There are more regulatory mechanisms in place besides just the control of cell division. A malignant tumor can result if some of these other regulatory systems break down as well. All mammalian cells are capable of moving over a substrate that they can attach to. One regulatory system keeps cells of a certain type in the place where they belong. Loss of this system can allow cells to move through basement membranes and invade the vasculature, where they can travel through the body and reverse the process at some remote location. An invasive cell that does not respond to growth regulation can form a metastase, or secondary tumor. This sort of malignant growth is usually fatal to a multicellular organism, so there have been many approaches used to remove or kill malignant cells before metastasis occurs.
One approach that has been tried, and is now being found to be particularly effective for certain cancers, is cryosurgery. The tumor is frozen in order to kill the cells, leaving the detritus for the body to clean up on its own. The crucial aspect of any treatment of a cancerous tumor is that all the cancer cells must be killed. Since these cells have escaped growth regulation, they also are no longer responsive to programmed cell death--the suicidal mechanism by which cells limit their lifetime in multicellular organisms. The consequence of leaving any cancerous cells alive after treatment is that a new tumor will form and metastatic disease becomes more likely.
Cryosurgery was introduced in the early 60's after the mechanisms of freezing injury started to be studied by cryobiologists. The procedure is analogous to the cryopreservation of organized tissues, but the many problems that make cryopreservation difficult are virtues, when the goal is to kill the tissue. The major impediments, however, are the difficulty in killing all of the cells without also damaging the normal tissue that surrounds the tumor. The injury that results from a cryosurgical procedure is complex and is still not well understood. In 1964, Cooper published some of his experimental results and stated that all of the tumor must be taken below -20°C for the procedure to be effective. Unfortunately, the variables that can be manipulated during a procedure are numerous, and the relative effects of changing these variables is still not firmly established. The idea of a critical isotherm of -20°C remains as dogma in the field, but with renewed activity and improvements in technology, cryosurgical treatment is likely to rapidly become a precise intervention, with optimization of the protocol for individual cases.
The same factors that confound tissue cryopreservation also act on the tumor tissue. Perhaps the most destructive is due to altered ice growth through porous materials such as the walls of blood vessels. Ice growth inside the blood vessels cannot easily penetrate the vessel walls, thus water moves from the interstitium into the blood vessels, enlarging the ice lens that exists within the vessel. This can cause mechanical damage to the blood vessels and the redistribution of solutes (only pure water migrates to join the ice crystal) will provide an osmotic stress to the interstitial cells upon thawing.
Before freezing, and during thawing, the cells are brought down to hypothermic storage conditions, without the additives that are commonly used for hypothermic preservation (mannitol, citrate, high K+, etc.). There may be cellular injury due to the metabolic imbalance that occurs with hypothermia as well as from cold shock, due to cooling.
The primary biological injury to the tumor following cryosurgery is thought to be due to vascular stasis. The loss of nutrients, vitamins, oxygen, etc. cause ischemic damage to the tumor begining shortly after the tissue is thawed. Cancer cells are metabolically very active, and thus do not survive long without a blood supply. Without patent blood vessels nearby, they are also prevented from being invasive, limiting the tumor from metastasizing even if all the cells are not killed.
The mechanism of vascular injury is not well understood. There may be damage to the endothelial cells that line the walls of the blood vessels, causing the capillaries to become more permeable. There may also be mechanical damage caused by ice lensing within the blood vessels. Whatever the cause, edema, platelet aggregation, and microthrombosis begin to occur within an hour of thawing. Once the tumor's blood supply is cut off, then ischemic damage will begin to accumulate.
Upon thawing, there will be little or no morphological evidence of damage due to freezing. Microscopic examination of the cells, especially if viewed with viability indicators, will reveal that there is an extensive direct cell injury due to freezing (at least with protocols that are effective clinically, this is usually the case) but it is far from complete. In fact, in many instances where the conditions are sufficient to destroy the tumor, there can be a significant proportion of viable cells present just after thawing. The spatial pattern of recovery is highly dependent on the freezing conditions. With a cryoprobe that can cool rapidly, there may be a zone of dead cells around the probe (due to intracellular freezing), a zone of mostly live cells around that (the optimal cooling rate), and a zone of dead cells farther out (due to solution effects injury).
Edema will begin shortly after thawing and will usually increase for a day. Tissue necrosis, usually toward the center of the tumor, is evident at about two days and increases steadily toward a sharp line of demarcation (the boundary between live and dead tissue) thereafter. The cells at the periphery of the iceball usually survive in high enough numbers to prevent that tissue from becoming necrotic.
Factors that affect the cooling rate consist of the cooling apparatus and any structures that bring heat close to the tumor. The cryogen that is used in the cryoprobe determines how cold it can get as well as how quickly it can be brought down to that temperature and how much heat it can remove during freezing. The material that the probe is made out of, and its size, affect heat transfer between the tissue and the probe. The number of probes being used, often there are several placed at various positions within the tumor, also affect the thermal history that the tumor is subjected to. Nearby blood vessels will continuously add heat to the region of the tumor and it is sometimes necessary to insert catheters through which warm water is passed near the tumor to preserve fragile structures (such as the urethra during prostate tumor cryotherapy).
Rapid cooling is reported to be the most effective at producing cellular destruction but it is unlikely that intracellular freezing occurs to a great extent throughout the tumor. There is little experimental evidence to support the idea of rapid cooling leading to more damage, but neither is there much evidence to refute the idea. Currently, technical limitations prevent the idea from being fully explored.
From cryobiological studies, we know that the same degree of cell death can be achieved by holding cells at -10°C for a longer period or by holding them at -20°C for a short period, yet the cryosurgical literature mostly ignores the possibility of holding tumors at higher temperatures for longer periods. Since slow cooling injury increases until the temperature reaches -40°C (at least with single cells in a glycerol solution), this value is now often cited as the critical isotherm (the temperature that will indicate total cell death).
The relationship between the kinetics of cell injury vs. the temperature (or salt concentration produced by ice formation) has not been studied for vascular damage during tissue cryopreservation; indeed, the very nature of the vascular injury during cryosurgery is not yet understood well enough to even make credible predictions. Some experimental tests have claimed that the demarcation between live and dead tissue corresponds to the critical temperature but this relationship has not yet been tested in a controlled manner where the other variables that are concomitant with reaching a certain temperature have been held constant. It may well be that the minimum temperature is sufficient to determine the outcome of cryosurgical interventions, but that would not be expected, based on what we know from cryobiology.
It is unfortunate that little work has been done to investigate the effects of varying the time/temperature profile during cryosurgery as it might provide a mechanism for increasing injury within the tumor without having the iceball penetrate as far into peripheral tissue. For example, if the tumor only needs to be cooled to -15°C rather than -30°C to achieve the same degree of cell killing (achieved by holding at -15°C for a longer period), then the damage to tissue beyond the tumor might be lessened.
Some investigators have looked at using three freeze-thaw cycles, reasoning that each additional cycle will add to the injury. Interestingly, these studies have found that in cases where two cycles was more effective than one, three cycles led to death of the animal. Death from the immediate complications of cryosurgery is called cryoshock, and is distinguished by systemic failure, coagulopathy, and acute renal failure. Cryoshock may be induced by increasing the direct cell kill beyond acceptable levels. Whereas the cells die over a period of days when vascular injury and ischemic damage are the primary targets of cryotherapy, it seems likely that multiple freeze-thaw cycles are more targeted toward increasing the direct cellular injury. Releasing the intracellular contents of so many cells into the circulation instantaneously might cause an innapropriate response from the cells of the blood and vasculature, as well as putting too great a strain on the kidneys. The actual mechanism is poorly understood, as yet, but it is imperitave that it be investigated with all possible haste, as there is a good deal of research underway that is aimed toward increasing the direct cellular injury as a means of improving cryotherapy.
Fig. 10.1.1 Typical Cryoprobes.
The size and temperature of the iceball; the time that the iceball is held at a certain temperature--indeed, the entire thermal history of the iceball; the number of freeze-thaw cycles (with the possibility of different thermal protocols on different cycles); adjuvant therapy, where chemicals (cryosensitizers) are added to increase the effectiveness of the procedure; these are all parameters that can be modified right now to tailor cryosurgery to specific ends. In addition, the improvements in monitoring what happens within the iceball by ultrasound, thermometry, infra-red thermography, CT scanning, and magnetic resonance imaging will all serve to improve the control available to the cryosurgeon.
Clinical cryosurgery is now in a position to be implemented as a treatment of optimization, tailored to the particulars of a case, rather than implemented as a standard procedure. In addition to examining the particulars of each case, such as tumor geometry, nearby blood vessels, nearby sensitive tissues, tumor type, etc.; the procedure as a whole is a problem in optimizing several competing goals. The procedure must achieve the complete destruction of malignant cells, although not necessarily immediately after thawing. Peripheral injury, especially to critical tissues, must be minimized. The vascular injury should be maximized and the direct cell injury should also be maximized up to the cryoshock limit, if an excessive cell death is the cause of cryoshock. Given the effectiveness of the procedure for certain types of tumors already (also given the right cryosurgeons), it is a given that with more research into this rapidly growing field, cryosurgery will become one of the dominant clinical procedures in surgical oncology.
The process of freezing cells can lead to two types of injury, one that occurs during slow-cooling and another that occurs when cooling is more rapid. Although the mechanisms by which the injuries occur are different, they both target the plasma membrane of the cell as the primary site of injury. Without an intact membrane, the cell dies in a very short time following thawing. The fact of these two types of injury, though, is what allows tissue cryo-engineering to be performed. Because one type of injury occurs when you cool slowly, and a different injury occurs when you cool quickly, there is an optimal cooling rate that results in the best recovery. Each cell type has a different optimal cooling rate, sometimes dramatically so, thereby allowing the recovery within a tissue to be tailored to a particular cell type. The growth of ice through organized tissues has also been found to be governed by complex interactions. This phenomenon allows another means to engineer the freezing process for differential recovery of cells based on location within the tissue. Thus, by the process of freezing and thawing, the recovery as well as injury of the cells within a tissue can be controlled to meet pre-specified criteria. The biological response following transplantation of such an engineered tissue can be exploited to enhance the recovery following joint reconstruction; a benefit that will have a widespread impact on orthopaedics and biomedical engineering.
In a recently published experiment, a cryoprobe was used to cause a discrete cryoinjury in cardiac tissue, and then transplanting myoblasts into the cryo-scar to regenerate new heart tissue. The interesting point was that they considered the cryo-scar a good place to transplant cells because of all the growth factors released by the fibrotic process. The scope for tissue cryo-engineering in vitro (for allogeneic transplants) and in vivo (like the heart regeneration experiment) is clearly increasing rapidly. Hopefully this new growth will complement the recent interest in cryosurgery to help us understand the freezing and thawing process better.