Cryotherapy, a method based on the cytotoxic effects of cold, consists in the therapeutic application of extremely low temperatures to living tissue in order to obtain their destruction. It represents a minimally invasive surgical technique that has expanded in applicability in recent years, in part because of the development of new and improved equipment. Cryosurgery has now a wide range of clinical applications: dermatology, gynecology, urology, neurology, pulmonary medicine, cardiology, oncology and many others... (see the other sections of the website). It is also used in veterinary medicine.
In the same way, fundamental research in cryobiology has received renewed attention as shown by recent in vitro and in vivostudies. Basic science is essential as it helps understand the mechanisms of action of cryotherapy and its biological effects at tissue, cellular and molecular levels. A better understanding of the biological events is also expected to lead to the optimization of the cryosurgical technique and consequently to improved clinical results.
The purpose of this section is not to present an exhaustive review of what have been done to date in this field (excellent reviews are available in the literature), but it aims at giving a general overview of current knowledge providing major references for further information. Most importantly, after this overview, is an update on current research in cryobiology. It is an open section where researchers are invited to expose and comment their latest findings. Finally is a link to a forum discussion where theoretical concepts as well as technical problems in fundamental research can be discussed, where anyone could ask and/or answer questions about recent research, making of this website a tool for sharing experience and diffusing information...
Dr Valérie Forest, PhD
Centre de Recherche du Centre Hospitalier de l’Université de Montréal,
Pavillon J.A De Sève, porte Y5626,
1560 rue Sherbrooke Est, H2L 4M1 Montréal (QC),
I. Overwiew of Basic Science in Cryobiology
The physical effect which is known as “direct cell injury” is immediate and consists in the formation of extra- and intra-cellular ice crystals. Cell structure and cell functions begin to be stressed when temperature falls into hypothermic range. But as temperature reaches the freezing range water crystallizes, first in the extracellular spaces. It creates a hyperosmotic environment, which in turn leads to ion and water movements and finally results in cellular dehydration (this is referred to as “solution-effect injury”). Consequently, cells shrink and membranes and cellular components (especially mitochondria) are damaged. With further cooling, ice crystals may also form within the cells as shown by Figure 1.
Figure 1 – Direct cell injury from freezing.
Ice crystal formation first occurs in the extracellular spaces, which withdraws water from the system and creates a hyperosmotic environment. This in turn draws water from the cells. Intracellular ice formation (IIF) occurs when the cooling rate is sufficiently rapid to trap water within the cells. Adapted from Theodorescu .
During thawing, ice crystals fuse to form larger crystals, this phenomenon, called recrystallizationoccurs especially at temperatures about -20 and -25°C. As ice melts, the extracellular environment rapidly becomes hypotonic allowing water to enter within the damaged cells, causing cell swelling until cell membrane disrupts.
Additional damages relate to the cytoskeleton which structure depends on bonds between membrane proteins and the cell scaffold. Lowering the temperature weakens these bonds and makes them particularly vulnerable to mechanical damage.
Moreover, ionic changes cause a pH decrease, damaging proteins and enzymes necessary to cell functions and consequently lead to metabolism failure.
Damages are additive, depending on time and are particularly expressed when cells return to their normal physiological temperature.
The vascular effect is delayed but intense.
Vascular stasis and cellular anoxia represent the main mechanisms of the cryoinjury. Vascular stasis is the outcome of a series of changes in the circulation: the initial response of a tissue to freezing is a vasoconstriction and thus a decrease in the flow of blood. The vascular endothelium is damaged, which results in increased permeability of the capillary wall, oedema, platelet aggregation and micro-thrombi formation, which in turn lead to stagnation of the circulation. The loss of blood supply deprives cells of any possibility of survival and results in ischemic necrosis.
When tissue thaws the circulation returns with a compensatory vasodilatation: because the tissue has been deprived of blood flow during freezing, the cells release vasoactive factors after thaw, causing the vasculature to dilate and the tissue to be hyperperfused. This hyperperfusion is theorized to induce free radical formation that can cause further endothelial damage by peroxidation of the lipids in the membrane.
Finally, concerning the long-term vascular effects of cryosurgery, a relative hypervascularization has been observed 15 days after cryotherapy.
To be complete, it should also be mentioned that a third effect of cryosurgery has been reported: animmunological effect. However it remains controversial as in some studies cryosurgery has been shown to produce a positive immune response whereas in other studies it had either no effect or even a negative immunologic consequence. The majority of evidences for cryoimmunologic responses comes from reports that have described patients with widely metastatic disease, who after palliative cryotreatment of the primary tumor, experienced shrinkage, or even resolution of their metastases [8, 9]. It was theorized that during cryosurgery the immune system of the host became sensitized to the tumor being destroyed by cryosurgery.
A wide range of studies have compared tumor growth rate following cryoablation or surgical excision of a primary tumor and have also investigated the incidence of metastases after these treatments.Joosten et al. have shown that cryoablation of tumor tissue resulted in a significant inhibition of secondary and metastatic tumor growth compared to animals treated by surgical excision in a mouse colon tumor model . Sabel et al. have further demonstrated that cryoablation resulted in increased protection against rechallenge compared to surgical resection . While these studies argue for a positive effect of cryoablation, Allen et al. have shown that though cryoablation of hepatomas in rats does not accelerate residual tumor growth, no evidence for the development of tumor immunity following cryosurgery was found . On the contrary, other studies claim that not only the development of antitumor immunity is delayed but also that secondary tumor growth and incidence of metastases are more likely to be enhanced by cryosurgery than by surgical excision [11-14].
The exact mechanisms of the cryoimmunologic effect remain unclear; it has been assumed thatantigen release from necrotic tumor tissue could play a role in the induction of specific or non specific antitumor response. It has been suggested that both cell-mediatedand humoral immunity could be implicated . Concerning the production of antitumor antibodies, the hypothesis is that the frozen tumor left behind after cryosurgery may serve as antigen for the development of tumor immunity . A second potential method of the immune system to affect tumor growth is by cytotoxic T-cell-mediated tumor killing. This effect could be related to the cryo-induced necrosis, as membrane disruption and cellular content release induced by freezing could behave as attracting signals to inflammatory cells (contrary to apoptosis which proceeds without inflammation and thus is unable to generate an immune response) [16, 17]. The pro-inflammatory chemokines produced stimulate the T-cells [9, 17].
Though aspecific immune responses have been described (increased activity of natural killer cells and macrophages), cryosurgery seems to increase immunity that is specifically directed against the tumor [9, 15, 18]. This was evidenced by rechallenge studies demonstrating only antitumor immunity against tumor tissue identical to cryotreated tumor tissue. Moreover, cryotreatment of normal tissue does not influence the growth rate of the secondary tumor .
Studies which attempted to evaluate the extent of the cryoimmunologic response produced conflicting results. A systemic antitumor response has been postulated after cryoablation of tumor tissue , and studies have demonstrated a multi-organ inflammation in response to cryosurgery through the release of pro-inflammatory cytokines [19, 20]. On the contrary, according to Sabel et al., the immunologic response is clearly limited as cryosurgery induces tumor-specific pre-effector cells regionally but not systemically . Additional information is needed on the mechanisms by which cryoablation may stimulate tumor immunity in order to amplify the response.
It has been well documented that cryotherapy is a method of spherical action as isotherms extend radially from the probe, as shown by Figure 2.
Figure 2 – Cartography of a cryolesion.
Temperatures in the tissue vary depending on the distance from the probe.
Histologically, three distinct areas can be clearly identified in a frozen tissue:
- the centre of the cryogenic lesion (closest to the cryoprobe impact site) is completely necrotic,
- this area is surrounded by a small margin of tissue where the freezing temperature was not cold enough to kill all cells (0 to –40°C), as temperatures warm further from the probe tip. This area ofpartially damaged tissue is constituted by a mixture of living and dying cells: some cells survive (though damaged to some extent), others die and some are in balance. It is in this border zone that apoptosis, a gene-regulated cell death has been reported to be implicated [2, 3].
- and finally, in periphery, intact tissue, unaffected by freezing, is found [21, 22].
Because of both vascular and tissue inhomogeneities, the thermal history will vary dramatically from point to point in the tissue . For instance, cryotoxicity decreases near the permeable vessels where some perivascular cells are protected from destruction.
- Technical considerations
The basic science of cryosurgery is an interdisciplinary research field involving both biology and engineering. This latter is focused on how to measure and predict the thermal and injury behavior usingengineering tools .
Cryosurgery should be tightly monitored, indeed, if freezing is not sufficient, recurrence of malignancies could occur, and inversely, if it is excessive it could affect adjacent healthy tissue.
The tissue-freezing process and the extent of the cryolesion can be monitored by local measurement techniques (thermometry or impedancemetry with thermocouples or electrodes placed inside or around the tissue that is being frozen) or by imaging (ultrasound, computed tomography, magnetic resonance imaging...) [5, 25, 26].
The choice of the freezing agent is crucial as the effects of cryosurgery are directly linked to the temperature achieved in the tissue. It is admitted that the temperature necessary for cell destruction should be between –20 and –40°C .
Very different cryogens can be used (liquid or gaseous). The main refrigerant agents currently used are liquid nitrogen, argon and nitrous oxide... Concerning the freezing device, the working of the probes is mainly based on the Joule-Thomson effect (cooling of a gas by sudden expansion from a high to a low pressure zone through a small orifice) [21, 25].
Freezing can be used for cell destruction as well as for cell preservation, so different parameters should be carefully taken into consideration. It is well known that a slow freezing followed by a rapid thawing will result in a better cell survival whereas rapid freezing and slow thawing will induce a maximal lethal effect because of recrystallisation [1, 2, 5].
Other parameters are important such as:
- the number of freeze/thaw cycles: it has been demonstrated that damages increase with the repetition of cycles, because cells are subjected to additional deleterious physicochemical changes after they are already weakened by damage sustained in the first cycle. In addition intracellular ice crystals are larger [1, 2, 5],
- holding (duration of freezing),
- the masse of frozen tissue,
- the nature of tissue: it is well admitted that lethal temperatures are highly cell-type dependent. For example muscle cells and melanocytes are very cryosensitive whereas cartilage and keratinocytes resist freezing temperatures. Cancer cells seem to be more sensitive to freezing than normal cells. Differences in sensitivity of malignant cells also exist, for instance Yang et al. have shown that the tolerance of cryolytic cell death varied in different cell lines, even though they were all derived from human colorectal tumors [1, 2, 23, 17, 27].
- the distance from the probe as previously discussed.
UII. Update on current research in Cryobiology and Latest News
Cryosurgery is a potent method of in situ tissue destruction, but it generally needs the support of adjunctive therapy su
ch as che
motherapy or radiotherapy. Combined treatments are important topics of current research. The aim is to inc
rease the rate of cell death in the peripheral zone of the cryolesion where cell survival is in balance. Indeed, in this area, less damaged cells can repair and survive, therefore requiring
further injury to complete the lethal effect [2, 22, 28].
Cryotherapy and radiotherapy or chemotherapy could present synergistic effects because the latter could affect the area of lower cryosensitivity but also because freezing induces hypervascularization and thus enhances the sensitivity of well-vascularized tissue.
A wide range of current studies are also particularly focused on how cell death occurs after cryosurgery. Apoptosis is an important mechanism of cell death when temperature does not fall sufficiently low enough to kill cells through direct ice rupture or necrosis. A better knowledge of the pathways of cell death involved will certainly improve the therapeutic outcome.
Cryotherapy and Radiotherapy:
It has been assumed that the hypervasularity observed after cryosurgery could enhance the radio-sensitivity of tissue .
The results of a study lead in patients suffering from inoperable lung cancers have suggested an efficient potentiation of irradiation by cryotherapy as the survival of the patients was increased after a combined treatment .
More recently, Znati et al. exposed PC-3 prostate cancer cells to freezing (-180, -100 or -20°C) and to irradiation (2 Gy) immediately after thawing or up to 4h later. Cells exposed to cryothermic conditions exhibited increased sensitivity to radiation for a short period of time after thawing but quickly recovered to baseline sensitivity with intervals of at least 1h between thawing and irradiation .
Burton et al. have investigated the effects that cooling might have on the radiosensitivity of a human cervical carcinoma cell line (HTB35) . Cells were subjected to hypothermia, but not freezing temperatures (0, 5 or 15°C) for up to 24h before irradiation. Results demonstrated that cooling-enhanced radiosensitivity was dependent on cooling temperature, duration and rewarming interval before irradiation.
The combination cryoradiotherapy has been poorly investigated; fortunately, more data on the association of cryosurgery and chemotherapy are available.
Cryotherapy and Chemotherapy:
The first study presenting the effects of a combined treatment of cryosurgery (performed with liquid nitrogen) and chemotherapy (5-FU) in the treatment of oral cancer was reported by Benson in the 70s. Results have shown that chemotherapy was more efficient when administered aftercryotherapy as it seemed that the anticancer agents concentrated in the tumor immediately after cryosurgery .
Later, Ikekawa et al. confirmed in a model of melanoma grafted into a murine model that some drugs (but not all kinds of antimitotic agents) could be trapped in the tumor immediately after freezing. But this trapping was observed only when the chemotherapeutic agents were injected after cryosurgery, because of the disturbance of the micro-circulation freezing induces .
Homasson et al. have also observed in patients suffering from inoperable lung cancer that bleomycin (BLM) could be trapped in tumors immediately after cryosurgery . Indeed, the uptake of BLM was significantly increased (about 30%) in the frozen tissue, when it was injected 2 to 6 hours after cryosurgery. Moreover the plasma clearance of BLM was accelerated which was in correlation with an increased accumulation of BLM in the frozen tissue. This could be explained by the vascular disrupt and the local vascular stasis caused by freezing.
More recently, the effects of cryochemotherapy have been investigated in vitro. Mir et al.  have demonstrated that cytotoxicity was increased when cells from melanoma were first exposed to -20°C and then to BLM. This is due to the fact that freezing induces a membrane destabilisation, allowing BLM to enter the cells whereas it hardly enters normally, under physiological conditions.
Concerning the schedule of a combined treatment, results reported by Clarke et al. [36, 37] seem to be in contradiction with the above-mentioned studies as the authors claim that chemotherapy is more efficient when followed by cryosurgery in a model of renal carcinoma cells. They observed that whereas the addition of 5-FU at the same time or 2 days after freezing resulted in a synergistic lethal effect, many cells survived this combination treatment. However, when cells were treated with 5-FU 2 days prior to freezing there was an apparent complete loss of cell viability.
The authors confirmed in a model of human prostate cancer cell line (PC3) that using 5-FU in combination with freezing enhanced loss of cell viability in comparison to either freezing or 5-FU alone . More interestingly, this study has implicated apoptosis in an underlying contributor to this increased efficacy. Further investigation was performed and molecular analysis indicated that freezing and chemotherapy differentially activated apoptotic cascades through modulating opposing members of the Bcl-2 protein family . Freezing (-15°C) resulted in a time-dependent increase of the anti-apoptotic Bcl-2 protein, while chemotherapy triggered an increase of the pro-apoptotic Bax protein. The cryo-induced increased expression of Bcl-2 was prevented by the addition of either 5-FU or cisplatin. Thus, pre-exposure of cells to a cytotoxic agent resulted in a shift of the Bcl-2 to Bax ratio from pro-survival to pro-death signalling, indicating that both Bcl-2 and Bax may play an important role in the efficacy of the cryochemotherapy combination and the balance between the two may determine the primary mode of cell death following treatment.
Interest in how cell death occurs after cryosurgery has grown and several studies have been recently published. For instance, Hanai et al. identified apoptotic cells when cells from a human colon carcinoma cell line (HT29) were exposed to freezing (-6°C to -36°C) followed by thawing (37°C) . Induction of apoptosis was found to be temperature-dependent: the percentage of apoptotic cells was increased when freezing temperature decreased; it reached its maximal level at -15°C, but decreased at -25°C (where cell death is rather due to necrosis). In this model, it was shown that the apoptotic stimulus from freezing/thawing was quick and short-lasting. It triggered the release of cytochrome c from mitochondria to cytosol followed by the activation of caspase-9 and the degradation of PARP. The activation of caspases and the cleavage of PARP are common hallmarks of apoptosis.Caspases are cystein-aspartate proteases that cleave various proteins (among which PARP) resulting in morphological and biochemical changes characteristic of apoptosis. The release of cytochrome c further implicates the mitochondrial pathway, one of the caspase cascade involved in apoptosis.
Yang et al. have further confirmed that cryo-induced apoptosis was associated with mitochondrial dysfunction in four human colorectal cancer cell lines . However, the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL and pro-apoptotic proteins Bax, Bcl-X, Bad and Bak in response to cryoinjury varied in the cell line panel tested. Bax level decreased in the cytosol and increased in mitochondria, followed by a loss of mitochondrial membrane potential, indicating that cryosurgery induced apoptosis via disruption of mitochondrial integrity. Cryo-induced apoptosis was also identified in vivo in a nude mouse tumor xenograft model.
Baust et al.  have shown that the addition of caspase-1 inhibitor to three human prostate and colorectal cancer cell lines prior to freezing resulted in an increase in cell survival in comparison to non-inhibited conditions, indicating the involvement of apoptosis in freezing-induced cell death. The maximal protective effect of the inhibitor was observed in the temperature range of -10°C to -20°C. Further evidences were provided by alterations in caspase-3 activity and caspase-9 expression, this latter involving more specifically the mitochondrial apoptotic pathway.
Wang et al. have demonstrated that combination of cryotherapy and 5-FU remarkably enhanced the apoptosis of G422 glioma cells, presumably through modulating Hsp90 alpha and p53 expression pattern .
In vivo studies are sparse. Steinbach et al. , through freezing the brain of mice with liquid nitrogen observed apoptosis at the periphery of the cryogenic lesion. The authors further proposed a model of the phases and mechanisms of the cryoinjury, which discriminates an early phase characterized by physical changes caused by hypothermia and their immediate consequences (transcriptional block), an intermediate phase where secondary changes lead to necrosis in the central area and a final phase of delayed apoptotic cell death in the periphery.
Similarly, Romaneehsen et al.  using a non-small-cell lung cancer model implanted into nude mice found histological evidence of apoptotic and necrotic cell death following freezing.
Latest news: in vivo studies
More recently, using a xenograft tumor model (human prostate tumor cells inoculated into nude mice), Le Pivert et al. have shown synergistic effects of cryosurgery and local chemotherapy . In this study, a single freeze/thaw cycle was applied to tumor and microcapsules of 5-FU were delivered at the outer margin of the frozen area. This combined treatment produced a dramatic inhibition of tumor growth, better and longer compared to that allowed by cryoablation or local microcapsule chemotherapy alone.
In a model of lung adenocarcinoma (A549 cell line) xenografted into SCID mice, Forest et al. have demonstrated that cryosurgery and chemotherapy (Vinorelbine) produced very different effects, both at histological and kinetic levels, suggesting complementary effects . And indeed, their combination resulted in an enhanced cell death either by necrosis or apoptosis, mainly during the early phase of the combined treatment . However, in this model, this benefit was not due to a concentration-dependent effect as the drug concentration was more important in tumors treated by chemotherapy than in tumors treated by cryochemotherapy.
More recently, the benefit afforded by the association of cryotherapy and chemotherapy previously observed at a molecular level was found to be correlated with a benefit on tumor growth as tumors treated by cryochemotherapy presented a significantly reduced volume and a lower T/C ratio compared to tumors treated either by cryosurgery or chemotherapy alone . Moreover, intratumoralangiogenesis was enhanced 8 to 15 days after cryosurgery, as shown by an increased expression of VEGF. To determine if this hypervascularization could enhance the efficiency of chemotherapy, the drug was injected 15 days after cryosurgery and the induction of cell death was investigated. Necrosis was increased but not apoptosis, suggesting that though a crucial parameter, intratumoral microvessel density is not the only factor to consider to reach an optimal efficiency of a combined treatment.
Useful tools for testing various cryoadjuvants can come from latest research in the engineering field.
Latest news: in the engineering field.
Fundamental research in this area concerns for instance the development of mathematical models to predict the extent of freezing during cryosurgery. Han et al. have recently developed a cryoinjury model where thermal thresholds of freezing-induced injury can be investigated in different cell types . This model can also potentially allow the study of cell death mechanisms, cell proliferation or migration and extracellular matrix (ECM) structural damage after a freeze/thaw cycle. This model is based on tissue engineering technology: tissue equivalents are produced by seeding and culturing cells (in this study rat and human prostate tumour cell lines were used) in a type I collagen matrix. It is interesting to develop such models because in vitro models (cell suspensions, cell monolayers) are relatively easy to control and manipulate but lack cell-cell and cell-ECM interactions and vascular structures. And in vivo models (native tissue, 2 and 3 dimensional tissues) are more realistic but present many difficulties for controlling experimental parameters. These new models, which are easy to control but still maintain tissue-like characteristics seem to be a good compromise.
Link to forum
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