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1. OSMOTIC INJURY: Rapid and sustained freezing of the tumor initially causes freezing of water and ice crystal formation in the extracellular space (the spaces between cells) at minus 15 Celsius, which dehydrates the extracellular space. Dehydration of the extracellular space concentrates the electrolytes and proteins between cells, which creates an osmotic or concentration gradient that forces water to flow from the interior 
   of the cells (which have yet to freeze) to the extracellular space to restore the water balance outside the cells. Forced withdrawal of water from within the cells causes the cells dehydrate, shrink or crinkle, creating cracks in the cell membrane or cell wall.  During the subsequent thaw cycle, ice crystals in the extracellular space melt to form water, which then flows back into the dehydrated cells causing them to swell and then rupture because the cell walls have been weakened from earlier cracking. In addition to causing cells shrinkage, the concentration of salts in the dehydrate cells impairs cellular enzyme function and directly destabilizes the cell membrane. As it turns out, the killing effect of a long thaw cycle seems to be as important as the killing effect of a long cold freeze cycle. (Figures A & B)
    

2. MECHANICAL INJURY: Although ice crystal formation is one of the earliest changes during the freeze cycle, sustained freezing causes formation of larger ice crystals inside of the cells that directly damage the walls of intracellular organelles like the DNA-containing nuclei and energy-creating mitochondria; damage the intracellular skeletons that help to maintain cell shape; and further destabilizes the cell membrane. With each subsequent cycle of freezing, the damaged tissue conducts the freezing with increasing efficiency, which progressively increases the diameter of the cryoablation zone. Intracellular ice crystal formation is most prominent during rapid freezing and during the thaw phase when temperatures reach minus 20-25 Celsius. (Figures A & B) 
    

3. VASCULAR INJURY: Cancer cells rely on blood vessels to deliver oxygen, supply nutrients, and eliminate waste. During cryoablation, the small and medium size vessels within and surrounding the tumor are killed in very much that same way that cancer cells are killed.  Intracellular ice crystal formation damages the cells that line the blood vessels. This causes blood clot formation in the vessels feeding the tumor, which deprives the cancer cells of oxygen and nutrients. In addition, restoration of blood flow around the cryoablation zone after thawing releases chemicals called "free radicals" that re-injure the blood vessel lining and cause further clot formation. Taking aspirin or anti-oxidants might inhibit blood clot and free radial formation.  Therefore, it is probably best to avoid aspirin or anti-oxidants 10 days before or after cryoablation.  Vascular injury is one of the major causes of tissue necrosis near the outer edge of the iceball where the temperature may be sublethal.
    

4. APOPTOTIC CELL DEATH OR PROGRAMMED CELL DEATH:  The tumor next to the cryoprobe in the center of the iceball reaches the coldest temperature, as low as minus 180 Celsius. The temperature near the outer portions of the iceball and farther from the cryoprobe do not reach such a low, direct-tumor killing temperature. Nonetheless, “warmer” sub-lethal temperatures in the range of 6 Celsius to minus 10 Celsius are capable of activating enzymes within the cancer cells that destroy the intracellular proteins and DNA.  This phenomenon, called apoptosis or programmed cell death, essentially causes the cancer cells to commit suicide typically 8-12 hours after the freezing injury.  Programmed cell death from cold temperature has been exploited as a weight loss method by plastic surgeons who now employ a new technology, called CoolSculpting, that uses cold temperatures in the range of 4 Celsius to induce apoptosis in fat cells.
    

5. IMMUNOGENIC INJURY: Although the strength of a generalized systemic immune response remains to be fully understood, experimental studies show that abnormal cancer cell proteins are capable of inducing an immune response at the tumor site in two key ways: 1) uptake of abnormal tumor proteins by granulocytes, monocytes, and macrophages that stimulate formation of antibodies (and possible immunity) that bind to cancer cells and target them for attack by T-cells, and 2) uptake of abnormal tumor proteins byantigen presenting cells like dendritic cells and macrophages that directly stimulate T-cells to attack the cancer as foreign cells. A third and increasing important immunogenic mechanism is immune checkpoint inhibition, a phenomenon in which cancer cells release "checkpoint" proteins that suppress the immune response. Killing cancer cells with cryoablation stops the release of immune-suppressing "checkpoint" proteins from the cancer cells. This permits the immune system to recognize and attack remaining cancer cells. There is now a whole new field of "immunotherapy" that is focused on developing immune checkpoint inhibitors to prevent cancer cells from suppressing natural immune responses.