Therapeutic Modification of Hypoxia.

Regions of reduced oxygenation (hypoxia) are a characteristic feature of virtually all animal and human solid tumours. Numerous preclinical studies, both in vitro and in vivo, have shown that decreasing oxygen concentration induces resistance to radiation. Importantly, hypoxia in human tumours is a negative indicator of radiotherapy outcome. Hypoxia also contributes to resistance to other cancer therapeutics, including immunotherapy, and increases malignant progression as well as cancer cell dissemination. Consequently, substantial effort has been made to detect hypoxia in human tumours and identify realistic approaches to overcome hypoxia and improve cancer therapy outcomes. Hypoxia-targeting strategies include improving oxygen availability, sensitising hypoxic cells to radiation, preferentially killing these cells, locating the hypoxic regions in tumours and increasing the radiation dose to those areas, or applying high energy transfer radiation, which is less affected by hypoxia. Despite numerous clinical studies with each of these hypoxia-modifying approaches, many of which improved both local tumour control and overall survival, hypoxic modification has not been established in routine clinical practice. Here we review the background and significance of hypoxia, how it can be imaged clinically and focus on the various hypoxia-modifying techniques that have undergone, or are currently in, clinical evaluation.

Molecular and biological rationale of hyperthermia as radio- and chemosensitizer.

Mild hyperthermia, local heating of the tumour up to temperatures <43 °C, has been clinically applied for almost four decades and has been proven to substantially enhance the effectiveness of both radiotherapy and chemotherapy in treatment of primary and recurrent tumours. Clinical results and mechanisms of action are discussed in this review, including the molecular and biological rationale of hyperthermia as radio- and chemosensitizer as established in in vitro and in vivo experiments. Proven mechanisms include inhibition of different DNA repair processes, (in)direct reduction of the hypoxic tumour cell fraction, enhanced drug uptake, increased perfusion and oxygen levels. All mechanisms show different dose effect relationships and different optimal scheduling with radiotherapy and chemotherapy. Therefore, obtaining the ideal multi-modality treatment still requires elucidation of more detailed data on dose, sequence, duration, and possible synergisms between modalities. A multidisciplinary approach with different modalities including hyperthermia might further increase anti-tumour effects and diminish normal tissue damage.

Realizing the Potential of Vascular Targeted Therapy: The Rationale for Combining Vascular Disrupting Agents and Anti-Angiogenic Agents to Treat Cancer.

Vascular targeted therapies (VTTs) are agents that target tumor vasculature and can be classified into two categories: those that inhibit angiogenesis and those that directly interfere with established tumor vasculature. Although both the anti-angiogenic agents (AAs) and the vascular disrupting agents (VDAs) target tumor vasculature, they differ in their mechanism of action and therapeutic application. Combining these two agents may realize the full potential of VTT and produce an effective therapeutic regimen. Here, we review AAs and VDAs (monotherapy and in combination with conventional therapies). We also discuss the rationale of combined VTT and its potential to treat cancer.

Targeting therapy-resistant cancer stem cells by hyperthermia.

Eradication of all malignant cells is the ultimate but challenging goal of anti-cancer treatment; most traditional clinically-available approaches fail because there are cells in a tumour that either escape therapy or become therapy-resistant. A subpopulation of cancer cells, the cancer stem cells (CSCs), is considered to be of particular significance for tumour initiation, progression and metastasis. CSCs are considered in particular to be therapy-resistant and may drive disease recurrence, which positions CSCs in the focus of anti-cancer research, but successful CSC-targeting therapies are limited. Here, we argue that hyperthermia - a therapeutic approach based on local heating of a tumour - is potentially beneficial for targeting CSCs in solid tumours. First, hyperthermia has been described to target cells in hypoxic and nutrient-deprived tumour areas where CSCs reside and ionising radiation and chemotherapy are least effective. Second, hyperthermia can modify factors that are essential for tumour survival and growth, such as the microenvironment, immune responses, vascularisation and oxygen supply. Third, hyperthermia targets multiple DNA repair pathways, which are generally upregulated in CSCs and protect them from DNA-damaging agents. Addition of hyperthermia to the therapeutic armamentarium of oncologists may thus be a promising strategy to eliminate therapy-escaping and -resistant CSCs.

Relevance of hypoxia in radiation oncology: pathophysiology, tumor biology and implications for treatment.

Tumors are characterized by an inefficient and disorganized vasculature which leads to tumor regions that are transiently or chronically undersupplied with oxygen (hypoxia) and nutrients (e.g., glucose). These adverse conditions are linked to treatment resistant and metastasizing disease with poor prognosis. Radiation sensitivity is dramatically lowered in hypoxic, yet viable and clonogenic, cells since oxygen is involved in the fixation of radiation-induced DNA damage (radiobiological hypoxia), and loco-regional tumor control is adversely affected in patients with hypoxic tumors. Hypoxia also leads to reduced sensitivity towards chemotherapeutics since drug delivery is reduced in hypoperfused hypoxic areas and hypoxic cells are quiescent, making drugs that target dividing cells ineffective. Fortunately, clinical attractive imaging and gene-expression based technologies that allows pre- and during treatment assessment of tumor hypoxia are now available. These technologies may identify patients suitable for established or emerging hypoxia-targeting treatments and, equally important; they allow us to monitor the efficacy of such intervention and may thus pave the way for effective individualized treatment. In the current review, we address 1) the causes and consequences of tumor hypoxia, 2) technologies that allow assessment of tumor hypoxia in individual patients and 3) current status of hypoxia-targeting treatments.

PET hypoxia imaging with FAZA: reproducibility at baseline and during fractionated radiotherapy in tumour-bearing mice.

PURPOSE: Tumour hypoxia is linked to treatment resistance. Positron emission tomography (PET) using hypoxia tracers such as fluoroazomycin arabinoside (FAZA) may allow identification of patients with hypoxic tumours and the monitoring of the efficacy of hypoxia-targeting treatment. Since hypoxia PET is characterized by poor image contrast, and tumour hypoxia undergoes spontaneous changes and is affected by therapy, it remains unclear to what extent PET scans are reproducible. Tumour-bearing mice are valuable in the validation of hypoxia PET, but identification of a reliable reference tissue value (blood sample or image-derived muscle value) for repeated scans may be difficult due to the small size of the animal or absence of anatomical information (pure PET). Here tumour hypoxia was monitored over time using repeated PET scans in individual tumour-bearing mice before and during fractionated radiotherapy. METHODS: Mice bearing human SiHa cervix tumour xenografts underwent a PET scan 3 h following injection of FAZA on two consecutive days before initiation of treatment (baseline) and again following irradiation with four and ten fractions of 2.5 Gy. On the last scan day, mice were given an intraperitoneal injection of pimonidazole (hypoxia marker), tumours were collected and the intratumoral distribution of FAZA (autoradiography) and hypoxia (pimonidazole immunohistology) were determined in cryosections. RESULTS: Tissue section analysis revealed that the intratumoral distribution of FAZA was strongly correlated with the regional density of hypoxic (pimonidazole-positive) cells, even when necrosis was present, suggesting that FAZA PET provides a reliable measure of tumour hypoxia at the time of the scan. PET-based quantification of tumour tracer uptake relative to injected dose showed excellent reproducibility at baseline, whereas normalization using an image-derived nonhypoxic reference tissue (muscle) proved highly unreliable since a valid and reliable reference value could not be determined. The intratumoral distribution of tracer was stable at baseline as shown by a voxel-by-voxel comparison of the two scans (R = 0.82, range 0.72-0.90). During treatment, overall tracer retention changed in individual mice, but there was no evidence of general reoxygenation. CONCLUSION: Hypoxia PET scans are quantitatively correct and highly reproducible in tumour-bearing mice. Preclinical hypoxia PET is therefore a valuable and reliable tool for the development of strategies that target or modify hypoxia.

Space allowance for gestating sows in pens: behavior and immunity.

Different floor space allowances for dry, pregnant sows in pens were evaluated to determine the impacts of space on sow behavior, immune, and cortisol measures. The experiment consisted of 6 replications (blocks 1 to 6; n = 20 sows/group), and within each replicate, physiological measurements were recorded for 2 consecutive pregnancies. A total of 152 sows were measured at 1 gestation, and 65 of those sows were measured at the successive gestation (n = 217). Groups of 5 sows/pen were assigned to 1.4, 2.3, or 3.3 m(2) of floor space/sow or of 5 sows in individual stalls (1.34 m(2)). Behavior measures were stand, sit, lie, walk, drink, oral-nasal-facial (ONF), sham chew, and aggression. Immune traits included both descriptive and functional aspects and cortisol. At d 90 ± 5 of gestation, the occurrence of ONF behaviors increased from 0300 to 1500 h, and lying behavior decreased from 0700 to 1100 h for sows kept at 2.3 m(2). Sows in stalls displayed more (P < 0.05) ONF from 1500 to 2300 h. Stand, sit, drink, ONF, and sham-chew behaviors were affected by floor space; sows in pens at 2.3 m(2) performed more ONF, and sows at 1.4 m(2) performed more sham chewing (P < 0.05). Standing (P = 0.05) and drinking (P = 0.06) were increased, but lying (P = 0.06) was reduced for sows in pens at 2.3 or 3.3 m(2). Sitting and drinking were greater but lying was less for sows in stalls compared with sows in pens (P < 0.01). Immune traits were affected by treatment (P < 0.05); neutrophils were less and lymphocytes were greater, resulting in a reduced neutrophil:lymphocyte (N:L) ratio (P < 0.05) for sows in pens at 3.3 m(2). Natural killer cell was greater but lymphocyte proliferation was less for sows in pens at 1.4 m(2) (P < 0.05). Sows in stalls had greater N:L ratio than sows in pens (P < 0.05). For sows in pens, linear and quadratic responses were detected for behavior and immune traits. As floor space increased, walking and aggression increased. As floor space decreased, neutrophils, N:L, and natural killer cell increased, but as floor space increased lymphocyte proliferation increased. On the basis of behavioral and physiological responses shown by sows in all 4 environments it is apparent that neither floor space nor stall environment provided adequate or quality of space to improve sow well-being. However, the differential behavioral and physiological mechanisms initiated by sows in response to their specific environment the sows were able to evoke the appropriate response(s) needed to adequately adapt to their environment.

Vascular targeting therapy: potential benefit depends on tumor and host related effects.

The growth and development of most solid tumors require that they form their own functional vascular supply, which they do from the host normal vascular network by the process of angiogenesis. The significance of this neo-vasculature makes it an excellent target and two forms of vascular targeting agents (VTAs) have evolved; those that inhibit the angiogenesis process (angiogenesis inhibitors, AIs) and those that damage the already established vessels (vascular disrupting agents, VDAs). Although both AIs and VDAs can have substantial anti-tumor activity, neither induce tumor control, thus for their full clinical potential to be achieved they will need to be combined with more conventional cancer therapies. Numerous pre-clinical studies have demonstrated the efficacy of combining AIs and VDAs with radio- and chemo-therapy and many of these approaches are now under clinical evaluation. Although the tumor is the target for VTA therapy the normal host cells play an important role in VTA efficacy. Host cells such as infiltrating macrophages, neutrophils, mast cells, platelets, endothelial cells, and stromal fibroblasts can all produce the important growth factors that initiate angiogenesis. Many of these host cells also actively participate in the physical steps of angiogenesis including destabilization of existing vessels, blood vessel sprouting, endothelial cell migration and proliferation, and vessel stabilization. There is some evidence that these host cells can also influence VTA treatment, either by helping to normalize tumor vessels when AIs are administered or stimulate tumor angiogenesis after treatment with VDAs. The host itself also plays a critical role. Cancer patients undergoing therapy are normally treated to tolerance, thus the normal tissue side effects actually control the effective dose given to the tumor. All VTAs currently in clinical development induce some form of systemic side effects, which range from being rather mild and tolerable to more severe and even life threatening. For more localised therapies there is also the issue of possible VTA-enhanced local tissue reactions. A few pre-clinical studies have investigated this with radiation, but failed to show any enhancement of radiation-induced normal tissue damage by VTAs. Clearly, the therapeutic benefit of VTA treatment will depend on a balance between tumor and host related effects, and in this review we will consider the contribution of each.

The new vascular disrupting agent combretastatin-A1-disodium-phosphate (OXi4503) enhances tumour response to mild hyperthermia and thermoradiosensitization.

PURPOSE: The aim of this study was to investigate the anti-cancer effect of the novel vascular disrupting agent (VDA), combretastatin-A1-disodium-phosphate (OXi4503), when combined with mild hyperthermia and/or radiation. MATERIALS AND METHODS: A C3H mammary carcinoma was grown subcutaneously in the rear right foot of female CDF1 mice, and treated when a volume of 200 mm(3) was reached. OXi4503 was administered intra-peritoneally at variable doses. Hyperthermia was administered locally to the tumour-bearing foot using a thermostat-controlled water bath. Radiation treatment was performed locally using a conventional X-ray machine. Tumour response was assessed with either a tumour growth time or a tumour control assay. RESULTS: The optimal delay between administration of 50 mg/kg of OXi4503 and hyperthermia was found to be 3 hours. The linear relationship between tumour growth time (TGT) and heating time at a specific temperature resulted in slope values between -0.003 days/min and 0.09 days/min at temperatures between 40 degrees C and 42.5 degrees C. When combined with OXi4503 this was significantly increased to 0.008 days/min and 0.03 days/min at temperatures between 39.5 degrees C and 41 degrees C, respectively. Above 41 degrees C, combined treatment did not result in significantly greater slope values. The radiation dose required to control 50% of the tumours (TCD50) was 52 Gy. Combining radiation with either heat treatment at 41.5 degrees C for 1 hour or OXi4503 reduced the TCD50 to 47 Gy and 41 Gy, respectively. Combining radiation with heat and OXi4503 further reduced the TCD50 to 37 Gy. CONCLUSIONS: OXi4503 is a highly potent VDA, which is capable of significantly enhancing the anti-cancer effect of mild hyperthermia. Mild temperature thermoradiosensitization was also enhanced.

Hyperthermia: a potent enhancer of radiotherapy.

Hyperthermia is generally regarded as an experimental treatment with no realistic future in clinical cancer therapy. This is totally wrong. Although the role of hyperthermia alone as a cancer treatment may be limited, there is extensive pre-clinical data showing that in combination with radiation it is one of the most effective radiation sensitisers known. Moreover, there are a number of large randomised clinical trials in a variety of tumour types that clearly show the potential of hyperthermia to significantly improve both local tumour control and survival after radiation therapy, without a significant increase in side-effects. Here we review the pre-clinical rationale for combining hyperthermia with radiation, and summarise the clinical data showing its efficacy.

Morphological muscle and joint parameters for musculoskeletal modelling of the lower extremity.

BACKGROUND: To assist in the treatment of gait disorders, an inverse and forward 3D musculoskeletal model of the lower extremity will be useful that allows to evaluate if-then scenarios. Currently available anatomical datasets do not comprise sufficiently accurate and complete information to construct such a model. The aim of this paper is to present a complete and consistent anatomical dataset, containing the orientations of joints (hip, knee, ankle and subtalar joints), muscle parameters (optimum length, physiological cross sectional area), and geometrical parameters (attachment sites, 'via' points). METHODS: One lower extremity, taken from a male embalmed specimen, was studied. Position and geometry were measured with a 3D-digitizer. Optotrak was used for measurement of rotation axes of joints. Sarcomere length was measured by laser diffraction. FINDINGS: A total of 38 muscles were measured. Each muscle was divided in different muscle lines of action based on muscle morphology. 14 Ligaments of the hip, knee and ankle were included. INTERPRETATION: The presented anatomical dataset embraces all necessary data for state of the art musculoskeletal modelling of the lower extremity. Implementation of these data into an (existing) model is likely to significantly improve the estimation of muscle forces and will thus make the use of the model as a clinical tool more feasible.

Experience with mouse hepatitis virus sanitation in three transplantable murine tumour lines.

Transmission of viral infection by tumour lines or other biological materials may have confounding effects on research. Many research organizations require screening for viral agents of all cell lines, tumours, sera and other biologicals before implantation or inoculation into animal models. Screening for viral contamination is done by the mouse antibody production (MAP) test, by cell culture, or alternatively by direct detection of the viral agents by polymerase chain reaction (PCR). The description of procedures for sanitation of infected cell lines or tumours is sparse. The present report describes the procedures used for sanitation of three transplantable murine tumour lines, which were transplanted in vivo in a mouse hepatitis virus (MHV)-infected colony of mice at the Department of Experimental Clinical Oncology (DECO). The tumours were frozen and serially transplanted three times in a quarantine colony of syngenic mice. Serological examination of the mice transplanted with tumours as well as their cage mates in the quarantine colony did not detect any antibodies against MHV. After repeated serial transplantation in seronegative animals, tumour material was frozen and thawed tumours were later used for transplantation into the newly established virus-free colony of mice at DECO. PCR-based detection of MHV did not reveal any contamination of the tumour examined by this technique, indicating that this murine tumour apparently did not transmit MHV or that MHV was eliminated from the tissue so fast after the infection that it could not be transmitted by the tumour tissue. It is concluded that MHV infection of mice with transplantable murine tumours does not necessarily cause the tumours to be contaminated.

The assessment of antiangiogenic and antivascular therapies in early-stage clinical trials using magnetic resonance imaging: issues and recommendations.

Vascular and angiogenic processes provide an important target for novel cancer therapeutics. Dynamic contrast-enhanced magnetic resonance imaging is being used increasingly to noninvasively monitor the action of these therapeutics in early-stage clinical trials. This publication reports the outcome of a workshop that considered the methodology and design of magnetic resonance studies, recommending how this new tool might best be used.

Tumour-specific enhancement of thermoradiotherapy at mild temperatures by the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid.

The effect of combining the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) with both radiation and hyperthermia treatments was investigated in a transplanted C3H mouse mammary carcinoma and a normal mouse tissue. Tumours were grown on the right rear foot of female CDF1 mice and treated when sized 200 mm3. The foot skin of non-tumour-bearing CDF1 mice was used to assess normal tissue damage. Radiation and hyperthermia were given locally to the tumour/skin of restrained non-anaesthetized animals. DMXAA (20 mg/kg) was dissolved in saline and injected intraperitoneally 1 h after irradiating and then heating started 3 h later. The endpoints were local tumour control within 90 days or the development of moist desquamation in skin between 11 and 23 days after treatment. The radiation dose (+/- 95% confidence intervals) producing local tumour control in 50% of treated animals was 53 (51-55) Gy for radiation alone. This value was significantly (Chi-squared test; p < 0.05) decreased to 47 (42-52) Gy by DMXAA and to 47 (44-51) Gy by heating (41.5 degrees C/60 min) 4 h after irradiation. Combining both DMXAA and heating further reduced this to 30 (26-35) Gy. When the heating temperature was decreased to 40.5 degrees C, the effect of the triple combination was decreased but was still significant compared with radiation + DMXAA or radiation + hyperthermia. However, this enhancement disappeared at 39.5 degrees C. Radiation damage of normal foot skin was not enhanced by combining DMXAA and hyperthermia at 41.5 degrees C. In conclusion, adding DMXAA to thermoradiotherapy at 40.5-41.5 degrees C significantly improved local tumour control without enhancing normal tissue damage. Thus, including a vascular targeting agent in a mild thermoradiotherapy treatment regimen is a useful approach that may lead to a re-evaluation of the use of hyperthermia in cancer treatment.

Intense inflammation in bladder carcinoma is associated with angiogenesis and indicates good prognosis.

The aim of this study was to investigate the prognostic influence of microvessel density using the hot spot method in 107 patients diagnosed with transitional cell carcinoma of the bladder. In each case, inflammation was found in the invasive carcinoma, therefore we classified the degree of inflammation as minimal, moderate or intense. Microvessel density was then reevaluated in each tumour in areas corresponding to these three categories. Median microvessel density irrespective of degree of inflammation was 71. Areas of minimal, moderate and intense inflammation were found in 48, 92 and 32 tumours. Microvessel density increased significantly with increasing degree of inflammation. Disease-specific survival was improved if areas of intense inflammation were present in the carcinoma (P=0.004). High microvessel density, irrespective of the degree of inflammation, was associated with a significantly better disease-specific survival (P=0.01). Multivariate analysis using death of disease as endpoint demonstrated an independent prognostic value of N-classification (N0, hazard ratio (HR)=1 vs N1, HR=2.89 (range, 1.52-5.52) vs N2, HR=3.61 (range, 1.84-7.08)), and intense inflammation, HR=0.48 (range, 0.24-0.96). Malignancy grade, T classification and microvessel density were not independent significant markers of poor outcome. In conclusion, inflammation was significantly correlated to microvessel density, and areas of intense inflammation were an independent marker of good prognosis.

Combretastatin A-4 disodium phosphate: a vascular targeting agent that improves that improves the anti-tumor effects of hyperthermia, radiation, and mild thermoradiotherapy.

PURPOSE: To investigate the effect of combining the vascular targeting drug combretastatin A-4 disodium phosphate (CA4DP) with hyperthermia, radiation, or mild thermoradiotherapy in a transplanted C3H mouse mammary carcinoma. METHODS AND MATERIALS: The C3H mammary carcinoma was grown on the rear foot of female CDF1 mice and treated when at 200 mm(3) in size. CA4DP was dissolved in saline and injected i.p. Hyperthermia and/or radiation were locally given to tumors in restrained nonanesthetized mice. Tumor response was assessed using either a tumor growth or a tumor control assay. Mouse foot skin was used to assess normal tissue damage. RESULTS: CA4DP significantly enhanced thermal damage in this tumor model. This effect was independent of drug doses between 25-400 mg/kg, but was strongly dependent on the time interval between drug injection and heating, with the greatest improvement seen when CA4DP preceded the heating by 1 h or less. There was also a suggestion of a temperature dependency with a 1.9-fold increase in heat damage at 42.5 degrees C and a 2.6-fold increase at 41.5 and 40.5 degrees C. Heat-induced normal tissue damage was also enhanced by combining CA4DP with heat, but the degree of enhancement was less than that seen in tumors. CA4DP (25 mg/kg) significantly increased radiation-induced local tumor control and this was further enhanced by combining CA4DP with mild temperature (41.5 degrees C, 60 min) heating. CONCLUSIONS: CA4DP improved the anti-tumor effect of hyperthermia, especially at mild temperatures. More importantly, it also increased the tumor response to mild hyperthermia and radiation, which suggests that CA4DP may ultimately have an important application in clinical thermoradiotherapy.

Potentiation of the anti-tumour effect of hyperthermia by combining with the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid.

The potential of the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) to enhance the effect of hyperthermia was investigated in a C3H mouse mammary carcinoma grown in the feet of female CDF1 mice and in normal foot skin. DMXAA, when injected intraperitoneally in restrained non-anaesthetized animals, reduced tumour perfusion, as measured using the RbCl extraction procedure, and increased necrosis in histological section, but these effects were dependent on the drug dose and time interval. At a dose of 20 mg/kg, it significantly enhanced the thermal damage of this tumour, when given 1 h or more before the start of heating, as assessed by a tumour growth assay. This enhancement became larger with increasing interval between the two treatments. No thermo-potentiation was seen at doses of 10 mg/kg or lower. These combined effects seem to be associated with the tumour vascular shut-down by DMXAA. Thermal potentiation by DMXAA was also dependent on the heating temperature, with a greater enhancement relative to hyperthermia alone obtained at the lower temperatures at 40.5 and 41.5 degreesC than at the higher temperature of 42.5 degrees C. DMXAA (20 mg/kg) also enhanced the heat damage of normal skin, and this could not be explained by any DMXAA-induced TNF-alpha production. The heat enhancement-ratio by DMXAA was larger in tumours (1.9) than in normal skin (1.3-1.5), thus giving rise to a therapeutic gain.

Improved tumor response by combining radiation and the vascular-damaging drug 5,6-dimethylxanthenone-4-acetic acid.

The interaction between 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and radiation was investigated in two different mouse tumor models and a normal mouse tissue. C3H mouse mammary carcinomas transplanted in the feet of CDF1 mice and KHT mouse sarcomas growing in the leg muscles of C3H/HeJ mice were used. DMXAA was dissolved in saline and injected intraperitoneally. Tumors were irradiated locally in nonanesthetized mice, and response was assessed using tumor growth for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma. DMXAA alone had an antitumor effect in both tumor types, but only at doses above 15 mg/kg. DMXAA also enhanced radiation damage, and again there was a threshold dose. No enhancement was seen in the C3H mammary carcinoma at 10 mg/kg and below, while in the KHT sarcoma, doses above 15 mg/kg were necessary. This enhancement of radiation damage was also dependent on the sequence of and interval between the treatments with DMXAA and radiation. Combining radiation with DMXAA at the maximum tolerated dose (i.e., the highest dose that could be injected without causing any lethality) of either 20 mg/kg (CDF1 mice) or 17.5 mg/kg (C3H/HeJ mice) gave an additive response when the two agents were administered simultaneously. Even greater antitumor effects were achieved when DMXAA was administered 1-3 h after irradiation. However, when administration of DMXAA preceded irradiation, the effect was similar to that seen for radiation alone, suggesting that appropriate timing is essential to maximize the utility of this agent. When such conditions were met, DMXAA was found to increase the tumor response significantly in the absence of an enhancement of radiation damage in normal skin, thus giving rise to therapeutic gain.