ArticlePDF Available

Abstract and Figures

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.
Content may be subject to copyright.
cancers
Review
Hyperthermia: The Optimal Treatment to Overcome
Radiation Resistant Hypoxia
Pernille B. Elming 1, Brita S. Sørensen 1, Arlene L. Oei 2, Nicolaas A.P. Franken 2,
Johannes Crezee 2, Jens Overgaard 1and Michael R. Horsman 1, *
1
Department of Experimental Clinical Oncology, Aarhus University Hospital, DK-8000 Aarhus C, Denmark;
pernille.elming@oncology.au.dk (P.B.E.); bsin@oncology.au.dk (B.S.S.); jens@oncology.au.dk (J.O.)
2Department of Radiation Oncology, Amsterdam University Medical Centers, University of Amsterdam,
1105AZ Amsterdam, The Netherlands; a.l.oei@amc.uva.nl (A.L.O.); n.a.franken@amc.uva.nl (N.A.P.F.);
h.crezee@amc.uva.nl (J.C.)
*Correspondence: mike@oncology.au.dk; Tel.: +45-78462622
Received: 12 November 2018; Accepted: 29 December 2018; Published: 9 January 2019


Abstract:
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.
Keywords: hyperthermia; radiation therapy; hypoxia
1. Introduction
Hypoxia is a hallmark of solid tumors [
1
,
2
]. It occurs because the tumor neo-vasculature that
develops from the host vascular supply via angiogenesis [
3
] is a primitive and chaotic system that is
unable to meet the oxygen demands of the growing tumor mass [
2
,
4
]. As a consequence, cells that are
distant from the blood vessels and at the limit of the diffusion distance of oxygen become chronically
hypoxic [
2
,
4
]. Flow through tumor vessels is also unstable and periodically fluctuates and this can
give rise to transient or acute hypoxia [
2
,
4
]. Regardless of the type of hypoxia, both pre-clinical and
clinical studies show that its presence in tumors is a major factor influencing malignant progression
and response to treatment, especially radiation therapy [
2
,
5
]. These observations have led to extensive
pre-clinical and clinical attempts during the last 5 decades or so to try to specifically target this hypoxia
and thereby improve patient outcome [
6
,
7
]. Examples of the approaches used are listed in Table 1.
They include improving oxygen availability, increasing the radio-sensitivity of the hypoxic cells, killing
the hypoxic population, or modifying the radiation treatment either by increasing the dose to the
hypoxic areas (dose painting) or utilizing radiation of a higher LET (linear energy transfer) in which
the oxygen enhancement ratio (OER) is reduced. It is interesting that hyperthermia (heat treatments of
Cancers 2019,11, 60; doi:10.3390/cancers11010060 www.mdpi.com/journal/cancers
Cancers 2019,11, 60 2 of 20
39–45
C) actually induces many of these effects and as such may have the potential to be one of the
best agents for eliminating hypoxia.
Table 1. Approaches for dealing with hypoxia.
Increasing oxygen delivery
- High oxygen content gas breathing (e.g., hyperbaric oxygen, carbogen)
- Altering hemoglobin (e.g., transfusion, erythropoietin, perflurochemical emulsions)
- Reducing fluctuations in flow (e.g., nicotinamide, pentoxifylline)
- Decreasing oxygen consumption (e.g., metformin, phenformin)
- Increasing blood flow (e.g., hyperthermia)
Radio-sensitizing hypoxic cells
-
Nitro-aromatic sensitizers (e.g., misonidazole, nimorazole, etanidazole, doranidazole)
-Hyperthermia
Preferentially killing hypoxic cells
-Hyperthermia
- Bioreductive drugs (e.g., tirapazamine, banoxantrone, PR-104, evofosfamide)
Vascular targeting therapies
- Angiogenesis inhibitors (e.g., avastin, DC101, tyrosine kinase inhibitors)
- Vascular disruptive drugs (e.g., combretastatin, OXi4503, vademezan, hyperthermia)
Radiation based approaches
- Dose painting
- High LET (linear energy transfer) radiation
The concept of using heat to treat cancer is actually not a new idea. In fact, it is probably one of
the oldest documented treatments, since the Edwin Smith Surgical papyrus dating back to 3000 years
B.C. describes a patient with a tumor in the breast treated with heat in the form of red-hot irons [
8
].
Early Greek (Hippocrates 400 B.C.) and Roman (Galen 200 A.D.) translations similarly recorded the
use of heat treatments [
9
]. After the 17th Century, there were numerous reports of tumor regression in
patients suffering with infectious diseases, which ultimately led to the application of fever-induced
treatment with Coley’s toxin to control tumors at the end of the 19th Century [
9
]. Fever-induced
treatments typically required temperatures of around 40
C for several days. Later studies utilized
primitive external heating techniques to achieve higher temperatures for shorter time-periods [
9
].
What is novel is that today we have new developments in technology (i.e., electromagnetic, ultrasound,
infrared, and nano-technology based techniques) available to us [
10
,
11
]. We can thus select the
appropriate technique for the specific tumor location and size, and with support from thermometry
and quality assurance, we can now apply reproducible and uniform high quality hyperthermia
treatments [
11
13
]. As a result, there have been numerous clinical studies showing the potential of
hyperthermia to improve radiotherapy outcome [
14
17
]. In this review, we will discuss the different
ways in which hyperthermia has been combined with radiation therapy, focusing on why this approach
is selective for hypoxia, and suggest the clinical potential of this therapeutic combination to significantly
improve patient outcome.
2. Combining Hyperthermia with Radiation
Tumor response to the combination of radiation and hyperthermia is dependent on the time
interval between the two modalities, the heating temperature, and the time of heating [
15
,
18
,
19
],
as illustrated in Figure 1. For the C3H mammary carcinoma model, shown in this example, the greatest
enhancement of radiation response by heat occurs when both treatments are given simultaneously
(Figure 1A). However, if any interval is introduced between the two modalities then the enhancement
decreases with this decrease becoming greater as the time interval increases, eventually reaching a
Cancers 2019,11, 60 3 of 20
plateau as shown. A recent clinical study in patients with cervical cancer confirms the superior benefit
of using a short rather than long interval between the radiation and hyperthermia treatments [
20
].
For the example shown in Figure 1A, the decreased response as the time interval increases is
independent of whether heating is applied before or after irradiating, and this seems to be a general
result for other tumor models [15]. Also shown in Figure 1A, is the effect of combining radiation and
heat in a normal tissue. A simultaneous application of radiation with heat results in an enhanced effect
that is identical to that seen in tumors. However, unlike the tumors, the drop-off is steeper when there
is an interval and the final plateau reached is lower. For heat given after irradiating, this drop-off
actually reaches a value where no enhancement occurs, but when heat is applied prior to irradiating,
a residue enhancement remains. Again, the trends seen with the model shown in Figure 1A has been
reported for other normal tissues [15,21].
The heating temperature and time of heating also influence the enhancement (Figure 1B). Generally,
the higher the temperature and the longer the heating period, the greater the enhancement [
15
,
18
,
19
].
This is true regardless of the interval between the two modalities [
15
,
18
,
19
]. Although, as shown in
Figure 1B, the degree of enhancement does depend on whether the radiation and heat are applied
simultaneously or if there is an interval.
Cancers 2019, 11, x 3 of 20
(Figure 1A). However, if any interval is introduced between the two modalities then the enhancement
decreases with this decrease becoming greater as the time interval increases, eventually reaching a
plateau as shown. A recent clinical study in patients with cervical cancer confirms the superior benefit
of using a short rather than long interval between the radiation and hyperthermia treatments [20].
For the example shown in Figure 1A, the decreased response as the time interval increases is
independent of whether heating is applied before or after irradiating, and this seems to be a general
result for other tumor models [15]. Also shown in Figure 1A, is the effect of combining radiation and
heat in a normal tissue. A simultaneous application of radiation with heat results in an enhanced
effect that is identical to that seen in tumors. However, unlike the tumors, the drop-off is steeper
when there is an interval and the final plateau reached is lower. For heat given after irradiating, this
drop-off actually reaches a value where no enhancement occurs, but when heat is applied prior to
irradiating, a residue enhancement remains. Again, the trends seen with the model shown in Figure
1A has been reported for other normal tissues [15,21].
The heating temperature and time of heating also influence the enhancement (Figure 1B).
Generally, the higher the temperature and the longer the heating period, the greater the enhancement
[15,18,19]. This is true regardless of the interval between the two modalities [15,18,19]. Although, as
shown in Figure 1B, the degree of enhancement does depend on whether the radiation and heat are
applied simultaneously or if there is an interval.
Figure 1. (A) Influence of time interval and sequence between radiation and hyperthermia (42.5 °C;
60 min) on tumor control in C3H mammary carcinomas () or moist desquamation in normal skin (). (B)
Effect of heating time and temperature on the thermal enhancement ratio (TER) for tumor control in
a C3H mammary carcinoma when radiation and hyperthermia were given either simultaneously
(solid symbols) or tumors irradiated and then heated 4 h later (open symbols); the heat temperatures
are indicated. For both figures the TERs were determined from full radiation dose-response curves
and represent the ratio of the radiation dose for radiation alone to that for radiation + heat to produce
a response in 50% of animals. (Modified from [18,19]).
Figure 1.
(
A
) Influence of time interval and sequence between radiation and hyperthermia (42.5
C;
60 min) on tumor control in C3H mammary carcinomas ( ) or moist desquamation in normal skin (
#
).
(
B
) Effect of heating time and temperature on the thermal enhancement ratio (TER) for tumor control
in a C3H mammary carcinoma when radiation and hyperthermia were given either simultaneously
(solid symbols) or tumors irradiated and then heated 4 h later (open symbols); the heat temperatures
are indicated. For both figures the TERs were determined from full radiation dose-response curves and
represent the ratio of the radiation dose for radiation alone to that for radiation + heat to produce a
response in 50% of animals. (Modified from [18,19]).
Cancers 2019,11, 60 4 of 20
3. Radio-Sensitization by Hyperthermia
The consensus opinion is that the enhancement of radiation response by hyperthermia, when both
treatments administered simultaneously, reflects radio-sensitization. This decreases as the interval
between the two modalities increases ultimately disappearing when the interval is long enough
(around 4 h in Figure 1A). The remaining enhancement seen as the plateau in Figure 1A is simply
the result of heat killing hypoxic cells. Since the effects of a simultaneous treatment occurs in both
the tumor and normal tissue, it would suggest that sensitization does not involve hypoxia. However,
the normal tissue shown in Figure 1A is skin and that is known to be somewhat hypoxic [
22
,
23
] and
found to have an increased radiation sensitivity when treated with classical nitro-aromatic hypoxic
cell sensitizers [
24
]. Similar peak enhancements have been reported by others in skin and other normal
tissues in which hypoxia is even absent [
15
,
21
], although in those studies no comparison was made
with tumor response under similar conditions, so it is impossible to state whether the peak normal
tissue response was equivalent to that for tumors or actually lower. What is interesting is that the
radio-sensitization seen with hyperthermia administered in the temperature range of 40.5–42.5
C at
the same time as irradiating is as good as, or even better than, that found using nitro-aromatic drugs
that specifically sensitize hypoxic cells to radiation (Figure 2). However, unlike the nitro-aromatic
radio-sensitizers in which there is only a small effect in normal tissues [
24
] with hyperthermia one sees
exactly the same effect in normal tissues and tumors [
15
,
18
,
19
], thus there is no therapeutic benefit for
sensitization unless the heat and radiation treatments specifically target the tumor.
Cancers 2019, 11, x 4 of 20
3. Radio-sensitization by Hyperthermia
The consensus opinion is that the enhancement of radiation response by hyperthermia, when
both treatments administered simultaneously, reflects radio-sensitization. This decreases as the
interval between the two modalities increases ultimately disappearing when the interval is long
enough (around 4 h in Figure 1A). The remaining enhancement seen as the plateau in Figure 1A is
simply the result of heat killing hypoxic cells. Since the effects of a simultaneous treatment occurs in
both the tumor and normal tissue, it would suggest that sensitization does not involve hypoxia.
However, the normal tissue shown in Figure 1A is skin and that is known to be somewhat hypoxic
[22,23] and found to have an increased radiation sensitivity when treated with classical nitro-aromatic
hypoxic cell sensitizers [24]. Similar peak enhancements have been reported by others in skin and
other normal tissues in which hypoxia is even absent [15,21], although in those studies no comparison
was made with tumor response under similar conditions, so it is impossible to state whether the peak
normal tissue response was equivalent to that for tumors or actually lower. What is interesting is that
the radio-sensitization seen with hyperthermia administered in the temperature range of 40.5–42.5 °C
at the same time as irradiating is as good as, or even better than, that found using nitro-aromatic
drugs that specifically sensitize hypoxic cells to radiation (Figure 2). However, unlike the nitro-aromatic
radio-sensitizers in which there is only a small effect in normal tissues [24] with hyperthermia one
sees exactly the same effect in normal tissues and tumors [15,18,19], thus there is no therapeutic
benefit for sensitization unless the heat and radiation treatments specifically target the tumor.
Figure 2. The radio-sensitizing effect of nitro-aromatic drugs and hyperthermia in a C3H mammary
carcinoma. The sensitizer enhancement ratios (SERs) were calculated from full radiation dose-
response curves of tumor control and represent the ratio of the radiation dose for radiation alone to
that for radiation + sensitizer to produce a response in 50% of animals. The drug treatments were
misonidazole (), nimorazole (), and doranidazole (), with different drug doses administered as a
single intraperitoneal (misonidazole and nimorazole) or intravenous (doranidazole) injection 30 min
prior to irradiating (Modified from [24]). The dashed lines represent the SER levels when tumors were
irradiated in the middle of a 60-min heating period at the indicated temperatures and are taken from
Figure 1B.
Interestingly, the enhancement of radiation response by hyperthermia can be further increased
by simply combining the radiation and heat treatment with the radio-sensitizer misonidazole [25–27].
This additional benefit was seen with both a simultaneous and sequential radiation and heat
treatment, and increased with temperature and drug dose [27]. Misonidazole specifically targets
hypoxic cells [5,6] and if we assume that this is also true for hyperthermia, then it suggests that they
may be acting on different hypoxic subpopulations. In fact, one study clearly showed that combining
nicotinamide with hyperthermia was an effective approach to improve radiation response by
Figure 2.
The radio-sensitizing effect of nitro-aromatic drugs and hyperthermia in a C3H mammary
carcinoma. The sensitizer enhancement ratios (SERs) were calculated from full radiation dose-response
curves of tumor control and represent the ratio of the radiation dose for radiation alone to that
for radiation + sensitizer to produce a response in 50% of animals. The drug treatments were
misonidazole (
#
), nimorazole (
), and doranidazole (
4
), with different drug doses administered
as a single intraperitoneal (misonidazole and nimorazole) or intravenous (doranidazole) injection
30 min prior to irradiating (Modified from [
24
]). The dashed lines represent the SER levels when
tumors were irradiated in the middle of a 60-min heating period at the indicated temperatures and are
taken from Figure 1B.
Interestingly, the enhancement of radiation response by hyperthermia can be further increased
by simply combining the radiation and heat treatment with the radio-sensitizer misonidazole [
25
27
].
This additional benefit was seen with both a simultaneous and sequential radiation and heat treatment,
and increased with temperature and drug dose [
27
]. Misonidazole specifically targets hypoxic cells [
5
,
6
]
and if we assume that this is also true for hyperthermia, then it suggests that they may be acting on
different hypoxic subpopulations. In fact, one study clearly showed that combining nicotinamide with
hyperthermia was an effective approach to improve radiation response by reducing hypoxia [
28
] and
Cancers 2019,11, 60 5 of 20
that since nicotinamide only affected acute hypoxia the additional benefit of heat must have been
through a reduction in chronic hypoxia.
Two different mechanisms are believed to account for the radio-sensitization by hyperthermia.
The first involves an improvement in oxygen delivery to the tissue. Over two decades ago, it was
proposed that the clinical benefit of hyperthermia was the result of mild hyperthermia temperatures
(less than 42
C) improving radiation response by increasing tumor oxygenation status, resulting in a
corresponding decrease in radiation resistant hypoxia [
29
]. Pre-clinical data clearly shows that mild
heat temperatures can improve tumor oxygenation [
30
34
] and this is most likely the result of changes
in tumor blood flow [
32
35
], perhaps mediated through a decrease in interstitial fluid pressure [
33
,
34
]
causing an increase in perfused vessels [
34
,
36
]. Interestingly, the improvement in tumor oxygenation
and radiation response observed with mild temperature hyperthermia, could be further increased if
animals were allowed to breathe carbogen gas immediately after heating and during the radiation
period [
37
]. Studies have generally shown that at mild heat temperatures tumor blood flow and
oxygenation status temporarily increases during the heating period but returns to normal values
afterwards [
31
,
32
,
35
,
38
]. However, the radio-sensitization by heat increases with temperature and
while temperatures above 42
C might produce a very transient increase in oxygenation during the
heating period, immediately after heating there is a rapid induction of vascular damage that would
be expected to significantly increase the level of hypoxia and thus radio-protect tumors [
32
,
35
,
38
].
Cellular oxygen utilization may play a role here. This actually increases at low temperatures but
decreases at high temperatures [
32
]. However, the increase at low temperatures of around 41
C is
only transient and with time will actually drop [
39
]. Any decrease in oxygen consumption, if it occurs
in tumors, can actually increase the diffusion distance of oxygen, thus improving oxygen availability
to hypoxic regions and so decreasing the level of hypoxia [
40
,
41
], even at the high temperatures.
The situation is made even more complicated by studies from one group reporting improved tumor
oxygenation that lasted for 1–2 days after heating at mild temperatures [
30
,
42
45
], an effect that is
difficult to explain. Clinical studies, in which the oxygenation status of soft tissue sarcomas [
46
] or
locally advanced breast cancer [
47
] was measured with oxygen electrodes after heating, also seemed to
indicate that mild temperature hyperthermia improved tumor oxygenation. However, in the sarcoma
study the improvement in oxygenation correlated with the degree of necrosis found in resected
specimens [
46
], suggesting that the apparent oxygenation effects simply reflected the damage caused
by higher temperatures. Indeed, an additional pre-clinical study in which tumors treated with high
thermal temperatures, known to induce physiological changes that cause a significant decrease in
tumor perfusion and oxygenation [
1
], resulted in substantial tumor control [
48
] while actually reporting
apparent improvements in tumor oxygenation 1–2 days after applying the heat. This suggests that
the apparent improvements in oxygenation were not the result of improved oxygen delivery. Clearly,
the role of heat-induced effects of tumor oxygenation status accounting for radio-sensitization is
somewhat controversial and unclear. What is clear from rapid-mix studies is that the improved oxygen
levels are only beneficial if they are present at the time of irradiation or within a few milliseconds after
irradiating [
49
,
50
]. Yet the enhancement of radiation by heat is the same whether the heat is applied
long before or after irradiating (Figure 1).
An alternative explanation for radio-sensitization by hyperthermia involves potential effects
on radiation-induced DNA damage repair. Ionizing radiation causes different DNA lesions that
include base damage, single strand breaks (SSBs) and double strand breaks (DSBs), the latter being
produced either directly by an ionizing event or indirectly when SSBs are produced close to each
other on both DNA strands [
51
] or indirectly when problems occur during DNA replication [
52
,
53
].
DNA DSBs are potentially the most toxic DNA lesions to cancer cells, repairable by two major pathways,
non-homologous end joining (NHEJ) or homologous recombination (HR) [
54
,
55
]. NHEJ rejoins the
broken ends of the DNA without the need for homology or a repair template and is active through the
cell cycle [
56
,
57
], whereas HR does require a template thus is only active during the S and G2 phases of
the cell cycle [
56
,
58
]. When a DSB occurs, kinases initially recognize the DNA break, and accumulate
Cancers 2019,11, 60 6 of 20
together with other kinases around the location of the break and attract other DNA repair proteins to
repair the break [
58
]. During this process, histone H2AX (
γ
-H2AX) is phosphorylated; which explains
its use as a marker for the induction and repair of DSBs [
59
]. Many studies demonstrated increased
levels of
γ
-H2AX after combined treatment with ionizing radiation and mild hyperthermia as compared
with ionizing radiation alone at 24 h after treatment, indicating that the number of residual DSBs was
increased [
60
,
61
]. Moreover, after heat treatment, decreased levels are reported of BP53 and Rad51;
these proteins are involved in recruiting other repair proteins of NHEJ and HR to the DNA break
ends, suggesting that hyperthermia interferes with both DNA repair mechanisms [
62
]. How the DNA
repair pathways are affected is only partially known. Multiple groups reported that hyperthermia
affects NHEJ pathway-specific proteins. Hyperthermia is suggested to affect NHEJ by heat-mediated
inactivation of Ku, decreased activity of DNA-PK and decreased levels of KU70, KU80, and Ligase
IV [
62
]. Hyperthermia has also been found to temporarily degrade the BRCA2 protein and reduce
BRCA1, thereby inhibiting the homologous recombination DNA repair pathway [
63
]. Interestingly,
as a result of the poor supply of oxygen and nutrients, hypoxic cells often exist in a quiescent state [
64
];
these quiescent cells are less sensitive to ionizing radiation because they have the time to repair the
DNA properly, as well as the known resistance due to the lower level of oxygenation. Hyperthermia
can push cells out of this quiescent state and make them more susceptible to ionizing radiation [65].
4. Hyperthermia as a Cytotoxic Agent
Hyperthermia can also kill hypoxic cells either directly or indirectly. Direct cell killing is strongly
dependent on the heating temperature and the time of heating; the higher the temperature and the
longer the heating period, the greater the effect [6668]. Typically, temperatures below 42 C actually
have little effect on cell killing, unless long exposure times are utilized [
66
68
], certainly longer
than a typical heating period of 1 h when combined with radiation [
14
17
]. The killing seen with
temperatures above 42
C increases significantly if the cells are maintained under conditions of oxygen
deprivation and/or low pH [
69
,
70
]. Such adverse micro-environmental conditions are those typically
found in hypoxic tumor regions [
1
,
2
] and the ability of heat to actually kill hypoxic cells in tumors has
been demonstrated [
28
,
71
]. The Overgaard study actually suggested that this heat-killing effect was
primarily in chronically hypoxic rather than acutely hypoxic cells [
71
]. This seemed to be confirmed in
the other study [
28
] and is supported by
in vitro
data, since long periods of hypoxic exposure were
necessary to obtain cell killing [
69
,
70
]. In tumors, it is also the chronically hypoxic cells, rather than
acutely hypoxic, that will be more likely associated with nutrient deprived conditions that also give
rise to heat sensitive low pH. Preferential killing of radio-resistant hypoxic cells probably explains the
plateau effect seen when heat and radiation are separated by greater than 4 h (Figure 1A). Analysis of
the time-temperature cell survival curves also suggest that the slopes of the curves were very different
above or below 42.5
C [
66
68
], either reflecting different cell killing mechanisms or that with the
longer heating times the cells developed resistance to heat, often referred to as thermo-tolerance [
72
].
Interestingly, the time-temperature response curves for tumors
in vivo
also shows different slopes
above and below 42.5
C [
73
] suggesting similar thermo-tolerance mechanisms as
in vitro
. However,
additional studies suggest that thermo-tolerance
in vivo
may also be vascular mediated resulting from
the induction of vessel normalization [
74
], a process that involves a decrease in micro-vessel density
and increase in pericyte coverage [75], and causes a decrease in tumor hypoxia [76].
Heat kills cells by a variety of mechanisms, including necrosis, apoptosis and modes related to
mitotic catastrophe [
77
80
]. The biological effects of heating cells include chromosomal aberrations,
mitotic dysfunction, cytoskeletal damage, changes in membrane fluidity and transport, and metabolic
changes [
81
]. But the most likely rate limiting step for killing by hyperthermia is protein denaturation
since this in a similar time-temperature relationship as for cell killing, especially at temperatures of
42.5
C and above, although some effects do occur with long heating times at lower temperatures [
82
].
At temperatures around 43
C and below apoptosis appears to predominate with necrosis seen at
higher temperatures [
77
,
80
], but whether these effects are mediated via denaturation of proteins
Cancers 2019,11, 60 7 of 20
associated with the cytosol, membrane, or nucleus, is unclear [
77
,
79
]. However, the fact that cell killing
is substantially increased if cells are heated under low pH conditions [
69
,
70
] would seem to support the
cell membrane as the primary target. Measurement of intracellular pH (pHi) and extracellular pH (pHe)
show that cells can maintain a neutral pHi even when pHe is acidic [
1
]). Increasing extracellular acidity
would put more stress on the membrane pumps responsible for maintaining neutral intracellular pH
and thus be more susceptible to heat damage.
Whatever the mechanism, one can significantly increase heat killing
in vivo
using agents that
decrease tumor blood flow and, thus, increase the adverse environmental conditions, especially
hypoxia, within tumors. This has been achieved using physiological modifiers, such as hydralazine,
sodium nitroprusside, or glucose [
83
]; the effects are often transient and hard to predict, yet have
been shown to enhance tumor response to heat [83]. More consistent, longer-maintained changes are
seen with so-called vascular disrupting agents (VDAs) that damage the established tumor vascular
supply [
83
85
]. Many of these VDAs, including tumor necrosis factor, chemotherapeutic drugs (e.g.,
arsenic trioxide and vinblastine), flavonoid compounds (e.g., flavone acetic acid and vandremycin)
and tubulin-binding agents (e.g., combretastatin and its analog OXi4503), have been combined with
hyperthermia to enhance the anti-tumor response [
85
]. Additional benefits were observed when VDAs
and hyperthermia were combined with radiation [
15
,
85
]. Moreover, such combinations were extremely
effective when using mild temperature heat treatments; the radiation induced tumor control reported
after systemic treatment of mice with VDAs and local tumor heating at 41.5 C was as good as, if not
better than, that seen with 43 C alone [15,85].
Hyperthermia itself can also induce vascular damage and, as a result, will kill tumor cells indirectly.
Although higher heat temperatures may transiently increase tumor blood flow during the heating
period, immediately after the cessation of heating a rapid decrease in tumor blood flow is seen that
is often prolonged (Figure 3), although the effects are tumor and temperature dependent. This rapid
and prolonged vascular collapse following heating is similar to that seen after treatment with the
drug based VDAs [
84
,
85
]. For such drugs, the target is the vascular endothelial cells [
86
], which when
damaged undergo a rapid shape change and eventually undergo apoptosis [
86
]. The initial effects will
cause an increase in vessel permeability and as fluid leaks out of the tumor vessels it will cause a rise
in interstitial fluid pressure that then collapses the vessels [
86
]. This is likely to be transient, but would
initiate a number of other effects such as reduced blood flow, increased blood viscosity and red cell
stacking, and these would cause coagulation and thus responsible for the prolonged effect. The result
of the vascular shutdown will deprive tumor cells downstream of the blockage of essential oxygen
and nutrients, resulting in rapid and widespread tumor necrosis and ischemia [
84
,
87
]. The cells that
die first are likely to be those that already exist under deprived conditions, especially hypoxic cells.
Interestingly, despite the massive necrosis induced by VDAs their potential as stand-alone therapeutic
agents is limited and for their full clinical potential to be realized they need to be combined with
more conventional therapies, especially radiotherapy [
84
,
87
]. This is exactly the same situation with
hyperthermia and is another strong argument for combining heat and radiation [15].
Cancers 2019,11, 60 8 of 20
Cancers 2019, 11, x 8 of 20
Figure 3. Effect of heating on perfusion in RIF-1 fibrosarcomas (A) or C3H mammary carcinomas (B).
Tumors were heated for 1 h (shown by the black bars) at the indicated temperatures and blood
perfusion in the tumors measured at different times before, during, or after heating by intravenously
injecting radioactive rubidium chloride; tumors were excised 90–120 min later and tracer uptake
measured on a gamma counter. Points are means (±1 S.E.) with the pre-treatment control values
shown by the open symbols (Modified from [38,88].
5. Hyperthermia and Alternative Radiation Approaches
The only category in Table 1 where hyperthermia is not listed is the use of novel radiation-based
approaches to overcome hypoxia. These involve either increasing the radiation dose to the hypoxic
areas identified from imaging analysis (dose painting) or utilizing high LET radiation (i.e., carbon
ions) leading to a lower OER. Dose painting approaches [89] have generally been limited because
they typically involve positron emission tomography (PET) based imaging technology that fails to
represent the hypoxic distributions within tumors [4,7], thus non-hypoxic areas could receive a higher
dose of radiation, while regions with hypoxia are missed. However, a recent pre-clinical study [90]
suggests that oxygen images obtained using electron paramagnetic resonance could identify hypoxic
tumor regions to which radiation was boosted to improve local tumor response. On the other hand,
effective application of hyperthermia should target all hypoxic cells regardless of where they are
located, thus making dose painting redundant.
There is clear pre-clinical evidence that as LET increases the OER decreases [91,92]. At
sufficiently high LET, as seen with heavy ions such as carbon ions, the OER is extremely low so
hypoxia becomes less of an issue, but in a clinically obtainable LET range hypoxia is not entirely
eliminated. The advantage of using heavy ions is the improved dose distribution to the tumor thus
reducing normal tissue complications. Unfortunately, there are currently only 11 heavy ion facilities
in the world (almost half located in Japan) [93]. Interestingly, proton facilities are for more common
(there are currently some 70 facilities operational around the world with other facilities in
development) [93] and even though protons have a LET that is significantly lower than carbon ions,
thus hypoxia is still a significant problem, protons and carbon ions actually share similar physical
advantages. It has been suggested that the combination of hyperthermia and protons may mimic
carbon ion therapy [94] and as a result a clinical trial (HYPOSAR) of hyperthermia and protons in
sarcoma patients is already underway [94]. This trial will involve applying heat temperatures of 41.5–
42.5 °C for 60 min some 90–150 min prior to the first of five daily irradiations given each week over
a seven-week period. However, this is a new concept based on theory and limited in vitro cell survival
data [95], so whether the planned tumor temperatures and time interval will be as effective as carbon
Figure 3.
Effect of heating on perfusion in RIF-1 fibrosarcomas (
A
) or C3H mammary carcinomas (
B
).
Tumors were heated for 1 h (shown by the black bars) at the indicated temperatures and blood perfusion
in the tumors measured at different times before, during, or after heating by intravenously injecting
radioactive rubidium chloride; tumors were excised 90–120 min later and tracer uptake measured on a
gamma counter. Points are means (
±
1 S.E.) with the pre-treatment control values shown by the open
symbols (Modified from [38,88]).
5. Hyperthermia and Alternative Radiation Approaches
The only category in Table 1where hyperthermia is not listed is the use of novel radiation-based
approaches to overcome hypoxia. These involve either increasing the radiation dose to the hypoxic
areas identified from imaging analysis (dose painting) or utilizing high LET radiation (i.e., carbon
ions) leading to a lower OER. Dose painting approaches [
89
] have generally been limited because
they typically involve positron emission tomography (PET) based imaging technology that fails to
represent the hypoxic distributions within tumors [
4
,
7
], thus non-hypoxic areas could receive a higher
dose of radiation, while regions with hypoxia are missed. However, a recent pre-clinical study [
90
]
suggests that oxygen images obtained using electron paramagnetic resonance could identify hypoxic
tumor regions to which radiation was boosted to improve local tumor response. On the other hand,
effective application of hyperthermia should target all hypoxic cells regardless of where they are
located, thus making dose painting redundant.
There is clear pre-clinical evidence that as LET increases the OER decreases [
91
,
92
]. At sufficiently
high LET, as seen with heavy ions such as carbon ions, the OER is extremely low so hypoxia
becomes less of an issue, but in a clinically obtainable LET range hypoxia is not entirely eliminated.
The advantage of using heavy ions is the improved dose distribution to the tumor thus reducing normal
tissue complications. Unfortunately, there are currently only 11 heavy ion facilities in the world (almost
half located in Japan) [
93
]. Interestingly, proton facilities are for more common (there are currently
some 70 facilities operational around the world with other facilities in development) [
93
] and even
though protons have a LET that is significantly lower than carbon ions, thus hypoxia is still a significant
problem, protons and carbon ions actually share similar physical advantages. It has been suggested
that the combination of hyperthermia and protons may mimic carbon ion therapy [
94
] and as a result a
clinical trial (HYPOSAR) of hyperthermia and protons in sarcoma patients is already underway [
94
].
This trial will involve applying heat temperatures of 41.5–42.5
C for 60 min some 90–150 min prior to
the first of five daily irradiations given each week over a seven-week period. However, this is a new
concept based on theory and limited
in vitro
cell survival data [
95
], so whether the planned tumor
Cancers 2019,11, 60 9 of 20
temperatures and time interval will be as effective as carbon ions is unclear. Clearly, detailed
in vivo
studies looking at the combination of hyperthermia and protons are required, but studies with photons
and hyperthermia may give some idea of the potential success of this approach. From single dose
studies in murine tumors, we know that carbon ions are 1.4–2.4 times more effective than photons [
96
].
Similar enhancement ratios are shown in Figure 1A with heating at 42.5
C for 60 min at time intervals
ranging from 0 to 240 min, and has also been seen with other tumor models [
15
], supporting the use of
this temperature and proposed time intervals in the clinical study. At a lower temperature of 41.5
C,
reduced enhancement ratios have been reported [
97
], decreasing from 1.7 with a simultaneous heat
and radiation treatment to 1.2 with an interval of only 120 min. This suggests that for the selected time
intervals between heating and irradiating, the lower temperature of 41.5
C may be close to the limit
of benefit. However, since protons have an increased relative biological effectiveness (RBE) compared
to photons [
98
], with a RBE of 1.1 currently generically applied for clinical use, the effect of protons at
41.5
C may actually be somewhat higher than predicted from photon studies. Obviously, pre-clinical
testing of protons and hyperthermia, using a range of temperatures and time intervals is required to
support the planned clinical studies.
Interestingly, there appear to be radiobiological differences between photon and proton
irradiation [
99
,
100
]. Although the initial number of DNA damage foci increases with LET [
101
,
102
],
the actual number are comparable between photons and protons when irradiating with protons
using therapeutic beams with relatively low LET [
103
,
104
]. What has been found to be different
is the residual number of unrepaired DSBs [
102
,
104
106
] suggesting that the repair processes are
different following photon and proton irradiation, which may be part of the RBE of 1.1 for proton
irradiation. It has been suggested that DSBs induced by high LET radiation, are preferentially repaired
by HR [
107
,
108
], possibly because the short DNA fragments induced by the high-LET clustered DNA
damage are unable to bind the Ku heterodimer. Several studies suggest this may also be the situation
with protons [
103
,
109
,
110
], although data from at least one study has indicated that NHEJ also plays
an important role in repairing DSBs induced by protons [
108
]. Interestingly, an
in vitro
study using
cells with normal repair capacity, or deficient in either NHEJ or HR, investigated the effect of heating
(42.5
C for 1 h) immediately after irradiating with X-rays or protons [
111
]. The authors reported an
enhancement of radiation-induced cell killing in the normal or NHEJ deficient cells, but not in the
HR deficient, thus if HR is the principal repair mechanism after proton irradiation it adds further
support to the potential combination of heat and proton irradiation. The authors also showed the
same effect for carbon ions. This taken together with other studies showing the benefit of combining
hyperthermia with high LET radiation [
112
115
], and the fact that hypoxia is less of an issue with high
LET radiation [
91
,
92
], suggest that the combination of high LET and hyperthermia may be the ultimate
approach for totally eliminating tumor hypoxia.
6. Clinical Relevance
Hyperthermia has been combined with radiation in a large number of clinical trials [
14
17
].
A meta-analysis of trials in which the patients were randomized to receive radiation alone or radiation
and heat is summarized in Figure 4[
116
129
]. Most of the studies involved conventional fractionated
radiation therapy although one study with cervix cancer patients applied brachytherapy [
119
].
The planned heat treatment was typically 42–43
C, for a period of 30–60 min, although in the
brachytherapy study the aim was for a slightly lower temperature of around 41
C [
119
]. Hyperthermia
was applied either once or twice weekly after radiotherapy in all the studies, but with a time interval
between the radiation and heat that varied from a few minutes up to 4 h. The endpoint for these
studies was complete response/local tumor control, which is understandable because the heat and
radiation were applied locally to the primary tumor, with sometimes a few local lymph nodes being
treated. Some of the studies also reported survival data [
117
119
,
122
,
123
] but the significance of this
when using local treatments is questionable. When considering tumor site, the analysis showed a
significant improvement in local tumor control when hyperthermia was combined with radiation for
Cancers 2019,11, 60 10 of 20
all sites except lung. This lung study was an International Atomic Energy Agency trial in non-small
cell lung cancer patients [
124
] and the failure to show any benefit may simply reflect poor quality
control resulting from poor communication infrastructure in developing countries rather than any
biological basis. Even including the results of this trial in the overall analysis, there was still a highly
significant better response for radiation and heat compared to radiation alone.
Cancers 2019, 11, x 10 of 20
rather than any biological basis. Even including the results of this trial in the overall analysis, there
was still a highly significant better response for radiation and heat compared to radiation alone.
Figure 4. Meta-analysis of all trials in which patients were randomized to receive radiation alone
(RAD) or radiation + hyperthermia (RAD + HEAT). The endpoint in each trial was complete response
(CR) measured by loco-regional control and shows the calculated Odds ratio with 95% confidence
intervals (95% CI). Data from [116–129] and observations from Overgaard, J. [130].
Today, patients typically receive chemotherapy with radiation as part of the standard treatment,
so one could question the validity of combining heat and radiation. Several studies have actually
shown that including hyperthermia in the radiation/chemotherapy schedule can improve outcome
[14,16]. Interestingly, a recent study in which patients with locally advanced cervical cancer were
randomized to radiotherapy + hyperthermia or radiotherapy + cisplatin, reported comparable
outcome and toxicity between the two treatment arms [131], suggesting hyperthermia might have a
role to play as an alternative treatment if chemotherapy tolerance issues arise. Furthermore, hypoxia
is often a source of resistance to many clinically used chemotherapeutic agents. This can be a
consequence of hypoxia per se, but could also be due to hypoxic cells being distant from blood
vessels; thus, creating a drug delivery problem, or because hypoxic cells are generally non-cycling
and exist at low pH, both of which can influence drug activity [2,5]. Thus, including hyperthermia in
a treatment schedule involving radiation and chemotherapy would seem to be a logical choice.
7. Future Perspectives
The future of hyperthermia involves technical developments and improved approaches for
applying current methodology that allows for better and homogeneous tumor heating or being able
to reduce the interval between the heat and radiation to the point where a truly simultaneous
radiation and heat treatment occur. However, there are a number of biological issues, which have
recently become hot topics, in which hypoxia and hyperthermia may play critical roles [132]. The first
of these involves immune response. Numerous reviews have addressed the issue of immune
modulatory effects of hyperthermia [133–135] and different mechanisms have been suggested that
involve both the innate and adaptive immune system. When heat is applied to tumor cells they
respond by producing heat shock proteins (HSPs) which when become extracellular act as danger
signals for the adaptive and innate immune systems [136]. These released HSPs activate NK cells and
antigen presenting cells (APCs) and thereby increase the cytotoxic T-cell response [135]. Heat itself
can also cause cellular damage and this will lead to the release of damage-associated molecular
patterns (DAMPs) which basically has the same effect as the HSPs [133]. There is evidence that
immune cells, such as NK cells, CD8+ T-cells, and dendritic cells already in the tumor also become
activated when heated [135]. Finally, immune cell trafficking into the tumor can be improved as a
result of the vascular effects of heating [134]. More recent studies have shown that the combination
Figure 4.
Meta-analysis of all trials in which patients were randomized to receive radiation alone (RAD)
or radiation + hyperthermia (RAD + HEAT). The endpoint in each trial was complete response (CR)
measured by loco-regional control and shows the calculated Odds ratio with 95% confidence intervals
(95% CI). Data from [116129] and observations from Overgaard, J. [130].
Today, patients typically receive chemotherapy with radiation as part of the standard treatment, so
one could question the validity of combining heat and radiation. Several studies have actually shown
that including hyperthermia in the radiation/chemotherapy schedule can improve outcome [
14
,
16
].
Interestingly, a recent study in which patients with locally advanced cervical cancer were randomized
to radiotherapy + hyperthermia or radiotherapy + cisplatin, reported comparable outcome and toxicity
between the two treatment arms [
131
], suggesting hyperthermia might have a role to play as an
alternative treatment if chemotherapy tolerance issues arise. Furthermore, hypoxia is often a source of
resistance to many clinically used chemotherapeutic agents. This can be a consequence of hypoxia
per se, but could also be due to hypoxic cells being distant from blood vessels; thus, creating a drug
delivery problem, or because hypoxic cells are generally non-cycling and exist at low pH, both of which
can influence drug activity [
2
,
5
]. Thus, including hyperthermia in a treatment schedule involving
radiation and chemotherapy would seem to be a logical choice.
7. Future Perspectives
The future of hyperthermia involves technical developments and improved approaches for
applying current methodology that allows for better and homogeneous tumor heating or being able to
reduce the interval between the heat and radiation to the point where a truly simultaneous radiation
and heat treatment occur. However, there are a number of biological issues, which have recently
become hot topics, in which hypoxia and hyperthermia may play critical roles [
132
]. The first of these
involves immune response. Numerous reviews have addressed the issue of immune modulatory
effects of hyperthermia [
133
135
] and different mechanisms have been suggested that involve both the
innate and adaptive immune system. When heat is applied to tumor cells they respond by producing
heat shock proteins (HSPs) which when become extracellular act as danger signals for the adaptive
and innate immune systems [
136
]. These released HSPs activate NK cells and antigen presenting cells
(APCs) and thereby increase the cytotoxic T-cell response [
135
]. Heat itself can also cause cellular
damage and this will lead to the release of damage-associated molecular patterns (DAMPs) which
Cancers 2019,11, 60 11 of 20
basically has the same effect as the HSPs [
133
]. There is evidence that immune cells, such as NK
cells, CD8+ T-cells, and dendritic cells already in the tumor also become activated when heated [
135
].
Finally, immune cell trafficking into the tumor can be improved as a result of the vascular effects of
heating [
134
]. More recent studies have shown that the combination of hyperthermia and radiation
can further enhance the immune response, probably by the induction of a greater proportion of
immunogenic cell death in the tumor [
137
,
138
]. The significance of hypoxia in this issue comes
from the finding that hypoxia in tumors can have a negative effect on immunogenicity by altering
the function of immune cells and/or increasing resistance of tumor cells to the cytolytic activity of
immune effectors [
139
,
140
]. The elimination of hypoxia by hyperthermia adds to its already established
immune modulatory effects. These effects on immune response will not only impact the primary tumor,
but should also induce an abscopal effect. Indeed, anti-tumor activity in contralateral tumors that
are not actually heated has been reported [
141
,
142
], but whether the same applies to truly metastatic
disease is not known. Clearly, additional studies into the role of hypoxia in the immune response and
its targeting by hyperthermia should be undertaken. Furthermore, these studies should be extended to
consider what happens when heat and radiation are combined.
Successful cancer therapy requires targeting both the primary tumor and metastases. The cells
within a tumor generally consist of non-stem cells that have a limited proliferative capacity but
constitute the bulk of the cancer cells and a subset of cancer stem cells (CSCs) that can both expand
the CSC population and differentiate into the various tumor cell populations [
143
,
144
]. CSCs are
not only important for the growth of the primary tumor, they also play a major role in influencing
metastatic spread [
145
,
146
]. Evidence exists that such CSCs are also an important factor in influencing
response to treatment [
144
,
146
]. This is especially true for radiation in which studies have shown that
the higher the proportion of CSCs, the greater the level of radiation resistance [
147
,
148
]. Part of this
resistance may be due to intrinsic factors, such as greater repair capacity, and protection from reactive
oxygen species-induced damage [
149
,
150
]. However, it is now becoming clear that additional extrinsic
factors can influence resistance, especially the micro-environmental parameter of hypoxia [
144
146
].
Although an association between CSCs and hypoxia has been identified, the exact details of this
relationship are unclear. It is not known whether CSCs and hypoxia simply co-exist, or whether
hypoxia causes recruitment of non-stem cells in to the CSC pool. There is also the issue of how CSCs
and hypoxia influence treatment resistance and malignant progression. Studies have reported that
CSCs are more radiation resistant than non-stem cells [
149
], and hypoxia is definitely a critical factor
in influencing resistance to radiation [
2
,
5
]. However, hypoxia may also increase radiation resistance by
preventing cell differentiation and thus maintaining tumor cells in a more resistant undifferentiated
“stem-cell-like” state [146,151].
It is also not clear as to how CSCs and hypoxia can influence malignant progression.
Undifferentiated cells are more malignant [
146
,
151
], and with the hypoxic microenvironment contributing
to the undifferentiated state of CSCs, this could partially explain how hypoxia and CSCs increase
metastatic spread. Proteases, especially cathepsins (CTSs), are functionally involved in cancer progression
including tumor invasion and metastases [
152
]. CTS expression, especially for CTS-L and K, have been
associated with CSCs [
153
,
154
]. Such expression is also elevated under hypoxic conditions [
155
]. Clearly,
targeting hypoxia using hyperthermia could be a novel approach for dealing with CSCs and thereby
influencing both local response to radiation as well as malignant progression [156].
8. Conclusions
Figure 5summarizes the critical issues in this review. Hypoxia is a characteristic feature of solid
tumors that is a clinically relevant problem because it is a major resistance factor for conventional
therapy, especially radiotherapy, and plays a significant role in malignant progression. Effectively
decreasing hypoxia would clearly improve response to therapy and reduce the likelihood of metastatic
spread. Despite numerous pre-clinical and clinical studies since the 1970s, only one agent has been
established as a treatment option against hypoxia, and that is the radio-sensitizer nimorazole and only
Cancers 2019,11, 60 12 of 20
for treating head and neck patients with radiotherapy in Denmark. Since hyperthermia can effectively
target hypoxia via a variety of different mechanisms, and has been shown to improve radiotherapy
treatment in a number of tumor sites, it would suggest the application of heat to combat hypoxia
should be adopted on a much wider basis, and even established as part of standard cancer therapy.
Cancers 2019, 11, x 12 of 20
application of heat to combat hypoxia should be adopted on a much wider basis, and even established
as part of standard cancer therapy.
Figure 5. Hypoxia in tumors causes resistance to conventional treatments (i.e., radiotherapy of
chemotherapy) and enhanced malignant progression (i.e., more aggressive growth of primary tumors
or metastatic spread). Attempts to decrease hypoxia, and thereby improve tumor response to therapy
as well as decrease the formation of metastases, have utilized a variety of different “traditional”
approaches, as listed in Table 1. Hyperthermia can also decrease tumor hypoxia by a variety of
mechanisms equivalent to all those seen with the more traditional methods.
Author Contributions: The concept for this review was developed by P.B.E. with help from M.R.H. The
manuscript was written by P.B.E. with help from M.R.H., B.S.S., A.L.O., N.A.P.F., J.C., and J.O.
Funding: This research was funded by the Danish Cancer Society, grant number R40-A2022-11-S2 and the
Danish Council for Independent Research: Medical Sciences, grant number DFF-4004-00362.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
References
1. Vaupel, P.; Kallinowski, F.; Okunieff, P. Blood flow, oxygen and nutrient supply, and metabolic
microenvironment of human tumors: A review. Cancer Res. 1989, 49, 6449–6465.
2. Horsman, M.R.; Vaupel, P. Pathophysiological basis for the formation of the tumor microenvironment.
Front. Oncol. 2016, 6, 66, doi:10.3389/fonc.2016.00066.
3. Folkman, J. How is blood vessel growth regulated in normal and neoplastic tissue? G.H.A. Clowes memorial
Award lecture. Cancer Res. 1986, 46, 467–473.
4. Horsman, M.R.; Mortensen, L.S.; Petersen, J.B.; Busk, M.; Overgaard, J. Imaging hypoxia to improve
radiotherapy outcome. Nat. Rev. Clin. Oncol. 2012, 9, 674–687, doi:10.1038/nrclinonc.2012.171.
5. Siemann, D.W.; Horsman, M.R. Modulation of the tumor vasculature and oxygenation to improve therapy.
Pharmacol. Ther. 2015, 153, 107–124, doi:0.1016/j.pharmthera.2015.06.006.
6. Overgaard, J.; Horsman, M.R. Modification of hypoxia induced radioresistance in tumours by the use of
oxygen and sensitizers. Semin. Radiat. Oncol. 1996, 6, 10–21.
7. Horsman, M.R.; Overgaard, J. The impact of hypoxia and its modification of the outcome of radiotherapy.
J. Radiat. Res. 2016, 57 (Suppl. 1), 90–98, doi:10.1093/jrr/rrw007.
Figure 5.
Hypoxia in tumors causes resistance to conventional treatments (i.e., radiotherapy of
chemotherapy) and enhanced malignant progression (i.e., more aggressive growth of primary tumors
or metastatic spread). Attempts to decrease hypoxia, and thereby improve tumor response to therapy
as well as decrease the formation of metastases, have utilized a variety of different “traditional”
approaches, as listed in Table 1. Hyperthermia can also decrease tumor hypoxia by a variety of
mechanisms equivalent to all those seen with the more traditional methods.
Author Contributions:
The concept for this review was developed by P.B.E. with help from M.R.H. The
manuscript was written by P.B.E. with help from M.R.H., B.S.S., A.L.O., N.A.P.F., J.C., and J.O.
Funding:
This research was funded by the Danish Cancer Society, grant number R40-A2022-11-S2 and the Danish
Council for Independent Research: Medical Sciences, grant number DFF-4004-00362.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
References
1.
Vaupel, P.; Kallinowski, F.; Okunieff, P. Blood flow, oxygen and nutrient supply, and metabolic
microenvironment of human tumors: A review. Cancer Res. 1989,49, 6449–6465. [PubMed]
2.
Horsman, M.R.; Vaupel, P. Pathophysiological basis for the formation of the tumor microenvironment.
Front. Oncol. 2016,6, 66. [CrossRef] [PubMed]
3.
Folkman, J. How is blood vessel growth regulated in normal and neoplastic tissue? G.H.A. Clowes memorial
Award lecture. Cancer Res. 1986,46, 467–473. [PubMed]
4.
Horsman, M.R.; Mortensen, L.S.; Petersen, J.B.; Busk, M.; Overgaard, J. Imaging hypoxia to improve
radiotherapy outcome. Nat. Rev. Clin. Oncol. 2012,9, 674–687. [CrossRef] [PubMed]
5.
Siemann, D.W.; Horsman, M.R. Modulation of the tumor vasculature and oxygenation to improve therapy.
Pharmacol. Ther. 2015,153, 107–124. [CrossRef] [PubMed]
6.
Overgaard, J.; Horsman, M.R. Modification of hypoxia induced radioresistance in tumours by the use of
oxygen and sensitizers. Semin. Radiat. Oncol. 1996,6, 10–21. [CrossRef]
Cancers 2019,11, 60 13 of 20
7.
Horsman, M.R.; Overgaard, J. The impact of hypoxia and its modification of the outcome of radiotherapy.
J. Radiat. Res. 2016,57 (Suppl. 1), 90–98. [CrossRef]
8.
Breasted, J.H. The Edwin Smith surgical papyrus. In Therapeutic Heat and Cold, 2nd ed.; Licht, S., Ed.; Waverly
Press: Baltimore, MD, USA, 1930; pp. 196–211.
9.
Storm, K.F. (Ed.) Early history. In Hyperthermia in Cancer Therapy, 1st ed.; G. K. Hall Medical Publishers:
Boston MA, USA, 1983; pp. 1–8.
10.
Dewhirst, M.; Stauffer, P.R.; Das, S.; Craciunescu, O.I.; Vujaskovic, Z. Hyperthermia. In Clinical Radiation
Oncology, 4th ed.; Gunderson, L.L., Tepper, J.E., Eds.; Elsevier: Philadelphia, PA, USA, 2016; pp. 381–398,
ISBN 978-0-323-24098-7.
11.
Trefná, H.D.; Crezee, J.; Schmidt, M.; Marder, D.; Lamprecht, U.; Ehmann, M.; Nadobny, J.; Hartmann, J.;
Lomax, N.; Abdel-Rahman, S. Quality assurance guidelines for superficial hyperthermia clinical trials: I.
Clinical requirements. Int. J. Hyperth. 2017,33, 471–482. [CrossRef]
12.
Crezee, H.; van Leeuwen, C.M.; Oei, A.L.; Stalpers, L.J.; Bel, A.; Franken, N.A.; Kok, H.P. Thermoradiotherapy
planning: Integration in routine clinical practice. Int. J. Hyperth. 2016,32, 41–49. [CrossRef]
13.
Bruggmoser, G.; Bauchowitz, S.; Canters, R.; Crezee, H.; Ehmann, M.; Gellermann, J.; Lamprecht, U.;
Lomax, N.; Messmer, M.; Ott, O. Guideline for the clinical application, documentation and analysis of clinical
studies for regional deep hyperthermia. Strahlenther. Onkol. 2012,188, 198–211. [CrossRef]
14.
Wust, P.; Hildebrandt, B.; Sreenivasa, G.; Rau, B.; Gellermann, J.; Riess, H.; Felix, R.; Schlag, P. Hyperthermia
in combined treatment of cancer. Lancet Oncol. 2002,3, 487–497. [CrossRef]
15.
Horsman, M.R.; Overgaard, J. Hyperthermia: A Potent Enhancer of Radiotherapy. Clin. Oncol.
2007
,19,
418–426. [CrossRef] [PubMed]
16.
Cihoric, N.; Tsikkinis, A.; van Rhoon, G.; Crezee, H.; Aebersold, D.M.; Bodis, S.; Beck, M.; Nadobny, J.;
Budach, V.; Wust, P. Hyperthermia-related clinical trials on cancer treatment within the ClinicalTrials.gov
registry. Int. J. Hyperth. 2015,31, 609–614. [CrossRef] [PubMed]
17.
Peeken, J.C.; Vaupel, P.; Combs, S.E. Integrating hyperthermia into modern radiation oncology: What
evidence is necessary? Front. Oncol. 2017,7, 132. [CrossRef] [PubMed]
18.
Overgaard, J.; Nielsen, O.S.; Lindegaard, J.C. Biological basis for rational design of clinical treatment with
combined hyperthermia and radiation. In Phisics and Technology of Hyperthermia; Field, S.B., Franconi, C.,
Eds.; Springer: Dordrecht, The Netherlands, 1987; pp. 54–79, ISBN 978-94-010-8109-2.
19.
Overgaard, J. The design of clinical trials in hyperthermia. In Phisics and Technology of Hyperthermia; Field, S.B.,
Franconi, C., Eds.; Springer: Dordrecht, The Netherlands, 1987; pp. 598–620, ISBN 978-94-010-8109-2.
20.
Van Leeuwen, C.M.; Oei, A.L.; Chin, K.W.; Crezee, J.; Bel, A.; Westermann, A.M.; Buist, M.R.; Franken, N.A.;
Stalpers, L.J.; Kok, H.P. A short time interval between radiotherapy and hyperthermia reduces in-field
recurrence and mortality in women with advanced cervical cancer. Radiat. Oncol.
2017
,12, 75. [CrossRef]
[PubMed]
21.
Field, S.B.; Bleehen, N.M. Hyperthermia in treatment of cancer. Cancer Treat. Rev.
1979
,6, 63–94. [CrossRef]
22. Hendry, J.H.; Sutton, M.L. Care with radiosensitizers. Br. J. Radiol. 1978,51, 927–928. [CrossRef]
23.
Hendry, J.H. Quantitation of the radiotherapeutic importance of naturally-hypoxic normal tissues from
collated experiments with rodents using single doses. Int. J. Radiat. Oncol. Biol. Phys.
1979
,5, 971–976.
[CrossRef]
24.
Murata, R.; Tsujitani, M.; Horsman, M.R. Enhanced local tumour control after single or fractionated radiation
treatment using the hypoxic cell radiosensitizer doranidazole. Radiother. Oncol.
2008
,87, 331–338. [CrossRef]
25.
Porschen, W.; Gartzen, J.; Geweher, K.; Mühlensiepen, H.; Weber, H.-J.; Feinedegen, L.
In vivo
assay of
the radiation sesnitivity of hypoxic tumour cells; influence of
γ
-rays, cyclotron neutrons, misonidazole,
hyperthermia and mixed modalities. Br. J. Cancer 1978,37 (Suppl. III), 194–197.
26. Stone, H.B. Enhancement of local tumour control by misonidazole and hyperthermia. Br. J. Cancer 1978,37
(Suppl. III), 178–183.
27.
Overgaard, J. Effect of misonidazole and hyperthermia on the radiosensitivity of a C3H mouse mammary
carcinoma and its surrounding normal tissues. Br. J. Cancer 1980,41, 10–21. [CrossRef] [PubMed]
28.
Horsman, M.R.; Overgaard, J. Combination of nicotinamide and hyperthermia to eliminate radioresistant
chronically and acutely hypoxic tumour cells. Cancer Res. 1990,50, 7430–7436. [PubMed]
29.
Oleson, J.R. Eugene Robertson Special Lecture: Hyperthermia from the clinic to the laboratory: A hypothesis.
Int. J. Hyperth. 1995,11, 315–322. [CrossRef]
Cancers 2019,11, 60 14 of 20
30.
Iwata, K.; Shakil, A.; Hur, W.; Makepeace, C.; Griffin, R.; Song, C. Tumour pO
2
can be increased markedly by
mild hyperthermia. Br. J. Cancer 1996,74 (Suppl. XXVII), S217–S221.
31.
Horsman, M.R.; Overgaard, J. Can mild hyperthermia improve tumour oxygenation? Int. J. Hyperth.
1997
,
13, 141–147. [CrossRef]
32.
Vaupel, P.W.; Kelleher, D.K. Pathophysiological and vascular characteristics of tumours and their importance
for hyperthermia: Heterogeneity is the key issue. Int. J. Hyperth. 2010,26, 211–223. [CrossRef]
33.
Sen, A.; Capitano, M.L.; Spernyak, J.A.; Schueckler, J.T.; Thomas, S.; Singh, A.K.; Evans, S.S.; Hylander, B.;
Repasky, E.A. Mild elevation of body temperature reduces tumor interstitial fluid pressure and hypoxia and
enhances efficacy of radiotherapy in murine tumor models. Cancer Res. 2011,71, 3872–3880. [CrossRef]
34.
Winslow, T.B.; Eranki, A.; Ullas, S.; Singh, A.K.; Repasky, E.A.; Sen, A. A pilot study of the effects of mild
systemic heating on human head and neck tumour xenografts: Analysis of tumour perfusion, interstitial
fluid pressure, hypoxia and efficacy of radiation therapy. Int. J. Hyperth. 2015,31, 693–701. [CrossRef]
35.
Song, C.W. Effect of local hyperthermia on blood flow and microenvironment: A review. Cancer Res.
1984
,
44, 4721–4730.
36.
Xu, Y.; Choi, J.; Hylander, B.; Sen, A.; Evans, S.S.; Kraybill, W.G.; Repasky, E.A. Fever-range whole body
hyperthermia increases the number of perfused tumor blood vessels and therapeutic efficacy of liposomally
encapsulated doxorubicin. Int. J. Hyperth. 2007,23, 513–527. [CrossRef] [PubMed]
37.
Griffin, R.J.; Okajima, K.; Barrios, B.; Song, C.W. Mild temperature hyperthermia combined with carbogen
breathing increases tumor partial pressure of oxygen (pO
2
) and radiosensitivity. Cancer Res.
1996
,56,
5590–5593. [PubMed]
38.
Song, C.W.; Patten, M.S.; Chelstrom, L.M.; Rhee, J.G.; Levitt, S.H. Effect of multiple heatings on the blood
flow in RIF-1 tumours, skin and muscle of C3H mice. Int. J. Hyperth. 1987,3, 535–545. [CrossRef]
39.
Durand, R.E. Potentiation of radiation lethality by hyperthermia in a tumor model: Effects of sequence,
degree, and duration of heating. Int. J. Radiat. Oncol. Biol. Phys. 1978,4, 401–405. [CrossRef]
40.
Ansiaux, R.; Baudelet, C.; Jordan, B.F.; Crokart, N.; Martinive, P.; DeWever, J.; Grégoire, V.; Feron, O.;
Gallez, B. Mechanism of reoxygenation after antiangiogenic therapy using SU5416 and its importance for
guiding combined antitumor therapy. Cancer Res. 2006,66, 9698–9704. [CrossRef] [PubMed]
41.
Zannella, V.E.; Dal Pra, A.; Muaddi, H.; McKee, T.D.; Stapleton, S.; Sykes, J.; Glicksman, R.; Chaib, S.;
Zamiara, P.; Milosevic, M. Reprogramming metabolism with metformin improves tumor oxygenation and
radiotherapy response. Clin. Cancer Res. 2013,19, 6741–6750. [CrossRef] [PubMed]
42.
Shakil, A.; Osborn, J.L.; Song, C.W. Changes in oxygenation status and blood flow in a rat tumor model by
mild temperature hyperthermia. Int. J. Radiat. Oncol. 1999,43, 859–865. [CrossRef]
43.
Song, C.W.; Park, H.; Griffin, R.J. Improvement of tumor oxygenation by mild hyperthermia. Radiat. Res.
2001,155, 515–528. [CrossRef]
44.
Song, C.W.; Shakil, A.; Osborn, J.L.; Iwata, K. Tumour oxygenation is increased by hyperthermia at mild
temperatures. Int. J. Hyperth. 2009,25, 91–95. [CrossRef]
45.
Vujaskovic, Z.; Song, C.W. Physiological mechanisms underlying heat-induced radiosensitization.
Int. J. Hyperth. 2004,20, 163–174. [CrossRef]
46.
Brizel, D.M.; Scully, S.P.; Harrelson, J.M.; Layfield, L.J.; Dodge, R.K.; Charles, H.C.; Samulski, T.V.;
Prosnitz, L.R.; Dewhirst, M.W. Radiation therapy and hyperthermia improve the oxygenation of human soft
tissue sarcomas. Cancer Res. 1996,56, 5347–5350. [PubMed]
47.
Jones, E.L.; Prosnitz, L.R.; Dewhirst, M.W.; Marcom, P.K.; Hardenbergh, P.H.; Marks, L.B.; Brizel, D.M.;
Vujaskovic, Z. Thermochemoradiotherapy improves oxygenation in locally advanced breast cancer.
Clin. Cancer Res. 2004,10, 4287–4293. [CrossRef] [PubMed]
48.
Hetzel, F.W.; Chopp, M.; Dereski, M.O. Variations in pO
2
and pH response to hyperthermia: Dependence on
transplant site and duration of treatment. Radiat. Res. 1992,131, 152–156. [CrossRef] [PubMed]
49.
Howard-Flanders, P.; Moore, D. The time interval after pulsed irradiation within which injury in bacteria can
be modified by dissolved oxygen. I. A search for an effect of oxygen 0.002 seconds after pulsed irradiation.
Radiat. Res. 1958,9, 422–437. [CrossRef] [PubMed]
50.
Michael, B.; Adams, G.; Hewitt, H.; Jones, W.; Watts, M. A post-effect of oxygen in irradiated bacteria:
A submillisecond fast mixing study. Radiat. Res. 1973,54, 239–251. [CrossRef] [PubMed]
51.
Cannan, W.J.; Pederson, D.S. Mechanisms and Consequences of Double-Strand DNA Break Formation in
Chromatin. J. Cell. Physiol. 2016,231, 3–14. [CrossRef] [PubMed]
Cancers 2019,11, 60 15 of 20
52.
Lydeard, J.R.; Lipkin-Moore, Z.; Sheu, Y.-J.; Stillman, B.; Burgers, P.M.; Haber, J.E. Break-induced replication
requires all essential DNA replication factors except those specific for pre-RC assembly. Genes Dev.
2010
,24,
1133–1144. [CrossRef]
53.
Kuzminov, A. Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proc. Natl.
Acad. Sci. USA 2001,98, 8241–8246. [CrossRef]
54.
Takata, M.; Sasaki, M.S.; Sonoda, E.; Morrison, C.; Hashimoto, M.; Utsumi, H.; Yamaguchi-Iwai, Y.;
Shinohara, A.; Takeda, S. Homologous recombination and non-homologous end-joining pathways of DNA
double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate
cells. EMBO J. 1998,17, 5497–5508. [CrossRef]
55.
Jackson, S.P. Sensing and repairing DNA double-strand breaks. Carcinogenesis
2002
,23, 687–696. [CrossRef]
56. Fleck, O.; Nielsen, O. DNA repair. J. Cell Sci. 2004,117 Pt 4, 515–517. [CrossRef]
57.
Davis, A.J.; Chen, D.J. DNA double strand break repair via non-homologous end-joining. Transl. Cancer Res.
2013,2, 130–143. [CrossRef] [PubMed]
58.
Jasin, M.; Rothstein, R. Repair of strand breaks by homologous recombination. Cold Spring Harb. Perspect. Biol.
2013,5, a012740. [CrossRef] [PubMed]
59.
Kuo, L.J.; Yang, L.-X. Gamma-H2AX—A novel biomarker for DNA double-strand breaks. In Vivo
2008
,22,
305–309. [PubMed]
60.
El-Awady, R.A.; Dikomey, E.; Dahm-Daphi, J. Heat effects on DNA repair after ionising radiation:
Hyperthermia commonly increases the number of non-repaired double-strand breaks and structural
rearrangements. Nucleic Acids Res. 2001,29, 1960–1966. [CrossRef] [PubMed]
61.
Van Leeuwen, C.; Oei, A.; Ten Cate, R.; Franken, N.; Bel, A.; Stalpers, L.; Crezee, J.; Kok, H. Measurement
and analysis of the impact of time-interval, temperature and radiation dose on tumour cell survival and its
application in thermoradiotherapy plan evaluation. Int. J. Hyperth. 2018,34, 39–48. [CrossRef] [PubMed]
62.
Ihara, M.; Takeshita, S.; Okaichi, K.; Okumura, Y.; Ohnishi, T. Heat exposure enhances radiosensitivity by
depressing DNA-PK kinase activity during double strand break repair. Int. J. Hyperth.
2014
,30, 102–109.
[CrossRef] [PubMed]
63.
Krawczyk, P.M.; Eppink, B.; Essers, J.; Stap, J.; Rodermond, H.; Odijk, H.; Zelensky, A.; van Bree, C.;
Stalpers, L.J.; Buist, M.R.; et al. Mild hyperthermia inhibits homologous recombination, induces BRCA2
degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition. Proc. Natl. Acad.
Sci. USA 2011,108, 9851–9856. [CrossRef]
64.
Gilbertson, R.J.; Rich, J.N. Making a tumour’s bed: Glioblastoma stem cells and the vascular niche.
Nat. Rev. Cancer 2007,7, 733–736. [CrossRef]
65.
Zölzer, F.; Streffer, C.; Pelzer, T. Induction of quiescent S-phase cells by irradiation and/or hyperthermia. II.
Correlation with colony forming ability. Int. J. Radiat. Biol. 1993,63, 77–82. [CrossRef]
66.
Dewey, W.C.; Hopwood, L.E.; Sapareto, S.A.; Gerweck, L.E. Cellular responses to combinations of
hyperthermia and radiation. Radiology 1977,123, 463–474. [CrossRef] [PubMed]
67.
Nielsen, O.S.; Henle, K.J.; Overgaard, J. Arrhenius analysis of survival curves from thermotolerant and
step-down heated L1A2 cells in vitro. Radiat. Res. 1982,91, 468–482. [CrossRef] [PubMed]
68.
Roizin-Towle, L.; Pirro, J.P. The response of human and rodent cells to hyperthermia. Int. J. Radiat. Oncol.
1991,20, 751–756. [CrossRef]
69.
Overgaard, J.; Bichel, P. The Influence of Hypoxia and Acidity on the Hyperthermic Response of Malignant
Cells In Vitro. Radiology 1977,123, 511–514. [CrossRef] [PubMed]
70.
Gerweck, L.E.; Nygaard, T.G.; Burlett, M. Response of cells to hyperthermia under acute and chronic hypoxic
conditions. Cancer Res. 1979,39, 966–972. [PubMed]
71.
Overgaard, J. Effect of hyperthermia on the hypoxic fraction in an experimental mammary carcinoma
in vivo
.
Br. J. Radiol. 1981,54, 245–249. [CrossRef] [PubMed]
72.
Field, S.B.; Anderson, R.L. Thermotolerance: A review of observations and possible mechanisms. Natl. Cancer
Inst. Monogr. 1982,61, 193–201.
73.
Lindegaard, J.C.; Overgaard, J. Factors of importance for the development of the step-down heating effect in
a C3H mammary carcinoma in vivo. Int. J. Hyperth. 1987,3, 79–91. [CrossRef]
74.
Dings, R.P.; Loren, M.L.; Zhang, Y.; Mikkelson, S.; Mayo, K.H.; Corry, P.; Griffin, R.J. Tumour thermotolerance,
a physiological phenomenon involving vessel normalization. Int. J. Hyperth. 2011,27, 42–52. [CrossRef]
Cancers 2019,11, 60 16 of 20
75.
Jain, R.K. Normalizing tumor vasculature with anti-angiogenic therapy: A new paradigm for combination
therapy. Nat. Med. 2001,7, 987–989. [CrossRef]
76.
Winkler, F.; Kozin, S.V.; Tong, R.T.; Chae, S.S.; Booth, M.F.; Garkavtsev, I.; Xu, L.; Hicklin, D.J.; Fukumura, D.;
di Tomaso, E.; et al. Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response
to radiation: Role of oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell
2004
,6, 553–563.
[CrossRef] [PubMed]
77.
Harmon, B.V.; Corder, A.M.; Collins, R.J.; Gobé, G.C.; Allen, J.; Allan, D.J.; Kerr, J.F. Cell death induced in a
murine mastocytoma by 42–47
C heating
in vitro
: Evidence that the form of death changes from apoptosis
to necrosis above a critical heat load. Int. J. Radiat. Biol. 1990,58, 845–858. [CrossRef] [PubMed]
78.
Kampinga, H.H. Thermotolerance in mammalian cells: Protein denaturation and aggregation, and stress
proteins. J. Cell Sci. 1993,104, 11–17. [PubMed]
79.
Roti Roti, J.L.; Kampinga, H.H.; Malyapa, R.S.; Wright, W.D.; vanderWaal, R.P.; Xu, M. Nuclear matrix as a
target for hyperthermic killing of cancer cells. Cell Stress Chaperones 1998,3, 245–255. [CrossRef]
80.
Vertrees, R.A.; Das, G.C.; Coscio, A.M.; Xie, J.; Zwischenberger, J.B.; Boor, P.J. A mechanism of
hyperthermia-induced apoptosis in ras-transformed lung cells. Mol. Carcinog.
2005
,44, 111–121. [CrossRef]
[PubMed]
81.
Lepock, J.R. Cellular effects of hyperthermia: Relevance to the minimum dose for thermal damage.
Int. J. Hyperth. 2003,19, 252–266. [CrossRef] [PubMed]
82.
Heacock, C.S.; Brown, S.L.; Bamburg, J.R.
In vitro
inactivation of actin by heat. Natl. Cancer Inst. Monogr.
1982,61, 73–75.
83. Horsman, M.R. Tissue physiology and the response to heat. Int. J. Hyperth. 2006,22, 197–203. [CrossRef]
84.
Horsman, M.R.; Siemann, D.W. Pathophysiological effects of vascular targeting agents and the implications
for combination with conventional therapies. Cancer Res. 2006,66, 11520–11539. [CrossRef]
85.
Horsman, M.R. Angiogenesis and vascular targeting: Relevance for hyperthermia. Int. J. Hyperth.
2008
,24,
57–65. [CrossRef]
86.
Tozer, G.M.; Kanthou, C.; Baguley, B.C. Disrupting tumour blood vessels. Nat. Rev. Cancer
2005
,5, 423–435.
[CrossRef] [PubMed]
87.
Siemann, D.W.; Chaplin, D.J.; Horsman, M.R. Realizing the potential of vascular targeted therapy: The
rationale for combining vascular disrupting agents and anti-angiogenic agents to treat cancer. Cancer Investig.
2017,35, 519–534. [CrossRef] [PubMed]
88. Horsman, M.R. (Aarhus University, Aarhus, Denmark). Personal communication, 2018.
89.
Ling, C.C.; Humm, J.; Larson, S.; Amols, H.; Fuks, Z.; Leibel, S.; Koutcher, J.A. Towards multidimensional
radiotherapy (MD-CRT): Biological imaging and biological conformality. Int. J. Radiat. Oncol. Biol. Phys.
2000,47, 551–560. [CrossRef]
90.
Epel, B.; Maggio, M.C.; Barth, E.D.; Miller, R.C.; Pelizzari, C.A.; Krzykawska-Serda, M.; Sundramoorthy, S.V.;
Aydogan, B.; Weichselbaum, R.R.; Tormyshev, V.M.; et al. Oxygen-guided radiation therapy. Int. J. Radiat.
Oncol. Biol. Phys. 2018. [CrossRef] [PubMed]
91.
Barendsen, G.W. Responses of cultured cells, tumours and normal tissues to radiations of different linear
energy transfer. Curr. Top. Radiat. Res. Q. 1968,4, 293–356.
92.
Wenzl, T.; Wilkens, J.J. Modelling of the oxygen enhancement ratio for ion beam radiation therapy.
Phys. Med. Biol. 2011,56, 3251–3268. [CrossRef] [PubMed]
93.
Particle Therapy Facilities in Clinical Operation (Last Update: January 2019). Available online: https://www.
ptcog.ch/index.php/facilities-in-operation (accessed on 7 January 2019).
94.
Datta, N.R.; Puric, E.; Schneider, R. Could hyperthermia with proton therapy mimic carbon ion therapy?
Exploring a thermo-radiobiological rationale. Int. J. Hyperth. 2014,30, 524–530. [CrossRef]
95.
Gerner, E.W.; Leith, J.T. Interaction of hyperthermia with radiations of different linear energy transfer. Int. J.
Radiat. Biol. 1977,31, 283–288. [CrossRef]
96.
Karger, C.P.; Peschke, P. RBE and related modelling in carbon-ion therapy. Phys. Med. Biol.
2018
,63, 01TR02.
[CrossRef]
97.
Horsman, M.R. The therapeutic potential of using the vascular disrupting agent OXi4503 to enhance mild
temperature thermoradiation. Int. J. Hyperth. 2015,31, 453–459. [CrossRef]
Cancers 2019,11, 60 17 of 20
98.
Lühr, A.; von Neubeck, C.; Pawelke, J.; Seidlitz, A.; Peitzsch, C.; Bentzen, S.M.; Bortfeld, T.; Debus, J.;
Deutsch, E.; Langendijk, J.A.; et al. “Radiobiology of Proton Therapy”: Results of an international expert
workshop. Radiother. Oncol. 2018,128, 56–67. [CrossRef] [PubMed]
99.
Ray, S.; Cekanaviciute, E.; Lima, I.P.; Sørensen, B.S.; Costes, S.V. Comparing Photon and Charged Particle
Therapy Using DNA Damage Biomarkers. Int. J. Part. Ther. 2018,5, 15–24. [CrossRef]
100.
Willers, H.; Allen, A.; Grosshans, D.; McMahon, S.J.; von Neubeck, C.; Wiese, C.; Vikram, B. Toward A
variable RBE for proton beam therapy. Radiother. Oncol. 2018,128, 68–75. [CrossRef] [PubMed]
101.
Suetens, A.; Konings, K.; Moreels, M.; Quintens, R.; Verslegers, M.; Soors, E.; Tabury, K.; Grégoire, V.;
Baatout, S. Higher Initial DNA Damage and Persistent Cell Cycle Arrest after Carbon Ion Irradiation
Compared to X-irradiation in Prostate and Colon Cancer Cells. Front. Oncol.
2016
,6, 87. [CrossRef]
[PubMed]
102.
Antonelli, F.; Campa, A.; Esposito, G.; Giardullo, P.; Belli, M.; Dini, V.; Meschini, S.; Simone, G.; Sorrentino, E.;
Gerardi, S.; et al. Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human
Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive
Cell Death. Radiat. Res. 2015,183, 417–431. [CrossRef] [PubMed]
103.
Grosse, N.; Fontana, A.O.; Hug, E.B.; Lomax, A.; Coray, A.; Augsburger, M.; Paganetti, H.; Sartori, A.A.;
Pruschy, M.; et al. Deficiency in homologous recombination renders mammalian cells more sensitive to
proton versus photon irradiation. Int. J. Radiat. Oncol. Biol. Phys. 2014,88, 175–181. [CrossRef] [PubMed]
104.
Chaudhary, P.; Marshall, T.I.; Currell, F.J.; Kacperek, A.; Schettino, G.; Prise, K.M. Variations in the Processing
of DNA Double-Strand Breaks Along 60-MeV Therapeutic Proton Beams. Int. J. Radiat. Oncol. Biol. Phys.
2016,95, 86–94. [CrossRef]
105.
Hojo, H.; Dohmae, T.; Hotta, K.; Kohno, R.; Motegi, A.; Yagishita, A.; Makinoshima, H.; Tsuchihara, K.;
Akimoto, T. Difference in the relative biological effectiveness and DNA damage repair processes in response
to proton beam therapy according to the positions of the spread out Bragg peak. Radiat. Oncol.
2017
,12, 111.
[CrossRef]
106.
Kavanagh, J.N.; Currell, F.J.; Timson, D.J.; Savage, K.I.; Richard, D.J.; McMahon, S.J.; Hartley, O.; Cirrone, G.A.;
Romano, F.; Prise, K.M.; et al. Antiproton induced DNA damage: Proton like in flight, carbon-ion like near
rest. Sci. Rep. 2013,3, 1770. [CrossRef]
107.
Wang, H.; Wang, X.; Zhang, P.; Wang, Y. The Ku-dependent non-homologous end-joining but not other
repair pathway is inhibited by high linear energy transfer ionizing radiation. DNA Repair
2008
,7, 725–733.
[CrossRef]
108.
Gerelchuluun, A.; Manabe, E.; Ishikawa, T.; Sun, L.; Itoh, K.; Sakae, T.; Suzuki, K.; Hirayama, R.;
Asaithamby, A.; Chen, D.J.; et al. The Major DNA Repair Pathway after Both Proton and Carbon-Ion
Radiation is NHEJ, but the HR Pathway is More Relevant in Carbon Ions. Radiat. Res.
2015
,183, 345–356.
[CrossRef] [PubMed]
109.
Fontana, A.O.; Augsburger, M.A.; Grosse, N.; Guckenberger, M.; Lomax, A.J.; Sartori, A.A.; Pruschy, M.N.
Differential DNA repair pathway choice in cancer cells after proton- and photon-irradiation. Radiother. Oncol.
2015,116, 374–380. [CrossRef] [PubMed]
110.
Liu, Q.; Ghosh, P.; Magpayo, N.; Testa, M.; Tang, S.; Gheorghiu, L.; Biggs, P.; Paganetti, H.; Efstathiou, J.A.;
Lu, H.M.; et al. Lung Cancer Cell Line Screen Links Fanconi Anemia/BRCA Pathway Defects to Increased
Relative Biological Effectiveness of Proton Radiation. Int. J. Radiat. Oncol.
2015
,91, 1081–1089. [CrossRef]
[PubMed]
111.
Maeda, J.; Fujii, Y.; Fujisawa, H.; Hirakawa, H.; Cartwright, I.M.; Uesaka, M.; Kitamura, H.; Fujimori, A.;
Kato, T.A. Hyperthermia-induced radiosensitization in CHO wild-type, NHEJ repair mutant and HR repair
mutant following proton and carbon-ion exposure. Oncol. Letts. 2015,10, 2828–2834. [CrossRef] [PubMed]
112.
Chang, P.Y.; Tobias, C.A.; Blakely, E.A. Protein synthesis modulates the biological effectiveness of the combined
action of hyperthermia and high-LET radiation. Radiat. Res. 1992,129, 272–280. [CrossRef] [PubMed]
113.
Kinashi, Y.; Masunaga, S.I.; Suzuki, M.; Ono, K.; Ohnishi, T. Hyperthermia enhances thermal-neutron-induced
cell death of human glioblastoma cell lines at low concentrations of 10B. Int. J. Radiat. Oncol. Biol. Phys.
1998
,
40, 1185–1192. [CrossRef]
114.
Takahashi, A.; Ohnishi, K.; Wang, X.; Kobayashi, M.; Matsumoto, H.; Tamamoto, T.; Aoki, H.; Furusawa, Y.;
Yukawa, O.; Ohnishi, T. The dependence of p53 on the radiation enhancement of thermosensitivity at
different let. Int. J. Radiat. Oncol. Biol. Phys. 2000,47, 489–494. [CrossRef]
Cancers 2019,11, 60 18 of 20
115.
Takahashi, A.; Ohnishi, K.; Ota, I.; Asakawa, I.; Tamamoto, T.; Furusawa, Y.; Matsumoto, H.; Ohnishi, T.
p53-dependent thermal enhancement of cellular sensitivity in human squamous cell carcinomas in relation
to LET. Int. J. Radiat. Biol. 2001,77, 1043–1051. [CrossRef] [PubMed]
116.
Huilgol, N.G.; Gupta, S.; Sridhar, C.R. Hyperthermia with radiation in the treatment of locally advanced
head and neck cancer: A report of randomized trial. J. Cancer Res. Ther. 2010,6, 492–496. [CrossRef]
117.
Datta, N.R.; Bose, A.K.; Kapoor, H.K.; Gupta, S. Head and neck cancers: Results of thermoradiotherapy
versus radiotherapy. Int. J. Hyperth. 1990,6, 479–486. [CrossRef]
118.
Valdagni, R.; Amichetti, M. Report of Long-term follow-up in a randomized trial comparing radiation
therapy and radiation therapy plus hyperthermia to metastatic lymphnodes in stage IV head and neck
patients. Int. J. Radiat. Oncol. Biol. Phys. 1994,28, 163–169. [CrossRef]
119.
Harima, Y.; Nagata, K.; Harima, K.; Ostapenko, V.V.; Tanaka, Y.; Sawada, S. A randomized clinical trial
of radiation therapy versus thermoradiotherapy in stage IIIB cervical carcinoma. Int. J. Hyperth.
2001
,17,
97–105. [CrossRef] [PubMed]
120.
Van der Zee, J.; González, G.D. The Dutch Deep Hyperthermia Trial: Results in cervical cancer. Int. J. Hyperth
2002,18, 1–12.
121.
Franckena, M.; Stalpers, L.J.; Koper, P.C.; Wiggenraad, R.G.; Hoogenraad, W.J.; Van Dijk, J.D.;
Wárlám-Rodenhuis, C.C.; Jobsen, J.J.; van Rhoon, G.C.; van der Zee, J. Long-term improvement in treatment
outcome after radiotherapy and hyperthermia in locoregionally advanced cervix cancer: An update of the
Dutch Deep Hyperthermia Trial. Int. J. Radiat. Oncol. Biol. Phys. 2008,70, 1176–1182. [CrossRef] [PubMed]
122.
Van Der Zee, J.; González, D.G.; van Rhoon, G.C.; van Dijk, J.D.P.; van Putten, W.L.J. Comparison of
radiotherapy alone with radiotherapy plus hyperthermia in locally advanced pelvic tumours: A prospective,
randomised, multicentre trial. Lancet 2000,355, 1119–1125. [CrossRef]
123.
Vernon, C.C.; Hand, J.W.; Field, S.B.; Machin, D.; Whaley, J.B.; van der Zee, J.; van Putten, W.L.; van Rhoon, G.C.;
van Dijk, J.D.; González González, D.; et al. Radiotherapy with or without hyperthermia in the treatment of
superficial localized breast cancer: Results from five randomized controlled trials. International Collaborative
Hyperthermia Group. Int. J. Radiat. Oncol. Biol. Phys. 1996,35, 731–744. [CrossRef] [PubMed]
124.
Mitsumori, M.; Zeng, Z.F.; Oliynychenko, P.; Park, J.H.; Choi, I.B.; Tatsuzaki, H.; Tanaka, Y.; Hiraoka, M.
Regional hyperthermia combined with radiotherapy for locally advanced non-small cell lung cancers: A
multi-institutional prospective randomized trial of the International Atomic Energy Agency. Int. J. Clin. Oncol.
2007,12, 192–198. [CrossRef]
125.
Overgaard, J.; Gonzalez Gonzalez, D.; Hulshof, M.C.; Arcangeli, G.; Dahl, O.; Mella, O.; Bentzen, S.M.
Randomised trial of hyperthermia as adjuvant to radiotherapy for recurrent or metastatic malignant
melanoma. Lancet 1995,345, 540–543. [CrossRef]
126.
Jones, E.L.; Oleson, J.R.; Prosnitz, L.R.; Samulski, T.V.; Vujaskovic, Z.; Yu, D.; Sanders, L.L.; Dewhirst, M.W.
Randomized Trial of Hyperthermia and Radiation for Superficial Tumors. J. Clin. Oncol.
2005
,23, 3079–3085.
[CrossRef]
127.
Berdov, B.A.; Menteshashvili, G.Z. Thermoradiotherapy of patients with locally advanced carcinoma of the
rectum. Int. J. Hyperth. 1990,6, 881–890. [CrossRef]
128.
Egawa, S.; Tsukiyama, I.; Watanabe, S.; Ohno, Y.; Morita, K.; Tominaga, S.; Onoyama, Y.; Hashimoto, S.;
Yanagawa, S.; Uehara, S.; et al. A randomized clinical trial of hyperthermia and radiation versus radiation
alone for superficially located cancers. J. Jpn. Soc. Ther. Radiol. Oncol. 1989,1, 135–140. [CrossRef]
129.
Perez, C.A.; Pajak, T.; Emami, B.; Hornback, N.B.; Tupchong, L.; Rubin, P. Randomized phase III study
comparing irradiation and hyperthermia with radiation alone in superficial measureable tumors. Am. J.
Clin. Oncol. 1991,14, 133–141. [CrossRef] [PubMed]
130. Overgaard, J. (Aarhus University, Aarhus, Denmark). Personal communication, 2018.
131.
Lutgens, L.C.; Koper, P.C.; Jobsen, J.J.; van der Steen-Banasik, E.M.; Creutzberg, C.L.; van den Berg, H.A.;
Ottevanger, P.B.; van Rhoon, G.C.; van Doorn, H.C.; Houben, R.; et al. Radiation therapy combined with
hyperthermia versus cisplatin for locally advanced cervical cancer: Results of the randomized RADCHOC
trial. Radiother. Oncol. 2016,120, 378–382. [CrossRef] [PubMed]
132.
Dewhirst, M.W.; Lee, C.-T.; Ashcraft, K.A. The future of biology in driving the field of hyperthermia.
Int. J. Hyperth. 2016,32, 4–14. [CrossRef] [PubMed]
Cancers 2019,11, 60 19 of 20
133.
Frey, B.; Weiss, E.M.; Rubner, Y.; Wunderlich, R.; Ott, O.J.; Sauer, R.; Fietkau, R.; Gaipl, U.S. Old and new
facts about hyperthermia-induced modulations of the immune system. Int. J. Hyperth.
2012
,28, 528–542.
[CrossRef] [PubMed]
134.
Repasky, E.A.; Evans, S.S.; Dewhirst, M.W. Temperature matters! And why it should matter to tumor
immunologists. Cancer Immunol. Res. 2013,1, 210–216. [CrossRef] [PubMed]
135.
Toraya-Brown, S.; Fiering, S. Local tumour hyperthermia as immunotherapy for metastatic cancer.
Int. J. Hyperth. 2014,30, 531–539. [CrossRef]
136.
Multhoff, G.; Pockley, A.G.; Streffer, C.; Gaipl, U.S. Dual role of heat shock proteins (HSPs) in anti-tumor
immunity. Curr. Mol. Med. 2012,12, 1174–1182. [CrossRef]
137.
Finkel, P.; Frey, B.; Mayer, F.; Bösl, K.; Werthmöller, N.; Mackensen, A.; Gaipl, U.S.; Ullrich, E. The dual
role of NK cells in antitumor reactions triggered by ionizing radiation in combination with hyperthermia.
Oncoimmunology 2012,5, e1101206. [CrossRef]
138.
Werthmöller, N.; Frey, B.; Rückert, M.; Lotter, M.; Fietkau, R.; Gaipl, U.S. Combination of ionising radiation
with hyperthermia increases the immunogenic potential of B16-F10 melanoma cells
in vitro
and
in vivo
.
Int. J. Hyperth. 2016,32, 23–30. [CrossRef]
139.
Barsoum, I.B.; Koti, M.; Siemens, D.R.; Graham, C.H. Mechanisms of hypoxia-mediated immune escape in
cancer. Cancer Res. 2014,74, 7185–7190. [CrossRef] [PubMed]
140.
Hatfield, S.M.; Kjaergaard, J.; Lukashev, D. Immunological mechanisms of the antitumor effects of
supplemental oxygenation. Sci. Transl. Med. 2015,7, 277ra30. [CrossRef] [PubMed]
141.
Wang, H.; Zhang, L.; Shi, Y.; Javidiparsijani, S.; Wang, G.; Li, X.; Ouyang, W.; Zhou, J.; Zhao, L.; Wang, X.; et al.
Abscopal antitumor immune effects of magnet-mediated hyperthermia at a high therapeutic temperature on
Walker-256 carcinosarcomas in rats. Oncol. Lett. 2014,7, 764–770. [CrossRef] [PubMed]
142.
Hoopes, P.J.; Mazur, C.M.; Osterberg, B.; Song, A.; Gladstone, D.J.; Steinmetz, N.F.; Veliz, F.A.; Bursey, A.A.;
Wagner, R.J.; Fiering, S.N. Effect of intra-tumoral magnetic nanoparticle hyperthermia and viral nanoparticle
immunogenicity on primary and metastatic cancer. Proc SPIE Int. Soc. Opt. Eng. 2017. [CrossRef]
143.
Clarke, M.F.; Dick, J.E.; Dirks, P.B.; Eaves, C.J.; Jamieson, C.H.; Jones, D.L.; Visvader, J.; Weissman, I.L.;
Wahl, G.M. Cancer stem cells—Perspectives on current status and future directions: AACR workshop on
cancer stem cells. Cancer Res. 2006,66, 9339–9344. [CrossRef] [PubMed]
144.
Baumann, M.; Krause, M.; Hill, R. Exploring the role of cancer stem cells in radioresistance. Nat. Rev. Cancer
2008,8, 545–554. [CrossRef] [PubMed]
145.
Hill, R.P.; Marie-Egyptienne, D.Y.T.; Hedley, D.W. Cancer stem cells, hypoxia and metastasis.
Semin. Radiat. Oncol. 2009,19, 106–111. [CrossRef]
146.
Marie-Egyptienne, D.T.; Lohse, I.; Hill, R.P. Cancer stem cells, the epithelial to mesenchymal transition (EMT)
and radioresistance: Potential role of hypoxia. Cancer Lett. 2013,341, 63–72. [CrossRef]
147.
Hill, R.P.; Milas, L. The proportion of stem cells in murine tumors. Int. J. Radiat. Oncol. Biol. Phys.
1989
,16,
513–518. [CrossRef]
148.
Yaromina, A.; Krause, M.; Thames, H.; Rosner, A.; Krause, M.; Hessel, F.; Grenman, R.; Zips, D.; Baumann, M.
Pre-treatment number of clonogenic tumour cells and their radiosensitivity are major determinants of local
tumour control after fractionated irradiation. Radiother. Oncol. 2007,83, 304–310. [CrossRef]
149.
Bao, S.; Wu, Q.; McLendon, R.E.; Hao, Y.; Shi, Q.; Hjelmeland, A.B.; Dewhirst, M.W.; Bigner, D.D.; Rich, J.N.
Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature
2006,444, 756–760. [CrossRef] [PubMed]
150.
Diehn, M.; Cho, R.W.; Lobo, N.A.; Kalisky, T.; Dorie, M.J.; Kulp, A.N.; Qian, D.; Lam, J.S.; Ailles, L.E.;
Wong, M.; et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature
2009,458, 780–783. [CrossRef] [PubMed]
151.
Kim, Y.; Lin, Q.; Glazer, P.M.; Yun, Z. Hypoxic tumor microenvironment and cancer cell differentiation.
Curr. Mol. Med. 2009,9, 425–434. [CrossRef] [PubMed]
152.
Verbovsek, U.; van Noorden, C.J.F.; Lah, T.T. Complexity of cancer protease biology: Cathepsin K expression
and function in cancer progression. Semin. Cancer Biol. 2015,35, 71–84. [CrossRef] [PubMed]
153.
Hira, V.V.; Ploegmakers, K.J.; Grevers, F.; Verbovšek, U.; Silvestre-Roig, C.; Aronica, E.; Tigchelaar, W.;
Turnšek, T.L.; Molenaar, R.J.; Van Noorden, C.J. CD133+ and nestin+ glioma stem-like cells reside around
CD31+ arterioles in niches that express SDF-1
α
, CXCR4, osteopontin and cathepsin K. J. Histochem. Cytochem.
2015,63, 481–493. [CrossRef] [PubMed]
Cancers 2019,11, 60 20 of 20
154.
Wang, W.; Long, L.; Wang, L.; Tan, C.; Fei, X.; Chen, L.; Huang, Q.; Liang, Z. Knockdown of cathepsin L
promotes radiosensitivity of glioma stem cells both
in vivo
and
in vitro
.Cancer Lett.
2016
,371, 274–284.
[CrossRef]
155.
Sudhan, D.R.; Siemann, D.W. Cathepsin L inhibition by the small molecule KGP94 suppresses tumor
microenvironment enhanced metastasis associated cell functions of prostate and breast cancer cells.
Clin. Exp. Metastasis 2013,30, 891–902. [CrossRef]
156.
Oei, A.L.; Vriend, L.E.; Krawczyk, P.M.; Horsman, M.R.; Franken, N.A.; Crezee, J. Targeting therapy-resistant
cancer stem cells by hyperthermia. Int. J. Hyperth. 2017,33, 419–427. [CrossRef]
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
... Maximal thermal enhancement of radiotherapy in pre-clinical and theoretical models occurs when the two modalities are given simultaneously or within a short time interval between the two [14]. The effect of time interval on radiosensitization is the result of the effects of HT on DNA damage repair [14]. ...
... Maximal thermal enhancement of radiotherapy in pre-clinical and theoretical models occurs when the two modalities are given simultaneously or within a short time interval between the two [14]. The effect of time interval on radiosensitization is the result of the effects of HT on DNA damage repair [14]. Retrospective analysis of the impact of time interval between HT and radiotherapy has been controversial for cervix cancer [15][16][17][18]. ...
... It has been speculated that reoxygenation rarely occurs hours to days after HT in human subjects; if it does occur, it has little to do with enhancing cell killing by radiotherapy [14]. Given the complexity of physiologic effects that occur in tumors in response to HT, this challenge requires rigorous and critical thought. ...
Article
Full-text available
Numerous randomized trials have revealed that hyperthermia (HT) + radiotherapy or chemotherapy improves local tumor control, progression free and overall survival vs. radiotherapy or chemotherapy alone. Despite these successes, however, some individuals fail combination therapy; not every patient will obtain maximal benefit from HT. There are many potential reasons for failure. In this paper, we focus on how HT influences tumor hypoxia, since hypoxia negatively influences radiotherapy and chemotherapy response as well as immune surveillance. Pre-clinically, it is well established that reoxygenation of tumors in response to HT is related to the time and temperature of exposure. In most pre-clinical studies, reoxygenation occurs only during or shortly after a HT treatment. If this were the case clinically, then it would be challenging to take advantage of HT induced reoxygenation. An important question, therefore, is whether HT induced reoxygenation occurs in the clinic that is of radiobiological significance. In this review, we will discuss the influence of thermal history on reoxygenation in both human and canine cancers treated with thermoradiotherapy. Results of several clinical series show that reoxygenation is observed and persists for 24–48 h after HT. Further, reoxygenation is associated with treatment outcome in thermoradiotherapy trials as assessed by: (1) a doubling of pathologic complete response (pCR) in human soft tissue sarcomas, (2) a 14 mmHg increase in pO2 of locally advanced breast cancers achieving a clinical response vs. a 9 mmHg decrease in pO2 of locally advanced breast cancers that did not respond and (3) a significant correlation between extent of reoxygenation (as assessed by pO2 probes and hypoxia marker drug immunohistochemistry) and duration of local tumor control in canine soft tissue sarcomas. The persistence of reoxygenation out to 24–48 h post HT is distinctly different from most reported rodent studies. In these clinical series, comparison of thermal data with physiologic response shows that within the same tumor, temperatures at the higher end of the temperature distribution likely kill cells, resulting in reduced oxygen consumption rate, while lower temperatures in the same tumor improve perfusion. However, reoxygenation does not occur in all subjects, leading to significant uncertainty about the thermal–physiologic relationship. This uncertainty stems from limited knowledge about the spatiotemporal characteristics of temperature and physiologic response. We conclude with recommendations for future research with emphasis on retrieving co-registered thermal and physiologic data before and after HT in order to begin to unravel complex thermophysiologic interactions that appear to occur with thermoradiotherapy.
... In some studies, HT was performed once or twice a week, but the frequency of HT was not the same for all patients, and the total number of the HT treatment session differed in each patient [23]. According to the quality assurance guidelines for HT [26,27], the general duration time of the HT treatment should be 30-60 min with a goal temperature of 40-44 • C, and the interval between HT and RT usually ranges from some minutes to 4 h [28]. However, most of these suggestions have until now not been evaluated for the immune effects of HT and, particularly, the influence of the sequence of HT and RT application on the immune phenotype of tumor cells is still unknown. ...
... Even though the sequence of application might affect several cellular processes [28,72], it does not significantly impact on the immune phenotype of the surface of breast cancer cells. In addition to the sequence, different time intervals between RT and HT should be analyzed in the future in vitro and particularly in vivo, also taking into account the oxygenation status of the tissues [73]. ...
Article
Full-text available
Citation: Sengedorj, A.; Hader, M.; Heger, L.; Frey, B.; Dudziak, D.; Fietkau, R.; Ott, O.J.; Scheidegger, S.; Barba, S.M.; Gaipl, U.S.; et al. The Effect of Hyperthermia and Radiotherapy Sequence on Cancer Cell Death and the Immune Phenotype of Breast Cancer Cells.
... The range of the tumour blood perfusion rate is also much larger than those of fibroglandular and fat breast tissue. This is possibly due to the increased vascularisation via angiogenesis typical of tumours, which presents a chaotic system of new blood vessels that supply the tumour with oxygen [82]. ...
Article
Full-text available
Electromagnetic thermal therapies for cancer treatment, such as microwave hyperthermia, aim to heat up a targeted tumour site to temperatures within 40 and 44 °C. Computational simulations used to investigate such heating systems employ the Pennes’ bioheat equation to model the heat exchange within the tissue, which accounts for several tissue properties: density, specific heat capacity, thermal conductivity, metabolic heat generation rate, and blood perfusion rate. We present a review of these thermal and physiological properties relevant for hyperthermia treatments of breast including fibroglandular breast, fatty breast, and breast tumours. The data included in this review were obtained from both experimental measurement studies and estimated properties of human breast tissues. The latter were used in computational studies of breast thermal treatments. The measurement methods, where available, are discussed together with the estimations and approximations considered for values where measurements were unavailable. The review concludes that measurement data for the thermal and physiological properties of breast and tumour tissue are limited. Fibroglandular and fatty breast tissue properties are often approximated from those of generic muscle or fat tissue. Tumour tissue properties are mostly obtained from approximating equations or assumed to be the same as those of glandular tissue. We also present a set of reliable data, which can be used for more accurate modelling and simulation studies to better treat breast cancer using thermal therapies.
... This is also evident in multiple in vitro experiments on specific prostate cancer cell lines [15][16][17]. Other than that, the ability of hyperthermia to increase perfusion, increase reoxygenation, and overcome radiation-resistant hypoxia [18] could enable a reinvestigation of single fraction treatments, since the lack of reoxygenation and hypoxic cells are presumed to be a possible cause of failure, according to Morton and Hoskin [19]. ...
Article
Full-text available
The road of acceptance of oncologic thermotherapy/hyperthermia as a synergistic modality in combination with standard oncologic therapies is still bumpy [...]
... HT, which has been shown to be effective in treating a wide variety of cancers, including GBM, increases the therapeutic effects of other cancer treatment modalities [33,34]. For example, HT has been found to increase the radiation sensitivity of radiation-resistant solid tumors by increasing oxygen delivery to hypoxic regions [35]. In addition, combinations of HT with radiation therapy and immunotherapy have been reported to inhibit the growth of cancers refractory to radiation therapy and immunotherapy [36]. ...
Article
Glioblastoma multiforme (GBM), the most common type of brain tumor, is a very aggressive and treatment-refractory cancer, with a 5-year survival rate of approximately 5%. Hyperthermia (HT) and tumor treating fields (TTF) therapy have been used to treat cancer, either alone or in combination with other treatment methods. Both treatments have been reported to increase the efficacy of other treatment techniques and to improve patient prognosis. The present study evaluated the therapeutic effects of combining HT and TTF on GBM cell lines. Cells were subjected to HT, TTF, HT+TTF, or neither treatment, followed by comparisons of cell proliferation, apoptosis, migration and invasiveness. Clonogenic assays showed that the two treatments had a synergistic effect. The levels of cleaved PARP and cleaved caspase-3 were higher and apoptosis was increased in cells treated with HT+TTF than in cells treated with HT or TTF alone. In addition, HT+TTF showed greater inhibition of GBM cell migration and invasiveness and greater downregulation of STAT3 than either HT or TTF alone. The stronger anticancer effect of HT+TTF suggested that this combination treatment can increase the survival rate of patients with difficult-to-treat cancers such as GBM.
... This is also evident in multiple in vitro experiments on specific prostate cancer cell lines [15][16][17]. Other than that, the ability of hyperthermia to increase perfusion, increase reoxygenation, and overcome radiation-resistant hypoxia [18] could enable a reinvestigation of single fraction treatments, since the lack of reoxygenation and hypoxic cells are presumed to be a possible cause of failure, according to Morton and Hoskin [19]. ...
Article
Full-text available
In high-dose-rate brachytherapy (HDR-BT) for prostate cancer treatment, interstitial hyperthermia (IHT) is applied to sensitize the tumor to the radiation (RT) dose, aiming at a more efficient treatment. Simultaneous application of HDR-BT and IHT is anticipated to provide maximum radiosensitization of the tumor. With this rationale, the ThermoBrachyTherapy applicators have been designed and developed, enabling simultaneous irradiation and heating. In this research, we present a method to optimize the three-dimensional temperature distribution for simultaneous HDR-BT and IHT based on the resulting equivalent physical dose (EQDphys) of the combined treatment. First, the temperature resulting from each electrode is precomputed. Then, for a given set of electrode settings and a precomputed radiation dose, the EQDphys is calculated based on the temperature-dependent linear-quadratic model. Finally, the optimum set of electrode settings is found through an optimization algorithm. The method is applied on implant geometries and anatomical data of 10 previously irradiated patients, using reported thermoradiobiological parameters and physical doses. We found that an equal equivalent dose coverage of the target can be achieved with a physical RT dose reduction of 20% together with a significantly lower EQDphys to the organs at risk (p-value < 0.001), even in the least favorable scenarios. As a result, simultaneous ThermoBrachyTherapy could lead to a relevant therapeutic benefit for patients with prostate cancer.
Article
Antibodies have become an important class of biological products in cancer treatments such as radiotherapy. The growing therapeutic applications have driven a demand for high-purity antibodies. Affinity chromatography with a high affinity and specificity has always been utilized to separate antibodies from complex mixtures. Quality chromatographic components (matrices and affinity ligands) have either been found or generated to increase the purity and yield of antibodies. More importantly, some matrices (mainly particles) and affinity ligands (including design protocols) for antibody purification can act as radiosensitizers or carriers for therapeutic radionuclides (or for radiosensitizers) either directly or indirectly to improve the therapeutic efficiency of radiotherapy. This paper provides a brief overview on the matrices and ligands used in affinity chromatography that are involved in antibody purification and emphasizes their applications in radiotherapy to enrich potential approaches for improving the efficacy of radiotherapy.
Article
Microscale oxygenation plays a prominent role in tumour progression. Spatiotemporal variability of oxygen distribution in the tumour microenvironment contributes to cellular heterogeneity and to the emergence of normoxic and hypoxic populations. Local levels of oxygen strongly affect the response of tumours to the administration of different therapeutic modalities and, more generally, to the phenomenon of resistance to treatments. Several interventions have been proposed to improve tumour oxygenation, being the elevation of the local temperature (hyperthermia) an important one. While other factors such as the metabolic activity have to be considered, the proficiency of the tumour vascular system is a key factor both for the tissue oxygenation and for its temperature maps. Consequently, the interplay of these factors has attracted considerable attention from the mathematical modelling perspective. Here we put forward a transport-based system of partial differential equations aimed at describing the dynamics of healthy and tumour cell subpopulations at the microscale in a region placed between two blood vessels. By using this model with diverse flow conditions, we analyse the oxygen and temperature profiles that arise in different scenarios of vascular status, both during free progression and under thermal therapy. We find that low oxygen levels are associated to elevations of temperature in locations preferentially populated by hypoxic cells, and hyperthermia-induced cell death, being strongly dependent on blood flow, would only appear under highly disrupted conditions of the local vasculature. This results in a noticeable effect of heat on hypoxic cells. Additionally, when pronounced cell death occurs, it is followed by a significant increase in the oxygen levels. Our results provide quantitative insight to the physiological and biological processes taking place at sub-voxel sizes, currently not accessible to standard functional imaging due to spatial resolution limitations.
Article
Nanoparticle mediated hyperthermia has been explored as a method to increase cancer treatment efficacy by heating tumours inside-out. With that purpose, nanoparticles have been designed and their properties tailored to respond to external stimuli and convert the supplied energy into heat, therefore inducing damage to tumour cells. Moreover, the combination of hyperthermia with chemotherapy has been described as a more effective strategy due to the synergy between the high temperature and the drug’s effects, also associated with a remote controlled and on-demand drug release. In this review, the methods behind nanoparticle mediated hyperthermia, namely material design, external stimuli response and energy conversion will be discussed and critically analysed. We will address the most relevant studies on hyperthermia and temperature triggered drug release for cancer treatment. Finally, the advantages, difficulties and challenges of this therapeutic strategy will be discussed, while giving insight for future developments.
Article
Transcription factors HIF1 and HIF2 are central regulators of physiological responses to hypoxia and important for normal functioning of tissue stem cells and maintenance of healthy microvasculature. Even modest decreases in HIF activity exert detrimental effects in tissues although it is unclear what factors can directly impair HIF functions. We hypothesized that the presence of functionally important, large intrinsically disordered regions in HIFα subunits of HIF1/2 could make them structurally vulnerable to protein-damaging conditions. We found that common protein-damaging agents such as endogenous/exogenous aldehydes (formaldehyde, acetaldehyde), moderate heat shock and the environmental toxicant cadmium cause inactivation of HIF1 and HIF2 due to structural damage to HIFα subunits. Aldehydes triggered a rapid and selective depletion of HIF1α and HIF2α, which occurred as a result of enhanced binding of Pro-hydroxylated/VHL-ubiquitinated HIFα by 26S proteasomes. In the absence of proteasomal degradation, aldehyde-damaged HIF1 and HIF2 were transactivation defective and HIFα subunits became insoluble/denatured when their VHL-mediated ubiquitination was blocked. Protein damage by heat shock and cadmium resulted in the insolubility of Pro-nonhydroxylated HIFα. Thus, VHL-dependent ubiquitination of damaged HIFα also acts as means to maintain their solubility, permitting capture by proteasomes. The observed control of HIFα stability at the point of proteasome binding may extend to several posttranslational modifications that occur in the conformationally flexible regions of these proteins. Our findings revealed vulnerability of HIF1 and HIF2 to direct inactivation by protein-damaging agents, which helps understand their tissue injury mechanisms and favorable responses of hypoxic tumors to hyperthermia.
Article
Full-text available
Treatment modalities for cancer radiation therapy have become increasingly diversified given the growing number of facilities providing proton and carbon-ion therapy in addition to the more historically accepted photon therapy. An understanding of high-LET radiobiology is critical for optimization of charged particle radiation therapy and potential DNA damage response. In this review, we present a comprehensive summary and comparison of these types of therapy monitored primarily by using DNA damage biomarkers. We focus on their relative profiles of dose distribution and mechanisms of action from the level of nucleic acid to tumor cell death.
Article
Full-text available
Carbon ion therapy is a promising evolving modality in radiotherapy to treat tumors, which are radioresistant against photon treatments. As carbon ions are more effective in normal and tumor tissue, the relative biological effectiveness (RBE) has to be calculated by bio-mathematical models and has to be considered in the dose prescription. This review (i) intro-duces the concept of the RBE and its most important determinants, (ii) describes the physical and biological causes of the increased RBE for carbon ions, (iii) summarizes available RBE-measurements in-vitro and in-vivo, describes the (iv) concepts of the clinically applied RBE models (Mixed beam model, Local effect model, Microdosimetric-kinetic model), and (v) the way they are introduced into clinical application as well as (vi) their status of experimental and clinical validation, and finally (vii) summarizes the current status of the use of the RBE-concept in carbon ion therapy and points out clinically relevant conclusions as well as open questions. The RBE concept has proven to be a valuable concept for dose prescription in carbon ion radiotherapy, however, different centers use different RBE-models and therefore care has to be taken, when transferring results from one center to another. Experimental studies significantly improve the understanding of dependencies and limitations of the RBE in clinical application. For the future more studies investigating quantitatively the differential effects between normal tissues and tumors are needed accompanied by clinical studies on effectiveness and toxicity.
Article
Full-text available
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.
Article
Full-text available
Background: Cellular responses to proton beam irradiation are not yet clearly understood, especially differences in the relative biological effectiveness (RBE) of high-energy proton beams depending on the position on the Spread-Out Bragg Peak (SOBP). Towards this end, we investigated the differences in the biological effect of a high-energy proton beam on the target cells placed at different positions on the SOBP, using two human esophageal cancer cell lines with differing radiosensitivities. Methods: Two human esophageal cancer cell lines (OE21, KYSE450) with different radiosensitivities were irradiated with a 235-MeV proton beam at 4 different positions on the SOBP (position #1: At entry; position #2: At the proximal end of the SOBP; position #3: Center of the SOBP; position #4: At the distal end of the SOBP), and the cell survivals were assessed by the clonogenic assay. The RBE10 for each position of the target cell lines on the SOBP was determined based on the results of the cell survival assay conducted after photon beam irradiation. In addition, the number of DNA double-strand breaks was estimated by quantitating the number of phospho-histone H2AX (γH2AX) foci formed in the nuclei by immunofluorescence analysis. Results: In regard to differences in the RBE of a proton beam according to the position on the SOBP, the RBE value tended to increase as the position on the SOBP moved distally. Comparison of the residual number of γH2AX foci at the end 24 h after the irradiation revealed, for both cell lines, a higher number of foci in the cells irradiated at the distal end of the SOPB than in those irradiated at the proximal end or center of the SOBP. Conclusions: The results of this study demonstrate that the RBE of a high-energy proton beam and the cellular responses, including the DNA damage repair processes, to high-energy proton beam irradiation, differ according to the position on the SOBP, irrespective of the radiosensitivity levels of the cell lines.
Article
Full-text available
Hyperthermia (HT) is one of the hot topics that have been discussed over decades. However, it never made its way into primetime. The basic biological rationale of heat to enhance the effect of radiation, chemotherapeutic agents, and immunotherapy is evident. Preclinical work has confirmed this effect. HT may trigger changes in perfusion and oxygenation as well as inhibition of DNA repair mechanisms. Moreover, there is evidence for immune stimulation and the induction of systemic immune responses. Despite the increasing number of solid clinical studies, only few centers have included this adjuvant treatment into their repertoire. Over the years, abundant prospective and randomized clinical data have emerged demonstrating a clear benefit of combined HT and radiotherapy for multiple entities such as superficial breast cancer recurrences, cervix carcinoma, or cancers of the head and neck. Regarding less investigated indications, the existing data are promising and more clinical trials are currently recruiting patients. How do we proceed from here? Preclinical evidence is present. Multiple indications benefit from additional HT in the clinical setting. This article summarizes the present evidence and develops ideas for future research.
Article
Full-text available
Background Combined radiotherapy and hyperthermia is a well-established alternative to chemoradiotherapy for advanced stage cervical cancer patients with a contraindication for chemotherapy. Pre-clinical evidence suggests that the radiosensitizing effect of hyperthermia decreases substantially for time intervals between radiotherapy and hyperthermia as short as 1–2 h, but clinical evidence is limited. The purpose of this study is to determine the effect of the time interval between external beam radiotherapy (EBRT) and same-day hyperthermia on in-field recurrence rate, overall survival and late toxicity in women with advanced stage cervical cancer. Methods Patients with advanced stage cervical cancer who underwent a full-course of curative daily EBRT and (4–5) weekly hyperthermia sessions between 1999 and 2014 were included for retrospective analysis. The mean time interval between EBRT fractions and same-day hyperthermia was calculated for each patient; the median thereof was used to divide the cohort in a ‘short’ and ‘long’ time-interval group. Kaplan-Meier analysis and stepwise Cox regression were used to compare the in-field recurrence and overall survival. Finally, high-grade (≥3) late toxicity was compared across time-interval groups. DNA repair suppression is an important hyperthermia mechanism, DNA damage repair kinetics were therefore studied in patient biopsies to support clinical findings. ResultsIncluded were 58 patients. The 3-year in field recurrence rate was 18% and 53% in the short (≤79.2 min) and long (>79.2 min) time-interval group, respectively (p = 0.021); the 5-year overall survival was 52% and 17% respectively (p = 0.015). Differences between time-interval groups remained significant for both in-field recurrence (HR = 7.7, p = 0.007) and overall survival (HR = 2.3, p = 0.012) in multivariable Cox regression. No difference in toxicity was observed (p = 1.00), with only 6 and 5 events in the short and long group, respectively. The majority of DNA damage was repaired within 2 h, potentially explaining a reduced effectiveness of hyperthermia for long time intervals. ConclusionsA short time interval between EBRT and hyperthermia is associated with a lower risk of in-field recurrence and a better overall survival. There was no evidence for difference in late toxicity.
Article
Full-text available
Purpose: Biological modelling of thermoradiotherapy may further improve patient selection and treatment plan optimization, but requires a model that describes the biological effect as a function of variables that affect treatment outcome (e.g. temperature, radiation dose). This study aimed to establish such a model and its parameters. Additionally, a clinical example was presented to illustrate the application. Methods: Cell survival assays were performed at various combinations of radiation dose (0–8Gy), temperature (37–42°C), time interval (0–4h) and treatment sequence (radiotherapy before/after hyperthermia) for two cervical cancer cell lines (SiHa and HeLa). An extended linear-quadratic model was fitted to the data using maximum likelihood estimation. As an example application, a thermoradiotherapy plan (23 × 2Gy+weekly hyperthermia) was compared with a radiotherapy-only plan (23 × 2Gy) for a cervical cancer patient. The equivalent uniform radiation dose (EUD) in the tumour, including confidence intervals, was estimated using the SiHa parameters. Additionally, the difference in tumour control probability (TCP) was estimated. Results: Our model described the dependency of cell survival on dose, temperature and time interval well for both SiHa and HeLa data (R²=0.90 and R²=0.91, respectively), making it suitable for biological modelling. In the patient example, the thermoradiotherapy plan showed an increase in EUD of 9.8Gy that was robust (95%CI: 7.7–14.3Gy) against propagation of the uncertainty in radiobiological parameters. This corresponded to a 20% (95%CI: 15–29%) increase in TCP. Conclusions: This study presents a model that describes the cell survival as a function of radiation dose, temperature and time interval, which is essential for biological modelling of thermoradiotherapy treatments.
Article
Background It has been known for over a hundred years that tumor hypoxia, a near-universal characteristic of solid tumors, decreases the curative effectiveness of radiation therapy. However, to date, there are no reports that demonstrate an improvement in radiation effectiveness in a mammalian tumor based on tumor hypoxia localization and local hypoxia treatment. Methods For radiation targeting of hypoxic subregions in a mouse fibrosarcoma, we used oxygen images obtained using electron paramagnetic resonance (EPR) pO2 imaging combined with 3D-printed radiation blocks. This achieved conformal radiation delivery to all hypoxic areas in FSa fibrosarcomas in mice. Results We demonstrate that the treatment delivering a radiation boost to hypoxic volumes has a significant (p=0.04) doubling of tumor control relative to boosts to well-oxygenated volumes. Additional dose to well oxygenated tumor regions minimally increases tumor control beyond the 15% control dose to the entire tumor. If we can identify portions of the tumor that are more resistant to radiation it may be possible to reduce the dose to more sensitive tumor volumes without significant compromise in tumor control. Conclusion This work demonstrates in a single intact mammalian tumor type that tumor hypoxia is a local tumor phenomenon whose treatment can be enhanced by local radiation. Despite enormous clinical effort to overcome hypoxic radiation resistance, this is the first such demonstration, even in preclinical models, of targeting additional radiation to hypoxic tumor to improve therapeutic ratio.
Article
In the clinic, proton beam therapy (PBT) is based on the use of a generic relative biological effectiveness (RBE) of 1.1 compared to photons in human cancers and normal tissues. However, the experimental basis for this RBE lacks any significant number of representative tumor models and clinically relevant endpoints for dose-limiting organs at risk. It is now increasingly appreciated that much of the variations of treatment responses in cancers are due to inter-tumoral genomic heterogeneity. Indeed, recently it has been shown that defects in certain DNA repair pathways, which are found in subsets of many cancers, are associated with a RBE increase in vitro. However, there currently exist little in vivo or clinical data that confirm the existence of similarly increased RBE values in human cancers. Furthermore, evidence for variable RBE values for normal tissue toxicity has been sparse and conflicting to date. If we could predict variable RBE values in patients, we would be able to optimally use and personalize PBT. For example, predictive tumor biomarkers may facilitate selection of patients with proton-sensitive cancers previously ineligible for PBT. Dose de-escalation may be possible to reduce normal tissue toxicity, especially in pediatric patients. Knowledge of increased tumor RBE may allow us to develop biologically optimized therapies to enhance local control while RBE biomarkers for normal tissues could lead to a better understanding and prevention of unusual PBT-associated toxicity. Here, we will review experimental data on the repair of proton damage to DNA that impact both RBE values and biophysical modeling to predict RBE variations. Experimental approaches for studying proton sensitivity in vitro and in vivo will be reviewed as well and recent clinical findings discussed. Ultimately, therapeutically exploiting the understudied biological advantages of protons and developing approaches to limit treatment toxicity should fundamentally impact the clinical use of PBT.
Article
The physical properties of proton beams offer the potential to reduce toxicity in tumor-adjacent normal tissues. Toward this end, the number of proton radiotherapy facilities has steeply increased over the last 10-15 years to currently around 70 operational centers worldwide. However, taking full advantage of the opportunities offered by proton radiation for clinical radiotherapy requires a better understanding of the radiobiological effects of protons alone or combined with drugs or immunotherapy on normal tissues and tumors. This report summarizes the main results of the international expert workshop "Radiobiology of Proton Therapy" that was held in November 2016 in Dresden. It addresses the major topics (1) relative biological effectiveness (RBE) in proton beam therapy, (2) interaction of proton radiobiology with radiation physics in current treatment planning, (3) biological effects in proton therapy combined with systemic treatments, and (4) testing biological effects of protons in clinical trials. Finally, important research avenues for improvement of proton radiotherapy based on radiobiological knowledge are identified. The clinical distribution of radiobiological effectiveness of protons alone or in combination with systemic chemo- or immunotherapies as well as patient stratification based on biomarker expressions are key to reach the full potential of proton beam therapy. Dedicated preclinical experiments, innovative clinical trial designs, and large high-quality data repositories will be most important to achieve this goal.