**3.5. Other selection factors**

There are numerous selection effects existing to distinguish the malignant cells. Here we comprehensively list some of them.

The missing cell-junctions and the characteristic isolation of the malignant cells from each other [247], [296] create a free extracellular pathway between the cells, definitely increasing the conductivity of the extracellular electrolyte. Furthermore, the decrease of the epithelial barrier function (tight-junction permeability changes) can be measured by electric impedance measurement, [297].

36 Hyperthermia

oncology, [291], [292], [293], [294].

**3.5. Other selection factors** 

comprehensively list some of them.

heating started in the late 19th century is called "galvanocautery" [263]. The method was further developed by D'Arsonval, introducing the impedance (alternating current [*AC*], later higher frequencies, even spark-generated currents) calling it "*Arsonvalization*", [264], and later a more modernized method was the "*fulguration*" [265]. The Arsonvalization method had fantastic popularity at the turn of the 19th-20th centuries, developing three different branches: the interstitial hyperthermia, including the galvanic heat-stimulation (*electrochemical-cancer-treatment*), the ablation techniques and the capacitive coupling. The first capacitive coupled device on conductive basis was the "*Universal Thermoflux*". It was launched on to the market by such a giant of the electric industry in that time as Siemens, which was later further developed, and the new device by the name "*Radiotherm*" was launched on the market in the early 1930s. The first start of the new capacitive-coupling technologies was in 1976 by LeVeen [266] and has been widely applied since then [267], [268], [74]. Many hyperthermia devices use capacitive coupling since its treatment is easy. An electric field application without an increase in temperature (using less than 5W power) has also been found effective against cancer [269], [270], [271], [272], [273], [274], by using galvanic (DC) current applications. The control of these treatments is the tissue-resistance and the quality parameter is the applied charge load, [275], [276]. Numerous devices were developed and applied widely, but the expected breakthrough result was missing. An entirely new line was started with Professors Rudolf Pekar, [277], [278], [279], Bjorn Nordenstrom [273], [274] and Xin You Ling [280], [281], [282]; and continued by others [283], [284], [285], [286], [287], [288], [289]. Remarkable results were produced by this method; and the biological mechanisms involved in electromagnetic field are intensively investigated [290], [72], and the effect of electric field is studied on various side of its complex behavior, [261], [262]. Recently, the effect of electric field has been used for special therapies in

A sophisticated study was performed to study the synergy of temperature and modulated electric field [295]. The model was HT-29 human colorectal carcinoma cell-line being xenografted to nude mice (BALB (nu/nu)). For comparison we performed low temperature oncothermia experiment, where the bolus of the upper-electrode was cooled down. The intensive cooling kept the tumor near the physiological temperature (38 oC) while the oncothermia field was identical with the heating conditions of 42 oC. The result is surprisingly interesting (see Figure 30). The effect of electric field made nano-range heating

There are numerous selection effects existing to distinguish the malignant cells. Here we

The missing cell-junctions and the characteristic isolation of the malignant cells from each other [247], [296] create a free extracellular pathway between the cells, definitely increasing the conductivity of the extracellular electrolyte. Furthermore, the decrease of the epithelial

only, the overall temperature had a minor role in the cell-killing mechanism.

**Figure 30.** Oncothermia was 3 times more effective than hyperthermia on the identically high (42 oC) temperature. However, cooling down the tumor during the treatment, the death-rate decreased only slightly, exceeding more than 2 times the classic hyperthermia on high temperature

The special membrane effect (rectification on membranes, [298]), is also a factor of the cellular selectivity of cancer by RF electric field. The impedance measurement is useful for the control of other treatment modalities. It adequately measures the distortion, made by irradiation [299], and the drug-effect can also be controlled, [300]. Such usual practice, like following the wound healing, is also objectively traceable [301]. Bioimpedance vector pattern can distinguish cancer patients without disease versus locally advanced or disseminated diseases [302].

The impedance measures selectively, differentiates between the cancerous and healthy tissue, and is able to distinguish the extra- and intra-cellular electrolyte. Selective impedance measurements are provided clinically as well. Many comparative studies have been provided for malignant tissues, however, the results are not identical; the measurements very much depend on the conditions. (This is a trivial consequence of many factors, which we listed above.) However, all the studies measured lower impedance in the tumor than in their healthy counterpart in all of the tissue and staging of the tumor, in vitro and in vivo as well.

Hyperthermia increases biochemical reaction rates [303] and therefore the metabolic rate as well. The metabolic heat production of tumor depends on the doubling time of its volume [304]. The high metabolic rate keeps the temperature for tumor tissues higher than its neighborhood, [305]. This works as selection factor for heating of tumor tissue. Therefore, in case of 6 oC increase the amount of growth will be 1.8-times higher [306] than its healthy counterpart.

It has been long known that hyperthermia can cause the softening or melting of the lipid bilayer [307], [308], [155], it can change lipid-protein interactions [309], and it can denature proteins [310]. All of these events can arrest the cell-cycle of the tumor.

Local Hyperthermia in Oncology – To Choose or not to Choose? 39

[335]. Development of the thermo-tolerance is one of the suppressors of hyperthermia efficacy [323]. From the point of view of the thermo-tolerance, one of the most prominent chaperone proteins is the HSP72. The concentration of this HSP is 5-10 times lower in the healthy cells than in the malignant ones, and both grow by the heat-treatment [336]. However, responding to the heat treatment, their concentration in healthy cells is: multiplied by 8 or 10, while during the same heat treatment the HSP72 multiplication

Like Nobel-laureate A. Szent-Gyorgyi formulated: from the point of view of life, it is not interesting how the monkey goes through the forest, but it is essential how the forest goes through the monkey [as nutrition]. In hyperthermia we have to have the same change of paradigm: it is not important how the incident energy increases the temperature, but that is essential how the incident energy changes the structure and the chemical bonds in the

The above described mechanism targets the membrane of the malignant cells, and excites numerous pathways essential for the cellular fate, (see Figure 31*.).* The excitations have

Rectificates, demodulates the modulated signal (stochastic effects are involved)

The effects in the cytoplasm are also remarkable. The transmembrane temperature gradient

These effects were shown in multiple, different experiments. The basic repeated phenomena are the time-delay of the action, selection and tumor destruction of oncothermia became trivial hours after the treatment (see Figure 32., [337]). The numerous apoptotic bodies show

creates only 1.2-1.5 times higher concentration in malignant cells, [323].

**4. Cellular effects – To kill or not to kill?** 

targeted tissue.

**4.1. Apoptotic cell killing** 

 Induces apoptotic signal Forms membrane-HSP

Damages the cell-membrane

 Activates apoptotic pathways Activates death receptors Suppresses the proliferation Arrests the DNA replication

Dilutes the cytoplasm

the character of the process.

*s*erious consequences on membrane:

 Makes the membrane more transparent Forms higher motility of membrane domains Rebinds the E-cadherin adherent connections

Develops higher intracellular pressure

Heat-treatment cause structural alteration in transmembrane proteins, causing a change in the active membrane transport and membrane capacity [311] leading to substantial changes in potassium, calcium, and sodium ion gradients [312], membrane potential [313], [314], cellular function [315], [316], and causing thermal block of electrically excitable cells [317], [303]. The thermo tolerable cells have significantly higher (~15%) membrane potential than the naïve cells [314**]**, and the difference rapidly grows by the elapsed time at 45°C.

In chemo-thermo-therapies the role of chaperone proteins is important. Chaperones (stressor heat-shock-proteins) are highly conserved proteins, which are vital in almost every living cell and on their surfaces during their whole lifetime, regardless of their stage in the evolution, [318]. Any kind of change in the dynamic equilibrium of the cell life (environmental stresses, various pathogen processes, diseases, etc.) activates their synthesis [319]. Excretion of the chaperones is the 'stress-answer' of the cells to accommodate themselves to the new challenges. As a consequence of the stressful 'life' of malignant cells, the molecular chaperones are present in all cancerous cells [320], [321], [322] to adapt the actual stress to help tumor-cell survival. Moreover, the shock-proteins are induced by every oncological treatment-method, which are devoted to eliminate the malignancy: after conventional hyperthermia [323], after chemotherapy [324], after radiotherapy [325] or even after photo-therapy [326] intensive HSP synthesis was shown. By the stress adaptation the induction or over-expression of the stress proteins generally provide effective protection of the cell against apoptosis [327], but their extracellular expression acts oppositely: it makes a signal to the immune system on the defect of the actual cell [328]. Furthermore, induction of various HSPs (HSP27, HSP70, and HSP90) was observed in numerous metastases and the HSP90 homologue, GRP94 may act as a mediator of metastasis generation. HSP generally degrades the effect of the hyperthermia therapy because it may increase the tumor cell survival, and its massive induction may generate the tumor thermo-tolerance and in parallel drug- and radiotolerance. Heat treatment can also lead to multi-drug resistance [329].

Non-temperature dependent effects (mainly field stresses) can also produce chaperonesynthesis [330]. The HSP manifestation in the biopsies might give a good clinical indication for the treatment response [331].

On the other hand, the chaperone HSP70 assists to freeze the actual dynamic equilibrium (the "status-quo") and so tries to re-establish the cellular communication in the extra-cellular electrolyte [328]. It is shown that their expression on the cell-membrane gains the apoptotic signals and enhances the immune reactions, [328]. HSP participates in the activation of the p53 tumor-suppresser [332] and has been associated with the tumor-suppresser retinoblastoma protein [333].

Recently, numerous scientific theories have also concentrated on the significance of thermally induced non-thermal effects, such as heat-shock protein (HSP) production [334], [335]. Development of the thermo-tolerance is one of the suppressors of hyperthermia efficacy [323]. From the point of view of the thermo-tolerance, one of the most prominent chaperone proteins is the HSP72. The concentration of this HSP is 5-10 times lower in the healthy cells than in the malignant ones, and both grow by the heat-treatment [336]. However, responding to the heat treatment, their concentration in healthy cells is: multiplied by 8 or 10, while during the same heat treatment the HSP72 multiplication creates only 1.2-1.5 times higher concentration in malignant cells, [323].
