Cancer therapies have rapidly developed in the last decades [1] . All classical treatments like chemotherapy (ChT), radiotherapy (RT), and surgery (Op) went through intense development. The new highly effective pharma products, the personalized and targeted approaches, the complex, versatile protocols, and the precise pointing of malignant lesions increased the success rate of the conventional treatment.

New therapies focus on extending patient survival with an acceptable quality of life. Despite the robust development, the estimated 2+ million new cases in 2024 [2] derail the optimistic and encouraging outlook for cancer care. Intensive efforts in drug development drive the progress of cancer therapies. Another challenge, however, is the present oncotherapies have severe adverse effects and toxicity, which limits their applications. There are 95% of the new cancer drugs applied in clinical studies have no approval because of their high toxicity [3] . Nonionizing electromagnetic therapies group different approaches without chemical toxicity. Like radiotherapy (ionizing radiation), electromagnetic energy absorption could cause thermal toxicity (burns), which differs from other adverse effects. This toxicity does not develop comorbidities. Avoiding its side effects or treating the accidentally appearing one is relatively easy. The electromagnetic impact on chosen reactions could increase the efficacy and decrease the toxicity of the complementary applied therapy [4] - [6] . The selection of malignancy by biophysical effects looks optional [7] [8] , and the thermal effects of the nonionizing could accelerate the excited chemical reactions (but chemically active) electromagnetic radiation [9] . These advantages support electromagnetic energy absorption as a helpful therapy, both standalone and in complementary applications.

The chemically active nonthermal electromagnetic effects are calculated and verified in vitro cell culture experiments [10] . The synergy of thermal and nonthermal effects had been measured in vitro but the selectivity considering intensive physiological feedback can be studied only in vivo. The present part of the series aims to show the in vivo research and review its results.

The modulated electro-hyperthermia (mEHT) technical solution is classical capacitive coupling with radiofrequency (RF) application at 13.56 MHz. A plan-parallel condenser provides the energy to the experimental animal between the electrodes, which are asymmetric [11] .

The mEHT is a hyperthermia method, which, in addition to the thermal energy absorption, uses nonthermal, electric field-dependent molecular excitations, increasing the tumor-destruction facility of the energy [7] . mEHT targets the electric and thermal differences between the malignant and healthy tissues and the micro heterogeneities at the tumor microenvironment (TME) [9] . The electric field selects the highly proliferative tumors by their high electric conductivity, and the low-frequency amplitude modulation active in polarization and molecular excitations of transmembrane proteins of the malignant cells [5] [6] . Various apoptotic signals are excited by external electric field impact. The frequencies are optimized fro the best performance [5] [8] . The electric field selects the tumor, and the low-frequency amplitude modulation polarizes and excites the transmembrane proteins of the malignant cells. The primary guidance of mEHT is biophysical using updated bioelectromagnetic considerations. The selective targeting modifies the conventional hyperthermia which intends to heat the entire mass of the tuor isothermally. The heterogeneous heating uses the thermal effects on the targeted molecules to accelerate the signal transduction which is triggered by the nonthermal impact [12] . The transmembrane molecular targeting of nonionizing radiation by a modulated electric field is like the ionizing radiation (radiotherapy, RT), which targets also molecular components in the strand of DNA.

The devices for in vivo use were the LabEHT100 and LabEHY200 (Oncotherm, Budaors, Hungary). The animal lies on the rectangular-shaped 6 × 12 cm size grounded electrode. To prevent the mice from cooling, the temperature of the counter electrode was controlled; it was kept at 37˚C during the treatment. The round-shaped opposing electrode (applicator) is above the animal, is 18 mm in size, and can be adjusted with a flexible holder to the necessary position above the treated tumor. A bolus with distilled water ensures optimal electromagnetic coupling. The applicator is cooled with circulating water. The allometric scaling estimates the speed of mice physiology (including the heartbeats) to be seven times quicker than that of humans. Due to the rapid physiological feedback, an ultrafast tuning system makes the prompt automatic adjustment of animal reactions possible. The temperature intratumoral, skin, and rectal measured, and the electric tuning status with the forwarded and reflected powers are also registered in real-time. The sampling frequency is 60/min. The temperature was controlled with specialized thermometers immune to RF radiation. The experimental conditions are discussed in detail in every referred article.

The animals are anesthetized during the treatment. The experimental setup is shown in Figure 1 .

Numerous cancer-bearing companion animals (dogs and cats) were successfully treated with mEHT [13] . These treatments better approach human tumorous cases because they have larger body weights than rodents. The larger body mass shifts the physiological timing to longer scales, better approaching human conditions. The naturally developed, not inoculated tumors also allow these treatments to be closer to human studies.

The technical details of the treatment are described elsewhere [14] - [17] . The treatment conditions mostly follow a standard protocol of 42˚C in the tumor for 30 min. The time from the inoculation of the tumor cells to the first mEHT treatment depends on the tumor size, which is approximately 2 g when the experimental process starts. In some experiments, Matrigel is used to control growth. The applied power varies by the animals and their tumors, ranging from 1 W to 8 W. The treated tumors were studied using various methods. The tumor in its 3D form was studied with size (caliper and ultrasound) and its activity (bioluminescence signals). The excised ex vivo tumors were characterized by their size/weight. The tumors are usually cut in half; one is frozen, formalin-fixed, and embedded in paraffin for further investigation. The frozen samples are helpful for genetic and protein analysis and Western blot, FACS, PCR, the paraffin-embedded samples for morphological studies, multiple immunohistochemistry, and signal pathway analyses; the most frequent processes with then excised samples are shown in Figure 2 .

The results are compared to sham mEHT treatment, infrared hyperthermia, or distant, untreated tumors in the same animal.

The publications on in vivo measurements are collected in Table 1 .

Most treatments were made on rodents. However, the thermal efficacy was measured in vivo on a healthy female pig (49.5 kg) liver, which is a model of a deep-seated, highly blood-perfunded organ [35] . The tumor focus was unavailable without the tumor in the liver, but the focus on the liver in the large-sized experimental animal was proven. The temperature dynamically grew in the liver mass while its surrounding temperature was behind ( Figure 3 ). The results show that mEHT can thermally impact the liver without raising the skin temperature; no surface overheating was observed, and the skin and the fatty tissue remained in a safe heating regime. Anesthesia influences temperature development in a short time. The physiological control actively regulates thermal homeostasis, having isothermal heating in the liver, while in a deep anesthetic state, the regulation is slower and less effective. The step-up heating condition in deep anesthesia allows for weaker physiologic reactions, making thermal regulation ( Figure 3(C) ).

The temperature development was measured in various experimental setups [50] . The typical temperature measurements in mice and dogs show good deep heating focus ( Figure 4 ) Noteworthy is the temperature in the lung ( Figure 4(D) ), which is well distinguishable high despite the cooling by the animal’s breathing Figure 4(D) . The power started high, but keeping the temperature enough was a fraction of the initial value because keeping the temperature only replaces the “lost” heat caused by the environmental conditions. Various treatment setups and tumor sizes influence temperature development, and the starting power is decided for the initial slope of the temperature plot.

The intratumoral temperature is usually fixed in the protocol for 42,(30 min treatment time), but the extratumoral temperatures vary by the animal, the tumor, and the environment Figure 5 .

The thermal component of mEHT heats the target. When the power adjusted step-up for a longer time (~1000 s) from 1.5 W to 2.5 W, the steady temperature growths proportionally reaching 42˚C in the tumor and ~33˚C in nearby tissues after 1000 s, when the power is downregulated to 2 W [33] . This high difference between the tumor and host temperatures is caused by the cooperative changes with thermal homeostasis and relatively high cooling, which requires 2 W power to keep the temperature constant. These special conditions were used for temperature mapping in a murine model, which agrees well with the simulations. In humans, the efficacy of mild temperature hyperthermia was studied in cervical cancer, which increases the peritumoral temperature to 38.5˚C, with proper blood flow for the complementary treatments [51] . The synergy of thermal and nonthermal processes induced by mEHT destroys the tumor, Figure 6 .

The tumor inhibition by oncothermia on human pancreatic adenocarcinoma cell line (BxPC-3) in female CD-1 mice (10-10 animals are involved). On Day 72, mice were sacrificed, and their tumors were excised and weighed. A significant difference was observed in the total weight of excised tumors between the control and treated groups. In summary, there were a total of ten treatment doses administered to each mouse (six doses in cycle 1 and four doses in cycle 2) using oncothermia [14] , Figure 7 . The mice responded well to the treatments, and no adverse side effects were observed. The treatment does not change the average body weight of mice. The tumor growth inhibition was 66% after the second cycle (p < 0.0005) measured on excised tumor weights.

It is noteworthy that the suppression of the tumor volume may be observed at temperatures as low as 39˚C. Figure 8 , measured in gynecological (ovary and cervix) tumor models. The tumor volume was significantly below the control in all cases.

Obtaining information about the synergy of thermal and nonthermal effects needs infrared heating [40] instead of the inapplicable water bath, which was in vitro a plausible pure thermal reference. The nonthermal addition to the thermal basis (42˚C) increased the dead tumor part ≈ 4.3 times larger than the only thermal effect in HT29 colorectal carcinoma [40] , Figure 9 . It is noteworthy to check that the nonthermal component, the difference between the mEHT treatment and control, both at 38˚C, shows a missing value to the mEHT 42˚C results are about the same as those of the measured thermal effect alone.

Apoptotic cell death dominates tumor cell destruction with intensive development of apoptotic bodies, Figure 10 . The volume of apoptosis depends on the treatment time and rapidly grows by the posttreatment time of the mEHT. The tumor destruction ratio (TDR, %) differs between the treated and distant, nontreated tumors. Notably, the dead area in the nontreated tumor decreases over time, while the treated tumor increases it. The ratio of the dead areas became constant from 48 h to 72 h, making the distant tumors’ interaction probable over time. In the early stages, the mitochondrial changes (decrease of membrane potential and opening transition pores); in the late stages, the caspase activation nucleus karyopycnosis, loss of cell membrane integrity, and DNA fragmentation occur.

The stress-induced antiapoptotic chaperones HSP70 and HSP90 are exhausted in the cytosol [20] and turn proapoptotic by relocating to the membrane and expressing in the tumor microenvironment (TME). The internal HSPs (iHSPs) exhaustion is connected to the subsequent cell destruction ( Figure 11 ). The shifts in the time course points of different tumor entities could be only the effect of the discrete point measurements ( Figure 11(A) ). The iHSP90 has its maximum much earlier than the iHSP70 ( Figure 11(B) ), and TDR follows the iHSP expressions in their baseline return [20] ( Figure 11(C) ). The identical dynamism of the destruction and cCas-3 shows the dominance of the caspase-dependent apoptotic pathways in 4T1 tumors [20] . Notably, the significant development of chaperoning iHSPs was a few hours posttreatment, indicating that the combined stress of the mEHT treatment excited intracellular processes, which induced the chaperone activity.

The gene study 4 h post-mEHT (42˚C, 30 min) treatment of HT29 tumor xenograft showed significant differences between the wHT and mEHT at the same 42˚C temperature, and both significantly altered from the sham control [24] ( Figure 12 ). The mEHT mostly upregulates the expression of iHSPs at the mRNA level. The HSPA1A, HSPA6, and HSPA8 genes encode various HSP70 family members (proteins 1A, 6, and 8, respectively). The HSPD1 and HSPH1 genes encode HSP60 and HSP105 chaperons, respectively. The death receptor D6 (TNFRSF21) is upregulated, and the surface receptor activator of osteoclasts, RANK (TNFRSF11), as well as Calpain 1 and 9, which support the cell autonomy and defecting the cytoskeleton, are downregulated. The BAG gene encodes the cochaperone of HSP70, and the EGR1 gene encodes a transcription factor protein, which is involved in multiple upregulated apoptotic processes. In general, we conclude from the gene map analysis that the mEHT strongly supports the apoptosis of the selected tumor cells and destroys the tumor “softly”. Nonnecrotic destruction helps liberate undeformed intracellular information from the dying cell, which could trigger antitumoral immune processes. Some other gene mapping [21] [22] [43] strongly support the above observations in vivo, which was also seen in vitro [53] . The gene map shows a distinct difference in the gene regulations between the homogeneous wHT and inhomogeneous mEHT treatment sat the same 42˚C temperature. Injection of NR4A3 or KLF11 siRNA into mouse A549 lung xenograft tumors knock down the effectiveness of RT and its combined application with mEHT [47] , revealing some aspects of the mechanism of sensitization of A459 tumors for RT and mEHT in xenograft experiments.

The protein expression (Proteome Profiler Human Apoptosis Kit) of HT29 tumor in xenograft experiments, 8 h, 14 h, and 24 h after mEHT treatment (42˚C, 30 min) shows many proteins, including pro and anti-apoptotic sets. 8 h posttreatment, the transmembrane excitation of the extrinsic pathway shows an increase of TRAILR2/DR5 and FAS-FADD complex and enhanced expression of antiapoptotic proteins, which activity drastically reduced at 14 h and further at 24 h posttreatment observations. The catalase protein remains active after 24 h, which probably regulates ROS production to the level necessary for proper apoptosis to avoid necrosis. All other proteins remain under the reference level after 24 h posttreatment of HT29 xenograft measurement ( Figure 13 ) [52] .

The cyclin-dependent kinase (CDK) inhibitors p21 and p27 are highly activated in the first 6 h after mEHT treatment and assist apoptosis by acting at different stages of the cell cycle as checkpoint controllers under the regulation of the p53 tumor-suppressor protein. The p27 CDK remains activated at 14 h posttreatment, preventing premature DNA synthesis. When the cell encounters stress like DNA damage, p53 becomes activated and plays a critical role in deciding cell fate. It can trigger cell cycle arrest to allow for DNA repair or initiate apoptosis (programmed cell death) if the damage is too severe. The activated p53 may trigger apoptosis by initiating proapoptotic genes and inhibiting antiapoptotic genes. The base form is the unphosphorylated p53 protein, which has modifications by the phosphate group (PO₄) bonded to serine (S) amino acids at positions 15, 46, or 392, modifying its regulatory mechanisms. Phospho-p53 (S15) increases the p53 protein stability and enhances the ability to bind to DNA and activate its target genes. The phospho-p53 (S46) promotes apoptosis, and the phospho-p53 (S392) is involved in transcriptional activation and potentially modulates its role in cell death pathways independent of transcription. The mEHT treatment significantly increases the expression of p53, p21, and p27 proteins in various tumors of treated mice, while the wHT effect is moderate, Figure 14 .

The mEHT extrinsically excites pathways for apoptosis [4] [55] . The extrinsic signal starts in the TRAIL death receptor with FADD and FAS complexes [38] . The TRAIL signaling, as usual in complex systems, has a dual effect, being pro-or antitumoral, depending on the TME conditions [56] . The membrane fluidity increases due to thermal effects [57] . Investigating the thermal and nonthermal complexity of apoptosis shows that the mEHT excites numerous apoptotic pathways in the cell, Figure 15 . The optimally modulated carrier signal directs the TRAIL to be proapoptotic. Massive apoptosis is noted with mEHT treatments, likely following the caspase-dependent extrinsic signal pathway with Caspase-8 (Cas8). The path may split towards the Cas3 followed by apoptosis ( $\text{Cas}8\to \text{Cas}3\to \text{apoptosis}$), or to mitochondria by signal transfer of BID protein. Mitochondria may ignite the intrinsic signals for apoptosis [20] . The intrinsic path has two options: it could be caspase-dependent (through Cas9), involving apoptosis-inducing factor (AIF) [23] [58] . AIF is usually located in the mitochondrial intermembrane space, but it is translocated to the nucleus, generating chromatin condensation and DNA degradation, providing an additional signal pathway for apoptosis. This process has the potential to affect untreated tumors as well. A notable factor is the arrest of the XIAP effect to block the main path of caspase-dependent apoptosis by the secretion of SMAC/Diabolo [38] and Septin4 [21] . The ignited nonthermal chemical processes are massively gained by the thermal conditions, producing a complex network resulting in

apoptosis. Thermal stress strongly supports the intrinsic apoptotic pathway through BAX and cytochrome c (point of no return), finishing the apoptosis through the cleaved Cas3 (cCas3) path. DNA fragmentation drives tumor-cell degradation [59] . The induced stress by mEHT upregulates the tumor suppressor p53 protein, one of the key cell-cycle regulation and DNA repair players. The p53 “master switch” drives the DNA fragmentation and the appearance of apoptotic bodies.

Noteworthy that mEHT also destroys the tumor stem cells in the U87-MG and A172 human glioma cells inoculated into BALB/c nude mice xenograft [22] .

The appropriately chosen modulation may trigger resonant excitations of the transmembrane proteins, excite the TRAIL R2 (DR5) death receptor, and activate the apoptotic pathway. Despite the increased expression of iHSPs, the mEHT inhibits tumor growth and supports apoptotic processes [25] . The expression of HSP70s has its maximum at around 12 h posttreatment. The extremely large complex thermal and non-thermal stresses exhaust the HSP protective response [20] . The intracellularly overproduced antiapoptotic HSPs relocate to the cell membrane or are released to the TME. The exhausted HSPs cannot block the apoptotic process [20] . Another antiapoptotic protein, the XIAP, is arrested by Septin-4 [21] , SMAC/Diabolo, and HTRA-2 proteins in HT29 xenograft treated with mEHT 42˚C 30 min. [52] . The development of the primary components was measured by immunohistochemistry of resected tumors of the sacrificed animals, Figure 16 .

The amplitude modulation of the carrier frequency signal (13.56 MHz) increases the effective value of the electric field of mEHT. It supports the cytoskeleton’s polymerization and the reorganization of the cytoskeletal network. Cytoskeletal polymerization has a significant effect on cancer cells, which inhibits cellular plasticity and cell migration. The 1/f fractal noise modulation introduces a huge amplitude increase at the carrier frequency, increasing its selective excitation facility. The noise modulation approach is like the harmonizing method [61] , whose application is emerging in physiology [62] . The modulation may promote the execution of the enzymatic processes [63] , modifying the transition between the initial and final states of molecules [64] . The applied modulation orchestrates the spatiotemporal order of the exported molecules to the TME and may trigger resonant excitations of the transmembrane proteins and extrinsically excite signal pathways for apoptosis. The benefits of modulation appear compared to the unmodulated (EHT) treatments ( Figure 17 ). [37] Modulation experiments with the mice having two distant tumors in their femoral regions also show the advantage of the modulation [65] [66] , Figure 17(F) , Figure 17(G) , Figure 17(H) .

The pulsing modulation pattern further improves tumor destruction [19] , Figure 18 . The pulsed signal significantly increases apoptosis (suppresses the Ki67 proliferating marker), decreases the growth rate, and develops fewer antiproliferative iHSP70 chaperones.

The thermal component of mEHT acts in synergy with the electric excitation, affecting the repair of DNA. The induced upregulation of cyclin-dependent kinase inhibitor protein ( $p{21}_{waf1}$) and the reduced Ki67 proliferation marker correlates with the expression of the $\gamma H2AX$, which is a sensitive molecular marker of DNA damage, Figure 19 . When p27 was unsuccessful in inhibiting premature DNA synthesis, the double-strand break (DSB) in DNA (shown by the $\gamma H2AX$) leads to apoptosis in mEHT-treated tumors [30] [42] . mEHT activates DSB production.

DNA damage by DSB could be promoted with an ionization beam of RT in complementary applications with mEHT. The TDR with RT + mEHT combination shows a significant decrease. The tumor volume increase is inhibited more in combined treatment than in standalone treatments, and survival decreases most rapidly in combined therapy of mice [44] , Figure 20 . The only thermal treatment with RT (RT + wHT) does not show the same inhibition of tumor growth as the RT + mEHT does.

The equivalent dose for apoptosis was determined [32] in Figure 21 for tumors of A549 and NCI-H1299 cells in BALB/c nude mice in a xenograft experiment. The amount of apoptosis was the same with RT and mEHT on tumors by inoculated A549 cells five days posttreatment (mEHT standard before RT (42˚C, 30 min)/session, RT standard 5 Gy/fraction, daily 2 times). The equivalent dose calculation used the in vitro determination of linear-quadratic equations from cell survival curves at standard RT + mEHT treatment: ${\alpha }_{A549}=0.5318$, ${\alpha }_{NCI-H1299}=0.5141$, and ${\beta }_{A549}=0.0283$, ${\beta }_{NCI-H1299}=0.0163$. The $\alpha$ value in vivo linearly depends on the temperature with a slope 0.005 $\frac{1}{Gy\cdot ˚\text{C}}$, starting from $\alpha =0.056$ at 40.5˚C. The apoptosis relative to the control did not show differences between the standard RT and mEHT treatments, but their combined treatment showed ~20+% more apoptosis than the addition of the two independently obtained values ( Figure 21(A) ), which supports that the combination is not additive but synergetic. The mEHT equivalent changes by temperature, starting with 20 Gy at 37˚C and almost doubling to 42˚C ( Figure 21(B) ).

The measurements of oxygenation (p ${\text{O}}_2$) before and right after the mEHT treatment showed a 78% increase in the p ${\text{O}}_2$ value on the treated side compared to untreated in Fisher rats with inoculated L9 glioma cell line in double tumors [68] . This increase supports the complementary therapy with RT. The mEHT generally increases blood flow in tumors, as shown in cervix tumors [51] , and the higher blood flow increases oxygenation. On the contrary, the RT decreases blood flow due to damaged vessels, consequently increasing hypoxia in addition to hypoxic tumors. Hypoxia activates the hypoxia-inducible factor-1 $\alpha$ ( $\text{HIF}-\text{1}\alpha$) and the homeostatic regulation induces vascular endothelial growth factor (VEGF) to revascularization and could promote tumor recurrence. HIF-1α can interact with the tumor suppressor protein p53, potentially leading to increased p53 activity and downstream pro-apoptotic signaling. While RT (15 Gy) alone upregulated the HIF-1 mEHT with 41˚C, 30 min treatment suppressed it together with the VEGF in FSalltumors of C3H mice [18] . The mEHT promoted the apoptosis of tumor cells and suppressed the tumor growth rate, Figure 22 .

Electrical stimuli increase the penetration of drugs like doxorubicin [69] in vivo, and the application of electromagnetic fields has been shown clinically to increase the sensitivity of cells to the effects of chemotherapies such as paclitaxel and doxorubicin [70] [71] . The complementary application of mEHT with chemotherapy improves the cellular destruction of the targeted tumors. The therapeutic effect of thermosensitive liposomal encapsulated doxorubicin (LTLD) was studied in BALB/c mice with tumors inoculated with 4T1 triple-negative breast cancer (TNBC) [46] . The variants of administered doxorubicin (free doxorubicin, (DOX); PEGylated liposomal DOX, (PLD); and LTLD) were compared by their DOX accumulation in tumors after mEHT. The TDR increased significantly by mEHT treatment and further grew by DOX, PLD, and LTLD in that order. The LTLD practically destroyed the complete tumor measured 24 h after the 3^rd mEHT treatment. The tumor destruction was massively apoptotic through the cCas-3 signal pathway, which was measured linearly with TDR. The apoptotic process was also indicated by the significant decrease of the Ki67 proliferation marker on the nuclear membrane of the dividing cells [46] , Figure 23(A) . The LTLD success shows the importance of the thermal effects for thermosensitive liposomes. In another model, murine colon carcinoma-inoculated CT26 cells were measured and treated with only thermal (wHT) and complex thermal and nonthermal (mEHT) effects [34] . While the thermal effects are effective, its synergy with nonthermal processes in mEHT doubles the release of doxorubicin. The mEHT significantly enhanced the uptake of liposome-encapsulated doxorubicin in BALB/c mice. Noteworthy that the liposomal envelope did not release the doxorubicin in the extracellular matrix but was directly liberated in the cytosol of tumor cells, Figure 23(B) . In consequence, the tumor growth was significantly suppressed by mEHT, indicating the efficacy of the nonthermal component of the energy absorption. The effect of the thermal factor was earlier also proven by wHT treatment at 39˚C - 40˚C [72] .

Other complementary applications with pharma products also show the advantage of the mEHT. The metformin increases the efficacy of mEHT (41˚C) +RT (15 Gy) complementary application in FSall fibrosarcoma in C3H mice. The apoptotic index and cCas-3 grow parallel with the metformin administration, showing the caspase-dependent apoptotic pathway in this tumor [31] . The metformin suppresses the expression of HIF-1α, VEGF and PD-L1, increasing the efficacy of the standalone treatments, Figure 24 .

Curcumin and resveratrol, effective antioxidants and immune activators were added as medications during the mEHT treatment period of inoculated CT26 cells forming allograft tumors in BALB/c mice. The added medication significantly induced cell cycle arrest and apoptosis of CT26 cells, significantly decreasing the weights of the excised tumors from the euthanized animals [29] , Figure 25 . The mEHT significantly elevated the serum concentrations of curcumin and resveratrol. The combination induced HSP70 expression while recruiting CD3⁺ T-cells and F4/80⁺ macrophages [29] .

Preclinical animal experiments on dogs and cats have a notable advantage because the tumors are naturally developed, not inoculated to the animal. The mEHT treatments of dogs show successful tumor degradation proven by various diagnostic methods, including imaging controls [13] . The treatments of dogs showed significant shrinking of tumors and considerable improvement in the quality of life of the animals [13] . These preclinical treatments, together with the validation of the method, help to optimize the technical solutions of mEHT because the body size of the animal is much larger than that of the rodents, and some of those approach human size, Figure 26 . Consequently, these studies can be transferred directly to human clinical applications to develop more precise treatment systems for clinical oncology.

Numerous successful veterinary cases were observed [73] . Many cases were treated with mEHT after first or second-line unsuccessful surgery and/or chemotherapy, Figure 27 .

The in vitro experiments in the previous part of the series about mEHT results had shown clear pieces of evidence of how the nonthermal electric field, compared to the only thermal treatment (wHT) sinergetically improved the cell destruction [10] . The primary message from the preclinical experiments in vivo is that electromagnetic energy absorption has thermal and nonthermal effects appearing synergistically in the mEHT. The strong synergy appears in many comparisons

The in vivo experiments reinforce the previous in vitro observations [10] , and the physiologic conditions make further support which drives the expectations from the clinical applications. The in vitro and in vivo proofs verify the usefulness of the mEHT making promises to a significant step forward in human oncology.

I am thankful to the listed authors for their diligent research.

This research received external funding from the Hungarian Government, grant number GINOP_PLUSZ_2.1.1-21-2022-00058.