Radiosensitization with hypertherrmia and chemotherapeutic agents

Hyperthermia refers to heat treatments if cells or malignancies in which the temperature is elevated in the range
of 39C to 45C. It is used in combination with chemo- and/or radiotherapy since it is has been shown to
enhance the anti-cancer effects of both therapies (Gonzalez Gonzalez et al., 1995; Van Der Zee et al., 2002;
Crezee et al. 2009). Many in vitro studies on the combination of hyperthermia and radiation have shown a
synergistic interaction between the two modalities, especially at higher temperatures (above 42ºC) (Dewey et
al., 1978; Roti Roti, 2004; Raaphorst et al., 1991). This interaction is believed to result from inhibition of repair
of radiation-induced DNA damage by hyperthermia (Kampinga et al., 2001; Hildebrandt et al., 2002). The
sequence of combined radiation and hyperthermia treatment is important. Optimal sensitization is obtained
when radiation and hyperthermia are applied simultaneously or with a short interval (Hall & Giaccio, 2006). In
the clinic this is not always possible. In our experiments hyperthermia was applied immediately after radiation
treatment
Recently it has been shown that
hyperthermia (42 ºC for 1h) transiently breaks down the BRCA2 protein (Krawczyk et al., 2011). In this paragraph the
effects of hyperthermia treatment for 1h at 41 or 43 C on the linear quadratic parameters are summarized. Several
different cell types have been studied Human colon carcinoma cell line.RKO is a poorly differentiated colon
carcinoma cell line developed
Plateau (or Stationary) Phase – cellular proliferation slows down due to the cell population becoming confluent This
paper describes the response of cultured mammalian cells to periods of elevated temperatures. It was found that 43° C
was necessary to produce substantial cell killing in aerated cells. Those made hypoxic in dense cultures were very
sensitive to hyperthermia, cell killing taking place even at 41° C. Survival curves were obtained for proliferating and
plateau phase cells exposed at 43° C for periods of 1 to 4 hours. There is no dramatic difference in sensitivity between
cells in these two metabolic states, although the survival curve may be slightly steeper for plateau phase cells.
Hyperthermia may be a useful adjunct to radiotherapy since these areas of the tumor which are most resistant to
conventional x rays are sensitive to heat
Chemotherapeutic agents, anticancer drug also referred to as antineoplastic agents, are used to directly or indirectly
inhibit the proliferation of rapidly growing cells, typically in the context of malignancy
the therapeutic use of chemical agents to treat disease especially : the administration of one or more cytotoxic drugs to
destroy or inhibit the growth and division of malignant cells in the treatment of cancer.
The linear-quadratic model is one of the key tools in radiation biology and physics. It provides a simple relationship
between cell survival and delivered dose: , and has been used extensively to analyse and predict responses to ionising
radiation both in vitro and in vivo

The identification of novel targets for developing synergistic drug–radiation combinations would pave the way to
overcome tumor radioresistance
Radiotherapy is one of the major therapeutic strategies for cancer treatment. In the past decade, there has been
growing interest in using high Z (atomic number) elements (materials) as radiosensitizers. New strategies in
nanomedicine could help to improve cancer diagnosis and therapy at cellular and molecular levels.
but the mechanism for selectivity against cancer cells compared with normal cells is not yet determined. The
identification of the enzymatic targets for these drugs offers the potential to develop predictive assays for response and
to develop methods of imaging the progress of therapy
It is not an exaggeration to state that most of the advances in curing cancer in the last decade have come from
successful combinations of conventional chemotherapeutic agents with radiation therapy. Further improvements in
therapy will depend on understanding the mechanisms by which chemotherapy improves the effectiveness of radiation
in model systems and in patients.  
Increased radiation sensitivity occurs in cells that progress inappropriately into S phase in the presence of drug,
suggesting a key role for dysregulation of S-phase checkpoints.
Radiosensitization remains an attractive mechanism for enhancement, but, again, clear evidence in its favor is lacking
although it has been ruled out specifically for bleomycin, mitomycin C, and spirohydantoin mustard
The use of a drug that makes tumor cells more sensitive to radiation therapy
ion. Radiosensitization is a physical, chemical, or pharmacological intervention that increases the lethal effects of
radiation when administered in conjunction with it
Radiosensitization is a physical, chemical, or pharmacological intervention that increases the lethal effects of radiation
when administered in conjunction with it. In experimental studies or in clinical practice, the aim of a radiosensitization
approach (or the use of  radiosensitizers) is to make tumor cells more sensitive to  radiation therapy. A clinical benefit
can be expected only if a differential effect can be demonstrated between tumors and normal tissues.
A radiosensitizer is an agent that makes tumor cells more sensitive to radiation therapy. It is sometimes also known as a
radiation sensitizer or radio-enhance
Hyperthermia refers to a group of heat-related conditions characterized by an abnormally high body temperature — in
other words, the opposite of hypothermia. The condition occurs when the body's heat-regulation system becomes
overwhelmed by outside factors, causing a person's internal temperature to rise
Hyperthermia occurs when the body's temperature raises to levels above normal (but is different from a fever due to an
illness or infection). It's usually caused by exertion in a hot environment and varies in severity based on how hot the
body gets
Hyperthermia is a group of conditions where the body becomes too hot and cannot regulate its temperature. ...
Severe dehydration, however, can strip the body of its ability to cool. Without treatment, this can result in dangerously
high body temperatures and life-threatening conditions, including organ failure and death
Hyperthermia is an abnormally high body temperature caused by a failure of the heat-regulating mechanisms of the
body to deal with the heat coming from the environment.
Hyperthermia is defined as elevated core temperature of greater than 38.5°C  (101.3°F). History and clinical examination
can help elucidate the etiology of hyperthermia and tailor treatment. The causes of hyperthermia include the
following [4] :
Increased ambient heat - Heat waves, humidity

(2019) 10:3134 | https://doi.org/10.1038/s41467-019-10966-8 www.nature.com/naturecommunications

R65L, N70H and P95R disrupt Tumor Suppressor Function

 WT OPCML completely abolished cell migration in SKOV3 and PEA1 (PEO1 cells did not migrate at all), and
invasion and growth in all three cell lines compared to control cells
 However, all the mutants showed loss of function in the three phenotypic assays
To evaluate the effect of these mutations on the tumor suppressor function of OPCML, we used three different ovarian
cancer cell lines: SKOV3 and PEA1, which have mesenchymal characteristics, and PEO1, which has epithelial
characteristics These were stably transduced with WT or mutant OPCML (R65L, N70H, P95R) or an empty control vector
(CTRL) and characterized
Compounds that increase cancer cell sensitivity to radiation ('radiosensitizers') have been used in an effort to optimize radiation
therapy.
Integrating Hyperthermia into Modern Radiation Oncology: What Evidence Is Necessary?
Hyperthermia (HT) is defined as an exogenous, supraphysiological elevation of tissue/body temperature. Nowadays, HT is either
administered independently or, more often, in combination with radiotherapy (RT) or chemotherapy (CT). HT alone is being used for
direct ablation of single tumor lesions with temperatures exceeding 50°C. Multiple techniques are being used to obtain necessary
temperature coverage such as high-intensity focused ultrasound and radiofrequency-, microwave-, or infrared laser-based
heating via ablation catheters directly inserted into the tumor (3).
In bimodal treatment schemes such as thermoradiotherapy (RTHT) and chemoradiotherapy (RTCT) as well as in trimodal
thermochemoradiotherapy (RTHTCT), HT is utilized for augmentation of treatment effects of the concomitant oncological therapy.
Necessary tissue temperatures are significantly lower ranging from 39 to 43°C (4, 5).
In this literature-based review, a brief introduction to HT physiology, cell biology, and immune response is given to examine the
underlying modes of action of HT. Currently used HT techniques for heat delivery and temperature control are described. The clinical
evidence of combining RT with HT is summarized and sorted per tumor entity.
Hypoxia
Unregulated cell reproduction and growth is the hallmark of many malignancies, and as a result of this unregulated growth, tumors
can often outgrow their blood supply and thus enter a relatively hypoxic state. Radioresistance due to hypoxia is a well-established
problem in cancer treatment. Radiation therapy damages tumor DNA in a variety of ways, but an important and often predominant
mechanism is the interaction of photons with water molecules to create reactive oxygen species. These species then damage tumor
DNA, which leads to cell apoptosis, necrosis, or inability to divide if unrepaired. Oxygen in the microenvironment allows for the
perpetuation of free radicals and is important in 'fixing' damaged DNA by formation of organic peroxides at damaged sites, which are
then resistant to cellular repair mechanisms (1). The importance of oxygen in the tumor microenvironment is quantified by the
oxygen
enhancement ratio (OER) and varies based on the tumor histology and on the type of therapeutic radiation (2). The hypoxic state
also results in stabilization of a transcription factor known as hypoxia-inducible factor 1 (HIF1), which is normally quickly degraded
under normoxic conditions (3). Activation of HIF1 is thought to lead to several downstream effects that seem to be associated with
higher rates of metastasis and poorer survival, at least in some types of cancer (4). Because of the adverse radiobiological and
clinical
consequences of hypoxia, improving tumor oxygenation or otherwise combating the hypoxic tumor microenvironment has been an
oncological strategy for over 50 years. Trials of hyperbaric oxygen were some of the earliest to explore hypoxic manipulation, and
although this intervention improved locoregional control and disease-specific survival, it has not translated into improvements in
overall survival based on a recent meta-analysis of head and neck cancer trials.
Changes in Perfusion and Oxygenation
Data and the respective interpretation of HT-induced changes in perfusion and oxygenation remain controversial and are briefly
described in the following. There is evidence that mild HT can increase blood perfusion of the heated tissue, preferentially at the
beginning of tumor heating (7, 8). It has been reported that this can lead to increased oxygen delivery via an improvement of
microcirculation (9). This is especially true in cases when the oxygen demand of the tissue is reduced. It has been proposed that
direct heat-dependent cell killing and loss of mitochondrial membrane potential contribute to this phenomenon (10, 11). On the
contrary, other studies showed increased oxygen consumption at elevated tissue temperature (van’t Hoff’s law!) counteracting the
oxygenating effect of increased perfusion (12). An increase in oxygen availability may favor oxygenation of hypoxic cells (7). The
effect appears to be preferentially in diffusion-limited, chronic hypoxia (13, 14). Whether the radiosensitizing effect outlasts the time
frame of increased perfusion remains so far unclear. Some studies have reported increased perfusion extending over 24 h after HT,
which would benefit following RT/CT sessions (15, 16). Other studies could not reproduce this result (17). As hypoxia is a central
causative factor for radioresistance, a decrease in hypoxia by HT may be responsible for the observed radiosensitization.
Induction of Cell Death
Hyperthermia has been shown to confer cell death by apoptosis or mitotic catastrophe (18, 19). It has been reported that HT triggers
unfolding of especially heat-labile non-histone nuclear proteins leading to aggregation, due to exposition of hydrophobic groups,
with surrounding proteins and subsequent association with the nuclear matrix. As consequence, basic nuclear matrix-dependent
functions such as transcription, replication, or DNA repair are impaired (20, 21). Malfunction of DNA replication finally causes
chromosome aberrations, genome instability, and cell death by mitotic catastrophe (22). Apoptosis may be mediated by cell death
membrane receptor activation and subsequent caspase 3 activation (23). The extent of apoptosis appears to differ among different
tumor types (24). In addition, the permeability of the cellular and mitochondrial membranes is altered leading to cellular Ca 2+-spikes
as well as mitochondrial depolarization with resulting bursts of reactive oxygen species. Both mechanisms may further enhance
protein instability and apoptosis (25–27).
Inhibition of DNA Repair Mechanisms
As mentioned above, there is sufficient evidence showing inhibition of DNA repair mechanisms upon HT. Krawczyk et al. have
demonstrated inhibition of homologous recombination at clinically achievable mild HT temperatures (41–42.5°C) associated with
BRCA2 degradation and its reduced accumulation at double-strand break sites (28). Further on, HT impairs the function of the Ku
heterodimer by reducing its DNA-binding capacity and preventing the initiation of non-homologous end joining at DNA double-
strand breaks sites (29). In addition, base excision after cell radiation has been shown to be reduced upon heat administration (30).
In summary, HT acts on multiple levels including excision repair, non-homologous end joining, and homologous recombination
influencing the repair of DNA lesions as well as single-strand and double-strand breaks (29–31). As a consequence, the effects of
DNA damaging treatments such as CT or RT are enhanced. A more detailed review was recently published discussing existing
evidence (32).
Breast Cancer
Breast cancer constitutes the most widely investigated malignant entity. Vernon et al. published the results of five randomized trials
conducted between 1981 and 1991 that were combined due to insufficient patient accrual. The pooled analysis of 306 patients with
inoperable primary or recurrent disease yielded a significantly better complete response (CR) (RTHT: 59%, RT alone: 41%, odds ratio
(OR): 2.3, p < 0.001) but no survival benefit. However, 50% of patients had metastases at the time of randomization. The effect was
most prominent in preirradiated recurrent lesions. Skin toxicities such as blisters, ulceration, and necrosis were higher in the HT
group, however with low impact on the patients well-being and generally treatable with conservative measures (60).
In cases of locoregional recurrence, breast surgery is recommended if possible. However, radical resection only appears to be
feasible in 65% of patients and may cause significant treatment-related morbidity (78). Thus, RT constitutes a significant alternative,
and depending on the time interval between first and second RT and other pretreatment characteristics offers substantial clinical
benefit. Since nowadays most patients receive RT in the primary situation, HT may help to enhance the effects of reirradiation in the
recurrence scenario, especially since in some clinical situations only reduced radiation doses may be prescribed. A recent meta-
analysis by Datta et al. included 8 two-arm studies and 24 single-arm studies involving 2,110 patients with locoregional recurrent
disease (79). CR was similar between one-arm studies (CR: 63.4%) and two-arm studies as well as significantly higher compared to RT
alone (RTHT: 60.2%, RT: 38.1%, OR 2.6, p < 0.001). In preirradiated patients (779 patients), a CR of 66.4% was achieved (mean re-RT
dose: 36.7 Gy). Treatment-related toxicity was overall not increased with mean acute grade 3/4 toxicities of 14.4%. Among the
analyzed studies, there was great heterogeneity in RT dose, HT fraction schedules, total number of HT fractions, HT duration, or
average achieved temperatures. However, no prognostic treatment variables could be identified in a subgroup analysis and meta-
regression. Similar results were recently published by Linthorst et al. In a retrospective study encompassing 248 patients with
unresectable recurrences, reirradiation + HT yielded a CR of 70% (80). In resectable cases, a regimen including surgery, RT, HT, and
partly CT or hormone therapy, a local control (LC) rate of 78% after 5 years was achieved (81). Notter et al. have treated patients
with locally recurrent breast cancer with RTHT using a hypofractionation scheme of 5 × 4 Gy with one fraction per week. A wIRA
system was used for superficial HT. A CR rate of 61% was achieved without any treatment-related toxicities (56). In summary, there
is sustained evidence demonstrating a value of adjunct HT in locoregional, recurrent breast cancer as definitive or adjuvant
treatment.
Melanoma
One multicentric randomized trial analyzed the benefit of adjunct HT in melanomas treated with RT. Patients either received RT (24
Gy or 27 Gy in three fractions) alone or with HT (43°C for 60 min). There was no significant difference in toxicity. CR (RT alone: 35%,
RTHT: 62%, p < 0.05) and LC after 2y were significantly increased (RT alone: 28%, RTHT: 46%, p = 0.008) (101). As these results are
very promising, more randomized trials would help to establish a distinct role for RTHT in the treatment of melanomas, for example
in combination with less hypofractionated treatment schemes.
Non-Small Cell Lung Cancer (NSCLC)
Only few studies have investigated the role of HT for the treatment of NSCLC. A multi-institutional prospective randomized trial
investigated the role of HT in addition to primary RT for locally advanced NSCLCs. No significant difference of OS, local response, or
treatment-related toxicity could be observed. However, with a significantly higher 1y-PFS, a certain benefit was apparent (67.5%,
29%, p = 0.036) (102). In a small case–control study encompassing 13 patients with direct bone invasion treated with RTHT (60–70
Gy) showed a possibly high efficacy in LC and survival under this unfavorable condition (103). The benefit of HT in addition to
reirradiation for recurrent NSCLC after primary RT was investigated in a small retrospective study involving 33 patients. Median
doses used initially and for reirradiation were 70 and 50 Gy, respectively. Toxicity was moderate and limited to a maximum toxicity
of grade 3 in 9% of patients. In patients with smaller tumors (<4 cm) and no distant metastases, long time survival was partly
achieved (104). All three studies employed radiofrequency capacitive heating systems. So far, only limited evidence exists showing a
true benefit of HT in NSCLC treatment. More studies are necessary to explore potential areas of application.
Sarcomas
Randomized trials have shown a significant benefit of HT in addition to CT (115). However, evidence of combined RTHT in sarcoma
treatment remains scarce. An early phase II study involving 17 soft tissue sarcomas (STS) patients neoadjuvant RTHT with twice
weekly HT showed significantly more extensive changes in histopathological examinations than the once weekly HT group (116). In
another study, 16 patients received irradiation with concurrent HT for the treatment of radiation-associated sarcomas
(predominantly angiosarcoma) showing a total response rate of 75%. Toxicity was mild except one grade 4 adverse event (117). First
clinical results show general feasibility of applying RTHT to sarcoma treatment. However, randomized trials are necessary to assess
whether a similar benefit exists as it has been shown for neoadjuvant HTCT.
RTHT in Indications with Limited Data
A Dutch retrospective trial analyzed the efficacy of hypofractionated (28/32 Gy with a single dose of 4 Gy) reirradiation with
concurrent HT for the palliation of painful unresectable recurrent rectal cancer with good to complete response in 72% of 47
patients (124). Similar results (70% pain relief) were reproduced in a prospective phase II trial by Milani et al. with
normofractionated RTCTHT (125). Klaver et al. proposed a novel treatment strategy in a case series for locally advanced rectal cancer
with concurrent peritoneal carcinomatosis by combing hyperthermic intraperitoneal chemotherapy with intraoperative RT showing
general feasibility (126).
Thermoradiotherapy was evaluated for the palliation of symptomatic locally advanced or recurrent hormone-refractory prostate
cancer in a small phase I/II study. All patients demonstrated partial response or complete response as well as complete symptom
relief (127). However, two of eight preirradiated patients developed grade IV toxicities.
Primary or recurrent locally advanced pancreatic cancer was subject to an open-label study comparing RTCT with RTHTCT
(gemcitabine ± 5FU/cisplatin/oxaliplatin) showing a significantly increased survival benefit without increased toxicity (mean OS:
RTHTCT: 15 months, RTCT: 11 months, p = 0.025) (128). Moreover, the influence of adjunct HT for the treatment of liver lesions has
been assessed. In a randomized Chinese trial of hepatocellular carcinoma patients, 1y-recurrence (RTHT: 10%, RT: 15%, p < 0.001)
and mortality rates (RTHT: 12.5, RT: 20%, p < 0.001) were significantly lower for combined RTHT compared to RT alone (129).
Chemorefractory colorectal cancer metastases were treated with whole-liver RT and concimittant HT showing partial response and
pain relief in 30% of treated patients, respectively (130). Vaginal cancers have been chosen as target for HT in small prospective
Dutch trial. Patients with vaginal carcinomas with a tumor size larger than 4 cm were treated with RTHT, whereas smaller tumors
were primarily treated with RT showing no significant difference in 5y-survival (131).
In a more general approach, Jones et al. performed a randomized prospective trial pooling superficial tumors of various entities
(breast carcinoma, melanoma, head and neck cancer, and others). Only tumors that appeared to be heatable on a pretest were
randomized. Addition of HT to RT lead to significantly increased CR (RTHT: 66.1%, RT 42.3%, OR 1.7, p = 0.02). In coherence with
many other studies, the highest difference was achieved in preirradiated patients (CR: RTHT: 68.2%, CR 23.5%) (64).
Despite the limited amount of evidence, substantial benefits of RTHT, especially for preirradiated, locally advanced and recurrent
tumors, became apparent. Since there is a lack of randomized trials, definite recommendations for treatment cannot be made. In
situations of recurrent or metastatic disease, RTHT may be justifiable as “individual treatment approach” on the basis of the existing
evidence. In order to reach necessary patient number in potential future randomized trials, multiple entities with a similar condition
(such as “recurrent,” “preirradiated,” or “locally advanced”) could be combined, following the approach of Jones et al. (64). In
addition, HT centers should work more closely together for the establishment of multicenter trials capable of gathering critical
patient numbers.
Combined Thermochemotherapy (CTHT)
Thermochemotherapy has been evaluated in multiple clinical trials. In contrast to RTHT, CTHT is also being combined with WBHT. A
limited number of phase II studies have shown feasibility of applying WBHT to CTHT treatment of various entities such as recurrent
ovarian cancer, malignant pleural mesothelioma, metastatic STS, melanoma, and pretreated metastatic colorectal cancer (132–136).
Up to date, no phase III trials exist. In contrast, regional HT has been evaluated in a large phase III trial (341 patients) of STS
performed as joint effort by the European Organization for the Research and Treatment of Cancer and European Society for
Hyperthermic Oncology. It showed a substantially and significantly improved local PFS, DFS, and OS after adding HT to EIA
(etoposide, ifosfamide, doxorubicin) CT (HR for local progression/death: 0.58, p = 0.003, local 2y-PFS: HTCT: 76%, CT: 61%; 2y-DFS:
CTHT: 58%, CT: 44%, p = 0.011; per-protocol OS: HR 0.66, p = 0.038) changing daily clinical practice in HT treatment centers (115).
Further randomized trials have evaluated neoadjuvant CTHT in esophageal carcinoma (40 patients; histologic effectiveness: CTHT
58.3, CT: 14.3, p < 0.05) and adjuvant CTHT after transurethral resection of bladder carcinomas (83 patients; 10y-DFS: CTHT: 53%, CT:
15%, p < 0.001) demonstrating increased treatment efficacy (137, 138). A randomized trial with NSCLC showed a small benefit
regarding “clinical benefit response” (80 patients, CTHT: 82.5%, CT: 47.5%, p < 0.05) (139). General feasibility of local CTHT has also
been shown in other entities such as refractory or recurrent non-testicular germ cell carcinomas, recurrent or persistent ovarian
cancer, breast carcinoma, or peritoneal carcinomatosis in several phase II studies (140–143). As alternative to regional HT,
hyperthermic isolated limb perfusion has been established for the treatment of STS and unresectable melanomas showing favorable
results (144, 145). In summary, the few existing randomized trials suggest substantial benefit by adding HT to CT. More randomized
trials are necessary to broaden the spectrum of CTHT.
Outlook
Review of the current literature has shown various retrospective and prospective trials exploring the value of adding HT to RT or
RTCT regiments in multiple tumor entities. As in some entities, the real benefit of HT remains elusive, in other malignancies
sustained evidence has been acquired. When considering the currently existing studies, a substantial part of evidence was gathered
between the 1980s and early 2000s. Apart from technical aspects of HT, RT techniques have dramatically evolved since then.
Nowadays, even better results in regard to treatment outcome as well as toxicity could be expected. Still, no widespread use of HT
has been established in the last decades. Several reasons, such as reimbursement issues in certain countries, technical complexities,
and challenges of homogenous heating and temperature monitoring, may have contributed to this fact. As more and more clinical
trials are being published, the willingness/memorandum to make the effort of establishing HT in a rising number of institutions has
increased.
The development of novel techniques with more exact heat delivery and temperature monitoring capacities may help to gain higher
acceptance among physicians. HT may as well be further improved by better planning techniques. Mathematical modeling of the
earlier mentioned HT effects on cell biology has paved the way to actual thermoradiotherapy planning (4). By integrating the
biological HT effect into the LQ-model, a more exact RTHT treatment planning becomes possible. By adding online temperature
control, as it can be achieved by MRI thermometry, temperature distribution could be optimized even further by real-time
adjustments.
In the trials performed so far, HT delivery specifications were highly heterogeneous. HT frequency varied between once weekly to
daily applications. Mean achieved temperature profiles differed vastly between 39 and 43°C. In trials using multiple HT specification,
higher temperatures or HT frequencies were associated with better outcome (88, 100, 116). This underlines the necessity of
optimizing HT schedules to optimize the treatment effect. To this end, larger randomized studies are necessary directly comparing
distinct HT specifications.
Even though there are still a lot of open questions, basic research has revealed many ways of action of HT. Regarding the biological
mechanisms of HT, combining HT with drugs exploiting underlying mechanisms may further increase radiosensitization. As an
example, HT has been combined with antiangiogenesis agents. HT itself appears to directly impair angiogenesis at least in part by
plasminogen activator inhibitor 1 induction (146). By combining HT with VEGFR2-inhibitor treatment, a synergistic antiangiogenesis
effect in vivo has been shown to inhibit tumor growth (147). Regarding the immunogenic effects of HT, adjunct immunotherapy,
such as checkpoint inhibition, constitutes a further interesting field of research. Before clinical trials can be designed, more basic
research is necessary to evaluate the effects of thermo-immunotherapy in preclinical models.
All studies mentioned above have used HT in combination with photon-based irradiation. However, all over the world, an increasing
number of particle beam facilities are being installed. Due to technical improvements, smaller and low-cost proton facilities may
become available in the future possibly enabling a further widespread use. In contrast, facilities capable of delivering heavy ion-
based irradiation with 12C remain scarce. In a brief summary, proton and 12C-ions share similar favorable dose distributions with low
entry dose, a high dose in the “Bragg peak” followed by a more or less steep dose decline (148). In contrast to protons, 12C-ions
inherit a significantly higher linear energy transfer and relative biological effectiveness (149). Several biological factors have been
identified such as a low oxygen enhancement ratio (OER), less cell-cycle-dependent cell killing, inhibition of non-homologous DNA
repair, cluster damages to the DNA, and more efficient cell killing of tumor stem cells (150). HT triggers killing of hypoxic, acidic as
well as energy-deprived tumor cells, decreases OER, and confers direct killing of S-phased cells. Therefore, it has been proposed that
combined therapy of proton irradiation with HT may have similar effectiveness as 12C beam therapy alone (151, 152). To the best of
our knowledge, no data of the clinical use of simultaneous thermo-particle therapy has been published. The HYPROSAR phase I/II
study is currently recruiting patients with unresectable STS at the Paul Scherrer Institut in Switzerland. Weekly HT is combined with
proton beam therapy to achieve tumor downstaging with subsequent resection. There has only been one randomized trial treating
151 patients with uveal melanoma with or without adjuvant transpupillary thermotherapy months after the end of proton
irradiation (153). Indeed, the rate of secondary enucleation was significant lower. Since the therapy was not conducted in direct
temporal proximity, the abovementioned factors would not have taken effect. Hence, clinical trials are necessary to explore the
benefit of combinational therapy. Direct comparison of thermo-particle therapy with 12C beam therapy should be performed in
randomized trials.
Different technical solutions of external HT delivery have been discussed. Advances in the field of nanomedicine have introduced a
novel approach for targeted HT by development of magnetic or superparamagnetic nanoparticles as recently reviewd by Datta et al.
(154). Particles with cores of iron oxide or gold shells have already made their way into clinical phase I trials. Bergs et al. recently
reviewed the existing particle constructs (155). Tumor-specific targeting might be achieved by passive accumulation in the tumor
due to the aberrant vasculature with increased leakage and simultaneous impairment of lymphatic drainage (156). Alternatively,
active targeting could be achieved by coating with tumor-specific antibodies or ligands (157). Heat can then be generated by
applying external magnetic fields with rapid field alternations (158). In clinical phase I trials, dispersed nanoparticles were directly
deposited at the tumor side either by percutaneous injection or intraoperatively. Only mild toxicities and quality of life impairments
were observed. A maximum temperature of up to 55°C was achieved but target coverage remained insufficient (159–161). The
advantage of nanoparticle-based HT may lead to a more selective heat delivery to the tumor with possibly higher temperatures and
lower toxicity to adjacent normal tissues. By carrying chemotherapeutic agents, antibodies, or gene silencing RNA residues,
nanoparticles may open completely new therapeutic opportunities (154). On the other side, tumor volume coverage is still far from
optimal. Poorly perfused regions of tumors, in which HT has its greatest potential for radiosensitization, tend to “collect” lower
particle numbers. Accumulation of particles in non-malignant tissues such as the reticuloendothelial system or by the glomerular
filter of the kidney carries the risk of side effects (162). Microscopic disease, e.g., in lymphatic tissue may not be reached by sufficient
high temperatures. Until safe appliance in the clinic becomes possible, more research is necessary to assess the biological risks and
to optimize particle distribution and targeting. If these problems can be addressed, nanoparticles may be a valuable alternative to
external HT.
Besides enhancement of RT effects, HT may also be used for radiation dose reduction. As described above, Notter et al. used a
hypofractionation treatment scheme (5 × 4 Gy, one fraction per week) to treat patients with locally recurrent breast cancer. A similar
CR rate of 61% was achieved compared to other studies using mean doses of 38.2 Gy (79). The relatively low dose enabled the
authors to perform re-reirradiations. 17 patients with re-recurrences of lymphangiosis carcinomatosa received re-reirradiation
following the same therapy scheme. A CR rate of 31% could be achieved (56).
Infections with human papilloma virus (HPV) are commonly found in carcinomas of the head and neck or cervix. HPV-positive tumors
appear to be more radiosensitive than HPV-negative tumors. HT has been shown to trigger the degradation of the oncogenic HPV-
derived E6 protein. As functional E6 binds and inactivates p53, HT may indirectly renew p53 activity favoring p53-dependent
apoptosis. Thus, a combination of RT and HT may be particularly effective. To evaluate this effect, future trials should include HPV
status for risk stratification (87). As a next step, clinical trials should test further RT dose de-escalation with concurrent HT in HPV-
positive patients as it is already performed for RT alone (163, 164).
Conclusion
There is abundant evidence demonstrating that HT constitutes a valuable supplement to currently performed RT or RTCT schemes
improving tumor response in tumor entities such as head and neck or cervix. It also inherits great potential to enhance RT-based
therapy of recurrent disease as it has been shown for breast cancer. Through continuous improvement of HT delivery, planning, and
monitoring techniques, treatment effects may further improve. Novel combinations with targeted therapy agents, immunotherapy,
nanomedicine, or particle therapy constitute promising fields of further research and interesting areas for future clinical application.

Biologic Rationale For Combining Heat With Radiation and Chemotherapy The biologic basis for benefit of hyperthermia with
modern cancer therapies including radiation and chemotherapy began to be appreciated by the 1970’s. In regards to radiation
therapy, hyperthermia was recognized as an ideal complementary treatment. Many conditions that contribute to radioresistance
including hypoxia, acidification, and S-phase of the cell cycle either enhance sensitivity to heat or do not temper it. (9-15) (FIGURE 1)
Hyperthermia also typically results in enhanced perfusion that may result in improved tumor oxygenation for subsequent radiation
treatments. (16-17) Chemosensitization with hyperthermia is dependent on the particular mechanism of effect for each agent. The
magnitude of effect ranges from none as is typical with anti-metabolites to synergistic effects such as with cisplatin. (FIGURE 2) The
mechanism of enhanced cytotoxicity can include increased intracellular drug accumulation, inhibition of DNA repair, abrogation of
cell cycling in S-phase when cells are most sensitive to heat, enhanced free radical production, and reversal of drug resistance. (18-
20)) Drugs for which synergistic effects have been noted include cisplatin, adriamycin, bleomycin, melphalan, cyclophosphamide,
nitrosoureas, nitrogen mustards, and mitomycin c. (19) Interaction of hyperthermia with other agents including taxanes and anti-
metabolites is possible although for many agents the complex interactions that contribute to enhanced treatment effects remain to
be fully defined. (20) In regards to timing and sequencing, simultaneous administration or chemotherapy followed immediately by
hyperthermia has been shown to have the greatest effects. Mechanism of Cell Death With Hyperthermia High temperatures
achieved with thermal ablation, typically in the range of 65-85°C result in instantaneous cell death. Temperatures above 45°C
sustained for sufficient time will lead to protein denaturization and cell death typically via necrosis. Clinical hyperthermia involves
temperature elevations in the range of 39-45°C Within this moderate temperature elevation range, tumor cell death occurs in a log-
linear manner with an initial shoulder region followed by a steeper decline in cell survival correlated with increasing temperatures
up to 45°C. While temperatures between 42- 45°C can result in cell death with sufficiently long exposure, use of hyperthermia alone
is not a practical nor particularly effective theraupeutic strategy as monotherapy. The primary benefit of hyperthermia with
moderate temperature elevation is therefore achieved through enhancement of anti-tumor effects through combination with either
radiation or chemotherapy. The precise mechanisms by which moderate temperature hyperthermia results in tumor cell death are
complex and highly dependent on the heating profile. In addition to complementary biologic effects with radiation and
chemotherapy, mechanisms for direct effect include protein denaturization including inhibition of sub-lethal and potentially lethal
damage repair through inactivation of DNA repair pathways leading to mitotic catastrophe, induction of senescence, apoptosis, and
necrosis. (21-28) Research has revealed that protein denaturization is a key biologic effect of hyperthermia at modest temperature
elevations.(29) The activation energeries for protein denaturization and heat induced cell death were noted to be within the same
range. Further research suggested that nuclear proteins are most sensitive (30-32) and a high degree of correlation of nuclear
protein aggregation and heat induced cell kill has been noted. Radiation sensitization is achieved in large part through nuclear
protein aggregation which inhibits the DNA repair process thus converting potentially lethal radiation induced DNA breaks into lethal
events. Specifically, heat induces changes in DNA repair foci such as the MRN complex which includes MRE11, rad 50, and nbs1, key
proteins involved in double strand break repair, the nucleolus and on the complexes that anchor DNA to the nuclear matrix thus
contributing to radiosensitization. (33-34) Chromosomal aberrations may also occur due to heat sensitivity of the centriole. (35)
Failure of DNA replication in S-Phase of the cell cycle results in mitotic catastrophe. Research to date has made it clear however that
other mechanisms including necrosis, apoptosis and senescence also have important roles in hyperthermic cell kill. Temperatures
above 44-45°C generally lead to cell death via extensive protein denaturization and necrosis. (36). Apoptosis has been noted at more
moderate temperature elevations such at 41.5°C sustained for one to two hours. (37) Stress induced premature senescence may
also occur in response to heat shock. (38) Heat Shock Proteins Heat shock proteins (HSP) play a central role in hyperthermia
response. Heat shock proteins (HSP), first identified in relation to thermal stress are now recognized as ubiquitous proteins involved
in general cellular stress response and also for their role in immune modulation. HSP were discovered in 1961 by Chanel and Maury
who described the effects of thermal shock on blood proteins in fish. (39) While initially identified in relation to heat shock and thus
named as such, HSP are now known to have a universal role in cellular stress response. In the intracellular milieu HSPs have a
primary role in stabilizing damaged proteins allowing for repair. This protective mechanism however has negative connotations for
treatments directed at killing cancer cells. This response which may last for several days has potential to abrogate the effects of
hyperthermia. This concept is referred to as thermal tolerance. The induction of thermal tolerance has been found to be associated
with HSP up regulation.(40-41) Due to concerns about thermal tolerance, hyperthermia is typically delivered no more than a few
times per week, generally with at least 72 hours between treatments. While serving a protective role for the cancer cell in the
intracellular environment, HSP have potentially beneficial anti-neoplastic effects in the extracellular milieu. HSPs have chaperokine
effects which include a central role in cellular immune regulation coupled with the ability to stimulate proinflammatory cytokine
production. (42) HSP-PC complexes with functional CD8+ cells and macrophages are all required for induction of immunity. (43) The
chaperone function of HSP relates to their role in shuttling immunogenic peptides onto MHCs for presentation to T cells. Notably,
HSP-PC complexes can induce tumor-specific immunity. (44-46) (FIGURE 3) Radiation has also been shown to have similar effects in
regard to immune response. In the clinical setting, a consistent increase in serum HSP70 over the duration of a standardly
fractionated course of radiation therapy for prostate cancer patients was noted. This increase in HSP70 was associated as expected
with increase in proinflammatory cytokines and components of the cellular immune response including CD8+ and natural killer cells.
Cell and animal modeling indicated this response is consistent with tumor specificity.(47) Optimization of immune response with
combination of hyperthermia with radiation and the ever increasing number of immunotherapeutics remains an active area of
emerging investigation. Thermal Enhancement Ratio The additional anti-neoplastic effect achieved with hyperthermia is referred to
as the thermal enhancement ratio or TER. TER is the ratio of radiation or chemotherapy doses required to produce a given level of
biological damage with versus without heat. The finding of a significant TER as first defined in the laboratory has subsequently been
demonstrated in clinical practice with most studies indicating a TER of approximately.

RTOG 81-04 compared radiation with or without hyperthermia for a variety of malignancies including breast, head and neck, trunk,
and extremity tumors. No difference was noted in complete response between groups; however, benefit was noted for tumors less
than 3 cm, suggesting that if tumors could be effectively heated meaningful clinical responses could be achieved. (75) A second
phase II trial, RTOG 84-19 was conducted to assess use of radiation with or without interstitial hyperthermia for persistent or
recurrent tumors after previous radiation or surgery. No difference in any of the study endpoints was noted; however, only 1 of 173
evaluable patients met the minimum accepted criteria for adequate hyperthermia. The authors concluded that the study ultimately
failed in its objective to assess the value of hyperthermia. (76) Coupled with other challenges including the typical hour or more
needed to administer hyperthermia, the difficulties in administering hyperthermia and lack of standards led many radiation
oncologists to abandon pursuit of hyperthermia. Additional Randomized Trials Following the RTOG trial experience, over 20
randomized trials assessing hyperthermia with radiation or chemotherapy have been published with most demonstrating benefit to
the addition of hyperthermia. These studies, while often of modest size, have shown benefit for hyperthermia in treatment of a wide
range of malignancies including glioblastoma multiforme, head and neck cancer, breast cancer, melanoma, esophageal malignancies,
and sarcoma.
Lung Cancer Data in lung cancer is relatively limited given the challenges of safely heating lung without inducing thermal damage. An
International Atomic Energy Agency (IAEA)-sponsored, multi-institutional prospective randomized trial was, however, successfully
completed. This trial was designed to assess whether the combination of hyperthermia and radiation improves the local response
rate of locally advanced non-small cell lung cancer (NSCLC) compared with radiation alone. 80 patients with locally advanced NSCLC
were randomized. The primary endpoint was local response rate. Secondary endpoints included local progressionfree survival and
overall survival. No significant differences between the two arms with regard to local response rate or overall survival rate was
noted. However, local progression- free survival was significantly better with the addition of hyperthermia. Toxicity was generally
mild and no grade 3 late toxicity was observed in either arm.(81) Breast Cancer Hyperthermia has been shown to improve complete
response and local control for women with breast cancer, particularly in the setting of recurrent chest wall disease. Vernon reported
combined results of five randomized breast cancer trials in Europe and North America including both locally advanced primary
disease and recurrent disease for which radiation therapy was indicated and surgery contraindicated.(82) Three distinct groups were
studied: patients with inoperable primary disease, recurrent disease in un-irradiated sites, and recurrent disease in previously
radiated areas. The primary endpoint in each trial was local tumor response. A decision to perform a combined analysis was made
after initiation of the individual studies due to slow accrual. A prospective statistical plan was generated to allow for assessment of
the initial objectives. A total of 306 eligible patients were enrolled. Treated lesions included chest wall (71%) and intact breast tissue
(26%). 48% of cases included multiple lesions. There were a greater number of patients who had received prior chemotherapy and
lesion size was greater in the hyperthermia plus radiation treatment arm. A highly significant difference in complete response was
note for combined radiation and hyperthermia, 59%, vs. radiation alone, 41%. On further analysis it was determined this difference
was driven by benefit in patients undergoing re-irradiation for whom radiation doses were limited. In this group there was nearly a
doubling of complete response with the addition of hyperthermia to radiation of 59% as compared to 31% with radiation alone.
Patients achieving a complete response who received hyperthermia had a reduced risk of recurrence with hazard ratio of 0.67
compared to patients who received radiation alone. Treatment with hyperthermia was well tolerated. Apart from acute occurrence
of skin blistering of 11% vs. 2% with vs. without hyperthermia no differences in either acute or late toxicity were noted. Researchers
at Duke University confirmed a dose response relationship for hyperthermia in a randomized trial in which patients with superficial
tumors ≤ 3 cm depth received either “low” vs “high” dose hyperthermia combined with radiation. As prior studies had shown a
thermal dose of CEM 43°C T90 (the number of cumulative equivalent minutes at 43°C exceeded by 90% of monitored points within
the tumor) equated with improved treatment outcome, (83-84) hyperthermia was prescribed to either CEM 43°C T90 10. 122
patients were enrolled of which 89% were determined at the time of first hyperthermia treatment to have tumors that could be
effectively heated. These 109 patients were randomized to receive either no further hyperthermia or twice weekly hyperthermia
over their course of radiation therapy up to 10 treatments. Patients were also stratified by whether they had received prior radiation
therapy and by site of disease. Median CEM 43°C T90 in the low and high dose hyperthermia groups was 0.74 and 14.3 respectively.
65% of patients had breast or chest wall disease, 13% head and neck, 11% melanoma, with the remainder having various other
malignancies, 36% of patients had received prior radiation. Median radiation dose was 41 Gy in previously treated patients and 60
Gy for those not previously irradiated. Higher dose hyperthermia was found to be beneficial. Complete response rate in the high
dose arm was 66% and in the low HT arm 42%. The odds ratio for complete response was 2.7. This improvement in complete
response translated into improved duration of local control of 48% vs. 25% at the time of death or last followup. Previously
irradiated patients had the greatest incremental gain in complete response, 23% in the no HT arm vs. 68% in the HT arm as
compared to 51% vs. 65% for patients without prior radiation therapy. The toxicity profile was also excellent. The study therefore
demonstrated that thermal dose can be prospectively prescribed and delivered, and correlates with outcome
The potential for hyperthermia to benefit cancer patients has long been recognized. New appreciation for the biologic and
physiologic mechanisms through which hyperthermia may improve outcomes of radiation and chemotherapy treatments led to
enthusiasm in the 1980’s that subsequently waned due to technical limitations and the need to develop clinical standards for heat
delivery. Despite these early challenges clinical trials for a wide range of malignancies have been completed with the vast
majority indicating clinical benefit. Technology for heat delivery, standards for quality assurance and thermal biology have all
significantly advanced in the 35 years since hyperthermia was introduced into the modern cancer clinic. Coupled with timely
opportunities to advance multi-disciplinary cancer care through combination with state-of-the-art radiation, chemotherapy, and
immunotherapy, renewed focus on hyperthermia in oncology is warranted by the broad cancer community

Cancer immunity and therapy using hyperthermia with immunotherapy, radiotherapy, chemotherapy, and surgery
hyperthermia is a type of medical modality for cancer treatment using the biological effect of artificially induced heat. Even though
the intrinsic effects of elevated body temperature in cancer tissues are poorly understood, increasing the temperature of the body
has been recognized as a popular therapeutic method for tumorous lesions as well as infectious diseases since ancient times.
Recently accumulated evidence has shown that hyperthermia amplifies immune responses in the body against cancer while
decreasing the immune suppression and immune escape of cancer. It also shows that hyperthermia inhibits the repair of damaged
cancer cells after chemotherapy or radiotherapy. These perceptions indicate that hyperthermia has potential for cancer therapy in
conjunction with immunotherapy, chemotherapy, radiotherapy, and surgery. Paradoxically, the anticancer effect of hyperthermia
alone has not yet been adequately exploited because deep heating techniques and devices to aggregate heat effects only in cancer
tissues are difficult in practical terms. This review article focuses on the current understanding concerning cancer immunity and
involvement of hyperthermia and the innate and adoptive immune system. The potential for combination therapy with
hyperthermia and chemotherapy, radiotherapy, and surgery is also discussed

Hyperthermia enhances immune systems in response to cancer

Body temperature elevation has been considered an important phenomenon associated with regulation in both innate and adaptive
immune responses[38]. Hyperthermia elicits various effects in several steps of the immune reaction for cancer. It up-regulates the
homing of immune cells and the function of adhesion molecules on both immune cells and endothelial cells, activating immune cells
including CTLs, DCs, and NK cells, and inhibiting immune suppression. In this section, we discuss how thermal stress up-regulates the
immune system.
Hyperthermia, especially whole body hyperthermia, has the potential to increase the homing of immune cells. Continuous secretion
of homeostatic chemokines including CCL21 and the expression of adhesion factors including selectin, integrin, and ICAM regulate
immune homeostasis by maintaining the homing of these immune cells. Thermal effect can enhance the expression of ICAM-1 and
CCL21 in high endothelial venules (HEVs)[39] and can up-regulate L-selectin- and integrin-dependent adhesive interaction to induce
the adhesion and migration of DCs and T-cells toward HEVs [40]. Additionally, increases in the migration capacity of DCs ex vivo has
been reported[41].
We reported previously that heat treatment stimulated cytokine production from peripheral T-cells in vitro and in vivo in fresh
peripheral venous blood obtained from 5 healthy volunteers [42]. We first incubated peripheral blood mononuclear cells (PBMCs)
separated from obtained blood samples at 37 °C or 39 °C for 2 h in a water bath, then PBMCs were co-cultured with anti-CD3/CD28
monoclonal antibodies for 24 h at 37 °C. To evaluate the secretory properties of cytokines in T-cells, IFN-γ and IL-2 levels in the
supernatant were measured. Results showed that both cytokine production levels were significantly increased (approximately
twofold) when PBMCs were cultured at 39 °C [Figure 2]. Next, the volunteers underwent whole body hyperthermia until the rectal
temperature reached 38.5 °C (generally it required 1 h of treatment). After terminating heating, volunteers were covered with a
leather tent for 60 min as a heat-retention phase. Blood samples were obtained four times: before the treatment, at the end of the
heat-retention phase, and then 24 and 48 h after treatment. IFN-γ and IL-2 levels in each supernatant were measured in response to
monoclonal antibody against CD3 and CD28. The results showed significant increases in the production of both IFN-γ and IL-2; these
were observed not only immediately after but also 24 h after whole body hyperthermia. At 48 h after whole body hyperthermia, the
production levels of both cytokines had returned to the pretreatment levels [Figure 3].
The potential mechanisms that stimulate cytokine production after hyperthermia may be explained by an increase in the membrane
fluidity of T-cells. It was reported that physiological heat stress enhanced the membrane fluidity of T-cells. It also showed an increase
in the cluster formation of the GM1+ CD-microdomain in CD8+ T-cells, clustering TCRβ and the CD8 co-receptor, and enhanced
conjugate formation between T-cells and APCs in mice [43]. These results suggest that a heat-stress-induced increase in membrane
fluidity is one of the primary events, and it subsequently triggers a cascade of molecular events that eventually make T cells crosstalk
more rapidly and efficiently with APCs. These cellular events, including the formation of TCR microclusters, consist of several
adhesion and signaling molecules[44], which accumulate at the immune synapse[45]. This is also known as the central supramolecular
activation complex[46].
Heat-shock proteins (HSPs) have been considered to play an important role in the effects of heat treatment on T-cell function.
Indeed, the synthesis of HSPs was shown to increase with elevated body temperature in fever-range whole-body hyperthermia [47].
The essential function of HSPs is known to involve their actions as molecular chaperones. As part of this function, HSPs are involved
in antigen presentation and cross-presentation in DCs by delivering chaperoned antigenic peptides to MHC class I molecules, thereby
inducing antigen-specific T-cell activation[48-50]. It was reported that the presence of recombinant Hsp60 allows antigen-dependent T-
cell activation with antigen-specific IFN-γ secretion in conditions when even stimulation is not sufficient to activate T-cells [51].
In contrast, Hsp70 is also expressed in cancers and acts as an effective inhibitor of apoptosis caused by heat stress, thereby
participating in tumor progression[52]. Hsp70 can prevent aggregation, remodel folding pathways, and regulate activity of cancer
cells[53]. However, the effects of HSPs on DCs and T-cells are still contradictory [54]. Thus, the function of HSPs must continue to be
investigated in order to clarify whether and how HSPs are involved in antigen presentation between T-cells and DCs during heat
stress.
NK cells can behave as a spearhead of the innate immune response toward exogenous antigens and can make an initial attack
against targets without prior exposure to the specific antigens. Basically, normal cells express MHC class I molecules, whereas
aberrant cells such as cancer cells extinguish the expression of MHC class I molecules on themselves [55]. This phenomenon was
especially observed in pancreatic[56], cervical[57], breast[58], prostatic[59], and penile cancer[60]. Down-regulation of MHC class I
molecules on cancer cells is one of the steps for immune escape from cancer-specific immune response by T-cells, because abnormal
antigens must be presented with MHC class I molecules when T-cells recognize them. In contrast to T-cells, NK cells target cells that
have lost the expression of MHC class I molecules, because NK cells express inhibitory receptors that engage with MHC class I
antigens. Hence, the anticancer ability of NK cells is exercised at this time to complement T-cell-based immune reactions owing to
attenuated inhibitory signals between NK cells and cancer cells [61]. Activated NK cells have nonspecific anticancer potential by
secreting cytotoxic molecules including perforin and granzyme [33] and death receptors such as FasL, TRAIL, and TNF-α[62,63].
Additionally, heat stress can enhance the distinct clustering of NK cell-activating receptors such as NKG2D on the surface of NK cells
and the expression of NK cell-activating ligands, including major histocompatibility complex class I-related chain A (MICA) [64,65].
Moreover, an increase in the expression of MICA and increased cytotoxicity of NK cells were also observed in several cancer cell
lines[66]. Concerning the contribution of NK cells to cancer-specific immune responses, after an initial attack, fragments including
cancer antigens of tumor cells are generated as a result of the interaction of the tumor and NK cells. These cancer antigens are
recognized by DCs for T-cell-based cancer-specific immune responses. Additionally, activated NK cells secrete cytokines including
IFN-γ and IL-2 to enhance acquired immune responses with immunoglobulin production from B-cells and activation of T-cells [55,61].
This perception leads to the conclusion that increasing the function of NK cells by hyperthermia could be expected to result in the
augmentation not only of nonspecific anticancer immune reactions regulated by NK cells but also of specific anticancer immune
reactions regulated by T-cells and DCs.
To avoid exaggerated immune responses that cause harmful effects on the body, the immune system is regulated to limit adaptive
immune responses and prevent autoimmune responses and auto-inflammatory reactivity in the normal situations. To achieve this,
our immune system combines immunological tolerance system. Regulatory T cells (Tregs) are a subpopulation of T-cells, expressing
CD4, CD25, and FOXP3, which negatively modulate both innate and adopted immune response by down-regulating or suppressing
induction and proliferation of immune cells including T cells, DCs, and NK cells [67-69]. Even though Tregs usually account for about 4%
of CD4+ T cells, they can make up as much as 20-30% of the total CD4 + population in the tumor microenvironment and are associated
with poor prognosis in many cancers, such as ovarian, breast, renal, and pancreatic cancer [70]. Depletion of Tregs in animal models
has been shown to increase the efficacy of immunotherapy. So, achieving the depletion of Tregs is one of the pivotal targets of
recent research and therapy associated with cancer immunology [71]. The potential effect of hyperthermia is considered to enhance
the cytotoxicity of NK cells against Tregs, and to inhibit the induction of Tregs while the apoptosis of Tregs is induced. A significant
decrease in the number of Tregs was observed while NK cell activity and the percentage of NK cells increased in peripheral blood
samples of healthy volunteers after irradiation of fever-range hyperthermia to the upper abdominal region [72]. Moreover,
combination therapy of intratumoral injection of immature DCs and local hyperthermia for patients with advanced malignant
melanoma demonstrated decreased infiltration of Tregs and increased infiltration of activated CTLs, even though there was no
statistical difference in overall survival time[73].
The efficacy of hyperthermia in down-regulating the expression of PD-L1 in some cancer cell lines was reported. In this study,
decreased expression of PD-L1 in cancer cell lines was shown when samples were exposed to temperatures between 40 °C and 43
°C[74]. Further accumulation of data associated with this new experimental model is eagerly awaited
Combination therapy with radiotherapy
The enhancement of anticancer efficacy of combination use of radiotherapy and hyperthermia was clinically recognized in cervical,
breast, and head and neck cancer and so on[77]. Even though radiological cytotoxicity induces DNA damage of cancer cells [78], some
cancer cells can come back into existence (termed sublethal damage repair or lethal damage repair) [79,80]. In the analysis of the cell
cycling, quiescent tumor cells were more resistant to irradiation because cells in this stage have the potential for lethal damage
repair[81]. In contrast, hyperthermia can inhibit the repair of radiation-induced damage in cancer cells, so that combination use of
hyperthermia can enhance the anticancer efficacy of radiotherapy [82,83]. Cells in the synthesis (S) phase are also relatively radio-
resistant, while they are the most sensitive to hyperthermia. Additionally, hypoxic cells in tumors are also radio-resistant, while
hyperthermia improves the anaerobic condition by oxygen delivery due to increased blood flow. These perceptions indicate the
synergetic effect of combination use of radiotherapy and hyperthermia [7]. Moreover, additional use of DNA repair inhibitors was
reported to further enhance its efficacy[84].
Combination therapy with chemotherapy
Chemotherapy is the most popular therapeutic method for patients with inoperative cancer and recurrent or metastatic cancer;
however, there are serious problems including its uncertain efficacy, drug resistance, and adverse effects. To improve therapeutic
results, combination use of hyperthermia was tested, and increased anticancer effect was reported in paclitaxel, docetaxel,
gemcitabine, oxaliplatin, and irinotecan [85]. The mechanism of interaction of chemotherapy and hyperthermia was considered as
follows: increased drug uptake into cancer cells by causing damage to the membrane of cancer cells and reducing oxygen radical
detoxification. Eventually, DNA damage increased while DNA repair decreased. Additionally, hyperthermia was reported to have a
potential ability to avoid drug resistance[86,87]. In addition, it is also expected that elevated blood flow could result in a relative
increase in anticancer drug concentration within the tumor. Moreover, adverse effects can be decreased because increased drainage
of the drugs may accelerate in normal cells due to the up-regulation of metabolism. On the other hand, some anticancer drugs,
including 5-fluorouracil, gemcitabine, and oxaliplatin, are considered to enhance cancer immunity by inducing the infiltration of CTLs
while reducing Tregs in the tumor[88]. Accordingly, enhancing the efficacy of chemotherapy will result secondarily in up-regulation of
cancer immunity.
By using hyperthermia in combination with chemotherapy or radiotherapy, the dose of these therapies may be reduced to ease
their side-effects without reducing therapeutic effects, because hyperthermia has the potential to augment the effect of
chemotherapy or radiotherapy in a less invasive manner.
This report shows that hyperthermia increases the advantage of the following biological features. Heat stress lowers the survival
rate of all cells, but normal tissues are better able to tolerate this than cancerous tissues. Heat has a potential to augment immune
responses while decreasing immune suppression. Heat inhibits the recovery of cancer cells from DNA damage. Heat enhances the
resorption of anticancer drugs into cancer cells. The sensitivity of cancer cells against heat and radiation differs depending on the
condition of cancer cells in the cell cycle. The anticancer efficacy of hyperthermia alone with currently available heating devices is
not enough to suggest its use as a standalone therapy. However, some studies have shown that combination therapy with
conventional methods including immunotherapy, radiotherapy, chemotherapy, and surgery improves its anticancer efficacy in
vitro and vivo
Hypoxia, pH
Blood perfusion in large solid tumors is generally poorer than that in normal tissue. 11 The vascular beds in tumors are chaotic and poorly
organized resulting in temporal and spatial unbalanced blood supply. 12 Therefore, many regions within tumors result hypoxic/acidic and
resistant to radiation and chemotherapy. The chronic or transient deficiency in tumor perfusion can generate state of chronic or acute
hypoxia.12 Chronic hypoxia develops when cancer growth outstrips its blood supply, reaching a critical mass > 1-2 mm 3 (106 cells) and a
distance from host nutritive vessel > of 100-200 μm (fig. 2). Regions of transient hypoxia can develop within tumor mass following a
temporary interruption of blood flow. Transient perfusional hypoxia is caused by various mechanisms. The most plausible ones seem to be:
i. Irregular expansion of tumour mass, whose three dimensional growth is subjected to a continuous remodelling, in a confined
space, causes a temporary compression or occlusion of some tumour capillaries (fig. 3).12-14
 ii. Transient stop of tumor blood flow or supply by platelets plug. 15,16 In our opinion, this intravascular thrombosis deserves to be
taken into much higher account than usually done. 16 In fact, the majority of cancer patients has coagulation abnormalities
associated to hypoxia.16,17 Recently, it has been demonstrated that hypoxia not only induces VEGF but also stimulates endothelial
cells to over express tissue factor (TF) and Plasminogen activator inhibitor (PAI-I). These factors induce endothelium to become
prothrombotic and cause fibrin formation and platelet activation. Furthermore, VEGF binds to fibrinogen and fibrin by stimulating
endothelial cell proliferation.17 Fibrin has been demonstrated to be essential to supporting endothelial cells spreading and
migration.15 The haemostatic system becomes, in a certain sense, a regulator of angiogenesis and can partially explain acute
hypoxia and its regional appearance and disappearance. In conclusion hypoxia becomes a self perpetuating mechanism able to
trigger angiogenesis, intratumoral fluid accumulation and thrombosis (fig. 3).16-18
In this figure the structural and functional effects of Hypoxia, HIF-1 and VEGF on tumor microcirculation, cancer metabolism and therapies,
are illustrated. (Modified with permission from: Baronzio et al. Anticancer Res 1994; 14:1145-1154.)
As just described, the two types of hypoxia have different origin and coexist together in a well-perfused zone of the tumor mass, causing a
functional disturbance of macro and microflow. 11 A more realistic vision shows that these two situations change continuously, because
tumor blood flow is time fluctuating. Furthermore in this low flow regions associated but occurring independently of hypoxia an acidic
environment is present.13,14,18,19 Hypoxia alone is the single strongest prognostic indicator of radiotherapy outcome. In fact, it has been
observed that larger tumor have a greater proportion of hypoxic cells and respond less to radiotherapy. 12,14,19 The precise nature by which
hypoxia exerts its adverse effects on cells radiosensitivity is not completely understood. 1 It is believed that oxygen enhance the efficacy of
radiotherapy by inducing free radicals production in the tumor area causing DNA damage. 2,18-20
Oxygen Enhancement Ratio (OER)
To better understanding the importance of oxygen on radiotherapy, survival curves of oxygenated and hypoxic cells treated with
radiotherapy are generally compared. The ratio of hypoxic to aerated dose needed to achieve the same biologic effect is called Oxygen
enhancement ratio (OER) and is defined by the Equation 1. 19 OER = Dose in hypoxic conditions/Dose in aerated conditions (to achieve same
cell survival fraction) (Eq. 1)
Gerweck et al,1 using the same methodology, compared the biological effect of hyperthermia on hypoxic and aerobic cells. The OER found
by these authors and other confirmed that hypoxic cells sensitivity to hyperthermia were equal or greater than oxygenated cells (OER ≈; ≤ 1
for HT; OER ≥ 2.5 -3 for RT),19,20 as shown in Figure 4. Acute and chronic hypoxic cells, submitted to heat, behave similarly. In fact, acute
hypoxic cells have an OER near 1, whereas chronically hypoxic cells have an OER slightly less. 21 Furthermore, cells cultured at lower pH have
a low extracellular pH, that is 0.2,0.4 units lower than blood. 22,23 These cells are more responsive to HT as shown in Figure 5, however the
effect seems not completely dependent from hypoxia. In fact cancer cells chronically deprived of nutrients, peculiarly of serum, are
extremely sensitive to heat as demonstrated by Hahn.24
Cells Cycle Stage
Beyond hypoxia, the reasons for combining hyperthermia and ionizing radiation are based on the following considerations:
I. Heat is effective against S phase cells a relatively radio resistant phase. In fact RT effect is maximal, in G2 phase. 24,25
II. Heat can interact with radiation and potentiates its cellular action by retarding the repair of radiation induced DNA damage; 25
III. HT can induce a cytotoxic killing effect by apoptosis an important mechanism of death not essentially determined by radiation. 24,25
Thermal Enhancement Ratio (TER) - Therapeutic Gain Factor - Heat Radiotherapy Sequence
The Radiosensitization effect of hyperthermia was quantified using the thermal enhancement ratio (TER). TER represents the ratio between the
minimal dose of radiation to induce a biological effect and the dose required when radiotherapy and hyperthermia are used combined, [Eq.
2].1,2 TER = Radiation dose alone/Radiation dose with heat (Eq. 2) (to achieve same end point) TGF = TER (Tumor)/TER (Normal Tissues) (Eq. 3)
A supplementary way to express the biologic effect of heat and radiation is the therapeutic gain factor (TGF) that is defined as the ratio of the TER
in tumor to the TER in normal tissues, [Eq. 3].2 However, this comparison is not obtained easily, because the tumor and normal tissues are not
equally heated. In fact, a greater heat washout is present in normal tissue compared to tumor tissue. 2,26 A practical aspect in order to obtain the
maximum effect by combining radiation and hyperthermia is the time and the sequence of their application. Studies by Overgaard 27 have shown
that an advantageous clinical TER was obtained when HT and RT are delivered concomitantly; however, for inherent clinical difficultness to operate
synchronously; a satisfactory TER is obtainable delivering HT and RT within a short period. Some investigators use a time interval of 2 - 4 hs
between radiation and heat to obtain a satisfactory TER (fig. 6).27 Interaction between RT and HT can be additive or super-additive. RT survival
curves are influenced by HT addition. Their behaviour has been studied on cells cultures and in animal experiments evidencing that temperature is
critical for the determination of their sequence and clinical uselfuness. Brief exposure to temperatures of about 45°C can steepen the x ray survival
curve reducing the D0. The predominant effect in this case is radiosensitization and the change of x-rays survival curve is minimal. For temperature
more clinically obtainable (≤ 43°C) (not involved in cell killing) the principal effect is the removal of shoulder effect from X rays survival curve: this
result proves that heat treatment after irradiation is the best sequence. The different effect obtained by higher (≥ 45°C) or lower temperatures (≤
43°C) may reflect different critical target or induced thermotolerance. Recently Song et al 26,28 have explained another enhancing factor that justifies
the effectiveness of HT and RT combination. These Authors 26,28 in accordance with others25,29 have reported that there is abundant evidence that
oxygenation improves after moderate heating (42°C). The improvement in blood flow and thereby of oxygen content may increase the
effectiveness of radiotherapy and chemotherapy. In every case, the clinical evidences concerning this moderate blood flow enhancement are not
sure and need further investigations.26
Several in vitro and in vivo animal studies have demonstrated that the combination of hyperthermia and
cytostatic drugs can be employed synergistically.30 However not all chemotherapeutic agents behave similarly at
elevated temperature.

Trimodality Therapy
Herman et al33 proposed in the late 1988 to combine HT RT and chemotherapy (Trimodality therapy) in order to obtain a better local
control of the disease. These and other Authors31,33,34 reported that the combination of anticancer drugs, such as cisplatin, radiation
and heat was better than the two separate modalities. These authors in a recent review have summarized many combinations of this
trimodality treatment and they concluded that chemotherapy is to be delivered following a right schedule to obtain the maximum
effect.33,34 Herman et al suggest to obtain a long lasting local control of tumor by using the maximal tolerated radiation dose followed
by adjuvant chemotherapy and hyperthermia. Radiation was used at the maximum dose because it is the single most effective
method of treatment.33,34 Clinical examples of trimodality association are treatments of Head & neck tumors, 46 esophageal47 and
brain tumors.48
Hyperthermia enhances the cytotoxicity of some chemotherapeutic agents. We have studied the effect of moderate hyperthermia
(41.5°C) on the cytotoxicity of five new chemotherapeutic agents (docetaxel, paclitaxel, irinotecan, oxaliplatin, and gemcitabine) and
mel-phalan against a spontaneous murine fibrosarcoma. The dose of ionizing radiation that can be given to the tumor is determined
by the sensitivity of the surrounding normal tissues. Strategies to improve radiotherapy therefore aim to increase the effect on the
tumor or to decrease the effects on normal tissues, which must be achieved without sensitizing the normal tissues in the first
approach and without protecting the tumor in the second approach. Hyperthermia is a potent sensitizer of cell killing by ionizing
radiation (IR), which can be attributed to the fact that heat is a pleiotropic damaging agent, affecting multiple cell components to
varying degrees by altering protein structures, thus influencing the DNA damage response. Hyperthermia induces heat shock protein
70 (Hsp70; HSPA1A) synthesis and enhances telomerase activity. HSPA1A expression is associated with radioresistance. Inactivation
of HSPA1A and telomerase increases residual DNA DSBs post IR exposure, which correlates with increased cell killing, supporting the
role of HSPA1A and telomerase in IR-induced DNA damage repair. Thus, hyperthermia influences several molecular parameters
involved in sensitizing tumor cells to radiation and can enhance the potential of targeted radiotherapy. Therapy-inducible vectors
are useful for conditional expression of therapeutic genes in gene therapy, which is based on the control of gene expression by
conventional treatment modalities. The understanding of the molecular response of cells and tissues to ionizing radiation has lead to
a new appreciation of the exploitable genetic alterations in tumors and the development of treatments combining pharmacological
interventions with ionizing radiation that more specifically target either tumor or normal tissue, leading to improvements in efficacy.

Besse, H.C., Bos, C., Zandvliet, M.M., Wurff-Jacobs, K.M., Moonen, C.T., & Deckers, R. (2018). Triggered radiosensitizer
delivery using thermosensitive liposomes and hyperthermia improves efficacy of radiotherapy: An in vitro proof of
concept study. PloS one.
Franken, Nicolaas & Hovingh, Suzanne & Oei, Arlene & Cobussen, Paul & Bergs, Judith & Bree, Chris & Rodermond, Hans
& Stalpers, Lukas & Kok, Petra & Barendsen, Gerrit & Crezee, Hans. (2012). Radiosensitization with Hyperthermia and
Chemotherapeutic Agents: Effects on Linear-Quadratic Parameters of Radiation Cell Survival Curves. 10.5772/34939.
Franken, Nicolaas & Hovingh, Suzanne & Rodermond, Hans. (2012). Radiosensitization with Chemotherapeutic Agents
and Hyperthermia: Effects on Linear-quadratic Parameters of Radiation Cell Survival Curves. Journal of Cancer Science &
Therapy. 01. 10.4172/1948-5956.S5-002.