Radiofrequency Ablation Induces Antigen-presenting Cell Infiltration and Amplification of Weak Tumor-induced Immunity

Abstract

Purpose: To evaluate the influence of subtotal radiofrequency (RF) ablation on a tumor-specific immune response in a murine tumor model and to explore the role of intratumoral dendritic cells (ITDCs) in mediating this effect.

Materials and Methods: Animal work was performed according to an approved protocol and in compliance with the National Cancer Institute Animal Care and Use Committee guidelines and regulations. A murine urothelial carcinoma (MB49) model expressing the male minor histocompatibility (HY) antigen was inoculated subcutaneously in female mice. Fourteen days later, splenic T cells were analyzed with enzyme-linked immunosorbent spot for HY immune response (n = 57). In subsequent experiments, mice were randomized into control (n = 7), RF ablation, ITDC (n = 9), and RF ablation + ITDC (n = 9) groups and monitored for tumor growth. Eleven days after treatment, tumors were harvested for histologic and immunohistochemical analysis. Animals demonstrating complete tumor regression were rechallenged in the contralateral flank.

Results: Animals treated with subtotal RF ablation showed significant increases in tumor-specific class I and II responses to HY antigens and tumor regression. RF ablation, ITDC, and combined groups demonstrated similar levels of antigen-presenting cell infiltration; all groups demonstrated greater levels of infiltration compared with untreated controls. ITDC injection also resulted in tumor regression. However, combination therapy did not enhance tumor regression when compared with either treatment alone. Rechallenged mice in RF ablation, ITDC, and combination groups demonstrated significant tumor growth inhibition compared with controls.

Conclusion: Subtotal RF ablation treatment results in enhanced systemic antitumor T-cell immune responses and tumor regression that is associated with increased dendritic cell infiltration. ITDC injection mimics the RF ablation effect but does not increase immune responses when injected immediately after RF ablation.

© RSNA, 2009

Thermal ablation techniques such as radiofrequency (RF), microwave, cryotherapy, and focused ultrasound have been increasingly used for minimally invasive management of local unresectable malignancies (1–3). Among these modalities, RF ablation is now the most widely used and has proved to be safe, with a minor complication rate of less than 10% (4,5). RF ablation employs an alternating RF current to generate heat and induce coagulation necrosis within a solid tumor and has been successfully used in the management of liver, bone, breast, kidney, and lung malignancies (6).

Previous work in preclinical models has illustrated that, in addition to producing local coagulation necrosis, RF ablation can also generate large amounts of cellular debris (7). This debris is a source of tumor antigens that can be targeted by the host's immune system and, when combined with the release of proinflammatory molecules, may generate an environment conducive to antitumor immunity, mediated by tumor-specific T cells in animal models (7,8). Indirect evidence suggests that this phenomenon may also occur in patients, with case reports of spontaneous regression of metastases following RF ablation of a primary tumor and enhancement of tumor-specific T-cell responses (9,10).

Dendritic cells (DCs) are potent antigen-presenting cells that are capable of either stimulating or inhibiting the immune response. DCs have not only been explored for their role in the treatment of malignancy but also for use in autoimmune diseases and transplant rejection (11). DCs serve as the basis for many cancer vaccination strategies and are capable of inducing tumor-specific T-cell responses, although the therapeutic efficacy of this approach has been limited thus far (12). DC-based vaccines typically involve the loading of tumor antigens ex vivo, and numerous strategies are being developed to augment the DC-initiated immune response through combination strategies with chemoembolization, thermal ablation, and photodynamic therapy (12–14). Given that RF ablation generates large amounts of cellular debris, stimulates necrotic cell death, and creates a proimmune environment both locally and systemically, it may be ideally suited for activation of DCs in vivo (7).

We hypothesized that (a) local RF ablation can produce systemic immunity, (b) subtotal RF ablation recruits DCs to the tumor, and (c) the addition of DCs directly to the tumor can improve tumor control over RF ablation alone. To test these hypotheses, we examined the influence of RF ablation, intratumoral DC (ITDC) injection, and a combination of the two treatments on quantitative immune responses measured by using interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISPOT), immune cell infiltration, and tumor growth in a murine urothelial carcinoma (MB49) model.

MATERIALS AND METHODS

Animal and Tumor Model

All animal work was performed according to an approved animal protocol and in strict compliance with the National Institutes of Health (NIH) National Cancer Institute (NCI) Animal Care and Use Committee guidelines and regulations. Ninety-one mice (C57BL/6; Animal Production Facility, NCI, Frederick, Md) were purchased and housed until 6 weeks of age in a pathogen-free facility (NIH, Bethesda, Md).

MB49, a tumor cell line that naturally expresses the male-specific minor histocompatibility (HY) antigen complex, was cultured in a medium (RPMI 1640; Invitrogen, Carlsbad, Calif) with 10% fetal calf serum (FCS; Gemini Bio-products, West Sacramento, Calif), 1% HEPES buffer, 1% nonessential amino acids, 1% sodium pyruvate, 1% penicillin-streptomycin, 1% l-glutamine (Invitrogen), and 2-mercaptoethnol (50 μmol/L; Sigma, St Louis, Mo) and incubated at 37°C in 5% CO₂. For tumor inoculations, the flanks of the mice were shaved, prepped with alcohol, and injected subcutaneously with 1 × 10⁶ cells for RF ablation experiments and with 3 × 10⁶ cells for the tumor rechallenge experiment. Mice were monitored daily and tumor volumes were measured three times per week with a digital caliper by using the sphere formula: (4/3 × 3.14 × L × W × (L + W)/2), where L and W are the tumor length and width, respectively. When tumors reached an approximate volume of 1700 mm³ ± 200 (largest diameter, 7.91 mm; smallest diameter, 3.46 mm), the mice were randomly assigned to one of the treatment or control groups. Mice were euthanized when tumor diameters reached 2 cm, in accordance with animal protocols.

RF Ablation Procedure

Animals were anesthetized by using isoflurane inhalation and the tumor area was shaved and cleaned. The mice were positioned prone on an electricity-conducting (grounding) pad. The ablation was performed by using a 22-gauge needle (Radionics SMK; Valleylab, Boulder, Co) with a 4-mm active tip that was inserted percutaneously and orthogonal to the skin in the center of the tumor. The probe tip was placed just beneath the skin for partial tumor ablation. Before RF ablation, the impedance and tumor temperature were measured with a 50-W RF lesion generator (Radionics RFG-3C Plus; Valleylab, Boulder, Colo). The mean impedance and mean temperature before RF ablation were approximately 670 ohms ± 260 (standard deviation) and 34.3°C ± 2.4, respectively. For T-cell response studies, treatments were administered for less than 70 seconds at approximately 90°C (as measured with the RF needle) by using a mean power output of 7.8 W ± 6.1. For tumor growth studies, tumors were treated with a similar power output but to a maximum temperature of 70°C for approximately 20 seconds in the center of the tumor. The shorter treatment regimen resulted in a smaller treatment volume and a greater reliance on systemic immune responses for tumor regression.

Infrared Imaging

Instant infrared imaging, performed with an infrared camera (ThermaCAM P65HS; FLIR Systems, Boston, Mass), allows for the measurement of superficial tumor temperatures at different times in a rapid, real-time, and noninvasive manner. The infrared camera has a thermal sensitivity of 50 mK at 30°C with an error of 0.05°C and a range of −40°C to 250°C. To confirm tissue heating and cooling during and after RF ablation, the infrared camera was placed approximately 75 cm above the animal with an angle of 75° between the imaging axis and the table throughout the treatment. RF ablation increased the temperature from the center of the tumor (measured with the RF ablation needle) to the skin (measured with the infrared camera).

ITDC Injection

DCs were cultured from the bone marrow of female mice and maintained in a medium (RPMI 1640; Invitrogen) with 10% fetal calf serum (FCS; Gemini Bio-products), 1% HEPES buffer, 1% nonessential amino acids, 1% sodium pyruvate, 1% penicillin-streptomycin, 1% l-glutamine (Invitrogen) and 2-mercaptoethnol (50 μmol/L; Sigma) for 8 days at 37°C and 5% CO₂, with cytokines (granulocyte monocyte colony-stimulating factor [1 μL/mL; PeproTech, Rocky Hill, NJ] and interleukin-4 [0.5 μL/mL; PeproTech]) added every 2 days. For intratumoral treatment, 1 × 10⁶ DCs were injected in the periphery of the tumor, with cells divided between three sites around the circumference of the tumor. Tumors treated with RF ablation were allowed to cool for at least 30 minutes before DC injection (verified with the infrared camera).

IFN-gamma ELISPOT and Phenotypic Analysis of Splenocytes

The ELISPOT assay measures cytokine production in response to in vitro stimulation such that each spot represents a single reactive cell. Splenocytes were assayed to measure the number of CD4⁺ and CD8⁺ T cells producing IFN-gamma in response to the major HY complex class I–dominant, class I–subdominant, and class II–dominant epitopes derived from the HY complex as previously described (15,16). The total number of activated CD4⁺ and CD8⁺ T cells were enumerated by using flow cytometry for 2 × 10⁶ freshly isolated red blood cell–depleted splenocytes preincubated with an anti-FcγIII/II receptor monoclonal antibody (clone 2.4G2). These cells were then stained at 4°C for 20 minutes with a monoclonal antibody cocktail containing CD44-FITC (Pharmingen, San Diego, Calif) and CD4-TC and CD8-APC (Caltag, San Diego, Calif).

Immunohistochemical Staining and Histologic Findings

Tumors were immediately harvested and divided in approximately equal halves. One-half of the tumor was fixed in 10% buffered formalin, embedded in paraffin, and sectioned for hematoxylin-eosin staining. The other half of the tumor tissue was directly embedded and frozen in an OCT matrix for cryosectioning and subsequent immunohistochemical analysis.

To identify specific DC infiltration in the tumors, immunohistochemical analysis was performed by using a polyclonal American hamster fluorescein isothiocyanate (FITC)-labeled CD11c antibody (5 μg/mL; BioLegand, San Diego, Calif). The antibody was replaced with nonimmune serum to serve as a negative control. Mouse skin Langerhans cells were used as a positive internal control in the same section. After CD11c antibody staining, the slides were counterstained and mounted with a medium containing 4′, 6-diamidine-2-phenylindole (Vectashield; Vector Laboratories, Burlingame, Calif). Observations were performed with a fluorescence microscope (Axioplan 2; Carl Zeiss MicroImaging, Thornwood, NY), and representative digital images were obtained.

Statistical Analysis

Data from ELISPOT assays were compared according to presence of treatment by using a stimulus antigen with a Wilcoxon rank sum test since the data were not normally distributed, with P values adjusted by using the Hochberg method.

For tumor growth studies following RF ablation or ITDC treatment, differences from baseline levels were analyzed because baseline tumor volume measurements differed for each treatment group (P = .003, Kruskal-Wallis test). For each of the five subsequent measurements after baseline (3, 5, 8, 10, and 12 days after treatment), the distributions of differences from baseline were analyzed two ways by using nonparametric methods because the data were not normally distributed. First, distributions of differences from baseline were compared by using the Kruskal-Wallis test; for a P value of less than .05, the control group was compared with each of the other three treatment groups. Second, each of the four distributions of differences from baseline was tested for a shift away from zero by using the Wilcoxon signed rank test.

Raw data from the tumor rechallenge study were analyzed, as baseline tumor volume measurements were identical (all zero). For each of the nine subsequent measurements after baseline (3, 5, 7, 10, 17, 18, 21, 23, and 25 days after treatment) the treatment group distributions were analyzed by using nonparametric methods because the data were not normally distributed. Group distributions were compared by using the Kruskal-Wallis test; for a P value of less than .05, the control group was compared with each of the other three groups. The Hochberg method was used to adjust all pairwise comparison P values (17).

RESULTS

RF Ablation Treatment Results in Increased Systemic CD8⁺ and CD4⁺ Antitumor Immune Responses

To investigate the magnitude of tumor-specific immune responses following RF ablation treatment, IFN-gamma ELISPOT assays were performed on splenocytes from mice with actively growing HY-expressing MB49 tumors (Fig 1). We have previously shown that naïve, nontumor-bearing mice do not demonstrate any evidence for HY immunity, as measured by using ELISPOT (15). Tumor-bearing mice that did not receive RF ablation treatment demonstrated weak responses against HY antigen peptides, although the immune response was insufficient to control progressive tumor growth, consistent with previous observations (15). Interestingly, RF ablation treatment of tumors resulted in significantly greater HY immune responses compared with untreated tumor-bearing mice (Fig 1a). Tumor antigen–specific CD8⁺ T-cell responses were significantly increased, as demonstrated by IFN-gamma ELISPOT responses against major HY complex class I–dominant and –subdominant CD8 peptide epitopes (P = .0001 and P = .037, respectively). CD4⁺ T-cell responses, measured by using responses against major HY class II–dominant responses, were also significantly increased in animals treated with RF ablation as compared with controls (P < .0001). The response to whole male splenocytes, assessing composite HY responses, was significantly greater in mice treated with RF ablation than in control animals (P = .001). There were no significant differences in the total number of CD4⁺ and CD8⁺ T cells in the spleens or lymph nodes of treated and untreated tumor-bearing mice as compared with nontumor-bearing mice (data not shown).

Figure 1a:

(a) RF ablation and (b) ITDC injection results in increased systemic antitumor immune responses. Fourteen days after RF ablation treatment, ITDCs, or no treatment, spleens were harvested from mice in each group (RF ablation, n = 23; no RF ablation, n = 25; ITDCs, n = 5; no ITDCs, n = 4). Splenocytes were assayed for IFN-gamma production by using ELISPOT. Both data sets were analyzed with Wilcoxon ranked sum test; medians shown with upper and lower quartiles. (a) RF ablation alone resulted in significant increases in IFN-gamma responses in treated mice compared with untreated mice. (b) ITDC treatment alone showed significant increases in responses to HY antigens compared with untreated mice. DBY = major HY complex (MHC) class II–dominant, RFA = RF ablation, SMCY = MHC class I–subdominant, UTY = MHC class I–dominant.

Figure 1b:

(a) RF ablation and (b) ITDC injection results in increased systemic antitumor immune responses. Fourteen days after RF ablation treatment, ITDCs, or no treatment, spleens were harvested from mice in each group (RF ablation, n = 23; no RF ablation, n = 25; ITDCs, n = 5; no ITDCs, n = 4). Splenocytes were assayed for IFN-gamma production by using ELISPOT. Both data sets were analyzed with Wilcoxon ranked sum test; medians shown with upper and lower quartiles. (a) RF ablation alone resulted in significant increases in IFN-gamma responses in treated mice compared with untreated mice. (b) ITDC treatment alone showed significant increases in responses to HY antigens compared with untreated mice. DBY = major HY complex (MHC) class II–dominant, RFA = RF ablation, SMCY = MHC class I–subdominant, UTY = MHC class I–dominant.

Subtotal RF Ablation

Given that different durations of RF ablation and the maximum temperatures reached during treatment could result in differential effects on antigen presentation, we sought to establish a treatment model that resulted in subtotal ablation of tumors, enabling us to investigate the effects of the systemic immune response generated by RF ablation on tumor growth. During RF ablation treatment, the centers of tumors reached temperatures above 70°C in less than 20 seconds (as measured with the RF ablation needle), while the periphery and skin remained at temperatures under 50°C (47.6°C, as measured with the infrared camera) for the duration of treatment (Fig 2). Infrared images taken 30 seconds after ablation demonstrated that the periphery and skin temperatures remained slightly elevated (35.8°C) as compared with pretreatment temperatures. However, within 30 minutes, the temperatures of the tumor periphery had returned to baseline levels (29.8°C). Thus, RF ablation treatment at a maximum temperature of 70°C allowed for subtotal ablation of MB49 tumors.

Figure 2a:

Infrared imaging shows confirmation of subtotal RF ablation. Imaging was performed before, during, and after RF ablation treatment to elucidate superficial temperature of tumor (green circle). (a) Baseline level shows tumor periphery and surrounding skin temperatures measured at 29.4°C. (b) Fifteen seconds after treatment (central temperature, 70°C as measured with RF ablation needle), tumor periphery temperature was 47.6°C. (c) Thirty seconds after treatment, temperatures were slightly lower (35.8°C). (d) Thirty minutes after treatment, tumor periphery temperatures returned to baseline (29.8°C).

Figure 2b:

Infrared imaging shows confirmation of subtotal RF ablation. Imaging was performed before, during, and after RF ablation treatment to elucidate superficial temperature of tumor (green circle). (a) Baseline level shows tumor periphery and surrounding skin temperatures measured at 29.4°C. (b) Fifteen seconds after treatment (central temperature, 70°C as measured with RF ablation needle), tumor periphery temperature was 47.6°C. (c) Thirty seconds after treatment, temperatures were slightly lower (35.8°C). (d) Thirty minutes after treatment, tumor periphery temperatures returned to baseline (29.8°C).

Figure 2c:

Infrared imaging shows confirmation of subtotal RF ablation. Imaging was performed before, during, and after RF ablation treatment to elucidate superficial temperature of tumor (green circle). (a) Baseline level shows tumor periphery and surrounding skin temperatures measured at 29.4°C. (b) Fifteen seconds after treatment (central temperature, 70°C as measured with RF ablation needle), tumor periphery temperature was 47.6°C. (c) Thirty seconds after treatment, temperatures were slightly lower (35.8°C). (d) Thirty minutes after treatment, tumor periphery temperatures returned to baseline (29.8°C).

Figure 2d:

Infrared imaging shows confirmation of subtotal RF ablation. Imaging was performed before, during, and after RF ablation treatment to elucidate superficial temperature of tumor (green circle). (a) Baseline level shows tumor periphery and surrounding skin temperatures measured at 29.4°C. (b) Fifteen seconds after treatment (central temperature, 70°C as measured with RF ablation needle), tumor periphery temperature was 47.6°C. (c) Thirty seconds after treatment, temperatures were slightly lower (35.8°C). (d) Thirty minutes after treatment, tumor periphery temperatures returned to baseline (29.8°C).

Subtotal RF Ablation Induces Local DC Infiltration

We hypothesized that the amplified HY immunity induced by using RF ablation may be a result of cross-presentation by antigen-presenting cells recruited to the tumor. To investigate DC populations in the tumor, hematoxylin-eosin and immunohistochemical slides were prepared from RF ablation–treated and untreated tumors 11 days after treatment. Hematoxylin-eosin staining confirmed partial ablation in treated tumors (Fig 3). Immunohistochemical analysis by using CD11c staining for antigen-presenting cells demonstrated pronounced reclusion of these cells in treated groups when compared with untreated tumor-bearing controls (Fig 4).

Figure 3:

Histologic confirmation of partial tumor ablation. Eleven days after RF ablation, ITDC, or combination treatment, tumors were harvested, divided longitudinally, and processed for hematoxylin-eosin staining. Note sharp demarcation between coagulation necrosis and viable tumor in RF ablation–treated lesions, indicative of partial ablation.

Figure 4:

Fluorescence microscopic images show presence of CD11c+ antigen-presenting cells (DCs) detected by using immunohistochemical analysis by means of FITC-labeled CD11c antibody after RF ablation, ITDC, and combination treatment. Tumors were harvested, divided longitudinally, and processed for immunohistochemical analysis 11 days after treatment. Antigen-presenting cells (green) and counterstained with 4′, 6-diamidine-2-phenylindole (blue). Controls show few antigen-presenting cells in tumor. In contrast, tumors treated with RF ablation, ITDCs, and combination therapy all show higher levels of CD11c staining, suggesting that treatment results in enhanced DC infiltration in tumors.

ITDC Injection Increases Systemic Immune Responses

Given the observation that RF ablation induced infiltration of DCs in the tumor microenvironment, we investigated whether ITDCs could also enhance systemic T-cell–mediated immune responses. As shown in Figure 1b, ITDCs significantly increased the magnitude of major HY complex class I–dominant, class I–subdominant, and class II–dominant responses. This further supports the fact that RF ablation–mediated DC infiltration contributes to the increase in systemic immune response observed as a result of RF ablation treatment.

Subtotal RF Ablation and ITDCs Induce Eradication of MB49 Tumors

We next evaluated the effect of RF ablation treatment, ITDCs, or ITDCs in combination with RF ablation on MB49 tumor growth by using subtotal RF ablation. Surprisingly, complete eradication of primary tumors was observed in several mice in all treatment groups by 18 days after treatment (four of nine mice in RF ablation and ITDC groups; five of nine mice in the combination therapy group), while the remaining mice in each group eventually died of progressive tumors (Fig 5). When the change from baseline tumor volume, or δs, of each group were compared on days following treatment, the change from baseline volume for animals that received any type of treatment was significantly different compared with that of controls, starting on day 5 after treatment. Mice that received RF ablation treatment or ITDC treatment demonstrated significantly enhanced control of tumor progression, and their δs generally decreased over time while control group δs increased. Combination therapy had a similar trend in tumor eradication to the RF ablation or ITDC group, however their changes in tumor volume, compared with that of controls, were not significant. Thus, subtotal RF ablation and ITDC treatment each resulted in significant improvement in tumor control and all treatment groups demonstrated complete MB49 tumor eradication in several mice.

Figure 5:

Graph shows median tumor volumes for treatments, enhanced control of tumor growth for 12 days following partial RF ablation and ITDC injection. On day 0, mice were treated with RF ablation (n = 9), ITDCs (n = 9), or both (n = 9), while controls (n = 7) were unmanipulated. Significant differences in changes from baseline tumor volumes (δs) were observed when comparing treatment groups with controls starting on day 5 by using Kruskal-Wallis test. Control δs tended to increase overtime, whereas RF ablation and ITDC δs generally decreased. Combination treatment δs increased early on and then decreased. Wilcoxon rank sum test performed with P values adjusted by using Hochberg method shows control and RF ablation δs differed significantly on days 5–12, whereas control and ITDCs δs differed significantly on days 8–12. Control and combination treatments did not differ significantly on any day tested. * P < .05.

Contralateral Tumor Challenge Following Complete Eradication of Primary Tumors with RF Ablation, ITDC, or Combination Therapy

Because ELISPOT results showed that RF ablation treatment elicited a tumor antigen–specific immune response, and subtotal RF ablation resulted in complete tumor regression in a subset of treated mice, we hypothesized that the eradication of primary MB49 tumors observed with RF ablation treatment was, in part, immunologically mediated. To test this hypothesis, we performed a contralateral secondary tumor rechallenge on mice with eradicated primary tumors.

Mice that demonstrated treatment-induced eradication of primary tumors from the three experimental groups (RF ablation, n = 4; ITDC, n = 4; and combination therapy, n = 5) were subsequently challenged with a high (3 × 10⁶) dose of MB49 cells in the contralateral flank. Secondary tumors were measured for 25 days following the secondary challenge, and the calculated volumes were transformed by using the cubed root to show all data on the graph (Fig 6).

Figure 6:

Graph shows median tumor volumes of each group compared with controls for 25 days after injections. Protection against tumor rechallenge following RF ablation, ITDC, or combination therapy mediated tumor eradication indicative of memory effect. Eighteen days after treatment, mice with eradicated primary tumors from each group (n = 4–5) were rechallenged with high (3 × 10⁶ cells) dose of MB49; naïve mice were challenged simultaneously as controls (n = 5). Cubed root of median tumor volumes used to show all data. Mice previously treated with RF ablation, ITDCs, or both exhibited growth and eradication of secondary tumors without additional treatment. Kruskal-Wallis and Wlicoxon rank sum tests showed significant difference between tumor volumes of all three treatment groups when individually compared with controls by day 5. For every subsequent measurement, differences in tumor volumes remained significant between all treatment groups and control mice. *P = .016.

For all mice in all three treatment groups, secondary tumors grew gradually and then regressed, becoming completely immeasurable by day 23 without additional therapy. On all days except day 3 after the secondary tumor challenge, there was a significant difference among tumor volume distributions, and the tumor volume distributions of previously treated mice were each significantly lower than that of the control group. In general, animals with primary tumors eradicated by using therapy demonstrated growth and subsequent elimination of high-dose secondary tumors without additional RF ablation or ITDC therapy, suggesting a tumor-specific memory response was established with initial therapy.

DISCUSSION

RF ablation could be a tool for complementary immunomodulation in immunotherapeutic treatment protocols. It has been well documented that heating (RF ablation) or freezing (cryoablation) of tumors is an effective way to induce local tumor destruction (18–20). After RF ablation–mediated necrosis, tumor debris remains in the treated area, which may help initiate antitumor immunity (24). By using a well-characterized antigen system and a tumor that naturally expresses this antigen, our goal was to first characterize the antigen response after RF ablation treatment and then test whether combination ITDC and RF ablation therapy further enhances tumor control. We have previously demonstrated that mice bearing the HY-expressing tumor MB49 exhibit weak HY immunity, which is insufficient to control tumor growth (15), and that vaccination of female mice with male DCs results in enhanced tumor protection (16). Furthermore, T cells from animals with progressive MB49 tumors are fully functional at transfer to nontumor-bearing hosts (15). Thus, it could be predicted that strategies to amplify this weak immunity would increase the likelihood of tumor control.

In these experiments, we examined whether RF ablation would enhance a systemic immune response, as mediated by local destruction of the tumor and subsequent inflammation. To carry this out, our RF ablation needle was limited to a 4-mm active tip length, which generated only partial ablation of the tumor. This ablation design intentionally provided a spectrum of local environments, including a volume of coagulation necrosis, a sublethal treated tumor zone, and a normal untreated (residual) tumor area. The elevation of tissue temperature to a range of 50°–60°C induces irreversible cellular damage in minutes. However, different tissues have different time-specific variable threshold levels of cell death as a result of elevated temperature (21,22). With higher temperature elevations (60°–100°C), as were achieved at the center of the tumor in these experiments, the irreversible damage is essentially instantaneous (23). However, at the tumor margin, sublethal heating occurred, thus creating a gradient of tissue destruction. In our model of a well-characterized antigen system, RF ablation resulted in significantly enhanced CD4⁺ and CD8⁺ T-cell responses. Furthermore, although only partial ablation of the tumor was performed, complete elimination of established tumors treated with RF ablation alone was observed. Immunohistochemical analysis demonstrated that RF ablation also resulted in increased DC infiltration. These data suggest that RF ablation enhanced the cross-presentation sufficiently to cause immune-mediated complete tumor eradication (7,13), although the exact mechanism is currently speculative. One possible explanation relies on the sublethal zone just outside the coagulation necrosis volume, which is exposed to slightly elevated temperatures, but not one high enough or for a long enough time to kill the tissue. This zone is known to contain increased amounts immunostimulatory and inflammatory factors after thermal stress (25) and could contribute to the enhanced systemic immune response. This scenario is supported by the secondary tumor challenge data, where RF ablation treatment and ITDC treatment both elicit a T-cell memory response that results in sustained protection against the contralateral tumor challenge. Further studies to delineate what costimulatory factors may be up- or downregulated would further elucidate the mechanism responsible for the enhanced immune response. Such mechanistic information could help determine how to best combine local ablative therapies with specific conventional immunotherapies or immunomodulators to maximize both local and systemic benefit.

To further stimulate the immune system and augment tumor antigen presentation, we injected DCs generated ex vivo into the tumor environment. We demonstrated in these experiments, and it has been observed by other groups, that after RF ablation, antigen-presenting cells such as DCs increase near tumor inflammation and debris (26). In these experiments, we provided exogenous DCs (by means of direct injection into the periphery of the ablation site in the RF ablation group and in the periphery of the tumor in ITDC group) to the tumor. The fact that ITDC injection alone could enhance systemic antitumor immunity and resulted in tumor regression further supports the conclusion that endogenous DCs recruited to the tumor in partial RF ablation treatments are responsible for RF ablation–mediated immune effects and complete tumor eradication.

The lack of synergy observed when ITDCs were combined with RF ablation should be explored. One potential reason why there was no apparent additive effect of combination therapy could be that the DCs were injected too quickly after RF ablation. Although infrared imaging confirmed that the tumor temperature was down to baseline level 30 minutes after RF ablation, at which time DCs were injected, there may be other local factors in the tumor microenvironment immediately after treatment that could affect the functionality of the DCs. We also chose to inject DCs that were not activated in culture, presuming that their activation would occur via the immune environment created by means of local RF ablation. However, it would be interesting to investigate the effect of RF ablation with ITDCs that have been activated in culture. Additionally, because RF ablation is strong enough alone to elicit complete tumor eradication as administered, it could be masking any potential additive effects of the injected DCs when the two therapies are combined. Perhaps administering ITDC treatment with a lower dose of RF ablation or in a metastatic tumor model could shed light on the potential synergistic effect of combination therapy. Although plausible, it is unlikely that a large tumor burden prevented additive effects of RF ablation and ITDCs, as all animals had similar tumor volumes at the time of treatment and tumor eradication was observed in groups that received RF ablation or ITDCs alone.

While our observations are compelling, there were a number of limitations to this study. First, MB49 is a murine bladder epithelial carcinoma, and though it is a useful tumor model for immunologic investigations because of its well-characterized tumor antigen, it may be less clinically relevant than other tumor models. Additionally, MB49 was the only tumor line used in the study. It would be beneficial to explore whether similar amplification of tumor-specific immunity and tumor eradication would occur in other, possibly immunogenic murine tumor models. However, it has already been demonstrated in other animal models that RF ablation induces a tumor-specific immune response (7,8). Our study extends this observation and provides important insight regarding the mechanism responsible for RF ablation–mediated enhancement of systemic antitumor immunity.

Clinically, it has been observed that despite progressive tumor growth, measurable immune responses are generated but are insufficient for a number of reasons, including, but not limited to, the magnitude of the immune response and local suppressive factors. In recent years, strategies to amplify the immune response, such as DC-based vaccines, have been explored but with limited success and will require further optimization. RF ablation enhances the tumor antigen quantity and produces an inflammatory reaction. With the combination of DC vaccines, RF ablation may contribute to a strong local immunologic and inflammatory response for in situ tumor destruction, thereby enhancing the systemic response initiated by the vaccine. Previous studies in animals and patients demonstrated activation of a tumor-specific T-cell response after RF ablation (8,27). While RF ablation appears to be one mechanism to help achieve this goal, studies that used other local thermal therapeutic resources, such as high-intensity focused ultrasound, suggest that there is enhancement of the systemic antitumor cellular immunity, in addition to local tumor destruction by using these modalities (28,29). Optimization of the deposition of thermal or mechanical image-guided energy toward focal regions with high levels of tumor antigens could define a new use for tumor ablation for cancer immunotherapy.

In summary, our study presents a mouse model that allows for elegant immunologic investigations following the application of RF ablation. We find that partial RF ablation of MB49 tumors results in an amplified tumor antigen–specific systemic immune response that is associated with increased DC infiltration and subsequent tumor eradication, and that the effects of RF ablation can be replicated by using ITDC injections. Conceptually, combining RF ablation treatment with ITDCs remains attractive, and the lack of synergy observed with combining the treatments in these experiments is probably a result of technical parameters. Thus, future experiments should explore their synergistic potential. Ultimately, in addition to local destruction of tumor, image-guided treatment of malignancy represents an attractive adjuvant to improve on current immunotherapeutic strategies.

ADVANCES IN KNOWLEDGE

• Radiofrequency (RF) ablation of a murine histocompatibility-expressing cell line derived from a urothelial carcinoma induces a systemic antitumor immune response.

• The antitumor immune system activation from RF ablation results in regression of the primary tumor and protects treated mice from tumor rechallenge in the contralateral flank.

• The injection of intratumoral dendritic cells (ITDCs) elicited regression of the primary tumor.

• Combination treatment (RF ablation + ITDCs) did not enhance antitumor immune responses, regression, protection against rechallenge, or immunohistochemical analysis compared with RF ablation or ITDCs alone.

Abbreviations

• DC = dendritic cell

• ELISPOT = enzyme-linked immunosorbent spot

• FITC = fluorescein isothiocyanate

• HY = histocompatibility

• IFN = interferon

• ITDC = intratumoral DC

• RF = radiofrequency