Cryosurgery and physical medicine in treatment of cancer

Background. At the present stage of oncology development, cryosurgery is keeping up with other surgical options of treating malignant tumors. However, especially when using cryoapplication technique, radical low-temperature destruction of large tumors is challenged with uneven freezing of the tissue inside and on the periphery of the cryoablation zone resulting in insufficient complete destruction of all tumor cells. The long-term strategy of increasing the efficiency of cryogenic treatment, which will maximize the potential for exposure of biological tissues to low temperatures, is its combination with other physical methods. On the other hand, the preliminary cryotherapy of the tumor tissue can be used to increase the efficiency of antitumor therapy by other methods, in particular, physical ones. Purpose – to summarize and analyze the literature data on applying low temperatures in combination with other physical medicine techniques in order to assess the efficiency and the potential of those in uptodate oncology. Materials and Methods. The paper deals with analyzing the studies published within the period from 2000 to 2020 outlining the data on the impact of low temperatures on the biological tissues in combination with other physical medicine techniques. After keyword search against international and Ukrainian databases, the papers, focused on this subject area, were sorted out, analyzed and included in the references. Results. The review presents pros and cons of using cryosurgery in medical practice with due regard for pathogenetic mechanisms of low temperature impact on biological structures. Based on experimental and clinical studies data, a positive effect of cryosurgical approaches in combination with other physical medicine techniques when performing surgical interventions for ablation of malignant neoplasms has been shown. The paper included the contributions describing the findings on combining cryosurgery with: preliminary hemodilution; radiofrequency and microwave hyperthermic ablation; laser and photodynamic therapy; electroporation. Additionally, a contemporary view of cryo-ultrasound therapy, cryonanoablation, cryoradiation treatment and cryoelectrolysis was presented. Conclusions. Cryosurgery is an effective minimally invasive surgical method keeping up with other methods of treating malignant tumors, despite the fact that these days the research on optimizing the method of exposure of biological tissues to low temperatures is still ongoing. Cryosurgery in combination with other physical medicine techniques in case of tumor ablation can enhance the effectiveness of treating cancer patients.


ІNTRODUCTION
The uptodate approach to treating cancer patients is based on generally accepted international standards, maintained according to the principles of evidence-based medicine (Evidence-Based Medicine) and aimed at improving the quality of cancer care. According to Order No 554 of the Ministry of Health of Ukraine "On approval of protocols for medical care in the specialty "Oncology" dated 17.09.2007, the adopted standards regulate the choice of method and treatment regimens for cancer patients. Methods of minimally invasive surgery, in particular, cryosurgical ones [1], are increasingly used to destroy pathological neoplasms.
The development of the cryogenic method results from collaborative study of cryobiologists, engineers, cryogenic equipment developers, clinicians, in particular, oncologists. Theoretical calculations and experience in applying low temperatures in experimental animal research have become the basis for introducing the cryosurgical method into practical medicine. Today, the cryogenic method is keeping up with other methods of treating malignant tumors. The main potential advantages of cryosurgical treatment include low incidence of complications, short bed-day, lower cost of treatment, the possibility of treating patients with severe comorbidities and, above all, reducing or preventing the risk of dissemination and metastasis of the tumor due to hemo-and lymphostatic effect of tissue freezing [2][3][4][5].
The major challenge of cryosurgery is the destruction of pathologically altered tissues within the healthy ones. A peculiarity of the cryosurgical method is the destruction of tumors without removing them. For complete cryodestruction of tumors, the target temperature at the tumor site should not be higher -40˚C. The mechanism of cryodamage of biological tissues is fulfilled during and after cryo-intervention at molecular, cellular, tissue and system levels. The coagulation necrosis of tissues is known to appear after cryosurgery. One of the factors of destructive action of low temperatures at the cellular level is extraand intracellular crystal formation leading to mechanical damage of cells. The changes in osmotic pressure, pH and dehydration are considered as additional factors of destruction, which lipoproteins of cell membranes are particularly sensitive to [6]. Due to reaching low temperatures in the depth of the tissues, the destruction of tumor cells by necrosis is highest possible, which leads to the release of intracellular contents, including DNA, RNA and specific tumor proteins [7]. The immune system recognizes these tumor antigens and provides an immune specific reaction in the form of proliferation and activation of T-and B-cells [9]. Studies have shown that low temperatures can sensitize dendritic cells, них білків пухлин [7]. Імунна система розпізнає ці антигени пухлини і дає імуноспецифічну реакцію, що проявляється в проліферації і активації Т-і В-клітин [9]. Дослідження показали, що дія низьких температур може сенсибілізувати дендритні клітини, підвищуючи їх здатність представляти антиген і сприяти секреції ними IL-4, IL-12 та інших цитокінів [10]. Є повідомлення про випадки, коли імуноспецифічна реакція може також впливати на ракові клітини поза первинною пухлиною, що відомо як абскопальний ефект [8]. Таким чином, кріоабляція новоутворень, крім деструкції пухлини, може підсилювати протипухлинну імунну відповідь організму, направлену на запобігання рецидиву захворювання та метастазуванню.
Для кріопошкодження суттєве значення має стан зв'язаної води в клітинних та тканинних системах. У зв'язаному стані вода зі зниженням температури increasing their ability to present antigen and promote secreting IL-4, IL-12 and other cytokines by them [10]. There have been reports on cases when immune specific reactions may also affect cancer cells outside the primary tumor, which is known as abscopal effect [8]. Thus, cryoablation of tumors, in addition to tumor destruction, can enhance the body antitumor immune response to prevent recurrence and metastasis.
The degree of cell damage depends on temperature and time modes. Thus, the most crucial parameters of cryodes-truction of the tumor are low temperatures level, exposure to cryotherapy, the rate of freezing and thawing of tissues, and the number of freezing-thawing cycles [3,5,11].
In oncology, depending on tumor location, cryoirrigation, application and penetration cryoablation are used. Cryoirrigation is most frequently indicated in dermatology, as well as in gastroenterology and ENT practice in comprehensive treatment of superficial neoplasms on the mucous membranes of organs by means of endoscopic equipment. Application method, i.e. the method of contact cryodestruction, makes it possible to destruct deeper pathologically altered tissues. Cryosurgery uses the penetration method to destroy large volumes of tissue, when a cryoprobe is immersed to the full depth of the tumor. The question of choosing a technical method of cryogenic treatment is decided for each case individually taking into account location, size, nature of growth and histological structure of the tumor, the general condition of a patient, his/her age, concomitant pathology, etc. In general, the cryogenic method of treatment refers to functionally safe methods of local exposure of the pathological focus [3,5].
The positive properties of low temperature impact on the blood vessels or hollow organs is the fact that under their influence there is a death of cellular elements while preserving the collagen framework. This effect prevents the risk of bleeding or perforation both during and after cryodestruction. Therefore, it is possible to perform surgery in hard-to-reach places and near large vessels [12,13].
However, especially when applying the cryoapplication method, radical low-temperature destruction of large tumors is challenged with uneven freezing of tissue inside and on the periphery of the cryoablation zone, leading to insufficient complete destruction of all tumor cells [14,15].
The state of bound water in cellular and tissue systems is essential for cryo-damage. In the bound state, water may not crystallize at all when the temperature decreases. This, to some extent, explains the resistance of tissues to the local action of low temperatures. If, in addition to the state of water, to take into account the heterogeneous structure of tissues and their metabolism, as well as the contribution of microcirculation, i.e. the blood supply intensity and heat flow activity, it becomes clear that the destruction of large volumes of tissues by cryopreservation is a significant issue requiring to change the tissue state, so that it becomes more sensitive to low-temperature destruction [16,17]. Applying additional physical methods of tissue exposure for this purpose can contribute to a more complete tumor cryodestruction.
Purpose -to summarize and analyze the literature data on applying low temperatures in combination with other physical medicine techniques in order to assess the efficiency and the potential of those in uptodate oncology.

MATERIALS AND METHODS
Information search was carried out against PubMed database (https://pubmed.ncbi.nlm.nih.gov/), ResearchGate database (https://www.researchgate.net/), Scientific Periodicals of Ukraine (http://www.irbis-nbuv.gov.ua/). The search depth: 2000-2020. Analyzing the summary of papers by keywords: cryosurgery and physical methods of ablation made it possible to specify the search. Full-text papers by the keywords: cryo-and hyperthermic ablation, cryo-ultrasound therapy, cryonanoablation, cryosurgery in combination with laser and photodynamic therapy, cryoradiation method, cryoelectrolysis, cryosurgery and electroporation were sorted out, analyzed and included in the references.

RESULTS AND DISCUSSION
Applying a combination of different treatment options was included in medical practice for a while now. The combined method principle consists in the sequential or simultaneous application of two methods of special treatment with local action. It is the combined use of different methods that increases the efficiency of treatment due to summation -additive action, potentiation of the effects of two or more therapeutic methods or, on the contrary, the combined use can reduce or eliminate the therapeutic effect of the methods.

Cryodestruction with preliminary hemodilution
One of the methods that would enhance the cryodestruction effect is pre-hemodilution. The use of acute normovolemic hemodilution in combination with the technology of controlled low central venous pressure in liver resection in patients with hepatocellular carcinoma, is known to reduce intraoperative hemorrhage and it does not significantly affect coagulation function [18]. Increasing the volume of circulating blood in the vessels ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)
On the isolated pig liver, O.I. Dronov et al. showed increased cryotherapy due to the preliminary administered distilled water into biological tissue. Carrying out hemodilution 5 minutes before the onset of local cryotherapy has a potentiating effect, which manifests in reaching lower temperatures in the areas remote from the cryoapplicator working surface. The average temperature in the liver tissue of animals of the study group, at a depth of 3 mm from the cryoapplicator working surface, at the end of the 10th minute of the second cycle of cryo-impact was by 36˚C lower than in the control group [19]. It should be taken into consideration that the findings, obtained via such simulaing system, i.e. in the absence of splanchnic blood flow, can not fully meet the conditions in vivo. However, the effect of previous hemodilution is probable, which requires further experimental and clinical studies.

Combination of cryo-and hyperthermic ablation
Hyperthermal ablation systems have been created and successfully used for local temperature rise in tumors: radiofrequency, microwave; moreover, electrodes can be inserted directly into the organ (after laparotomy) as well as percutaneously under ultrasound or radiation control [20,21]. The energy used to heat tumors includes microwave energy (in the frequency range 433-2450 MHz), radio frequencies (100 kHz-150 MHz). Hyperthermia is a long-term method of treating malignant neoplasms, which consists in the fact that a part of the body or individual organs are exposed to high temperatures (above 39°C, up to 44-45°C) [22][23][24]. Numerous studies have shown that the thermal sensitivity of tumor tissue in ultrahighfrequency (UHF) hyperthermia depends on the degree of vascularization and blood flow in it, the level of pO 2 and pH, etc. [23][24][25]. A distinctive feature in treatment with the use of microwave hyperthermia is that thermal energy is released in the tissues themselves, and is not supplied convectively from the outside, thus eliminating overheating of surface tissues.
A great number of papers have been focused on combined use of the cryogenic method and microwave hyperthermia in experiment and clinic [16,21]. V.V. Shafranov showed that preliminary intervention on the freezing zone by microwaves (ultrahigh-frequency electromagnetic field) significantly increases the volume of cryonecrosis and makes it possible to use this treatment option for large deep tumors of particularly complex anatomical location, when other treatments are not possible. One of the mechanisms of substantial enhancement of cryonecrosis in combined use of microwaves is to increase the thermal conductivity of biological tissues [16]. Under microwaves, polar molecules become excited, resonant phenomena ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)
Despite the availability of more than 900 clinical trials over the past 50 years, hyperthermia has not been included in practical oncology. Studying randomized clinical trials on electromagnetic hyperthermia performed after 1990 [26] showed that out of 14 studies, the results of 9 were considered to be negative by the authors themselves [27][28][29]. However, these days, studies on comparing the effects and exploring the mechanisms of the combined action of cryoand hyperthermic ablation do not become less of an issue.
Via experiments on pigs, a comparative analysis of the results of cryo-and microwave liver ablation was performed. Computed tomography showed that immediately after microwave ablation, the lesion area was significantly larger than the area after cryosurgery, while the depth of the necrotic area was similar [30]. Seven days later, the area of damage after microwave ablation increased vs the sizes obtained on day 1, but the area after cryoablation did not differ from the original size. Due to the study, it has been found that for a long time after both treatment options, complete tissue necrosis can be achieved, but the degree and depth of necrosis differ: foci of necrosis after cryoablation under CT image do not change, while after microwave ablation they increase over time. The authors conclude that these two methods can be used in combination, but the parameters of the procedures need further study [30].
The paper [23] presents an experimental substantiation of combined use of local cryodestruction and subsequent UHF hyperthermia on simulated sarcoma 37 in mice and Guerin's carcinoma in rats. Combination of cryodestruction of the primary tumor site with subsequent local microwave hyperthermia was revealed to have a significantly greater antitumor effect than monotherapy, since there were 30% more fully cured animals compared with monotherapy, and the life expectancy of animals increased 1.5-2-fold times, regardless of primary tumor histogenesis. The antitumor effect is driven by the fact that after cryodestruction, there are optimal conditions for further UHF hyperthermia: severe hypoxia of tumor tissue, microcirculation disorders and development of persistent vascular stasis, pH shift to the alkaline side [23].
In clinical practice, after treatment of patients with oral mucosal cancer using cryosurgery and local UHF hyperthermia, the best functional and aesthetic results were obtained without additional restorative interventions. Combination of two physical factors has made it possible to significantly increase the degree of tumor destruction, reduce the recurrence rate and, thus, increase the radical nature of treatment. Comprehensive treatment (cryodestruction of the tumor with further, in 24 hours, local UHF hyperthermia in 1.0-1.5 months after chemotherapy) substantially reduced the frequency of recurrences and regional metastases of the tumor. During the first 12 months, the recurrence rate decreased from 31.6 to 7.7%, for ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)
The 4T1 mouse breast cancer model showed that local cryothermal therapy (alternating cooling with liquid nitrogen and heating with radiofrequency waves) was significantly reducing distant lung metastases and improving long-term survival. Moreover, the results of tumor re-triggering experiments indicated the formation of strong tumor-specific immune memory after local treatment of primary tumors. Further research has shown that cryothermal therapy promotes increasing extracellular release of heat shock protein Hsp70. After a while, Hsp70 induced differentiating Myeloid derived suppressor cells (MDSCs) into mature DC dendritic cells, facilitating MDSCs-mediated immunosuppression easing and, ultimately, the activation of a strong antitumor immune response [22]. In general, despite criticizing the method of hyperthermia, combined use of cryo-and hyperthermia can significantly increase the degree of destruction of malignant neoplasms, reduce the frequency of their recurrence and, thus, increase the radical nature of treatment.

Cryo-ultrasound therapy
Among surgical methods of treating patients with malignant tumors of the ENT organs, cryo-ultrasound therapy is preferred as a relatively optimal method of treating tumors both in terms of cosmetic and functional effect, and achieving maximum local destructive effect on tumor tissue with minimal damage to surrounding healthy tissues [31]. Molotov O.V et al. have shown in their paper that in case of applying local cryo-ultrasound destruction on the background of modification by leukinferon, the increased number of immunocompetent cells is observed, providing intensive resorption and elimination of necrotized tumor masses, acceleration of local reparative processes in the area of cryonecrosis along with the formation of connective structures. Within three years after combination therapy, recurrences, regional and distant metastases were detected in 13.5% of patients, and in control -in 33.6% of patients.
of necrosis than after isolated cryosurgery. Tissues are most damaged in modes when insonification occurs both in freezing and thawing processes. In addition, ultrasound in combination with low temperatures affects the speed of reparative processes in the direction of reducing the regeneration time. Thus, after combined cryo-ultrasound intervention with preliminary insonification, a white elastic scab was formed, which was rejected independently on day 6-8, in contrast to the simultaneous cryo-ultrasound intervention, in which the scab was rejected on day 10-14. According to the authors, the strengthening of cryodestruction effect may be associated with the impact of ultrasound on formation and increase of ice crystal size, appearance of fine crystalline structure (at freezing stage) and possible intensification of recrystallization in the ultrasonic field (at thawing stage) [32].
M.V. Merzlikin shows in his study that using ultra-low temperatures for resection of liver tumors reduces intraoperative blood loss, increases the ablasticity of operations due to tissue destruction along the line of liver dissection. Cryotherapy in operations for malignant and benign liver tumors can be performed in three versions: cryoresection by means of cryoscalpel, cryo-ultrasonic scalpel and cryovibroscalpel; normal resection with cryodestruction of the stump of the liver along the line of dissecting; cryodestruction of metastases and small hemangiomas. In these cases, it is possible to perform the operation via laparoscopic access [33].
The possibility of remote exposure of the tumor has spread due a new non-invasive method, i.e. ultrasonic ablation (High Intensive Focused Ultrasound -HIFU) [20]. The principle of therapeutic action of ultrasound is the same as in diagnosis, and is based on the ability of the ultrasonic wave to pass through the tissues without damaging them. As it passes through the tissue, some energy is converted into heat. When focusing the ultrasonic wave with a special lens under HIFU-exposure, the temperature rises quite rapidly (1s) to a minimal level of 56˚C, which creates a cytotoxic effect, causing irreversible changes in tissues -coagulation necrosis. During HIFU exposure, the temperature can rise above 80°C, at which tumor tissues are effectively destroyed [34,35].
The destructive effect of HIFU-ablation is achieved due to the combined, almost simultaneous action of three processes: 1 -conversion of mechanical energy into heat energy, 2 -cavitation start, 3 -direct damage to the vessels supplying the tumor. As a result of HIFU-ablation there is a small local center of damage and necrosis, while the superficial and surrounding tissues remain intact. In tissues, these processes occur extremely quickly and simultaneously, so coagulation necrosis caused by focused high-intensity ultrasound is due to the total biological effect of all factors [20]. The literature provides only data on comparing the effectiveness of cryo-and HIFU-ablation in treatment of tumors, such as prostate cancer [36,37]. Literature review В тканинах ці процеси відбуваються надзвичайно швидко і одночасно, тому коагуляційний некроз, викликаний фокусованим ультразвуком високої інтенсивності, зумовлений сумарним біологічним ефектом усіх факторів [20]. У літературі наведено лише дані щодо порівняння ефективності кріо-та HIFU-абляції в лікуванні пухлин, наприклад, раку простати [36,37].

Cryonanoablation
One of the approaches to solve the issue of uneven tissue freezing is the local introduction of nanoparticles (NP) into the tumor before cryoablation procedure [38][39][40]. The use of NP is driven by the ability of some species to increase the likelihood of intracellular ice formation, which leads to damage to the cytoskeleton, organelles and membranes and, as a consequence, to cell death [10]. The results of experiments on differential scanning calorimetry confirmed that administering NP can enhance the heterogeneous formation of the ice core and promote the clustering of ice. NPs are known to also show unique thermally conductive properties, due to which the efficiency of combined cryoablation by means of NPs usually exceeds the results of standard cryosurgical operations [14,40,41].
A wide range of materials are used to produce NPs, including inorganic metals, biodegradable polymers, liposomes, micelles, and semiconductors. In order to improve the nucleation kinetics and enhance the thermodynamic parameters, Fe 3 O 4 is used, while to enhance cryodestruction, polymer NPs are used, which change the morphology of ice crystals and increase thermal conductivity. Nanoparticles of aluminum oxide (Al 2 O 3 ) are able to increase the rate of formation of an ice ball, thereby significantly increasing the efficiency of tissue freezing [14,41,42]. Magnesium oxide nanoparticles (MgO) increase the rate of freezing by increasing thermal conductivity. In addition to excellent thermal properties, MgO NPs are biocompatible substances and are capable of biodegradation, which allows them to be more widely used in cryosurgical practice [14].
It is worth pointing out that the use of cryosurgical interventions requires the determination of cryoablation parameters, which is essential for effective cryodestruction of tumors. This mainly concerns the freezing rate. When the freezing rate is high, no ice is formed in the solution and the cells do not suffer large losses [40]. Therefore, the optimal cooling rate should be maintained to achieve intensive formation of ice crystals. J. Wang in an experiment on cell cultures showed that the maximum probability of formation of intracellular ice was observed at a freezing rate of 30°C/min [40]. Similar results have been obtained in human embryonic kidney cells and microvascular endothelial cells [40]. Up to date, the processes that occur during freezing in tissues and large organs, as well as cryostability of tumors depending on the organs where they are located are not sufficiently studied, and, as a result, the questions on the parameters of tumor freezing in self-cryoablation as well as cryonanosurgery remain open. Sun Zi et al. [43] present an attempt to estimate the distribution of tissue freezing zone in the ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)

Комбінація фотодинамічної терапії та кріоабляції
Важливо зазначити, що в онкохірургії досить часто використовується метод фотодинамічної терапії, який дозволяє отримувати селективне руйнування пухлини з мінімальним пошкодженням навколишніх здорових тканин. Завдяки тому, що речовини для фотодинамічної терапії мають властивість виборчого накопичення в пухлині, уражені патологічним процесом тканини опромінюють світлом із довжиною хвилі, що experiment. Nanocryosurgical operations were simulated in a model of isolated muscle tissue, liver and heart of a pig. A 5% aqueous solution of NP Fe 3 O 4 was used, which was injected into the tissues. Temperature fields were monitored by thermocouples and an infrared thermometer. Ice balls were found to have different sizes due to tissue heterogeneity, and therefore the parameters of cryosurgical interventions should depend on the actual situation. Muscles, liver and heart differ in terms of porosity, water content, density, blood supply, thermal conductivity, specific heat, etc., so the number of NPs to be injected into the tissue should be predicted in advance. This will help to perform high-quality controlled ablation of target tumor tissues during surgery [43].
The ability of non-metallic nature of NPs to reduce the thermal conductivity of tissue make it possible to use them in cryonanosurgery [38]. Such NPs are injected along the edges of a solid tumor to "protect" healthy tissues before cryosurgery.

Laser therapy and cryoablation
The results of applying cryosurgical methods of treatment in combination with laser irradiation in ENT practice are well-argued and indicative [44]. The capabilities of modern surgical lasers (small size, autonomy, lack of special external cooling systems, a wide range of power and wavelength) allow them to be used in almost all oncosurgery areas [44]. A. Miehke et al. described the main mechanisms of interaction of СО 2 -laser with human tissues, indications for laser surgery of the larynx, in particular chordectomy and dissection of the ligament stenosis, and identified prospects for its expansion [44].
A high efficiency of combination of laser therapy and cryosurgery is shown in palliative veterinary practice. According to the outcomes of treatment, it was found that combined СО 2 -laser and cryosurgical ablation is reasonable, cost-effective long with providing a high aesthetic effect in dogs with squamous cell carcinoma of the nasal cavity as well as acceptable relief of local manifestations of the disease [45].
Combination of cryosurgery and photodynamic therapy is an alternative to conventional surgery for treatment of multifocal Paget disease of the vulva with substantial or recurrent manifestations in the elderly. C. Boularda et al. [49] described the treatment of two patients with Paget disease and presented positive outcomes of the use of cryosurgical techniques in combination with photodynamic therapy using СО 2 -laser. Complete remission of the disease in patients occurred 12 months after treatment [49].
The paper of Hou Yi et al. [50], presenting the development of a hybrid platform that includes a paste of liquid metal containing gallium and copper, is especially noteworthy. The paste has a high thermal conductivity, while liquid metal nanoparticles have a high photothermal conversion, to enhance the therapeutic effect of multimodal therapy. The liquid metal paste acts as a bandage on the skin with high thermal conductivity, improving the heat transfer of tumor tissue during cryoablation. With the use of the paste, the final temperature of the tumor tissue was lower, and the temperature distribution was more uniform than without the paste, which probably increased the effect of cell destruction. Liquid metal nanoparticles acted as photothermal agents that increased temperature changes during laser irradiation [50]. Thus, the hybrid platform can simultaneously mediate dual action and fulfill synergistic effects in cancer therapy.
З використанням оптичних вимірювань та вимірювань струмів в експериментальних дослідженнях було показано, що електроліз може також відбуватися під час заморожування фізіологічного розчину при мінусових температурах, вище евтектичної температури для розчину солі, і що поширення фронту рН The clinical study by J.M. Vergnon et al. [56] enrolled 38 patients with symptomatic obstructive tumor of the trachea or main bronchus, who were treated first with cryotherapy under general anesthesia, and then exposed to external irradiation. Cryotherapy, assessed by bronchoscopy, was found to be effective in 26 out of 38 patients. No residual bronchial tumor was detected after irradiation in 17 out of 26 patients. The best survival was associated with both the efficacy of primary tumor destruction by cryotherapy and radiation-induced treatment (median, 560 days). A positive result was obtained in 65% (17/26) cases, i.e., it was better than 35% usually reported only after irradiation. These outcomes indicate the effectiveness of enhanced irradiation by cryotherapy [56]. The results of experimental and clinical studies have shown that cryodestruction of a malignant neoplasm under certain regimens sensitizes cells to further radiation exposure, which may increase the effectiveness of treatment [56,57].

Cryoelectrolysis
Current trends in cryosurgery include cryoelectrolysis which is a new minimally invasive surgical technique of tissue ablation, combining electrolytic ablation with cryosurgery [58]. The electrochemical lysis method consists in the destruction of tumor tissue located between a pair of electrodes due to the lytic action of alkali (sodium hydroxide) and acid (hydrochloric acid), which are formed in the cathode and anode zone, respectively. Besides, the electric field causes a flow of water (electroosmosis) from the anode to the cathode and, accordingly, there is swelling near the cathode and dehydration of the tissues around the anode. Lysis is enhanced by direct exposure to direct electric current. Electrolytic ablation requires very low direct currents (from dozens to hundreds of mA) and very low voltages (from one to several dozens of volts) [58]. The affected area can expand significantly due to increasing the number of electrodes inserted into the tumor [59].
A special nature of changes in temperature and electric currents in the process of heating the frozen tissues, is attributed by the authors to large amount of energy that must be removed at the outer edge of the frozen area via transformation of solid/liquid phase at this border.
The concept of cryoelectrolytic ablation suggested by F. Lugnani et al. [61] is that the target organ is first exposed to electrolysis to produce cytotoxic electrolysis products in a specified volume; after that the tumor is exposed to cryotherapy to increase the local concentration and action on the inner part of the cell of electrolysis products. This leads to reducing the time for electrolysis and, as a consequence, to decreasing concentrations of cytotoxic electrolysis products and shortening the freeze-thaw cycles during cryoablation.
Experiments with animal tissues have shown that cryoelectrolysis is more effective in cell ablation than cryosurgery or electrolytic ablation alone [60], contributing to the effectiveness of cryosurgery.

Cryosurgery and electroporation
None of the treatment options in the world, from concept development to clinical use, has been introduced into medical practice as quickly as electroporation. The electroporation technique uses pulsed electric fields aimed at damaging tissues. The effect of electric shock consists in creating nanoscale pores in the cell membrane, disrupting intracellular homeostasis [62]. Fulfilling the electroporation occurs at the following stages: charging and polarization of the membrane; destabilization of the membrane structure and creation of hydrophilic pores; increase and stabilization of the pore radius; re-closure of pores and cell survival or cell death due to the appearance of large defects [63]. The formation of nanopores, depending on the influence of electric current, can be shortterm or permanent, so there is reversible and irreversible electroporation.
In the experimental paper, J. Edd et al. [64] studied the effect of irreversible electroporation caused by a single impulse lasting 20 μs with 1000 V/cm of voltage, in vivo in the rat liver. In histological examination three hours after the impulse on the treated areas of the liver with fixed perfusion, the authors observed microvascular occlusion, endothelial cell necrosis and diapedesis, which led to ischemic damage to the parenchyma and a massive accumulation of erythrocytes in the sinusoids. However, the architecture of large blood vessels was preserved. The authors also performed a mathematical analysis that showed that this damage was non-thermal in nature. Another advantage of irreversible electroporation is faster wound healing and less connective tissue formation.
Аналіз літературних даних показав, що незважаючи на потенційний ефект різних фізичних методів в temporary mode of impulse delivery. The best outcomes were obtained after applying 80 impulses of 100 μs at 0.3 Hz with voltage/distance ratio of 2500 V/cm. This protocol caused complete regression in 12 out of 13 tumors.
A higher percentage of tumor regression was observed in patients with a mean tumor size of 1 cm in areas with increased risk for surgery: perivascular area or tumors located close to the bile ducts. A low regression rate was observed in patients treated for liver metastases and tumor size greater than 5 cm [62].
In the future, cryosurgical treatment is being planned to be combined with electroporation. This approach is due to the fact that when the temperature of the tissue decreases, the action of the electric field is limited to the cooled zone, and, in addition, cooling increases the electric field strength. On the other hand, the increased osmolality of the external cellular environment, caused by the action of low temperatures, helps to reduce the voltage of electric current during electroporation [62].
In other words, the development of this direction, if there are opposite directed effects of pre-freezing of t he tissue on the electric field strength, requires further theoretical and experimental adjustment of cryoaction and electroporation modes to achieve optimal results.

CONCLUSIONS
Cryosurgery is an effective minimally invasive surgical method keeping up with other methods of treating malignant tumors, despite the fact that these days the research on optimizing the method of exposure of biological tissues to low temperatures is still ongoing. Having a number of advantages over existing ablation methods, especially due to the cryoimmunological component, cryosurgery is an alternative method of traditional surgical treatment.
The combination of hemodilution, nanoablation, electrolysis, electroporation, ultrasound and photodynamic therapy can potentially increase cryodestruction efficiency, including application cryodestruction of large deep-seated tumors. However, most of these approaches remain at the level of simulated experiments.
Despite the clinical experience in combination of cryotherapy with UHF-hyperthermia, laser, ultrasound, photodynamic or radiation therapy, the methods of cryoultrasound and, especially, cryoradiation therapy were quite widely introduced into practical oncology. Pre-cryotherapy can increase the radiosensitivity of malignant cells and partially compensate the negative side effects of radiation.
Literature data analysis has shown that despite the potential effect of different physical methods in combination with cryosurgery to confirm the effectiveness of