Radiosensitization of murine tumors by Fluosol-DA 20%

The effect of perfluorochemicals in combination with carbogen breathing on the response of SCK tumors of mice to fractionated irradiation was investigated. The SCK tumors of A/J mice were irradiated twice a day at 3 Gy per fraction (6 Gy per day), with a total dose of 18 Gy over 3 days. When the host animals were treated with an intravenous (iv) injection of 12 ml/kg of Fluosol-DA 20% before the first daily tumor irradiation and carbogen breathing during every X irradiation with Fluosol-DA 20% injection without carbogen breathing. The hypoxic cell fraction, as determined by an in vivo-in vitro cloning assay, decreased significantly, and the intratumor pO2, as determined with microelectrodes, was markedly increased by Fluosol-DA 20% injection and carbogen breathing. It was concluded that oxygenation of hypoxic cells in SCK tumors during the course of fractionated irradiation was improved by the iv injection of Fluosol-DA 20% and carbogen breathing.     

Effect of a perfluorochemical emulsion on the radiation response of BA1112 rhabdomyosarcomas

The effect of treatment with a perfluorochemical emulsion (Fluosol DA, 20%), carbogen, or the combination of these two agents on the radiation response of BA1112 tumors in WAG/rij rats was examined. Fluosol and carbogen as single agents had only small effects on the tumor cell survival curve. The combination of Fluosol plus carbogen had a larger effect on tumor cell survival, reducing the hypoxic fraction of the tumor from 23 to 1.6%. The amount of sensitization was a function of the Fluosol dose, with maximal augmentation of the radiation response obtained at doses of 7.5-15 ml/kg. Carbogen pretreatments ranging from 5 to 60 min in duration all had similar effects on tumor radiosensitivity. The effect of the perfluorochemical emulsion plus carbogen on the survival of irradiated tumor cells appears to reflect changes in tumor oxygenation, rather than cytotoxic or immunological effects, since the perfluorochemical emulsion (with or without carbogen) had no effect on the viability of cells in unirradiated tumors. These experiments extend previous studies by ourselves and others using mouse tumors to show that the combination of a perfluorochemical emulsion and carbogen breathing can also increase the radiation response of a nonimmunogenic rat tumor.     

Improvement of tumor oxygenation by mild hyperthermia

There is now abundant evidence that oxygenation in rodent, canine and human tumors is improved during and for up to 1-2 days after heating at mild temperatures. An increase in tumor blood perfusion along with a decline in the oxygen consumption rate appears to account for the improvement of tumor oxygenation by mild hyperthermia. The magnitude of the increase in tumor pO(2), determined with oxygen-sensitive microelectrodes, caused by mild hyperthermia is less than that caused by carbogen breathing. However, mild hyperthermia is far more effective than carbogen breathing in increasing the radiation response of experimental tumors, probably because mild hyperthermia oxygenates both (diffusion-limited) chronically hypoxic and (perfusion-limited) acutely hypoxic cells, whereas carbogen breathing oxygenates only the chronically hypoxic cells. Mild hyperthermia is also more effective than nicotinamide, which is known to oxygenate acutely hypoxic cells, in enhancing the radiation response of experimental tumors. The combination of mild hyperthermia with carbogen or nicotinamide is highly effective in reducing the hypoxic cell fraction in tumors and increasing the radiation response of experimental tumors. A primary rationale for the use of hyperthermia in combination with radiotherapy has been that hyperthermia is equally cytotoxic toward fully oxygenated and hypoxic cells and that it directly sensitizes both fully oxygenated and hypoxic cells to radiation. Such cytotoxicity and such a radiosensitizing effect may be expected to be significant when the tumor temperature is elevated to at least 42-43 degrees C. Unfortunately, it is often impossible to uniformly raise the temperature of human tumors to this level using the hyperthermia devices currently available. However, it is relatively easy to raise the temperature of human tumors into the range of 39-42 degrees C, which is a temperature that can improve tumor oxygenation for up to 1-2 days. The potential usefulness of mild hyperthermia to enhance the response of human tumors to radiotherapy by improving tumor oxygenation merits continued investigation.     

Enhanced effectiveness of radiochemotherapy with tirapazamine by local application of electric pulses to tumors

Tumor hypoxia is associated with resistance to radiotherapy and anticancer chemotherapy. However, it can be exploited to therapeutic advantage by concomitantly using hypoxic cytotoxins, such as tirapazamine (TPZ). Tumor electroporation offers the means to further increase tumor hypoxia by temporarily reducing tumor blood flow and therefore increase the cytotoxicity of TPZ. The primary objective of this work was to determine whether electric pulses combined with TPZ and radiotherapy (electroradiochemotherapy) was more efficacious than radiochemotherapy (TPZ + radiation). In these studies using the SCCVII tumor model in C3H mice, electroradiochemotherapy produced up to sixfold more tumor growth delay (TGD) than TPZ + radiation. In these studies, (1) large tumors (280 +/- 15 mm3) responded better to electroradiochemotherapy than small tumors (110 +/- 10 mm3), (2) TGD correlated linearly with tumor volume at the time of electroradiochemotherapy, (3) electric pulses induced a rapid but reversible reduction in O2 saturation, and (4) the electric field was highest near the periphery of the tumor in a 3D computer model. The findings suggested that electroradiochemotherapy gained its therapeutic advantage over TPZ + radiation by enhancing the cytotoxic action of TPZ through reduced tumor oxygenation. The greater antitumor effect achieved in large tumors may be related to tumor morphology and the electric-field distribution. These results suggest that electro-pulsation of large solid tumors may be of benefit to patients treated with radiation in combination with agents that kill hypoxic cells.     

Effects of Fluosol DA 20% and carbogen on the radioresponse of SCK tumors and skin of A/J mice

The effect of Fluosol DA 20%, an emulsion of perfluorochemicals, in combination with carbogen (95% O2 and 5% CO2) breathing on the response of mouse tumors to radiation was studied. When A/J mice bearing SCK tumors in the right hind limb were injected iv with Fluosol DA 20% at 12 ml/kg and exposed to carbogen for 1 h before and during the irradiation of tumors, the response of tumors to a single dose of X irradiation was significantly enhanced. The dose modification factors for growth delay and cure of SCK tumors were 2.10 +/- 0.01 (SE) and 1.86 +/- 0.18 (SE), respectively. Such a treatment slightly increased the radiation-induced skin damage by a factor of 1.17 +/- 0.02 (SE), resulting in a therapeutic gain of 1.79 +/- 0.01 (SE) for the growth delay and 1.59 +/- 0.09 (SE) for the curability. Carbogen breathing alone also increased the response of tumor and skin to radiation, but it was far less effective than the combination of Fluosol DA 20% and carbogen breathing. It was concluded that iv injection of Fluosol DA 20% in conjunction with carbogen breathing significantly increased the O2 transport to hypoxic areas in the SCK tumors and thus significantly enhanced the tumoricidal effect of radiation on SCK tumors.     

The range of oxygenation in SiHa tumor xenografts

Tumor cells at very low oxygen tensions are known to be about three times more resistant to killing by ionizing radiation. Since cells at intermediate oxygen tensions (defined here as greater than 0.1% and less than 2% O(2)) show partial radioresistance, they should be a consideration in tumor treatment. In an effort to estimate the extent and range of oxygenation in SiHa human cervical carcinoma xenografts, patterns of cell killing and DNA damage by radiation and two bioreductive drugs, PD-144872 and RSU-1069, were compared to those seen in SiHa cells grown as spheroids. These drugs produce DNA interstrand crosslinks that are largely responsible for cell killing, and the degree of crosslinking increases as the oxygenation is reduced. About 60% of the cells in SiHa xenografts exhibited drug-induced crosslinks, but only about 35% showed extensive crosslinking indicative of hypoxia below 0.1% oxygen. Patterns of toxicity and DNA damage in xenografts were comparable to those of spheroids equilibrated with about 2% oxygen, indicating that most cells in the xenografts exhibit some radioresistance due to lack of oxygen. Similarly, pimonidazole binding indicated that about 60% of the cells in SiHa xenografts were either intermediate in oxygenation or hypoxic, but only about half of those were consistent with extreme oxygen depletion. The apparent size of the population of "intermediately hypoxic" cells has implications for the use of ionizing radiation, hypoxic cell cytotoxins, and other antitumor agents whose cytotoxicity is dependent on cellular oxygen content.     

Comparisons among pimonidazole binding, oxygen electrode measurements, and radiation response in C3H mouse tumors.

Pimonidazole binding was compared with oxygen electrode measurements and with measurements of the radiobiologically hypoxic fraction in C3H mammary tumors in which oxygenation was manipulated by means of subjecting tumor-bearing CDF1 mice to air breathing, carbogen breathing, oxygen breathing, hydralazine injection or tumor clamping. Hypoxia measured by pimonidazole binding could be correlated with both pO2 (r2 = 0.81) and radiobiologically hypoxic fraction (r2 = 0.85) in this system. The scope and limitation of pimonidazole as an immunohistochemical marker for tumor hypoxia is discussed.     

Modulation of hypoxia in murine liver metastases of colon carcinoma by nicotinamide and carbogen

There is increasing evidence that modulation of tumor hypoxia may improve therapy outcome. However, most preclinical data are derived from subcutaneous rather than orthotopic tumor models. We investigated the effect of the hypoxia-modulating agents nicotinamide and carbogen on tumor hypoxia, tumor blood perfusion, and proliferative activity in liver metastases of the murine colon carcinoma line C26a. In untreated C26a liver metastases, we observed a considerable amount of hypoxia, similar to the amount in liver metastases of patients with colorectal cancer. Compared to untreated mice, we observed a significantly smaller hypoxic fraction in the liver metastases of mice treated with nicotinamide and carbogen breathing as single treatments or in combination. In the group of mice that underwent carbogen breathing, perfusion was significantly lower than in the untreated group, but the decrease was only marginal. The proliferative activity was similar in all groups. In C26a subcutaneous tumors, a similar effect on hypoxia has been observed that was, however, combined with a decrease in proliferative activity. The different effects of nicotinamide and carbogen on parameters of the tumor microenvironment in liver metastases and subcutaneous tumors suggest that the host tissue influences the mechanism by which nicotinamide and carbogen exert their effects. Since tumor hypoxia may be a clinical problem in colorectal liver metastases, our results open possibilities for further research on the effect of hypoxia modifiers on colorectal liver metastases to improve therapy outcome.     

Intravascular oxygen distribution in subcutaneous 9L tumors and radiation sensitivity

Cerniglia, George J., David F. Wilson, Marek Pawlowski,Sergei Vinogradov, and John Biaglow. Intravascular oxygendistribution in subcutaneous 9L tumors and radiation sensitivity.J. Appl. Physiol. 82(6):1939-1945, 1997.Phosphorescence quenching was evaluated as atechnique for measuring PO₂ in tumors and for determining the effect of increasedPO₂ on sensitivity of the tumors toradiation. Suspensions of cultured 9L cells or small pieces of solidtumors from 9L cells were injected subcutaneously on the hindquarter ofrats, and tumors were grown to between 0.2 and 1.0 cm in diameter.Oxygen-dependent quenching of the phosphorescence of intravenouslyinjected Pd-meso-tetra-(4-carboxyphenyl) porphine was used to image thein vivo distribution of PO₂ in thevasculature of small tumors and surrounding tissue. Maps (512 × 480 pixels) of tissue oxygen distribution showed that thePO₂ within 9L tumors was low(2-12 Torr) relative to the surrounding muscle tissue (20-40Torr). When the rats were given 100% oxygen or carbogen (95%O₂-5%CO₂) to breathe, thePO₂ in the tumors increasedsignificantly. This increase was variable among tumors and was greaterwith carbogen compared with 100% oxygen. Based on irradiation andregrowth studies, carbogen breathing increased the sensitivity of thetumors to radiation. This is consistent with the measured increase inPO₂ in the tumor vasculature. It isconcluded that phosphorescence quenching can be used for noninvasivedetermination of the oxygenation of tumors. This method for oxygenmeasurements has great potential for clinical application in tumoridentification and therapy.

     

Measuring tumor cycling hypoxia and angiogenesis using a side‐firing fiber optic probe

Hypoxia and angiogenesis can significantly influence the efficacy of cancer therapy and the behavior of surviving tumor cells. There is a growing demand for technologies to measure tumor hypoxia and angiogenesis temporally in vivo to enable advances in drug development and optimization. This paper reports the use of frequency‐domain photon migration with a side‐firing probe to quantify tumor oxygenation and hemoglobin concentrations in nude rats bearing human head/neck tumors administered with carbogen gas, cycling hypoxic gas or just room air. Significant increase (with carbogen gas breathing) or decrease (with hypoxic gas breathing) in tumor oxygenation was observed. The trend in tumor oxygenation during forced cycling hypoxia (CH) followed that of the blood oxygenation measured with a pulse oximeter. Natural CH was also observed in rats under room air. The studies demonstrated the potential of the technology for longitudinal monitoring of tumor CH during tumor growth or in response to therapy. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A paradigm for therapy-induced microenvironmental changes in solid tumors leading to drug resistance

Intrinsic alterations in the tumor microenvironment are known to contribute to various forms of drug resistance. For example, tumor hypoxia, due to abnormal or sluggish blood flow within areas of solid tumors, can result in both microenvironment-mediated radiation and chemotherapeutic drug resistance. In contrast, acquired resistance to chemotherapy is generally considered to be the result of the gradual selection of mutant subpopulations having genetic mutations and biochemical alterations responsible for the resistant phenotype. Here we present a paradigm for therapyinduced microenvironment-mediated acquired drug resistance. It is based on the results showing that tumor cells appear to be heterogeneous in their relative dependence on adjacent tumor-associated vasculature for survival. Some tumor cells are highly vessel dependent, whereas some are significantly less so, and thus can survive in more hypoxic regions of tumors, distal from such tumor vessels. Hence, it is possible that variant tumor cells that are less vessel dependent may therefore be selected for over time by successful antiangiogenic drug therapies. This results in loss of response or attenuated responses to the therapy. Preliminary evidence is summarized in support of this hypothesis, using paired human colon cancer (HCT116) cell lines that contain two copies of either the wild-type or the disrupted p53 tumor suppressor gene. The mutant cells were found to be less responsive to antiangiogenic therapy, compared to the wild-type cells, and could be progressively selected for in mixed cell populations. Because p53 inactivation can lead to resistance to hypoxia-mediated apoptosis, the results suggest that a protracted and successful antiangiogenic therapy may create more hypoxic tumor microenvironments, thereby creating the necessary conditions to accelerate the selection of mutant tumor cells that are more adept in surviving and growing in such environments. As such, consideration might be given to the combined use of bioreductive hypoxic cell cytotoxic drugs and angiogenesis inhibitors to prolong the efficacy of antiangiogenic therapeutics.     

Effects of a perfluorochemical emulsion on the response of BA1112 rat rhabdomyosarcomas to continuous low-dose-rate irradiation

The effects of the combination of a perfluorochemical emulsion (Fluosol DA, 20%) and carbogen (95% O2, 5% CO2) on the response of BA1112 rat rhabdomyosarcomas to continuous low-dose-rate irradiation were examined. Tumors were irradiated locally in unrestrained, unanesthetized rats at a dose rate of 0.98 Gy/h, using a specially designed 241Am irradiator system. Cell survival was measured using a colony formation assay. The tumor cell survival curves were fitted to linear relationships of the form ln S = - alpha D, where alpha for air-breathing rats was 0.104 +/- 0.005 Gy-1, as compared to 0.137 +/- 0.009 Gy-1 for rats treated with Fluosol plus carbogen. The increase in the slope of the survival curve produced by the treatment with Fluosol and carbogen was highly significant with a P value of 0.0015. The radiosensitization factor for the combination of Fluosol/carbogen plus continuous low-dose-rate irradiation was 1.32 +/- 0.11. Slightly less radiosensitization was observed with continuous low-dose-rate irradiation than in previous experiments using acute high-dose-rate irradiation. The diminished sensitization with Fluosol/carbogen during continuous low-dose-rate irradiation probably reflects the intrinsically lower oxygen enhancement ratio (OER) of low-dose/low-dose-rate irradiation, reoxygenation of the tumors during the prolonged treatment times used for continuous low-dose-rate irradiation, and the decrease in the levels of circulating perfluorochemicals during the 30-h irradiations. More importantly, the significant level of radiosensitization observed in the experiments with continuous low-dose-rate irradiation suggests that hypoxic cells persist in BA1112 tumors during continuous low-dose-rate irradiations and that the response of these tumors to continuous low-dose-rate irradiation can be improved by adjunctive treatments which oxygenate these radioresistant hypoxic tumor cells.     

Determining hypoxic fraction in a rat glioma by uptake of radiolabeled fluoromisonidazole

The usefulness of radiolabeled nitroimidazoles for measuring hypoxia will be clarified by defining the relationship between tracer uptake and radiobiologically hypoxic fraction. We determined the radiobiologically hypoxic fraction from radiation response data in 36B10 rat gliomas using the paired cell survival curve technique and compared the values to the radiobiologically hypoxic fraction inferred from mathematical modeling of time-activity data acquired by PET imaging of [(18)F]FMISO uptake. Rats breathed either air or 10% oxygen during imaging, and timed blood samples were taken. The uptake of [(3)H]FMISO by 36B10 cells in vitro provided cellular binding characteristics of this radiopharmaceutical as a function of oxygen concentration. The radiobiologically hypoxic fraction determined for tumors in air-breathing rats using the paired survival curve technique was 6.1% (95% CL = 4.3- 8.6%), which agreed well with that determined by modeling FMISO time-activity data (7. 4%; 95% CL = 2.5-17.3%). These results are consistent with the agreement between the two techniques for measuring radiobiologically hypoxic fraction in Chinese hamster V79 cell spheroids. In contrast, the FMISO-derived radiobiologically hypoxic fraction in rats breathing 10% oxygen was 13.1% (95% CL 7.9-8.3%), much lower than the radiobiologically hypoxic fraction of 43% determined from the radiation response data. This discrepancy may be due to the failure of FMISO to identify hypoxic cells residing at or above an oxygen level of 2-3 mmHg that will still confer substantial protection against radiation. The presence of transiently hypoxic cells in rats breathing reduced oxygen may also be under-reported by nitroimidazole binding, which is strongly dependent on time and concentration.     

Frataxin participates to the hypoxia-induced response in tumors

Defective expression of frataxin is responsible for the degenerative disease Friedreich''s ataxia. Frataxin is a protein required for cell survival since complete knockout is lethal. Frataxin protects tumor cells against oxidative stress and apoptosis but also acts as a tumor suppressor. The molecular bases of this apparent paradox are missing. We therefore sought to investigate the pathways through which frataxin enhances stress resistance in tumor cells. We found that frataxin expression is upregulated in several tumor cell lines in response to hypoxic stress, a condition often associated with tumor progression. Moreover, frataxin upregulation in response to hypoxia is dependent on hypoxia-inducible factors expression and modulates the activation of the tumor-suppressor p53. Importantly, we show for the first time that frataxin is in fact increased in human tumors in vivo. These results show that frataxin participates to the hypoxia-induced stress response in tumors, thus implying that modulation of its expression could have a critical role in tumor cell survival and/or progression.     

Factors influencing the thermosensitivity of two rodent tumors.

When SCCVII or KHT tumors (150 mm3) growing in the dorsum of the hind feet of mice were heated in a water bath at 44 degrees C for 60 min, the local control rate was 75 or 5%, respectively. To investigate factors responsible for the differential thermosensitivity between SCCVII and KHT tumors, the intratumor temperature distributions during heating and the thermosensitivities of the tumor cells were studied. Significant temperature heterogeneity was observed in heated tumors. The thermal dose distribution during heating for the sensitive SCCVII tumors was found to be more homogeneous than that for the resistant KHT tumors. For cells grown and heated in culture, SCCVII and KHT cells had similar thermosensitivities. However, when heated in vivo, both SCCVII and KHT cells were more sensitive than their counterparts grown in culture and SCCVII cells were more sensitive than KHT cells. If cells dispersed from the tumors were cultured in medium for 6 h and then heated, both types of cells became as resistant as cells grown in culture. One possible reason for tumor cells to be more sensitive to heating in vivo than in vitro, the temperature of unheated tumors, was examined. It was found that the temperature in the same region in unheated tumors varied temporally by several degrees with an average temperature of 31-32 degrees C. We found no evidence that the temperature during tumor growth could greatly influence the thermosensitivity of the tumor cells. Our findings indicate that a more homogeneous distribution of temperature in the tumor during heating and higher in vivo thermosensitivity of the tumor cells are characteristics of the more heat-sensitive tumor.     

Oxygen dependence of the metabolic activation and cytotoxicity of tirapazamine: implications for extravascular transport and activity in tumors

The hypoxic cytotoxin tirapazamine (TPZ) is currently in phase III clinical trial and appears to have clinical activity. One hypothesis as to why TPZ has been used more successfully in the clinic than most other bioreductive drugs is that its unusual O(2) dependence allows killing of radioresistant cells at "intermediate" O(2) concentrations. We have determined the O(2) dependence of the metabolism of TPZ to its reduction product SR 4317, and its cytotoxicity, in stirred suspensions of HT29 colon carcinoma cells while monitoring O(2) in solution with an Oxylite trade mark probe. The O(2) dependence of the cytotoxicity of TPZ is entirely accounted for by its inhibition of the metabolism of TPZ, with a K(O(2)) value (O(2) concentration for 50% inhibition) of 1.21 +/- 0.09 (SEM) microM. We used this experimental O(2) dependence to extend a recent (Hicks et al., Cancer Res. 63, 5970-5977, 2003) pharmacokinetic/pharmacodynamic model for the cytotoxicity of TPZ in anoxic HT29 multicellular layers to model cell killing in tumors. The model indicates that the O(2) dependence of killing by TPZ complements that of radiation well during fractionated radiotherapy. It predicts that lowering K(O(2)) would decrease killing in radioresistant cells at intermediate O(2) concentrations, while higher K(O(2)) values would exacerbate metabolic consumption of TPZ and thus further impede its penetration into hypoxic regions. Raising K(O(2)) would also increase metabolic activation at physiological O(2) concentrations, thereby compromising hypoxic selectivity. We conclude that the K(O(2)) value of TPZ is indeed close to the optimum for a bioreductive drug of this class (i.e. one that kills only cells in which it is reduced).     

The change in hypoxic and chronically hypoxic cell fraction in murine tumors treated with hyperthermia

The effect of hyperthermia on the size of hypoxic and chronically hypoxic cell fractions in murine tumors was studied. The chronically hypoxic cell fraction was defined as a fraction of tumor cells which were not oxygenated under hyperbaric oxygen. Animals were C3Hf/Sed mice derived from our defined flora mouse colony. Tumors were FSa-II and MCa which were early generation isotransplants of a spontaneous fibrosarcoma and a mammary carcinoma, respectively. TCD50 (50% tumor control dose) or the radiation dose which yields a local tumor control in half the treated animals and TG (tumor growth) time or the time required for half the treated tumors to reach 1000 mm3 from the first treatment day were experimental end points. Hyperthermia was given by immersing animal feet into a water bath maintained at 43.5 +/- 0.1 degrees C. Animal tumors were irradiated with a 137Cs unit under hypoxic conditions, in air or under O2 30 psi. The hypoxic cell fraction increased immediately after hyperthermia in both MCa and FSa-II tumors. The chronically hypoxic cell fraction was, on the other hand, decreased following hyperthermia. The decrease was more substantial in the MCa than in FSa-II.     

The use of needle biopsies for radiobiological assessment of oxygen levels in KHT-C tumors.

Hypoxia affects the sensitivity of cells to radiation. Hence there is considerable interest in the development and assessment of techniques for measuring oxygen levels. In the work described here, we explore the use of tumor needle biopsies (fine needle aspirates) in an assay that is standard in the field of radiation biology: the paired survival assay. We found that needle biopsies are a feasible option for estimating cell survival when conducting this assay, and that the variability in cell survival between tumors was greater than that between different biopsies from the same tumor. Using this technique, we then compared measurements of tumor hypoxia using the paired survival assay and the growth delay assay in the same individual tumors. We found a significant correlation between these two techniques.     

Growth of V79 cells as xenograft tumors promotes multicellular resistance but does not increase spontaneous or radiation-induced mutant frequency

A Chinese hamster V79 xenograft model was developed to determine whether cells subjected to a hypoxic tumor microenvironment would be more likely to undergo mutation at the HPRT locus. V79-171b cells stably transfected with VEGF and EGFP were grown subcutaneously in immunodeficient NOD/ SCID mice. V79-VE tumors were characterized for host cell infiltration, doubling time, hypoxic fraction, vascular perfusion, and response to ionizing radiation. When irradiated in vitro, the mutant frequency for a given surviving fraction did not differ for cells grown in vivo or in vitro. Similar results were obtained using HCT116 human colorectal carcinoma cells grown as xenografts. However, V79-VE cells grown as xenografts were significantly more resistant to killing than monolayers. The background mutant frequency and the radiation-induced mutant frequency did not differ for tumor cells close to or distant from blood vessels. Similarly, tumor cells from well-perfused regions showed the same rate of strand break rejoining and the same rate of loss of phosphorylated histone H2AX as cells sorted from poorly perfused regions. Therefore, deleterious effects of the tumor microenvironment on DNA repair efficiency or mutation induction could not be demonstrated in these tumors. Rather, development of multicellular resistance in V79-VE tumors acted to reduce mutant frequency for a given dose of radiation.