Cryoablation Induces Necrosis and Apoptosis in Lung Adenocarcinoma in Mice

Technology in Cancer Research and Treatment ISSN 1533-0346 Volume 6, Number 6, December 2007 Adenine Press (2007) Cryoablation Induces Necrosis and Apoptosis in Lung Adenocarcinoma in Mice www.tcrt.org This study evaluated cryoablation on subcutaneously transplanted tumors of lung adenocarcinoma LA795 in T739 mice in vivo, in an effort to assess the feasibility of cryoablation in treatment of NSCLC. Subcutaneously transplanted lung adenocarcinoma LA795 was implanted into T739 mice yielding tumors of approximately 2.5 cm in diameter. Following cryoablation, the various modes of cell death were studied: necrosis in the central frozen zone by light microscopy and apoptosis in periphery of the frozen zone by in situ end labeling (TUNEL). Bc1-2 and bax expression were detected by immunohistochemical SABC procedures, and the cleavage and activation of Caspase 3 and PARP in peripheral zone by Western blot. We find that in central cryoablated zone, necrosis was the dominant mode of cell death occurring at three hours and four days post-thaw. The first three-hour necrosis peak involved approximately 47% of the tumor while the four-day peak increased in volume to 68% of the tumor. In peripheral cryoablation zone, definite cell apoptosis could be observed by morphological examination under light microscope and TUNEL staining, peaking at 8-16 h after cryoablation. Immunohistochemical results yielded little change in bcl-2 protein expression before and after cryoablation. However, bax protein expression was up-regulated significantly after cryoablation. In addition, cleavage and activation of Caspase-3 and PARP occurred in the peripheral freeze zone after the treatment. It indicated that Cryoablation efficiently induces cell death both by necrosis and apoptosis. Cryoablation appears to induce apoptosis in the peripheral freeze zone through the intrinsic mitochondrial caspase pathway based on bax upregulation. This observation allows us to suggest that cryoablation may be combined with chemotherapy to increase cancer destruction. JuYi Wen, M.D., Ph.D. 1,* YunYou Duan, M.D., Ph.D. 2 YueKun Zou, M.D. 2 Zhoushan Nie, M.D. 2 HuaSong Feng, M.D., Ph.D. 2 Franco Lugnani, M.D., Ph.D. 3 John G. Baust, M.D., Ph.D. 4 1 Department of Oncology 2 Department of Respiratory Medicine Navy General Hospital Beijing 100037, China 3 President of International Society of Cryosurgery Casa di Cura Salus, Trieste, Italy 4 Institute of Biomedical Technology Binghamton University State University of New York Binghamton, NY 13902-6000, USA Key words: Cryotherapy; Cryoablation; Adenocarcinoma; and Apoptosis. Introduction Lung cancer is a common malignant tumor. Since 1950s, the morbidity and mortality of lung cancer rapidly increased world-wide with a high fatality rate (~90%). In China, lung cancer deaths represent 20% of all cancer-related deaths with rapidly increasing incidence and mortality and a 5-year survival lower than 10%. At present, even with effective early diagnosis, therapeutic options for treatment are limited. With lung cancers, non-small cell lung cancer (NSCLC) accounts for 75%-80% of the total. Within this cohort, only 20% of the patients in phase (stage) I-II have opportunity for surgery with a 5-year survival rate of 50%-80% after surgery. For patients staged in phase III-IV (75-80%) the 5-year survival rate is only 12% following palliative treatments. While patients in phase IIIA may still have the * Corresponding Author: JuYi Wen, M.D., Ph.D. Email:wenjuyi@hotmail.com 635

636 Wen et al. possibility of surgical intervention, recurrence is common with a 5-year survival of 10%-30%. In recent years, second line chemotherapy protocols have been adopted in the treatment of late stage NSCLC. Sequenced radio-/chemotherapy and synchronous radio-/chemotherapy have improved the response rate to some extent (only about 30%), but this treatment accompanied by severe adverse reaction. In an effort to improve life span and quality of life (QOL) for patients with NSCLC, minimally invasive targeted therapies, including intra-tumoral chemotherapy, intra-tumoral injection of absolute alcohol, radio frequency ablation (RFA), microwave therapy, and cryoablation, have been developed. Beginning in 1998, cryoablation of NSCLC tumors has been investigated as a minimally invasive therapeutic technology. Cyoablation (CA) technology is safe, efficient, and minimally invasive and has with few side effects. CA may play a role in locally alleviating tumor load, killing anoxic cells and cells in the rest stage that are typically insensitive to chemotherapy CA is considered a comprehensive therapy finding applications in the treatment of various solid tumors, such as carcinoma of prostate, lung cancer, liver cancer, et cetera, (1) with demonstrated clinical effectiveness. One problem with CA, however, relates to the difficulty in postcryosurgical pathological biopsy retrieval for ethical reasons. Without a post-surgical biopsy, the effectiveness of CA can only be judged by imaging methodologies, which may be compromised by tumor irregularity and incomplete freezing of a safe margin beyond the visual tumor boundary. In light of this concern and the temperature gradient between the cryoprobe and the edge of the freeze zone, we established an animal tumor model of subcutaneously transplanted lung adenocarcinoma LA795 to study the distribution of necrosis and apoptosis within the freeze zone. Materials and Methods Establishment of Tumor Model Healthy T739 mice (6-week old, average weight = 20 ± 2.05 g) were purchased from Institute of Laboratory Animal Science, Peking Union Medical College. LA795 cell line was cultured to log phase. Cell suspensions (0.2 ml) of lung adenocarcinoma LA795 was injected subcutaneously (2.5 10 6 cells/mouse) in the dorsal right leg. After two weeks, the long diameter of tumor equaled 25.64 ± 1.98 mm and short diameter equaled 23.41 ± 1.45 mm. Tumors developed in 91% of the injected mice. Cryoablation Control Group: 34 mice, half males and half females, of which ten were used for life span observations. Experimental Group: 34 mice received cryoablative treatment, half males and half females, of which ten would be used for life span observations, and three were sacrificed at each time interval (3h, 5h, 8h, 16h, 24h, 2d, 4d, 8d) following cryoablation. Three mice from the control group was sacrificed at each experimental time point simultaneously. The 24 mice in the cryoablation group were anesthetized using Sumianxin injection. After surgical preparation cryoablation was carried out with an argon-helium cryosurgical device (Cryocare Surgical System, Endocare, Inc., USA) with 2.4 mm cryoprobes inserted in to the center of tumor. A double freeze-thaw method was adopted (i.e., probe temperature was lowered to -120º C for 10 s and then thawed to 0º C. The cryoablation cycle was repeated. The long diameter of ice ball was 23.5 ± 1.06 mm, while the short diameter of 18.04 ± 0.96 mm. After cryoablation, animals were sacrificed as described above. Tumor tissue was then excised and samples were preserved according to different detection requirements. Morphological Observation Tumor tissue was fixed in 10% formalin, imbedded in imbedded paraffin, sectioned, and observed under light microscopy following after H&E staining. Apoptosis Detection by In Situ End Labeling (TUNEL) Samples from the experimental time points were analyzed. Apoptosis was detected using in situ Terminal deoxynucleotidyl transferase mediated dutp nick end labeling (TUNEL in situ immunohistochemical kit (Roche Co., USA) according to the manufacturer s instructions. Labeling was revealed by reaction with the DAB peroxidase substrate (3, 30-diaminobenzidine, Sigma, USA). A positive reaction was characterized by a brown coloration of the nuclei. Positive cell (apoptotic) population was randomly counted in five different visual fields (400 ) (three animals at each time point) and values averaged. Bcl-2 and Bax Expression Detected by Immunohistochemical SABC Bcl-2 and bax immunohistochemical kits were both purchased from Wuhan Boster Biological Engineering Co. Ltd. and analysis completed according to manufacturers recommendations. PBS was used as the negative control. Bcl-2 appeared as brownish yellow granules in the cytoplasm and bax stain is defined as positive cells. Five visual fields were randomly selected under high power using a Leica Qwin Image Analysis System to measure the optical density of positively staining cells. Apoptosis is Detected by DNA Electrophoresis Tissues in central necrotic zone and peripheral zone of ice ball

Cryoablation-Induced Necrosis and Apoptosis 637 were extracted 16-hours following freezing, and DNA was isolated in phenol/chloroform (1:1). 50 μl TE buffer solution containing 100 μg/ml RNase A was added at 65º C for 30 min. A 20 μl sample was added to 2 ~ 5 μl of a DNA Sample Buffer (0.25% bromphenol, 0.25% xylene cyanol, 30% glycerol). Gel electrophoresis (1.8% agarose) was run at 60V for 1 h. Results were observed under UV lamp and photographed. Caspase-3 Expression and PARP Cleavage in the Peripheral Zone of Ice Ball Detected by Western-blot Tissues in peripheral zone of ice ball of mice in cryoablation group and tissues in the corresponding zone of mice in control group were extracted 16-hours post-thaw, protein isolated (2), and sampling and electrophoresis conducted by standard methods. Protein was transferred to nitrocellulose membrane from the DNA gel and the membrane was slightly oscillated at 18C-24C for 1 h using 1 PBS, 5% skimmed milk powder, 0.1% Tween-20. 1:2000 anti-parp antibody (4338-MC-50, Santa Cruz, USA). Caspase-3 antibody was diluted by 1 PBS, 5% skimmed milk powder, 0.1% Tween- 20 at 4º C overnight. The membrane was washed (3X) with 1 PBS, 0.1% Tween-20. HRP labeled secondary antibody was diluted with bleaching buffer and added to blot and incubated for 1-hour. The membrane was washed (3X) with 1 PBS, 0.1% Tween-20. Finally visualization was accomplished using ECL lamination kit. Statistical Analysis SPSS11.0 software was utilized. Kaplan-Meier survival analysis generated, χ2 test was adopted for comparison of rate and t-test for comparison of paired data. P values less than 0.05 were defined as significant. Results Effect of Cryoablation on Life Span of Tumor-bearing Mice The life span of the cryoablation group was 9-27 days starting from the day of treatment (median = 20.6 ± 2.51 days). Control group life span equaled 7-14 days (median = 11.8 ± 1.79 days) (Figure 1). The life span in the experimental group was prolonged significantly (P<0.05). Ablation and necrosis rate of tumor foci at different time points after cryoablation Three distinct areas were observed after cryoablation (Figure 2). The central necrotic area (located near the cryoprobe) was characterized by cell ghosts and nuclear fragmentation, a condition often referred to as coagulation necrosis. In the periphery of this area we observed shrunken and condensed cells with compact nuclei and pyknotic chromatin, typical characteristics of apoptosis. Local vascular congestion, embolism, and exudate could also be seen. This cryodamaged zone was surrounded by intact tumor cells. The volume of necrotic tissue in the cryoablation group was far greater than that in the control group (P<0.01). The first necrosis peak (volume = 47%) occurred 3 h after freezing while the 2nd necrotic peak (volume = 685) occurred four days after cryoablation (Figure 3). Apoptosis Detected by In Situ End Labeling TUNEL Following cryoablation, apoptotic cells were distributed mainly in the area around or distal to the central necrotic area. Typical morphological changes of cells undergoing apoptosis included brownish yellow nuclei, karyopyknosis, chromatin densely stained like bean or new moon around karyotheca. In tumor tissues of the control group, small number of apoptotic cells was sparsely distributed throughout the tumor. The number of apoptotic cells in the cryoablation group, however, was significantly higher than in the control group (P<0.05). The number of apoptotic cells in the cryoablation group increased 3 h after cryoablation and reached the peak in 8-16 h (Figures 4 and 5). Figure 1: Kaplan-Meier Survival Curve.

638 Wen et al. Figure 2: The H&E stained images of T739 mice tumors after cryoablation ( 200). erally cryodamaged zone. Bax was sparsely distributed in the control group and was up-regulated after cryoablation (P<0.01) (Figure 6) (Table I). Immunohistochemical Staining of bax and bcl-2 100.0% 90.0% 80.0% 70.0% 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% 0.0% Apoptosis Detected by DNA-ladder DNA gel electrophoresis revealed the typical apoptotic DNA ladder in the peripheral cryodamaged zone (B) indicating that DNA was not randomly cleaved (Figure 7). 100.0% control group 90.0% Apoptosis Rate Surface Ratio Necrotic Areas/Whole Tumor Bcl-2 was observed by light microscope to mainly in the cytoplasm as brownish yellow particles distributed sparsely or foci-like. No bcl-2 expression was evident in the center of cryoablation zone. Expression of bcl-2 protein in peripherally cryodamaged zone of each cryoablation group was slightly lower when compared with the control group (yet showed no statistical significance P>0.05). Bax, also located in the cytoplasm, and was observed as brownish yellow particles. Bax expression was increased in periphcontrol group cryo group cryo group 80.0% 70.0% 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% 0.0% 3h 5h 8h 16h 24h 2d 4d 3h 8d 5h 8h 16h 24h 2d 4d 8d Time of Analysis Time of analysis Figure 5: The level of apoptosis in cells of the freeze zone periphery following cryoablation. Figure 3: Volume of necrotic tissue in each experimental group. A bax B bcl-2 Figure 6: The immunohistochemical staining of bax and bcl-2 in cells. Table I Post-Freeze Time Course of Bax to Bcl-2 Ratio Alterations Following Cryoablation Figure 4: Illustration of apoptosis in tumors after cryotherapy (TUNEL stain) ( 400). Control Cryo 5 Hours 0.73 21.2 8 Hours 0.79 25.7 12 Hours 0.93 19.3 48 Hours 0.99 20.3 96 Hours 1 18

Cryoablation-Induced Necrosis and Apoptosis 639 M A B Cleavage and Activation of Caspase-3 and PARP Experimental results showed PARP from tissues in cryodamaged zone was cleaved from an integral 116Kda to 85Kda after cryoablation but not in the control group. Caspase-3 in the cryoablation group was cleaved from 32Kda to 20Kda as well but not in the control group (see Figure 8). Discussion Figure 7: Agarose gel electrophoresis of DNA from cells located in central necrotic zone and peripheral cryodamaged zone after cryoablation Cryoablation is an effective means of local tumor control. The efficacy of targeted cryoablation therapy in treatment of tumors has been preliminarily demonstrated in a variety of clinical applications. The mechanism of cryoablation has historically been linked to changes in cellular membrane permeability, intracellular and extracellular osmotic pressure, and the formation of intracellular and extracellular ice crystal at lower temperature, which, in combination, result in disintegration of cells, and the appearance of post-thaw necrosis in target area following damage to the tumor vasculature (1, 3). More recently, in vitro studies demonstrated that the mechanisms of cell death include apoptosis following cryoablation (3-8). We now report that recent in vivo studies have confirmed these in vitro observations (3-8). In our study we confirmed the observation of a dual necrotic A B C D cleavage fragment of PARP 116KD 85KD activation of caspase3 peak following cryoablation. The first necrosis peak occurred 3 h after freezing and the 2nd necrosis peak followed at four days. The first necrosis peak is a direct response of cell damage following cryoablation while the 2nd necrotic peak is due to the effects of vascular stasis. Besides these direct cryoablation effects, the induction of apoptosis is now recognized a primary form for cell death after cryoablation. Cellular apoptosis was detected 3 h after cryoablation peaking 8-16 h later. Apoptotic cells were found mostly in the cryodamaged zone distal to the necrotic region, though with a margin between apoptotic and necrotic zones was not so obvious. It is the combination of these multiple modes of cell death that determines the extent of damage (1, 3). Earlier studies have shown that the temperature at the central freeze zone adjacent to the might reach about -125º C. With increasing distance from the cryoprobe, the temperature gradually increases. The temperature of tissues 0.7 cm from exterior wall of the cryoprobe may drop to about -40º C, while that 1 cm from the cryoprobe may be 0º C (This site is taken as physical edge of ice ball) (9). Also, due to the difference in tissue density and blood perfusion, heterogeneity of cryoablation might be further intensified. It is generally accepted that cells exposed to -40º C may be completely necrotic. During the cryoablation process, blood circulation in the frozen tissue mass is obstructed. It is commonly observed that in a short time following cryoablation, edema occurs in the edge of cryoablated zone. Within several hours after thawing, endothelial cells are be stripped from the capillaries causing an increasing permeability of capillary walls, platelets aggregate, and blood flow stagnation. Within several hours after cryoablation, smaller blood vessels are completely blocked leading to complete tissue necrosis. Meanwhile, due to a secondary inflammatory response co-induced by necrosis, hypoxemia, et cetera, delayed response following freezing occurs resulting in the induction of continued cellular apoptosis (10). 32KD 20KD Figure 8: Cleavage and activation of caspase 3 & PARP in different experimental group (lane A, C: control group; lane B, D: cyro group). Apoptosis was first described following cryosurgery by Hollister et al. in 1998 in a human prostate cancer cell model. In 1999, Hoffmann and Bischoff challenged this observation when the failed to observe apoptosis in an in vivo model. However, it is now recognized that this later study failed to recognize the chronology of the sequenced apoptotic and necrotic events and, therefore, misinterpreted their data. It is now widely accepted (and we confirm with this study) that cryoablation results in the a sequence of apoptotic and necrotic events related to both the level of stress experienced by cells within the frozen tumor mass and degree of vascular stasis. Apoptosis following freezing may be initiated by both extrinsic (membraneinduced) and intrinsic (mitochondrial-induced) pathways. In this study we confirm the existence of an intrinsic mediated cell death pathway following cryoablation.

640 Wen et al. Overexpression of bcl-2, an anti-apoptotic protein, occurs as a protective mechanism in various tumors (11). Bax, a family member of bc1-2, is a pro-apoptotic protein that exists in a cell specific levels under homeostatic conditions. Bcl-2 and bax have opposing functions (12). The up-regulated expression of bcl-2 and down-regulated expression of bax protein existed in tissue of lung cancer (13), yielding a survival and proliferative state (14). Results of this experiment showed a trend toward the increased expression of bc1-2 after cryoablation that was consistent with an increased in control levels. However, we observed significant up-regulated expression of bax protein after cryoablation. This was indicative of a bax-related role in the promotion of apoptosis following cryoablation. PARP is a nuclear protein that functions in DNA duplication, recombination, repair, regulation and control of cell cycle, cell differentiation, and cellular apoptosis. Caspase enzymes represent important effectors in the mechanism of apoptosis. More than 14 kinds of Caspase family members have been identified with 2/3 associated with cellular apoptosis (15). The caspase cascade includes the activation of caspase 3, the terminal executioner enzyme in caspase-dependent apoptotic pathway. Activation of caspase 3 requires cleavage into two segments. Following activation it participates in activating PARP, thus inducing cellular morphological changes and DNA degradation. The activity of caspase 3 is also regulated by other apoptosis regulating and controlling factors, such as p53, bcl-2, and bax, et cetera. Results of our study show that caspase-3 in the cryoablation group was cleaved from 32Kda to 20Kda in cryodamaged zone around ice ball while no cleaved fragments were observed in the control group. PARP was cleaved from an integral 116Kda to 85Kda after cryoablation while no PARP cleavage occurred in the same sites of the control group. 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Received: May 1, 2007; Revised: September 21, 2007; Accepted: September 22, 2007 Acknowledgements This work was supported by funds from the 11th Five-Year Plan of Military Medical Research, China (NO. 06MB021). The authors would like to thank Prof. Jiren Zhang for his inputs and discussions.