10.1245/ASO.2004.08.013
Annals of Surgical Oncology 11:399-406 (2004)
© 2004 Society of Surgical Oncology
Heat Shock Protein 70 Is Induced in Mouse Human Colon Tumor Xenografts After Sublethal Radiofrequency Ablation
Weng-Lang Yang, PhD,
Deepak G. Nair, MD,
Ryouji Makizumi, MD,
George Gallos, MD,
Xuemei Ye, BS,
Rohit R. Sharma, MD and
T.S. Ravikumar, MD
From the Department of Surgery, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York.
Correspondence: Address correspondence and reprint requests to: T. S. Ravikumar, MD, Department of Surgery, Albert Einstein College of Medicine/Montefiore Medical Center, 3400 Bainbridge Avenue, 4th Floor, Bronx, NY 10467; Fax: 718-798-1883; E-mail: travikum{at}montefiore.org
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ABSTRACT
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Background: Radiofrequency ablation (RFA) destroys tumor cells by generating high temperatures through ionic vibration. Tumor recurrence may be a direct function of sublethal temperature. Further, a set of proteins called heat shock proteins (HSPs) can be synthesized under heat stress to facilitate recovery of tumor cells from heat damage.
Methods: Subcutaneous xenografts were induced in nude mice by injection with HT29 human colon cancer cells. The tumors were exposed surgically and subjected to RFA. The tumors were randomly assigned to achieve a target tumor temperature of 42°C, 45°C, or 50°C. Total RNA and cell lysates were isolated from tumor tissues and subjected to reverse transcription-polymerase chain reaction and Western blot analyses, respectively, at various time points after treatments for assessing HSP expression. For in vitro experiments, HT29 cells were subjected to variable temperatures, and HSP expression was assayed.
Results: During a 50-day follow-up, the recurrence rates were 0% at 50°C, 30% at 45°C, and 100% at 42°C. The messenger RNA and protein levels of HSP90 and HSP27 remained unchanged after RFA at 45°C; however, HSP70 was induced at 4 and 10 hours after RFA. In vitro HT29 culture cells subjected to a heated water bath exhibited a cellular sensitivity to heat and change of HSP expression similar to those in tumor xenografts subjected to RFA.
Conclusions: Our data establish the requisite heat parameters during RFA for human colon tumors in vitro and in vivo. Because HSP70 plays an important role in protecting cell death from a variety of stresses, HSP70 could be a potential target for enhancing the efficacy of RFA.
Key Words: Radiofrequency ablation • Colon cancer • Hyperthermia • Heat shock protein
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INTRODUCTION
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Colorectal cancer strikes approximately 130,000 patients and accounts for more than 55,000 deaths each year in the United States. For the vast majority of patients who die of colorectal cancer, the liver is the most common site of metastasis.1,2 Left untreated, these lesions have a poor prognosis, with a median survival ranging from 4 to 21 months and a 3-year survival <3%.3 Surgical resection is a gold standard for the treatment of hepatic metastases. Hepatic resection of limited metastases from colorectal cancer is now an established modality, with 5-year survival rates of up to 30%.4 However, only 10% to 20% of patients fulfill selection criteria and are amenable to curative resection.5 Because so few patients are eligible for surgical resection, alternative therapies, including systemic and regional chemotherapy and local ablative therapies, have been applied to the treatment of unresectable colorectal liver metastases. The ablative techniques include percutaneous ethanol injection, focused ultrasound, cryoablation, microwave tumor coagulation, direct current fulguration, laser photocoagulation, and radiofrequency ablation (RFA).
Recently, RFA has gained popularity and works by converting radiofrequency waves into heat. A high-frequency alternating current (100–500 kHz) passes from an uninsulated electrode tip into the surrounding tissues and causes ionic vibration as the ions attempt to follow the change in the direction of the rapidly alternating current.6,7 This ionic vibration causes frictional heating of the tissue surrounding the electrode, rather than the heat being generated from the probe itself. The goal of RFA is to achieve local temperatures >50°C such that tissue destruction occurs.8,9 Tissue heating also drives extracellular and intracellular water out of the tissue and results in further destruction of the tissue because of coagulative necrosis.8
Local recurrence after RFA has been an issue, especially with larger tumors. During the RFA treatment, because of a cooling effect from blood vessels, the heterogeneity of tumor composition, or thermal gradient formation by thermodynamics at the treated tumor margin, the temperature at treated tumors may not uniformly reach >50°C, and tumor cells may survive RFA treatment, resulting in recurrence. Recently, a report with 447 ablations showed that 20.9% of patients who underwent RFA for the treatment of unresectable primary and metastatic hepatic malignancies had developed recurrence at a mean follow-up of 11 months.10 To enhance tumors’ responses to RFA and reduce recurrences, identification of the cellular events in cancer cells that survive RFA is necessary.
Heat shock proteins (HSPs) are a unique group of proteins induced by diverse kinds of stress, such as supraoptimal temperatures, anoxia, or chemical agents, in prokaryotic and eukaryotic cells.11 HSPs encompass several groups of proteins and can be divided into six subfamilies according to their molecular weights: the HSP110, HSP90, HSP70, HSP60, HSP40, and small HSP families.11 Some HSPs are constitutively expressed and increase in response to stress, whereas others’ expression is induced only after the exposure of cells to environmental and physiological stresses. Many of the HSPs function as chaperon proteins that act in the transport, assembly, and proper folding of proteins that ultimately affect survival of the cell.12 Recently, it has been recognized that HSPs also regulate apoptosis. For example, the expression of small HSPs (especially HSP27) and inducible HSP70 has been shown to enhance the survival of mammalian cells exposed to many types of stimuli that induce stress and apoptosis.13,14
In this study, we established a nude mouse model for studying the cellular responses of human colon cancer cells to RFA at different treatment temperatures. We then examined the changes of the transcript and protein levels of HSP27, HSP70, and HSP90 in nude mouse tumor xenografts after RFA. To further elucidate whether the cellular response and the change of HSP expression patterns after RFA are mainly due to a thermal effect, we compared the heat sensitivity and the changes of the transcript and protein levels of these three HSPs in cultured human colon cancer cells after heat treatment by immersion into a water bath at different temperatures.
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METHODS
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Cell Culture
A human colon adenocarcinoma cell line (HT29) was obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in McCoy’s 5A medium (Invitrogen, Rockville, MD) containing 10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA) and supplemented with penicillin and streptomycin in a humidified incubator at 37°C with a 5% CO2 atmosphere. Cells were kept in culture under exponential growth conditions and were harvested with trypsin/ethylenediaminetetra-acetate solution. Only single-cell suspensions of >90% viability, as determined by trypan blue exclusion assays, were used for inoculation into nude athymic mice.
Radiofrequency Ablation
Pathogen-free female athymic nude mice, 4 to 6 weeks old, were obtained from the National Cancer Institute. The mice were maintained in sterile conditions in microisolator cages and given autoclaved food and water. All encounters were performed in an air-filtered laminar flow hood. All animal procedures used in this study were approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine. The exponentially growing HT29 cells (1 x 106 cells in .1 mL of phosphate-buffered saline) were inoculated subcutaneously into the flank area of nude mice. When tumors grew to 6 to 8 mm in diameter, they were exposed surgically and subjected to RFA under isoflurane anesthesia. A 15-gauge RITA Starburst multiarray electrode (RITA Medical Systems, Mountain View, CA) was inserted into the tumor. Radiofrequency energy was set at 5 W by using a RITA model 1500 radiofrequency generator. The ablation temperature was monitored by a peripherally placed thermocouple on the tumor. The tumors were randomized into three groups with the following target temperatures achieved for 2 minutes: 42°C, 45°C, or 50°C. After RFA, the skin was sutured over the tumor, and the mice were allowed to recover in a warm pocket. Animals with tumors subjected to the same procedure without RFA served as sham controls. Tumor volume was calculated with the formula a x b2/2, where a is the longest diameter and b is the shortest diameter.
Western Blot Analysis
HT29 cells were lysed on ice for 30 minutes in RIPA buffer (10 mM of Tris-HCl [pH 7.5], 120 mM of NaCl, 1% NP-40, 1% sodium deoxycholate, and .1% sodium dodecyl sulfate) containing a protease inhibitor cocktail (Sigma, St. Louis, MO) and then centrifuged at 10,000 x g for 10 minutes. A Bio-Rad (Hercules, CA) protein assay was used to determine the protein concentration. Protein was electrophoresed on sodium dodecyl sulfate/polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in TTBS buffer (.1% Tween-20, 20 mM of Tris-HCl [pH 7.5], and 140 mM of NaCl). Membranes were incubated with primary antibody against HSP27, HSP70, HSP90ß, or ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA), followed by secondary antibody/horseradish peroxidase conjugate (Pierce, Rockford, IL) and detected with chemiluminescence (Pierce) and autoradiography.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction Analysis
Tumors dissected from nude mice were homogenized by Pellet Pestles (Kontes, Vineland, NJ). Cultured cells were harvested by trypsinization. The total RNA of the harvested cells was isolated by using a RNeasy isolation kit (Qiagen, Valencia, CA). A total of .2 µg of isolated total RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis with the Qiagen OneStep RT-PCR kit. The RT reaction was performed at 50°C for 30 minutes, followed by an initial PCR activation step at 95°C for 15 minutes. PCR conditions were denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute for 27 cycles, followed by a final step at 72°C for 10 minutes. A total of .3 µM of HSP90ß, HSP70, and HSP27 and .6 µM of ß-actin primer pairs were applied together to each RT-PCR reaction. The oligonucleotide sequences of the primer pairs used in this study have been described previously.15 The PCR products were separated on a 1.6% agarose gel and stained with ethidium bromide. Bands were visualized via UV transillumination and recorded by Polaroid (Waltham, MA) photography. Semiquantitative levels of band intensity were determined by scanning densitometry and analysis by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Relative HSP messenger RNA (mRNA) expression was calculated as ratio of HSP to ß-actin expression. The number of PCR cycles and the amount of total RNA for each product were determined after the linear exponential portion of the amplification was defined (Fig. 1).
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FIG. 1. Optimization of multiplex reverse transcription-polymerase chain reaction (RT-PCR) assays. (A) Determination of the optimal PCR cycle number. A total of .1 µg of total RNA isolated from HT29 cells was reverse-transcribed and PCR-amplified between 20 and 35 cycles with the primers containing heat shock protein (HSP)27, HSP70, HSP90, and ß-actin. A total of 10 µL of RT-PCR product from each cycle condition was electrophoresed. The relative intensity of each RT-PCR product, based on densitometric analysis, was plotted as a function of the PCR cycle number. (B) Determination of the optimal amount of input total RNA. The input amount of total RNA ranged from .05 to 1.0 µg and was amplified at 27 cycles with the primers containing HSP27, HSP70, HSP90, and ß-actin. The intensity of each RT-PCR product was plotted as a function of the amount of input total RNA.
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In Vitro Heat Treatment and MTT Assay
The T25 flasks containing HT29 cells at approximately 70% confluence or 96-well plates were sealed with parafilm and exposed to heat treatment at 37°C, 42°C, 45°C, 47°C, and 50°C for 15 minutes by immersion into a water bath. Cells subjected to 37°C served as controls. All temperatures were maintained within ±.05°C by testing for accuracy with a thermocouple (Fischer Scientific, Pittsburgh, PA). After heat treatment, the parafilm was removed, and flasks and plates were returned to a 37°C incubator. Cell viability was measured by MTT assay 72 hours after heat treatment. A total of 150 µL of medium containing .5 mg/mL of MTT (Sigma) was added to each well and incubated at 37°C for 3 hours. The formazan product was dissolved in 150 µL of isopropanol, and the plates were read at 570 and 690 nm (background). All measurements were performed in quadruplicate, and each experiment was repeated at least three times.
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RESULTS
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A Mouse Human Tumor Xenograft Model for RFA
Fig. 2 describes our human colon cancer xenograft model to investigate the effects of RFA. HT29 human colon cancer cells were inoculated subcutaneously into the flank to generate human tumor xenografts. During RFA, a thermocouple was placed on the surface of tumors away from the entry site of the electrode to monitor the temperature at the peripheral zone. The tumors were heated to a temperature of 42°C, 45°C, or 50°C and were maintained at the target temperature for 2 minutes to examine the tumor response to different RFA conditions. The mean size of tumors subjected to RFA in the 42°C, 45°C, and 50°C groups was 169.10 ± 65.71 mm3 (n = 6), 164.02 ± 59.39 mm3 (n = 7), and 167.34 ± 65.82 mm3 (n = 7), respectively (P = not significant). All tumors subjected to 42°C demonstrated progressive growth 3 to 13 days after RFA (Fig. 3A). Among tumors subjected to 45°C, two mice had tumor recurrence 3 days after RFA, two tumors recurred 10 days after RFA, one tumor recurred 29 days after RFA, and two mice were tumor free up to 50 days (Fig. 3A). At 50°C, all tumors were cured, and no tumor recurrence was observed in this group, with a 50-day follow-up (Fig. 3A).
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FIG. 2. Radiofrequency ablation (RFA) on a nude mouse carrying a subcutaneous tumor composed of HT29 cells. The mouse was placed on a dispersive electrode pad under mask anesthesia with isoflurane. The anatomized tumor was inserted with a radiofrequency probe (left) and a thermocouple (right).
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We also examined the effect of sublethal RFA on tumor growth kinetics by measuring the tumor size in the 42°C group. As shown in Fig. 3B, all tumors were smaller after RFA. After a lag period, the tumors grew exponentially. Two tumors had a very short lag period after RFA.
These results indicate that a temperature >50°C is necessary to effectively kill human colon cancer cells during RFA treatment. At sublethal temperatures (42°C to <50°C), variable recurrence are observed, suggesting that temperature-mediated biological events in tumor cells may contribute to cell survival.
HSP Expression in Tumor Xenografts After RFA
We next examined the expression of several HSPs after RFA at 45°C. Tumors were induced in nude mice by subcutaneous inoculation of HT29 cells and were subjected to 45°C RFA for 2 minutes. They were removed from mice at 4, 10, and 24 hours after RFA and subjected to Western blot analysis for assessment of HSP protein levels. There was no significant change in expression of HSP90, HSP27, or HSC70 (the constitutively expressed form of HSP70) in mouse tumor xenografts during the entire time course, whereas HSP70 was induced starting at 4 hours after RFA (Fig. 4A). To learn whether the change of HSP expression was regulated at the transcription level, we further examined the mRNA levels of HSP27, HSP70, and HSP90 at 4, 10, and 24 hours after RFA by using RT-PCR analysis. The mRNA levels of HSP90 and HSP27 remained unchanged after RFA, and this corresponded to their protein expression (Fig. 4B). The mRNA levels of HSP70 were increased to 2.9-fold and 3.2-fold at 4 and 10 hours, respectively, and returned to basal levels at 24 hours in comparison to controls (Fig. 4C).
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FIG. 4. Changes in heat shock protein expression in tumor xenografts after radiofrequency ablation (RFA). Total cell lysate and RNA were isolated from tumor xenografts at the indicated time points after RFA at 45°C. (A) A total of 30 µg of cell lysate was subjected to Western blot analysis against an indicated anti–heat shock protein (HSP) antibody. (B) A total of .2 µg of the isolated total RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) with the primers of HSP90, HSP70, HSP27, and ß-actin, as described in Methods. The products from RT-PCR were separated by 1.6% agarose electrophoresis and stained with ethidium bromide. (C) Densitometric scanning was performed on an agarose gel shown in (B). The relative intensity of HSP70 at different time points was calculated after correction of the background and by means of normalization versus actin. Actin was used as a loading or internal control. Similar results were observed in two independent experiments.
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HSP Expression in Culture Cells After Heat Treatment
To isolate the physical effects of heat from other factors in the tumor microenvironment in vivo that may contribute to tumor responses to RFA, we examined the growth of HT29 cells and the expression pattern of HSPs in culture cells after heat treatment with temperature-controlled water baths. We first determined the survival of HT29 cells after exposure to different temperatures. Cells were subjected to 37°C, 42°C, 45°C, 47°C, and 50°C for 15 minutes and then returned to a 37°C incubator. After 72 hours of incubation, cell growth was determined by MTT assay. As shown in Fig. 5, the growth of cells heated at 42°C was similar to that of control cells; whereas 60.7% of cells survived at 45°C, only 27.4% of cells survived at 47°C when compared with control cells. There was no cell survival at 50°C.
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FIG. 5. Effects of different temperatures on cell viability. HT29 cells were subjected to a water bath at the indicated temperatures for 15 minutes and then returned to a 37°C incubator. Cell viability was determined by MTT assay 72 hours after heat treatment. Each graph represents at least three experiments. Data are shown as mean ± SEM.
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We then conducted kinetic analysis of transcript levels and protein expression of HSP90, HSP70, and HSP27 after heat treatment at 43°C and 45°C. By RT-PCR analysis, we observed increased HSP70 transcript levels up to 3.7-fold and 3.6-fold at 3 and 6 hours, respectively, at 45°C, and they returned to the basal levels at 24 hours, in comparison to 37°C controls (Fig. 6). The HSP70 transcript levels at 42°C did increase in the entire course. There was a slight increase in HSP27 transcript levels at 3 hours at 45°C (Fig. 6). HSP90 transcript levels remained unchanged both at 42°C and 45°C. We further assessed the protein levels of HSP27, HSP70, and HSP90 in culture cells after heat treatment at 45°C. As shown in Fig. 7, the protein levels of HSP70 started to increase at 4 hours and maintained these high levels for 48 hours. HSP27 expression showed a slight decrease at 4 hours and started an increase at 10 hours. The HSP90 protein levels did not change after heat treatment.
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FIG. 6. Kinetic analysis of transcript levels of various heat shock proteins (HSPs) in culture cells after heat treatment. Total RNA was isolated from HT29 cells at the indicated time points after heating at different temperatures. (A) A total of .2 µg of the isolated total RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) with the primers of HSP90, HSP70, HSP27, and ß-actin, as described in Methods. The products from RT-PCR were separated by 1.6% agarose electrophoresis and stained with ethidium bromide. M, 100-base pair DNA marker. (B) Densitometric scanning was performed on the agarose gel shown in (A). The relative intensity was calculated after correction of the background and by means of normalization versus actin. The intensity of 37°C was used as a reference. Similar results were observed in two independent experiments.
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FIG. 7. Kinetic analysis of protein levels of various heat shock proteins (HSP) in culture cells after heat treatment. Total cell lysate was isolated from HT29 cells at the indicated time points after heating at 45°C. A total of 30 µg of cell lysate was subjected to Western blot analysis against the indicated anti-HSP antibody. Similar results were observed in two independent experiments.
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DISCUSSION
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RFA is an important component of the regional therapies used to treat unresectable hepatic tumors. RFA has gained popularity on the basis of ease of use, safety, reasonable cost, and applicability via percutaneous, laparoscopic, or open approach. RFA has also been demonstrated to be promising for treating a variety of solid tumors, such as those in bone, kidney, lung, prostate, pancreas, and breast.16 Clinical long-term outcomes for RFA in the treatment of hepatic metastases have not been conclusive. One concern is local recurrence. The most significant factor contributing to local recurrence is tumor size, as demonstrated in several studies.10,17–19 Recurrence seems to be very low in tumors <3 cm. It also has been reported that percutaneous procedures are associated with more recurrences than other procedures, perhaps because of imprecise monitoring and inadequate thermal effects.10,19 Another factor may be tumor pathology. Hepatic metastases from colorectal carcinoma have a higher recurrence rate than those from other primary tumor sites.10,19 This observation implies that diverse characteristics among tumor types may exhibit variability in response to RFA and may result in disparate clinical outcomes.
To design a rational strategy to enhance the sensitivity of tumor cells to RFA and reduce the escape of tumor cells from RFA-induced cell death, we need to have a better understanding of the cellular response of tumor cells to RFA at the molecular level. We have demonstrated the use of nude mouse tumor xenografts for investigating the in vivo effects of RFA. The RFA procedure is well tolerated in nude mice, and all mice survived in this study. Furthermore, nude mice can be used for developing human tumor xenografts. Our laboratory is also in the process of growing heterotransplants in nude mice from resected human colorectal liver metastases to study their responses to RFA with clinical correlation in individual patients.
In the clinical setting, although the protocol attempts to achieve a target temperature of at least 50°C in all areas of the tumor, it is quite likely that temperature heterogeneity within different parts of the tumor (especially those 
3 cm) results in cells subjected to <50°C. Therefore, we assessed the recurrence rates of tumors subjected to 42°C to 50°C. We have shown that the efficacy of RFA on tumor treatment is a function of temperature. During RFA, we used a peripherally placed thermocouple as an end point for monitoring. This setup was to simulate the clinical condition wherein tumor cells located at the margin of a target area may not achieve a lethal temperature. The temperatures near the electrodes, monitored by the radiofrequency generator, were 8°C to 17°C higher than the peripheral temperature, depending on the tumor size and final temperature. The effective temperature for cell killing in the tumor xenograft was the same as that in in vitro culture cells: >50°C. At 42°C, only a delay in the tumor growth in xenografts was observed, and this was paralleled by the in vitro results, wherein 42°C did not affect the cell survival in culture cells.
At 45°C, some tumors recurred, whereas others were totally eliminated, indicating that this was a transition point of two opposing pathways, either for survival or for cell death. The balance between these two pathways will ultimately determine the fate of the cells. If we can further activate some molecules in favor of upregulating cell death pathways, such as apoptosis, then cancer cells subjected to a marginal temperature may die during RFA and reduce the chance of recurrence. The model that we have established will allow us to examine whether the addition of other adjuvant therapies can more effectively eradicate tumor cells and reduce tumor recurrence at sublethal temperatures in comparison to RFA alone.
To further understand the potential molecular mechanisms of tumor cells that survive RFA, we examined the expression of various HSPs at transcriptional and protein levels kinetically after RFA in tumor xenografts; this longitudinal kinetic study is not feasible in patients. HSPs are important in maintaining cellular viability under a variety of stress conditions. Among the examined HSPs (HSP27, HSP70, and HSP90), HSP70 is the major one induced after RFA. The increase of HSP70 expression has been correlated with the development of thermotolerance in Chinese hamster fibroblasts,20 and the inhibition of HSP70 expression can block the induction of thermotolerance in leukemia cells.21 HSP70 can function as a chaperon for inhibiting the aggregation of peptides and can assist in the refolding of denatured peptides; thus, the chaperoning ability of HSP70 may be responsible for protecting cells from thermal stress by neutralizing the toxic effects of denatured proteins.22
Moreover, HSP70 can inhibit apoptosis and thereby increase the survival of cells exposed to a wide range of lethal stimuli.23 The mechanism of this inhibition may be ascribed to neutralizing interaction with several proapoptotic effectors, including Apaf-1 and AIF, and perhaps signaling molecules such as JNK-1, p53, or c-myc.24 HSP70 has been shown to render cells resistant to several anticancer drugs, such as gemcitabine, topotecan, cisplatin, doxorubicin, and 5-fluorouracil.25–27 Studies have demonstrated increased expression of HSP70 to be linked to malignant phenotypes in breast cancer and renal cell tumors.28,29 Therefore, tumor cells that survive RFA with the induction of HSP70 expression may alter their biological activities and become more malignant, increasing resistance to anticancer drugs in follow-up treatment.
We have compared the HSP expression pattern of tumor xenografts in response to RFA with that in in vitro culture cells after heat treatment. HSP90 expression remained unchanged both in in vivo RFA treatment and in in vitro heated water bath treatments. HSP27 expression remained constant in RFA, whereas it slightly decreased at early time points and returned to basal levels at 10 hours in vitro. Both tumor xenografts and culture cells had a similar kinetic pattern of induction of HSP70 at 45°C. Taken together, these results indicate that the in vivo cellular response to RFA on HSP expression is similar to that contributed from heat effects in vitro.
In this study, we have demonstrated the feasibility of using a mouse model for the study of cellular responses of human colon tumors to RFA at the molecular level. We were able to control precisely the target treatment temperature at the periphery of the tumor by thermocouple monitoring. The induction of HSP70 after RFA and in vitro heat treatment may be a mechanism for protecting cells from RFA or heat-induced injury; therefore, HSP70 can be a target to enhance the efficacy of RFA and prevent increased drug resistance to chemotherapy. Elucidating the mechanisms of how tumor cells escape during RFA will definitely provide a basis for developing a rational approach in conjunction with RFA and benefit the treatment of patients with unresectable colorectal liver metastasis.
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ACKNOWLEDGMENTS
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Supported by the Albert Einstein Cancer Center and the Department of Surgery Research Fund. The authors thank Dinak G. Nair for technical assistance.
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FOOTNOTES
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W.-L.Y. and D.G.N. contributed equally to this work.
We have developed a human colon cancer zenograft model to study cellular responses to radiofrequency ablation. We demonstrate the rate of tumor recurrence as a function of treatment temperature and the implications of HSP70 expression in treated tumors.
Received for publication August 20, 2003.
Accepted for publication December 22, 2003.
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ABSTRACT
INTRODUCTION
; n = 7), and 50°C (
; n = 7), as described in Methods. After RFA, mice were examined twice a week to monitor the tumor recurrence. (B) Growth of each tumor after being subjected to RFA at 42°C. Tumor growth was recorded twice a week by measuring two diameters with a caliper. The RFA operation day was designated as day 0.