting that it was influenced by intrinsic properties of the tumor cells such as rate of respiration and ability to survive under hypoxia. Similarly, there was no correlation between bioenergetic status and fraction of radiobiologically hypoxic cells across the four tumor lines. This indicates that 31P-NMR spectroscopy data have to be supplemented with other data, e.g., rate of oxygen consumption, cell survival time under hypoxic stress, and/or fraction of metabolically active, nonclonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.     

Patterns and levels of hypoxia in head and neck squamous cell carcinomas and their relationship to patient outcome

PURPOSE: EF5, a 2-nitroimidazole hypoxia marker, was used to study the presence, levels, and prognostic significance of hypoxia in primary head and neck squamous cell tumors. METHODS AND MATERIALS: Twenty-two patients with newly diagnosed squamous cell carcinoma of the oral cavity, oropharynx, or larynx with at least 2 years of clinical follow-up were included in this study. Quantitative analyses of EF5 immunofluorescence was carried out, and these data were compared with patient outcome. RESULTS: EF5 immunostaining showed substantial intra- and intertumoral hypoxic heterogeneity. The majority of cells in all tumors were well oxygenated. Three patterns of EF5 binding in cells were identified using criteria based on the cellular region that was stained (peripheral or central) and the relationship of binding to necrosis. We tested the association between EF5-binding levels with event-free and overall survival irrespective of the pattern of cellular binding or treatment regimen. Patients with tumors containing EF5-binding regions corresponding to severe hypoxia (< or =0.1% oxygen) had a shorter event-free survival time than patients with pO(2) values greater than 0.1% (p = 0.032). Nodal status was also predictive for outcome. CONCLUSIONS: These data illustrate the potential utility of EF5 binding based on quantitative immunohistochemistry of tissue pO(2) and provide support for the development of noninvasive hypoxia positron emission tomographic studies with fluorine 18-labeled EF5.     

Detection of individual hypoxic cells in multicellular spheroids by flow cytometry using the 2-nitroimidazole,EF5, and monoclonal antibodies

PURPOSE: The purpose of this work was to evaluate EF5, a 2-nitroimidazole compound, and anti-EF5 antibodies as a method to quantify radiobiologically hypoxic cells. METHODS AND MATERIALS: Multicellular spheroids of EMT6 mammary sarcoma cells were used as a model to identify hypoxic cells that were resistant to radiation damage. This was accomplished by incubating the spheroids with the 2-nitroimidazole (EF5), which forms hypoxia-dependent adducts with cellular macromolecules that are detected by fluorescent monoclonal antibodies. RESULTS: Cells from spheroids grown for 2 days in sealed flasks had an increased surviving fraction following radiation as compared to fully reoxygenated spheroids, indicating the presence of radiobiological hypoxia. Treatment of the spheroids with EF5 and subsequent immunohistochemical staining of cryosections with an anti-EF5 fluorochrome conjugated monoclonal antibody allowed for the identification of EF5-adduct containing cells. Spheroids grown under hypoxic conditions in the presence of EF5 showed limited staining of the peripheral cell layers, intense staining of the interior, and an absence of staining within the necrotic center. In contrast, there was minimal staining in reoxygenated spheroids and no staining in control spheroids incubated in the absence of EF5. Flow cytometric analysis of single cells dissociated from spheroids allowed for the calculation of the percentage of stained cells, as well as the intensity of staining. A comparison of the intensity of staining of EF5 treated hypoxic spheroids with the intensity of staining of single cells incubated with EF5 under controlled oxygen concentrations was used to estimate the oxygen concentration range within spheroids. Selective dissociation of spheroids provided a direct demonstration that the cells containing the highest level of EF5 binding were also the cells with increased radiation resistance. CONCLUSION: This technique provides an excellent means of detecting and quantifying hypoxia, which should be directly applicable in tumors.     

Enhancement of the anti-tumor effect of melphalan in experimental mice by some vaso-active agents

A comparison is made between the vaso-active agents hydralazine, nifedipine, and verapamil for their ability to increase the anti-tumor effectiveness of melphalan. Treatment of mice with hydralazine (5 mg/kg) 15 mins after melphalan increases by a factor of approximately 2.5 melphalan-induced delay in growth of either the RIF-1 or KHT tumors. Similar enhancements are obtained when measurement is made of the surviving fraction of tumor cells in vitro following treatment in vivo with hydralazine and melphalan. Further, tumor cell kill is also increased when nifedipine is administered with melphalan compared with the effect of melphalan alone. These enhanced effects are observed if the vaso-active agents are given before or after melphalan. Hydralazine (5 mg/kg) induces close to 100% radiobiological hypoxia in the RIF-1 and KHT tumors. In contrast, Nifedipine has no effect on tumor hypoxic fraction at a dose of 10 mg/kg although the anti-tumor effectiveness of melphalan is substantially increased. However, a higher dose of 50 mg/kg nifedipine causes a large increase in tumor hypoxic fraction, an effect that persists for several hours. Verapamil causes no change in the fraction of hypoxic cells in the KHT tumor and increases, only slightly, the anti-tumor effect of melphalan.     

The effects of vasodilating drugs on pH in tumors

Hydralazine has been used widely to reduce tumor blood flow and thereby to induce hypoxia and to reduce extracellular pH (pHe) in tumors. Here we have investigated and compared the effects of the vasodilating drugs hydralazine, captopril, nifedipine, prazosin, sodium nitroprusside, and labetalol to reduce pHe in EMT-6 and KHT tumors of mice and to cause antitumor effects. After a single injection, captopril was most effective in reducing pHe in EMT-6 tumors with a decrease in mean pHe from 6.93 to 6.67 at 2 h after injection, while nifedipine was most effective for KHT tumors with a decrease in mean pHe from 6.96 to 6.75 at 1 h after injection. During 72 h of chronic administration into mice bearing tumors, nifedipine was ineffective in reducing pHe, but both captopril and hydralazine caused a small but significant reduction of pHe. Captopril caused significant delay in growth of the tumors, but had only a small effect on clonogenic cell survival. Captopril appears to be the most effective vasodilating drug to enhance tumor acidity.     

YC-1改善乏氧增强人肺腺癌A549细胞放射敏感性的体外研究

目的 探讨3-(5′-羟甲基 -2′-呋喃基)-1-苯甲基吲唑(YC-1)对乏氧人肺腺癌A₅₄₉细胞的放射增敏作用。方法 用噻唑蓝比色法检测YC-1对细胞增殖活性的影响,以及YC-1对细胞作用24 h的20%药物抑制浓度(IC₂₀)。细胞克隆形成实验计算细胞存活分数,多靶单击模型拟合细胞存活曲线并计算放射增敏比(SER)。流式细胞仪检测细胞凋亡率和细胞周期。结果 YC-1对A₅₄₉细胞有增殖抑制作用,在一定剂量范围内呈时间—剂量依赖性。在常氧和乏氧培养24 h下aIC₂₀分别为16.7 μmol/L和39.2 μmol/L。乏氧加YC-1组的SER_D0=1.11,SER_Dq=1.26。在乏氧培养下,YC-1联合单次2 Gy放射能明显促进A₅₄₉细胞凋亡和G₂+M期阻滞\[(30.17±1.21)%∶(15.44±0.96)%,P=0.000和(21.56±0.47)%∶(6.16±0.16)%,P=0.000\]。结论 YC-1对乏氧的A₅₄₉细胞有放射增敏作用。     

Continuous irradiation of the lewis lung carcinoma in vivo at clinically-used “ultra” low-dose-rates

The in vivo response of the transplantable Lewis lung tumor to continuous irradiation (CI) at the clinical “ultra” low-dose-rates of 10–30 cGy/h was investigated. Tumor-bearing mice were housed in a dedicated ¹³⁷Cs unit for up to three weeks while receiving Cl. The horizontal ¹³⁷Cs beam allowed secondary shielding of the lower body, but full irradiation of the dorsally-placed tumor and part of the upper body. Radiation survival curves were measured using an excision assay and tumor-cell colony formation in soft agar. The relative cell survival per tumor decreased exponentially with time and the irradiation periods required to reach a surviving fraction of 10^?3 at the three dose-rates (10, 15 and 30 cGy/h) were 15.4, 10.2 and 4.3 days. As a function of dose, the survival curves were exponential and indistinguishable betwen the three dose-rates, the aveage D₀ value being 5.04 Gy. Neither the hypoxic fraction of the tumor nor the intrinsic cellular radiosensitivity were altered in the tumor cells surviving Cl. The effects of the three dose-rates on the growth of Lewis lung tumors differed considerably, with clear volume regression at the highest dose rate (30 cGy/h) and exponential decreases in clonogenic cells per tumor with time at 15 and 30 cGy/h. Since the overall effect of CI is determined by the competition between cell killing and cell repopulation, the response of slowly-growing human tumors should be proportionately greater.     

Are Direct Measures of Tumor Oxygenation Reflective of Changes in Tumor Radiosensitivity Following Oxygen Manipulation?

The present study investigates the correlation between tumor oxygen availability and radiosensitivity following oxygen manipulation. Previous work has shown that tumors may contain both diffusion- and perfusion-limited hypoxic cells. Recently, the combination of nicotinamide (NIC) administration plus carbogen breathing has been proposed as a means of targeting both hypoxic cell subpopulations. Intravascular HbO₂ saturations were measured for KHT murine sarcomas following either NIC, carbogen breathing, or the combination, and compared with determinations of tumor cell survival under matched conditions. The percentage of vessels ≥ 25% HbO₂ increased significantly for both the carbogen and NIC-carbogen combination, while remaining unchanged from controls following NIC. These findings contrast with the survival data, where all treatments showed identical cell survival. A possible explanation is that different proportions of clonogenic versus nonclonogenic cells may be oxygenated by the alternative treatments. Thus direct determinations of alterations in tumor oxygenation may not reflect corresponding changes in radiosensitivity.