The effects of thermal ablation on a targeted tumor are determined by increased temperatures, thermal energy deposition, rate of heat removal and the specific thermal sensitivity of the tissue. As the tissue temperature rises, the time required to achieve irreversible cellular damage decreases exponentially. At temperatures between 50 and 55 °C, cellular death occurs instantaneously in cell culture (Wheatley et al. 1989). Protein denaturation, membrane rupture, cell shrinkage, pyknosis and hyperchromasia occur ex-vitro between 60 and 100 °C, leading to immediate coagulation necrosis (Wheatley et al. 1989). Additional to this necrosis, tissue vaporization and boiling occur at temperatures greater than 105 °C. Carbonization, charring and smoke generation occur when the temperature is over 300 °C (Heisterkamp et al. 1997).

In addition, acoustic cavitation, one of mechanical effects induced by HIFU ablation, is the most important non-thermal mechanism for tissue disruption in the ultrasound field (Germer et al. 1998a, b). The presence of small gaseous nuclei within subcellular organelles and tissue fluids are the source of cavitation. These bubbles can expand and contract under influence of the acoustic pressure. During the collapse of bubbles, the acoustic pressure is more than several thousand Pascals, and the temperatures reach several thousand degrees Celsius, resulting in the local destruction of the tissue (Maris and Balibar 2000).

Histological changes are evident in tumor tissue after thermal ablation (Clement 2004). In addition to HIFU ablation, four cellular change zones are described in the liver after thermal ablation as follows: Application, central, transition and reference tissue zones (Ozaki et al. 2003a, b; Germer et al. 1998a, b; Ohno et al. 2001). The application zone is where the heat source contacts the tissue. The central zone immediately surrounds the application zone and consists of damaged tissue. The transition zone contains apparently undamaged tissue, but exhibits signs of subacute hemorrhage. The reference zone refers to normal tissue surrounding the transition zone.

Structural and functional changes are directly observed in tumor blood vessels after thermal ablation. These changes are not as well described as the thermal effects on tissues, but they do rely on varying temperatures. At temperatures between 40 and 42 °C, there is no significant change in tumor blood flow after 30–60 min exposure (Ozaki et al. 2003a, b). Beyond 42–44 °C, there is an irreversible decrease in tumor blood flow, with vascular stasis and thrombosis resulting in heat trapping and progressive tissue damage (Emami and Song 1984). While temperatures exceed 60 °C, immediate destruction of tumor microvasculature occurs (Tranberg 2004). It cuts the blood supply to the tumor directly through the cauterization of the tumor feeder vessels, leading to nutrient and oxygen deprivation. Thus, tissue destruction can be enhanced by the damage caused by thermal ablation to tumor blood vessels.

Indirect injury is a secondary damage to tissue that progresses after the cessation of thermal ablation stimulus (Muralidharan et al. 2004). It is based on histological evaluation of tissue damage at various time points after thermal ablation (Matsumoto et al. 1992). The full extent of the secondary tissue damage becomes evident 1–7 days after thermal ablation, depending on the model and energy source used (Wiersinga et al. 2003; Benndorf and Bielka 1997). The exact mechanism of this process is still unknown. However, it may represent a balance of several promoting and inhibiting mechanisms, including induction of apoptosis, Kupffer cell activation and cytokine release.

Cellular apoptosis may contribute to progressive tissue injury after thermal ablation. It is well established that apoptosis increases in a temperature-dependent manner, and temperatures between 40 and 45 °C cause inactivation of vital enzymes, thus initiating tumor cell apoptosis (Barry et al. 1990; Hori et al. 1989). Thermal ablation creates a temperature gradient that progressively decreases away from the site of probe insertion. The induction of apoptosis at a distance from the heat source may potentially contribute to the progression of injury. An increased apoptosis rate is observed in the liver 24 h after microwave ablation (Ohno et al. 2001). The stimulation of apoptosis may be directly induced by temperature elevations, alterations in tissue microenvironment and the release of various cytokines after thermal ablation. Kupffer cell activity may be one of the major factors involved in progressive injury after thermal ablation (Heisterkamp et al. 1997). Heat induces Kupffer cell IL-1 (Decker et al. 1989) and tumor necrosis factor-α (TNF-α) (Adams and Hamilton 1984) secretion, which are known to have in-vivo antitumor activity and increase cancer cell apoptosis (Hori et al. 1989). Kupffer cells also induce the production of interferon that augments liver-associated natural killer cell activity (Kirn et al. 1982).

Thermal ablation may induce both regional and systemic production of cytokines through activation of inflammatory cells. Compared with controls, the circulating level of IFN-γ and vascular endothelial growth factor levels markedly increase after RFA (Napoletano et al. 2008; Evrard et al. 2007a, b). The increased level of IL-1 and TNF-α is also observed after RFA (Ali et al. 2005). These cytokines may have direct cytotoxic effects, such as inducing tumor endothelial injury and rendering tumor cells more sensitive to heat-induced damage (Watanabe et al. 1988; Isbert et al. 2004). However, contrasting results are obtained for TNF-α levels in two studies (Evrard et al. 2007a, b; Schell et al. 2002) and IL-1 level in one study (Schell et al. 2002), where levels remain unchanged after thermal ablation. Cryoablation may cause pathophysiological changes, which are similar to those observed after endotoxin administration (Chapman et al. 2000a, b, c; Wudel et al. 2003). These changes cause significant increases in capillary permeability in the lung, leading to secondary injury (Washington et al. 2001). It is generally believed that all alterations may be associated with post-cryosurgery activation in the lungs of the nuclear factor-κB factor and derived cytokines, including TNF-α and macrophage inflammatory protein-2, along with an increase in serum thromboxane levels (Seifert et al. 2002; Sadikot et al. 2002).

As shown in Table 8.2, there is increasing evidence from animal studies that indicate that HIFU may modulate host antitumor immunity after tumor ablation. Yang and colleagues (Yang et al. 1992) used HIFU to treat C1300 neuroblastoma implanted in mouse flanks, followed by the re-challenge of the same tumor cells. A significantly slower growth of re-implanted tumors was observed in these mice compared with the controls. After HIFU treatment, the cytotoxicity of cytotoxic T lymphocytes (CTLs) and the number of activated tumor-specific CTLs was significantly increased in the H22 tumor bearing mice treated with HIFU. Adoptive transfer of the activated lymphocytes could provide better long-term survival and lower metastatic rates in the mice re-challenged by the same tumor cells when compared with sham-HIFU and control groups. This is indicative that HIFU ablation could activate tumor-specific T lymphocytes, thus inducing antitumor cellular immunity in the mice (Xia et al. 2012). Similar results were confirmed in the mice implanted with MC-38 colon adenocarcinoma and melanoma after HIFU ablation.

HIFU treatment could also induce an enhanced CTL activity in-vivo, thus providing protection against subsequent tumor re-challenge (Xing et al. 2008). In addition, HIFU could enhance infiltration of dendritic cells (DCs) in the treated tumor and subsequent migration to the draining lymph nodes. Compared to thermal HIFU treatment, antitumor immunity induced by mechanical HIFU treatment (being a pulsed HIFU exposure with no significantly elevated temperature increase in tumor tissue and thermal necrosis) was significantly stronger in terms of DC and CTL activation, and a superior protection against tumor re-challenge was reported (Hu et al. 2007).

After HIFU ablation, large amounts of tumor debris remain in-situ, and the host gradually reabsorbs the debris as the normal process of the healing response. Using a murine hepatocellular carcinoma model, Zhang and colleagues (Zhang et al. 2010) demonstrated that the remaining tumor debris induced by HIFU could be immunogenic, thus an effective vaccine to elicit tumor-specific immune responses. In this study, these included induction of CTL cytotoxic activity, enhanced activation of DCs and protection against lethal tumor challenge in naïve mice. When the tumor debris was loaded with immature DCs, it could significantly induce DC maturation, increase cytotoxicity and CTL TNF-α and IFN-γ secretion, thus initiating a host-specific immune response after H22 challenge in the vaccinated mice (Deng et al. 2010). Immediately after HIFU exposure to MC-38 colon adenocarcinoma cells in-vitro, the release of endogenous danger signals, including HSP60, was observed from the damaged cells. These signals could subsequently activate antigen-presenting cells (APCs), leading to an increased expression of co-stimulatory molecules and enhanced secretion of IL-12 by DCs and TNF-α by macrophages (Hu et al. 2005). In addition, HIFU could upregulate in-vitro and ex-vitro molecular expression of HSP70 (Kruse et al. 2008; Hundt et al. 2007). This is an intracellular molecular chaperone that can enhance tumor cell immunogenicity, resulting in potent cellular immune responses.

The potency of DC infiltration and activation following mechanical lysis and sparse-scan HIFU was much stronger than that from thermal necrosis and dense-scan HIFU exposure, suggesting that optimization of a HIFU ablation strategy may help in enhancing immune responses after treatment (Liu et al. 2010). Heat and acoustic cavitation are two major mechanisms involved in HIFU-induced tissue damage, while cavitation is a HIFU-unique effect when compared with other thermal ablation techniques. It causes membranous organelles to collapse, including mitochondria and endoplasmic reticulum, as well as cell and nuclear membranes. This breaks tumor cells up into small pieces, by which the tumor antigens can remain intact, or it may lead to the exposure of an immunogenic moiety that is normally hidden in tumor antigens. Zhou and colleagues (Zhou et al. 2007) used either heat-exposed or HIFU-treated H22 tumor vaccines to inoculate naïve mice. The vaccination times were four sessions once a week for four consecutive weeks, and each mouse was challenged with H22 tumor cells 1 week after the last vaccination. They found that the HIFU-treated tumor vaccine could significantly inhibit tumor growth and increase survival rates in the vaccinated mice, suggesting that acoustic cavitation could play an important role in stimulating the host antitumor immune system.

Emerging clinical results revealed that systemic cellular immune response was observed in cancer patients after HIFU treatment, as shown in Table 8.3. Rosberger and colleagues (Rosberger et al. 1994) reported five consecutive cases of posterior choroidal melanoma treated with HIFU. Three patients had abnormal, and two patients normal CD4⁺/CD8⁺ ratios before treatment. One week after treatment, the ratio in two patients reverted to normal, while another was noted to have a 37 % increase in CD4⁺ T cells relative to CD8⁺ cells. Wang and Sun (2002) used multiple-session HIFU to treat 15 patients with late stage pancreatic cancer. Although there was an increase in average numbers of NK cells, T lymphocytes and subsets in ten patients after HIFU treatment, a significant statistical difference was only observed in NK cell activity before and after HIFU treatment (p < 0.05). Wu and colleagues (Wu et al. 2004) observed changes in circulating NK, T lymphocyte and subsets in 16 patients with solid malignancy before and after HIFU treatment. The results showed a significant increase in the CD4⁺ T lymphocyte population (p < 0.01) and the ratio of CD4⁺/CD8⁺ cells (p < 0.05) after HIFU treatment. The abnormal levels of CD3⁺ lymphocytes returned to normal in two patients, CD4⁺/CD8⁺ ratio in three patients, CD19⁺ lymphocytes in one patient and NK cells in one patient, in comparison to the values in the control group. In addition, serum levels of immunosuppressive cytokines, including VEGF, TGF-β1 and TGF-β2, were significantly decreased in peripheral blood of cancer patients after HIFU treatment, indicating that HIFU may decrease tumor-induced immunosuppression and renew host antitumor immunity (Zhou et al. 2008).

Clinical evidence suggests that HIFU treatment may also enhance local antitumor immunity in cancer patients. Kramer and colleagues (Madersbacher et al. 1998; Kramer et al. 2004) found that HIFU treatment could alter the presentation of tumor antigens in prostate cancer patients, which was most likely to be stimulatory. Histological examination showed significantly upregulated expression of HSP72, HSP73 and glucose regulated protein (GRP) 75 and 78 at the border zone of HIFU treatment in prostate cancer. Heated prostate cancer cells exhibited increased Th1-cytokine (IL-2, IFN-γ, TNF-α) release, but decreased Th2-cytokine (IL-4, -5, -10) release from tumor infiltrating lymphocytes (TIL). The upregulated expression of HSP70 was confirmed in the tumor debris of breast cancer after HIFU ablation (Wu et al. 2007a, b), indicating that HIFU may modify tumor antigenicity to produce a host immune response.

Xu and colleagues (2009) found the number of tumor-infiltrating APCs, including DCs and macrophages, increased significantly along the margin of HIFU-treated human breast cancer, with an increased expression of HLA-DR, CD80 and CD86 molecules. Activated APCs may take up the HSP-tumor peptide complex, which remains in the tumor debris and present the chaperoned peptides directly to tumor-specific T lymphocytes with high efficiency, resulting in potent cellular immune responses against tumor cells after HIFU treatment.

Furthermore, HIFU could induce significant infiltration of TILs in human breast cancer, including CD3⁺, CD4⁺, CD8⁺, B lymphocytes and NK cells. The number of the activated CTLs expressing FasL⁺, granzyme⁺ and perforin⁺ significantly increased in the HIFU-treated tumor, suggesting that specific cellular antitumor immunity could be locally triggered after HIFU treatment (Lu et al. 2010).

With regards to minimally invasive therapies, RFA is only one technique that has been widely used in the clinical management of solid tumors, particularly in hepatocellular carcinoma (HCC). As coagulative necrosis is immediately induced in a targeted tumor after thermal ablation, necrotic cell death can be recognized by the immune system as a result of dangerous events, according to the “danger” model of immunity by Matzinger (Gallucci et al. 1999; Matzinger 2002). It is also accompanied by the release of “danger signals” from the heat-stressed cells, such as acute phase proteins, pro-inflammatory cytokines and heat shock proteins (HSPs), thus developing a temporary inflammatory stress. This stress may be associated with positive processes similar to the healing of injured tissues, but could also lead to the stimulation of tumor growth (Gravante et al. 2009). After RFA treatment, a moderate and temporary systemic inflammatory response has been observed in cancer patients, as demonstrated by the increase in plasma levels of pro-inflammatory cytokines and acute phase reactants (Evrard et al. 2007a, b; Schell et al. 2002; Meredith et al. 2007; Schueller et al. 2003; Fietta et al. 2009).

HSPs are families of highly conserved proteins involved in mechanisms of cell repair. They are intracellular molecular chaperones that physiologically bind tumor peptide antigens and enhance tumor cell immunogenicity (Pockley 2003). APCs take up HSP-tumor peptide complex and present the chaperoned peptides directly to tumor-specific T lymphocytes with high efficiency, resulting in potent cellular immune responses against tumor cells (Todryk et al. 2003). Around the necrotic ablated area, RFA produced sub-lethal injury in the zone of transition that showed apoptosis, and increased HSP70 expression in the liver of normal swine (Schueller et al. 2004). Schueller and colleagues found that there was an increased synthesis and cell surface expression of HSPs (HSP70, 90) after RFA in nude rats bearing human hepatocellular carcinoma (Rai et al. 2005). In addition, large amounts of tumor debris could induce local infiltration of activated DCs, the most potent APC for induction of adaptive immunity against cancer (Melief 2008). Activating signals, including necrotic tumor cells and HSPs, could induce the progression of infiltrating DCs from an immature to a mature stage, resulting in the presentation of tumor antigens by mature DCs to naïve T lymphocytes in a MHC-restricted fashion (Lutz and Schuler 2002). Ali and colleagues demonstrated that a transient function of myeloid DCs could be activated in HCC patients 7–14 days after RFA, with an increased ability to stimulate CD4⁺ T cells (Ali et al. 2005). Up to 7 % of DCs present in the draining lymph nodes contained tumor antigens in the ablated tumor after RFA. Compared to untreated HCC and normal liver tissue, expression of costimulatory molecules, such as CD80 and CD86, was significantly enhanced by incubation with RFA-treated HCC (den Brok et al. 2006). Similar results were also demonstrated by Zerbini and colleagues (2008) in HCC patients; indicative that local tumor ablation could lead to efficient antigen loading, migration and maturation of APCs, including DCs and monocytes. Direct evidence has recently been found that RFA could induce weak tumor-induced immunity in a murine tumor model harboring APC infiltration and amplification, and that enhanced systemic antitumor T cell immune responses and tumor regression was associated with increased infiltration of DCs after subtotal RF ablation (Dromi et al. 2009). These results suggest that the generation of heat-altered tumor antigens, in combination with the “dangerous signals”, may help to overcome immune tolerance or allergy towards the remaining tumor.