Angiogenesis and vascular targeting: Relevance for hyperthermia

Abstract

The creation of a functional blood supply from the normal tissue vasculature via the process of angiogenesis is critical for the continued growth and development of solid tumours. This importance has led to the concept of targeting the tumour vasculature as a therapeutic strategy, and two major types of vascular targeting agents (VTAs) have developed; those that inhibit the angiogenic process–angiogenesis inhibiting agents (AIAs)–and those that specifically damage the already established neovasculature–vascular disrupting agents (VDAs). The tumour vasculature also plays a critical role in influencing the response to hyperthermia. Generally, the poorer the blood supply the better the heat effect, thus combining VTAs with heat would appear to be a logical approach. Numerous pre-clinical studies have now demonstrated the benefits of combining AIAs or VDAs with heat to improve tumour response. VDAs have also been shown to enhance thermoradiosensitisation, especially at mild hyperthermia temperatures. Although some enhancement of normal tissue response has been observed when VDAs are given with heat, the effects are always less than those seen in tumours, and no increased normal tissue reactions are seen when radiation, VDAs and heat are combined, thus a therapeutic benefit exists. These pre-clinical studies clearly encourage clinical trials with these combinations.

• Hyperthermia
• angiogenesis
• vascular targeting agents
• angiogenesis inhibitors
• vascular disrupting agent

Introduction

The growth and development of most solid tumours requires that they develop their own functional blood supply [1], [2] which they do from the already established normal tissue vasculature by the process of angiogenesis [3], [4]. This tumour neovasculature is not only critical for survival of the tumour cells it is also probably one of the most important factors influencing the therapeutic application of hyperthermia. The vasculature is known to be a target for hyperthermia damage [5], [6]. In addition, blood flow is one of the major means by which heat is dissipated from tissues, and thus influences the ability to heat tumours; generally, the lower the rate of blood flow the easier it is to heat [7], [8]. The tumour vasculature also controls the microenvironment within tumours [9], [10] and this plays an important role in determining the response of the tumour cells to heat. Typically, the tumour neovasculature is very different from the normal vessels from which it arises, being both structurally and functionally abnormal [9], [10]. As a result, it is unable to meet all the oxygen and nutrient demands of the growing tumour mass and areas develop within the tumour that are oxygen deprived, nutrient deficient, and highly acidic [9], [10]. Cells existing in such conditions are more sensitive to the cytotoxic action of heat [11], [12].

In recent years, the concept of specifically targeting tumour vasculature has developed as a therapeutic approach. This can be achieved either by inhibiting the angiogenic process or damaging the already established tumour vessels. Both approaches have undergone extensive pre-clinical testing and many of these vascular targeting agents (VTAs) are now in clinical evaluation [13], [14]. Additional studies have demonstrated that physiological modification of tumour blood flow, either by simply clamping [15], [16], or physiological agents [17–19], can significantly increase tumour sensitivity to heat. This suggests that the anti-vascular effects of VTAs may make them promising new candidates for improving the efficacy of hyperthermia.

Angiogenesis and vascular targeting approaches

Angiogenesis is the formation of new blood vessels from pre-existing vessels. In adults, it is normally only associated with certain physiological processes including the female reproductive cycle, pregnancy, muscular hypertrophy and wound healing [20]. However, it is seen with a number of pathological conditions, such as rheumatoid arthritis, diabetes, macular degeneration, psoriasis, cardiovascular disease and cancer [20]. The need for angiogenesis in the successful growth and development of most solid tumours is known [1], [2], but the actual triggers that initiate this process are not fully established. Hypoxia is clearly one factor that plays a significant role [21], but other ‘non-hypoxia factors’ have also been implicated including loss of tumour suppressor gene function and oncogene activation [21–23]. Whatever the trigger, the angiogenic process begins with the release of angiogenic factors, primarily vascular endothelial growth factor (VEGF), by the tumour cells [23]. These factors then set in motion a number of physical steps that include enzymatic destruction of the basal membrane by the endothelial cells, migration of endothelial cells into the extracellular matrix to form sprouts, and endothelial cell division away from the sprout tip [4]. Solid strands of endothelial cells are then formed in the extracellular matrix, lumen develops within those strands, neighbouring sprouts fuse to form loops, and from the primary loops new buds and sprouts emerge [4]. Functional vessels are finally established (Figure 1).

Figure 1. Schematic illustration of how the growth of most solid tumours is dependent on the development of its own neovasculature from the normal tissue vessels by the process of angiogenesis. Also shown is how vascular targeting agents (VTAs) can be used as a therapeutic approach against the tumour vasculature. They include AIAs (angiogenesis inhibiting agents) and VDAs (vascular disrupting agents), which although similar are distinctly different in their mode of action.

The importance of the tumour neovasculature makes it a potential target for therapy and two major approaches have emerged. One is based on controlling the blood vessel development by inhibiting the angiogenesis process (angiogenesis inhibiting agents; AIAs), and the other involves compromising the function of the already existing blood vessels (vascular disrupting agents; VDAs). Although AIAs and VDAs both target the tumour vascular supply they are two distinct approaches [24], [25]. AIAs are designed to prevent further development of the tumour neovascular network. As such they are likely to be given as a chronic administration and are probably best suited for early stage or metastatic disease. VDAs are agents that cause direct damage to the already established tumour endothelium. Thus, their administration is most likely to be of a more acute type to induce substantial vascular shutdown. There is also good evidence that VDAs have a superior effect on bulky disease. Numerous AIAs capable of inhibiting new blood vessel formation have been identified, and each affects at least one of the several important stages of angiogenesis. The primary targets are the angiogenic factors because they play the most significant role in neo-vascularisation [23]. Of these angiogenic factors, the most potent and specific is VEGF, which is not only crucial for endothelial cell proliferation and blood vessel formation, but also induces significant vascular permeability and plays a key role in endothelial cell survival signalling in newly formed vessels [21], [23]. VEGF has been targeted by a variety of strategies [25–28], including monoclonal antibodies, e.g., bevacizumab/avastin and DC101, inhibitors of endothelial cell receptor-associated tyrosine kinase activity, e.g., SU5474, SU6668, ZD6474 and PTK787/ZK 222584, and antisense. Other approaches, including those targeting basement membrane degradation, endothelial cell migration, endothelial cell proliferation, and tube formation, have also been actively considered [26–28]. Many of these anti-angiogenic therapies are currently under clinical evaluation [13], [26–28].

VDAs include physical treatments like hyperthermia or photodynamic therapy, which have been well documented to induce direct tumour cell killing and an indirect effect through the induction of vascular damage [5], [6]. They also include biological response modifiers or cytokines like tumour necrosis factor (TNF) and interleukins; certain established chemotherapeutic drugs such as vinka alkaloids and arsenic trioxide (ATO); and various ligand-based approaches that use antibodies, peptides or growth factors that can selectively bind to tumour vessels [5], [29–31]. But, more commonly VDAs involve the use of small molecule drugs [31], of which there are two major classes of agents. The first includes flavone acetic acid (FAA) and its derivative 5,6-dimethylxanthenone-4-acetic acid (DMXAA), which have a complex mechanism of action that is poorly understood, but their main effect on vascular endothelial cells is thought to involve a cascade of direct and indirect effects, the latter involving the induction of cytokines, especially TNF-α, leading to the induction of haemorrhagic necrosis [32]. A second group includes the tubulin-binding agents combretastatin A-4 disodium phosphate (CA4P), ZD6126, AVE8062, NPI2358, MN-029, and OXi 4503 [31], [32]. These tubulin depolymerising agents are primarily believed to selectively disrupt the cytoskeleton of proliferating endothelial cells, resulting in endothelial cell shape changes and subsequent thrombus formation and vascular collapse [32]. Since they preferentially target dividing endothelial cells this accounts for their tumour specificity. Both types of small molecular drugs have been shown to have potent anti-vascular and anti-tumour efficacy in a wide variety of preclinical models and the lead agents are undergoing clinical evaluation [14]. Whether AIAs or VDAs are given alone or in combination, the effect on tumours generally does not lead to tumour regression. Thus, their potential benefit is likely to be when combined with other therapies such as radiation, chemotherapy or hyperthermia.

VTAs and hyperthermia

A limited number of studies have investigated the combination of AIAs with hyperthermia (Table I). One of the first agents to be tested was TNP-470, the synthetic analogue of fumagillin first isolated from Aspergillus fumigatus, a potent inhibitor of tumour angiogenesis [33]. Repeated administration of TNP-470 to mice bearing murine or human tumour xenografts significantly inhibited tumour growth [34], [35] and such a schedule given after heating tumours further significantly enhanced the heat response in those same studies. However, this effect was temperature dependent, being observed at 43° and 44°C, but not at 42°C [34], [35]. This enhancement was attributed to TNP-470 inhibiting angiogenesis that occurred following heat-induced vascular damage, the latter effect being temperature dependent. Similar studies in a BT₄An rat glioma model using batimastat, a broad spectrum inhibitor of metalloproteinases [36], failed to show any enhancement of heat damage using a temperature of 44°C that produced substantial vascular damage [37]. This lack of effect could be attributed to the fact that batimastat alone at the dose and schedule used in this study, was ineffective at inhibiting growth in this tumour model. Clearly, AIAs can be used to improve the heat response of tumours, but do so via mechanisms that do not involve any improvement in tumour heating, nor tumour sensitisation to heat resulting from any AIA-induced microenvironmental changes. All that is required is that the heat treatment is sufficient to induce vascular damage and that the AIAs are effective as angiogenesis inhibitors. With the large number of AIAs currently in clinical testing as potential anti-cancer agents [13] it is surprising that there are so few studies combining them with hyperthermia.

Table I.  Preclinical studies combining VTAs and hyperthermia.

VTA^1	Tumour model^2	Treatments^3	Reference
TNP-470	ESO-2 human oesophageal cancer	TNP-470 (30 mg/kg; s.c.; 3x/week for 2 weeks starting day 0)	34
		Waterbath heating (43°C for 30 min; shortly after each drug treatment)
	NSC-8 human gastric cancer	TNP-470 (30 mg/kg; s.c.; 3x/week for 2 weeks starting day 0)	34
		Waterbath heating (43°C for 30 min; shortly after each drug treatment)
	SCVII carcinoma	TNP-470 (100 mg/kg; s.c.; days 0 + 3 or 0, 3, 7 + 10)	35
		Waterbath heating (42° or 44°C for 30 min; day 0)
BMT	BT4An glioma*	BMT (30 mg/kg; i.p.; days 0-7 or 0-14)	37
		Waterbath heating (44°C for 60 min; days 0 or 0 + 7)
PDT	SMT-F mammary carcinoma	PDT (drug-24 hours-light on day 0)	48
		Microwave heating (40.5°, 41.5° or 44.5°C for 30 min; immediately before/after light)
	RIF-1 fibrosarcoma	PDT (drug-24 hours-light on day 0)	49
		Microwave heating (44°C for 30 min; immediately before/after light)
	R-1 rhabdomyosarcoma*	PDT (drug-24/48 hours-light on day 0)	51
		Radiofrequency heating (44°C for 30 min; 30 min after light)
	Squamous cell carcinoma	PDT (drug-83 hours-light on day 0)	50
		Microwave heating (43°C for 30 or 60 min; before/after light)
	C3H mammary carcinoma	PDT (drug-24 hours-light on day 0)	52
		Waterbath heating (43.5°C for 30, 60 or 90 min; 4 hours after light)
	C26 colon carcinoma	PDT (drug-1 hour-light on day 0)	45
		Infrared light heating (39°–43°C; simultaneously with PDT)
	DS-carcinosarcoma*	PDT (light-10 min-drug on day 0)	46, 47
		Water-filtered infrared-A radiation heating (43°C for 60 min; simultaneous with PDT)
TNF	DS-carcinosarcoma*	TNF (0.2 or 1.0 mg/kg; i.v.; days 5, 8, 11, 14, 17 + 20 after tumour implantation)	64
		Waterbath heating (43° or 44°C for 40 min; 3 hours after each TNF treatment)
	SCK mammary carcinoma	TNF (50 μg/kg; i.p.; day 0)	65
		Waterbath heating (42.5°C for 60 min; 4 hours after TNF)
ATO	SCK mammary carcinoma	ATO (2–8 mg/kg; i.p.; day 0)	59, 63
		Waterbath heating (41.5° or 42.5°C for 60 min; 2 hours after ATO)
	FsaII fibrosarcoma	ATO (2–8 mg/kg; i.p.; day 0)	59, 63
		Waterbath heating (42.5°C for 60 min; 2 or 6 hours after ATO)
VBL	BT4An glioma*	VBL (3 mg/kg; i.p.; day 0)	69
		Waterbath heating (44°C for 60 min; 0 + 3 hours before and 0, 3, 6, 12 + 24 hours after VBL)
FAA	C3H mammary carcinoma	FAA (50–200 mg/kg; i.p.; day 0)	55, 57, 73
		Waterbath heating (40.5°, 41.5° or 42.5°C for various times; from 0–120 hours after FAA)
	B16 melanoma	FAA (200 mg/kg; i.p.; day 0)	56
		Waterbath heating (43°C for 15 min; up to 8 hours before/after FAA)
DMXAA	C3H mammary carcinoma	DMXAA (1–20 mg/kg; i.p.; day 0)	60
		Waterbath heating (40.5°, 41.5° or 42.5°C for various times; from 90 min before to 6 hours after DMXAA)
CA4P	BT4An glioma*	CA4P (50 mg/kg; i.p.; day 0)	58, 62
		Waterbath heating (44°C for 60 min; up to 3 hours before and 24 hours after CA4P)
	C3H mammary carcinoma	CA4P (25–400 mg/kg; i.p.; day 0)	61
		Waterbath heating (40.5°, 41.5° or 42.5°C for various times; from 90 min before to 6 hours after CA4P)

¹The VTAs were TNP-470, batimastat, BMT; photodynamic therapy, PDT; tumour necrosis factor, TNF; arsenic trioxide, ATO; vinblastine, VBL; flavone acetic acid, FAA; 5,6-dimethylxanthenone-4-acetic acid, DMXAA; and combretastatin A-4 disodium phosphate, CA4P. ²Tumour models were either murine or rat*. ³Treatments were started when tumours had reached a specific size (day 0) or at indicated times after implantation. Drugs were injected either s.c. (subcutaneously), i.p. (intraperitoneally), or i.v. (intravenously).

This is not the situation with VDAs and many different agents have been combined with hyperthermia, as shown in Table I. The majority of these studies have involved the combination of photodynamic therapy (PDT) and heat. PDT involves the administration of a photosensitising dye with subsequent activation by visible light [38], [39]. Tumour cell killing by PDT in vivo has been suggested to be the result of both a direct effect on tumour cells [40], [41] as well as a result of damage to tumour vasculature [42–44]. When combined with hyperthermia, different schedules have been used, including simultaneous treatments [45–47], the PDT given immediately before or after the heat [48–50], or with a short 30-minute [51] or longer 4-hour [52] time interval. Of the four studies in which different schedules were compared [48–50], [52], only two found the PDT before heat to be superior [49], [52]. Furthermore, several studies have shown that PDT and heat are an effective combination in vitro, especially when the PDT is administered prior to heating [53], [54]. Thus the role of the vascular damaging component of PDT in the sensitisation of tumours to heat remains unclear.

Hyperthermia has also been combined with the cytokine TNF, and various drugs including ATO, vinblastine, FAA, DMXAA and CA4P (Table I). With all these vascular disrupting drugs there was an enhancement of the heat response and this effect was time and schedule dependent, with the maximum response generally observed if the heating was started 1–6 hours after VDA injection [55–63] and this clearly corresponded to the time of maximal reduction in tumour blood flow in those studies. For the TNF studies [64], [65] the heat was only administered 3–4 hours after injecting the VDA, but at this time the decrease in tumour blood flow was substantial. With vinblastine the decrease in tumour blood flow took almost 12 hours to reach a nadir, yet the maximal enhancement of heat response occurred much earlier [62]. Injecting VDAs after heating has been shown to be substantially less effective at enhancing the heat response [55], [56], [60], [61] and in most cases did not appear to be greater than a simple additive effect of the VDA and heat alone.

Several studies have investigated the temperature dependency for the enhancement of hyperthermia by VDAs. Using CA4P in a C3H mammary carcinoma [61], [71] or ATO in the SCK tumour [59], [63] these enhancements of tumour response were generally independent of heating temperatures between 40.5° to 42.5°C. Moreover, the effects obtained with CA4P were very similar to those found in the same tumour model when heated after injecting hydralazine, a transient physiological modifier of blood flow [17]. Interestingly, although measurements of tumour perfusion after treatment with CA4P [72] or ATO [59] were prolonged when compared to hydralazine [17] they were still somewhat transient with substantial recovery within 24 hours. FAA and DMXAA induce a maximum reduction in perfusion in a C3H mammary carcinoma [57], [72] that is actually maintained for 24 hours or more. With these VDAs the enhanced heat response at 42.5°C is similar to CA4P, but at temperatures below 42.5°C the enhancements are greater [57], [60], [71], [73]. Additional studies with hydralazine demonstrated that the reduction in blood flow leads to both a better tumour heating [17], [66], [67] and an increase in tumour damage [17], [67], [68], the latter effect presumably occurring because the reduction in tumour blood flow results in an increase in tumour hypoxia and associated tumour acidosis, and several in vitro studies have shown that prolonged oxygen deprivation of cells will increase their sensitivity to heat, an effect which is due to hypoxia-induced metabolic perturbations (leading to a decrease in pH) rather than hypoxia per se [11], [12]. With the VDAs there is evidence that similar mechanisms are operating, since studies with CA4P, FAA and DMXAA have shown both an improved tumour heating [56], [69] and a decrease in tumour pH [56], [70]. However, the greater enhancements seen with FAA and DMXAA at mild hyperthermia temperatures suggest that factors unrelated to a simple reduction in tumour blood flow are involved. Exactly what these factors are is not clear, although they may simply reflect increased cell killing as a result of the prolonged reduction in tumour blood perfusion.

Enhancing tumour response to heat is of no therapeutic benefit if the same effect occurs in normal tissues. Unfortunately, this important aspect has not been investigated with AIAs and only to a small degree with VDAs. Studies using rats indicated that the uptake of the photosensitiser was greater in a DS-carcinosarcoma than various normal tissues and this would be expected to reduce the effect of PDT in those normal tissues, as well as any enhancement of heat damage [46]. Indeed, in an additional study in which cortical lesions in rat brains were measured following PDT treatment, absolutely no increase was seen with heat, albeit using a mild temperature of 41°C [74]. Eikesdal and colleagues reported on the incidence of lethality and the development of oedema in heat treated feet of rats given CA4P and 44°C heating [58], [62]. Lethality was not influenced by the combination. A moderate increase in oedema was seen within 1–2 days after treatment, but this had fully recovered by day 5. Results from additional experiments into the development of skin damage in the normal feet of mice [57], [60], [61] demonstrated a significant increase in heat damage by VDAs. Neither DMXAA nor CA4P caused any significant effect on blood perfusion of skin [72], so this cannot explain the enhancement seen. Previous studies with FAA suggested that the increase in skin damage was the result of an FAA-induced increase in the concentration of TNF-α in the circulation [57]. However, there was no apparent correlation between TNF-α production after DMXAA treatment and the enhanced skin response to heating [60]. Furthermore, studies with CA4P failed to detect stimulation of TNF-α production [75], yet it was still capable of enhancing heat response of skin [61]. Although the reasons for the enhanced response of heat in normal skin remain to be elucidated, the most important finding is that in those studies in which skin reactions and tumour response were measured with the same drugs in the same mouse strains [57], [60], [61], then the enhancement of the heat response in skin was always less than in tumours, thus combining VDAs with heat does lead to a therapeutic benefit.

Thermoradiotherapy and VTAs

The use of hyperthermia as a curative treatment of cancer in humans is only possible when it is combined with more conventional therapies [76]. This is especially true for radiation, in which preclinical studies have demonstrated a sound rationale for the combination with heat [77] and significant benefit reported in a number of randomised clinical trials [77]. VTAs have also been shown to be effective at enhancing radiation response in animal tumours [25], so the combination of thermoradiotherapy with VTAs seems logical. Additional support for such a triple combination regime comes from the findings that the tumour vasculature plays an important role in the ability of heat to enhance radiation response [77]. Very few studies have actually investigated the combination of radiation, heat and VTAs [25]. None have been done with AIAs, and using VDAs only a limited number of agents have been used.

When combining VDAs with thermoradiotherapy, timing and sequencing of the drug, radiation and heat treatment will be critical to the response obtained. For radiation and heat the greatest anti-tumour effect is obtained when these treatments are given simultaneously, which is primarily because the heat acts as a radiation sensitiser [77]. However, an identical effect is also seen in normal tissues, thus no therapeutic benefit results. When radiation is administered prior to heating in a sequential schedule an enhancement is observed, but since it only results from the heat killing the hypoxic radiation resistant population the overall anti-tumour effect is less than with the simultaneous schedule. The advantage here is that no enhancement of normal tissue damage occurs, thus therapeutically a sequential schedule is superior. A sequential schedule fits very nicely with the application of the VDAs, because one would want to give the VDA prior to heating so that the heat is applied at a time when vascular shut-down is maximal, but with the VDA administered after the radiation treatment to avoid any possible induction of radiation resistant hypoxia. CA4P [61], FAA [73], and DMXAA [78] have all been combined with radiation and heat using such a sequential schedule and the results obtained with DMXAA in a C3H mammary carcinoma using the most clinically relevant endpoint of local tumour control are illustrated in Figure 2. In these studies, radiation alone resulted in a TCD50 dose (radiation dose necessary to control 50% of tumours) of 53Gy. For hyperthermia alone, one only begins to see any enhancement of radiation response at temperatures above 41.5°C and this effect gets larger as the temperature increases. With the VDA there is an effect of the drug alone on radiation response, but when combined with heat the radiation response is enhanced at temperatures as low as 40.5°C. In fact, at this temperature the enhancement is as good as one finds with 43°C alone. An even greater effect was found with the VDA and heat as the temperature increased to 41.5°C. However, this temperature appears to be optimal for the maximal enhancement, with no further sensitisation at 42.5°C. VDAs have been shown to have absolutely no effect on the thermoradiosensitisation response of normal skin using a temperature of 41.5°C [61], [78], thus the enhancement of tumour response at this temperature is a pure therapeutic benefit.

Figure 2. Effect of DMXAA and heating on the radiation response of a C3H mammary carcinoma. (Top panel) Tumours were locally irradiated with graded radiation doses and the percentage of mice in each treatment group showing local tumour control 90 days after treatment recorded. Mice were given radiation alone (○); local heating (41.5°C; 60 minutes) starting 4-hours after irradiation (•); DMXAA (20 mg/kg) injected intraperitoneally 1-hour after irradiating (△); or treated with radiation, DMXAA and heat (▴). Points are from an average of 13 mice/group. (Bottom panel) TCD50 values (with 95% confidence intervals) determined from data similar to that shown in the top panel for heat alone (○); or DMXAA with heat (•). Data modified from references 61 and 78.

Conclusions and future aspects

The importance of the tumour vasculature makes it an excellent target for therapy. Numerous VTAs have been developed, many of which are in clinical testing, and the list of agents is continually expanding. However, when used as solitary treatments their overall effects on tumour growth are generally considered modest and so for their full potential to be realised they will probably have to be combined with other therapies. In this context, hyperthermia is an excellent choice because of the role that the tumour vasculature also plays in influencing the response to heat treatment.

Numerous preclinical studies have now demonstrated that combining VTAs with heat can be an effective approach for improving tumour response. This effect is clearly dependent on a number of factors including the heat temperature, the VTA dose, and the timing and scheduling between the two therapies. For AIAs the best effect is generally seen if the AIA follows heat, which could be the result of the AIA influencing the repair of cellular damage by heat or preventing angiogenesis occurring after heat-induced vascular damage. Whatever the mechanism the effect is clearly temperature dependent, only being observed at high temperatures, and appears to require that the dose of AIA is sufficient to have anti-tumour activity on its own. With the VDAs, the greatest enhancement is generally seen when the heat is applied at the time of maximal vascular shutdown by the VDA. There is no requirement that the VDA show anti-tumour activity alone and significant enhancements are seen at all hyperthermia temperatures, with the greatest enhancement actually occurring at mild temperatures. Limited normal tissue studies suggest tumour specificity.

Pre-clinical studies have extensively shown that radiation and hyperthermia are an effective combination. Since VTAs work with either heat or radiation, the combination of all three modalities should be beneficial. No such combinations have been investigated with AIAs. A few studies have looked at VDAs with radiation and heat, and substantial improvements in local tumour control reported. This is seen without any influence on normal tissue response, although this is only in early responding skin and clearly needs to be confirmed in more clinically relevant late responding normal tissues. Timing and scheduling are clearly critical issues for maximal benefit and would certainly need to be considered when clinically relevant fractionated schedules are employed. VDA dose and heating temperature do not appear to be so critical.

Clinical testing of a whole range of different VDAs is ongoing [14] and many of these would be suitable candidates for combination with heat and radiation. In fact, several of the VDAs in clinical development are those that have been shown to be the most effective in preclinical studies. Hyperthermia has already undergone extensive clinical evaluation and a number of studies have demonstrated significant improvements in local control and survival when combined with radiation in specific tumour sites [77]. Unfortunately, most studies show little benefit and this is most likely the result of a failure to adequately heat tumours to effective temperatures; while mild hyperthermia temperatures of around 40.5°–41.5°C are easier to obtain clinically, such temperatures are not very efficient at enhancing radiation damage and temperatures of around 42.5°–43°C are required [77]. However, the preclinical data clearly demonstrates that when VDAs are included in the treatment schedule then the tumour response to a temperature of only 40.5°C was as good as that seen with a much higher temperature of 43°C in the absence of any VDA, and a far greater enhancement of radiation damage was seen with the VDAs when the temperature was increased to only 41.5°C. This suggests that if future clinical trials with hyperthermia and radiation were to include a VDA in the treatment regime, we may overcome the problem of ineffective heating of tumours and many more trials should be beneficial.

Acknowledgements

This review was made possible by financial support from the Danish Cancer Society.