Regions of low oxygenation (hypoxia) are a characteristic feature of solid tumors, and cells existing in these regions are a major factor influencing radiation resistance as well as playing a significant role in malignant progression. Consequently, numerous pre-clinical and clinical attempts have been made to try and overcome this hypoxia. These approaches involve improving oxygen availability, radio-sensitizing or killing the hypoxic cells, or utilizing high LET (linear energy transfer) radiation leading to a lower OER (oxygen enhancement ratio). Interestingly, hyperthermia (heat treatments of 39–45 °C) induces many of these effects. Specifically, it increases blood flow thereby improving tissue oxygenation, radio-sensitizes via DNA repair inhibition, and can kill cells either directly or indirectly by causing vascular damage. Combining hyperthermia with low LET radiation can even result in anti-tumor effects equivalent to those seen with high LET. The various mechanisms depend on the time and sequence between radiation and hyperthermia, the heating temperature, and the time of heating. We will discuss the role these factors play in influencing the interaction between hyperthermia and radiation, and summarize the randomized clinical trials showing a benefit of such a combination as well as suggest the potential future clinical application of this combination.

Exercise has long been known to be beneficial to human health. Studies aimed at understanding the effects of exercise specifically focus on predetermined exercise intensities defined by measuring the aerobic capacity of each individual. Many disease models involving animal training often establish aerobic capacity by using the maximal lactate steady state (MLSS), a widely used method in humans that has frequently been used in rodent studies. The MLSS is defined as the highest exercise intensity at which blood lactate concentration remains constant and is roughly equivalent to 70–80% of maximal aerobic capacity. Due to our up-coming experiments investigating the effect of different exercise intensities in specific strains of tumor-bearing mice, the aim of the present study was to determine the MLSS in athymic nude (NCr nu/nu and NMRI), CDF1, and C3H mice by treadmill running at increasing speeds. However, despite thorough exercise acclimation and the use of different exercise protocols and aversive stimuli, less than half of the experiments across strains pointed towards an established MLSS. Moreover, gently prodding the mice during low to moderate intensity running caused a 30–121% (p<0.05) increase in blood lactate concentration compared to running without stimulation, further questioning the use of lactate as a measure of exercise intensity. Overall, MLSS is difficult to determine and large variations of blood lactate levels were observed depending on the exercise protocol, mice handling strategy and strain. This should be considered when planning experiments in mice using forced exercise protocols.

Immune therapy improves cancer outcomes, yet many patients do not respond. This pre-clinical study investigated whether vascular disrupting agents (VDAs) could convert an immune unresponsive tumor into a responder. CDF1 mice, with 200 mm3 C3H mammary carcinomas in the right rear foot, were intraperitoneally injected with combretastatin A-4 phosphate (CA4P), its A-1 analogue OXi4503, and/or checkpoint inhibitors (anti-PD-1, PD-L1, or CTLA-4 antibodies), administered twice weekly for two weeks. Using the endpoint of tumor growth time (TGT5; time to reach five times the starting volume), we found that none of the checkpoint inhibitors (10 mg/kg) had any effect on TGT5 compared to untreated controls. However, CA4P (100 mg/kg) or OXi4503 (5–50 mg/kg) did significantly increase TGT5. This further significantly increased by combining the VDAs with checkpoint inhibitors, but was dependent on the VDA, drug dose, and inhibitor. For CA4P, a significant increase was found when CA4P (100 mg/kg) was combined with anti-PD-L1, but not with the other two checkpoint inhibitors. With OXi4503 (50 mg/kg), a significant enhancement occurred when combined with anti-PD-L1 or anti-CTLA-4, but not anti-PD-1. We observed no significant improvement with lower OXi4503 doses (5–25 mg/kg) and anti-CTLA-4, although 30% of tumors were controlled at the 25 mg/kg dose. Histological assessment of CD4/CD8 expression actually showed decreased levels up to 10 days after treatment with OXi4503 (50 mg/kg). Thus, the non-immunogenic C3H mammary carcinoma was unresponsive to checkpoint inhibitors, but became responsive in mice treated with VDAs, although the mechanism remains unclear.

Objectives: The objectives were to investigate 1) the effect of acute aerobic exercise on tumour hypoxia and blood perfusion, 2) the impact of exercise intensity, 3) the duration of the effect, and 4) the effect of prolonged training on tumour hypoxia as well as tumour growth. Methods: Female CDF1 mice were inoculated with the C3H mammary carcinoma either in the mammary fat pad or subcutaneously in the back. For experiments on the effect of different intensities in a single exercise bout, mice were randomized to 30 min treadmill running low, moderate, or high intensity speeds or no exercise. To investigate the prolonged effect on hypoxia and tumour growth, tumour-bearing mice were randomized to no exercise (CON) or daily 30 min high-intensity exercise averaging two weeks (EX). Tumour hypoxic fraction was quantified using the hypoxia marker Pimonidazole. Results: Initially, high intensity exercise reduced tumour hypoxic fraction by 37% compared with CON (p=0.046; 95% CI: 0.1; 10.3) in fat pad tumours. Low and moderate intensities did not. Following experiments investigating the duration of the effect - as well as experiments in mice with back tumours - failed to show any exercise induced changes in hypoxia. Interestingly, prolonged daily training significantly reduced hypoxic fraction by 60% (p=0.002; 95% CI: 2.5;10.1) compared with CON. Conclusion: Despite diverging findings on the acute effect of exercise on hypoxia, our data indicate that if exercise has a diminishing effect, high-intensity exercise is needed. Prolonged training reduced tumour hypoxic fraction - cautiously suggesting a potential clinical potential.

Background: This pre-clinical study was designed to refine a dissection method for validating the use of a 15-gene hypoxia classifier, which was previously established for head and neck squamous cell carcinoma (HNSCC) patients, to identify hypoxia in prostate cancer. Methods: PC3 and DU-145 adenocarcinoma cells, in vitro, were gassed with various oxygen concentrations (0–21%) for 24 h, followed by real-time PCR. Xenografts were established in vivo, and the mice were injected with the hypoxic markers [18F]-FAZA and pimonidazole. Subsequently, tumors were excised, frozen, cryo-sectioned, and analyzed using autoradiography ([18F]-FAZA) and immunohistochemistry (pimonidazole); the autoradiograms used as templates for laser capture microdissection of hypoxic and non-hypoxic areas, which were lysed, and real-time PCR was performed. Results: In vitro, all 15 genes were increasingly up-regulated as oxygen concentrations decreased. With the xenografts, all 15 genes were up-regulated in the hypoxic compared to non-hypoxic areas for both cell lines, although this effect was greater in the DU-145. Conclusions: We have developed a combined autoradiographic/laser-guided microdissection method with broad applicability. Using this approach on fresh frozen tumor material, thereby minimizing the degree of RNA degradation, we showed that the 15-gene hypoxia gene classifier developed in HNSCC may be applicable for adenocarcinomas such as prostate cancer.

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Introduction: The combination of hyperthermia with low LET (linear energy transfer) radiation may have similar anti-tumor effects as high LET radiation alone. This pre-clinical study determined the optimal heating temperature and time interval between radiation and heat to achieve this equivalent effect. Methods: C3H mammary carcinomas (200 mm 3 in size) growing in the right rear foot of CDF1 mice was used in all experiments. Tumors were locally irradiated with graded doses of either 240 kV orthoor 6 MV mega-voltage X-rays to produce full dose-response curves. Heating (41.0-43.5 C; 60 min) was achieved by immersing the tumor bearing foot in a water-bath applied at the same time, or up to 4hours after, irradiating. The endpoint was the percentage of mice showing local tumor control at 90 days, with enhancements calculated from the ratios of the radiation doses causing 50% tumor control (± 95% confidence intervals). Results: Previous published results in this tumor model reported that carbon ions were 1.3-1.7 times more effective than low LET radiation at inducing tumor control. Similar enhancements occurred with a temperature of only 41.0 C with a simultaneous heat and radiation treatment. However, higher temperatures were needed with the introduction of any interval; at 42.5 C, the enhancement was 2.5 with a simultaneous treatment, decreasing to a value within the carbon ion range with a 4hour interval. Conclusions: Combining hyperthermia with low LET radiation can be as effective as high LET at inducing tumor control, but the temperature needed depended on the time interval between the two modalities.

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