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DOI: 10.1148/radiol.2463070228
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(Radiology 2008;246:796-803.)
© RSNA, 2008

Experimental Studies

Combination of Sensitizing Pretreatment and Radiofrequency Tumor Ablation: Evaluation in Rat Model1

Brent D. Weinberg, PhD, Tianyi M. Krupka, BS, John R. Haaga, MD, and Agata A. Exner, PhD

1 From the Departments of Biomedical Engineering (B.D.W., T.M.K.) and Radiology (T.M.K., J.R.H., A.A.E.), Case Western Reserve University, 11100 Euclid Ave, Cleveland, OH 44106-5056. Received February 2, 2007; revision requested April 5; revision received May 9; accepted June 11; final version accepted July 31. A.A.E. supported by National Institutes of Health grants R21 EB002847 and R01 CA118399. B.D.W. supported in part by National Institutes of Health grant T32 GM07250 to the Case Western Reserve University Medical Scientist Training Program and by Department of Defense predoctoral fellowship BC043453. Address correspondence to A.A.E. (e-mail: agata.exner{at}case.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
Purpose: To prospectively determine, in an animal tumor model, if the block copolymer Pluronic P85 (BASF, Shreveport, La) sensitizes cancer cells to hyperthermia and if intratumorally or intravenously administered copolymer improves the therapeutic outcome of radiofrequency (RF) ablation tumor treatment.

Materials and Methods: The effects of Pluronic P85 and mild hyperthermia in vitro were tested in DHD/K12/TRb rat colorectal carcinoma cells. Cells were incubated at 37°C or 43°C for 15–60 minutes with 0%, 7%, or 10% wt/wt Pluronic P85, and cell viability was assessed by using a mitochondrial enzyme assay. In vivo experiments were performed as approved by the Institutional Animal Care and Use Committee at Case Western Reserve University and according to all applicable guidelines on animal use. Bilateral subcutaneous tumors in rats were treated with either intratumoral (13 tumors) or intravenous (15 tumors) Pluronic P85 followed by ablation or with ablation alone (14 tumors) and were monitored for 14 days by using volumes estimated from caliper measurements of tumor diameter. Acute effects of Pluronic P85 on the size of ablation-induced coagulation were measured after 24 hours in additional tumors (six tumors each treated according to the protocol for the ablation-only, intratumoral injection, and intravenous injection groups). Statistical testing was performed by using linear regression analysis and two-sided t tests with a significance level of .05.

Results: At 43°C, 7% and 10% Pluronic P85 reduced in vitro cell viability by 22% ± 5 (standard error of the mean) (P < .001) and 28% ± 5 (P < .001), respectively, compared with the viability of control cells. At day 14, the volume of tumors ablated after local and systemic Pluronic P85 pretreatment changed by –55% ± 14 (P = .03) and –59% ± 14 (P = .02), respectively, compared with an increase of 16% ± 28 for tumors treated with ablation alone. Coagulation area at 24 hours was reduced by 44% relative to that in control tumors (P = .03) after intratumoral Pluronic P85 but was unchanged after systemic Pluronic P85.

Conclusion: Tumor pretreatment with Pluronic P85 improved the outcome of RF ablation by decreasing the tumor volume and residual tumor in an experimental carcinoma model.

© RSNA, 2008


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
Radiofrequency (RF) ablation has become an increasingly powerful tool for treatment of primary or metastatic malignancy in a variety of sites, such as the liver (1), pancreas (2,3), and kidneys (4), in patients who are otherwise unsuitable candidates for surgical tumor resection. Despite the successes of RF ablation, some unresolved issues remain, such as peripheral cooling of tumor tissue near blood vessels and the restricted size of energy deposition. Both of these issues can result in a sublethal dose of heat delivered to the tumor and can lead to local tumor regrowth (5,6).

Studies of RF ablation combined with chemotherapeutic agents either administered intravenously or injected directly into the tumors have been performed in an attempt to improve treatment outcome and have indeed revealed increased tissue coagulation and tumor shrinkage compared with RF ablation alone (7,8). However, the cytotoxic drugs included in the combined treatment are most often nonspecific, and undesirable side effects may result from collateral damage to normal cells. An ideal agent for coadministration with RF ablation would facilitate the effects of tumor ablation with minimal damage to normal tissue. Pluronic block copolymers (also known as poloxamers) have been shown to have a variety of interactions with cancer cells, such as increasing their responsiveness to chemotherapeutic drugs (9) and general susceptibility to injury.

Thus, the purpose of our study was to prospectively determine, in an animal tumor model, if the block copolymer Pluronic P85 (BASF, Shreveport, La) sensitizes cancer cells to hyperthermia and if intratumorally or intravenously administered copolymer improves the therapeutic outcome of RF ablation tumor treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
All animal experiments were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University and followed all applicable guidelines on animal use.

Pluronic Solutions
Pluronic solutions were created by dissolving Pluronic P85 paste (donated to A.A.E. by BASF) in Roswell Park Memorial Institute (RPMI) medium 1640 with L-glutamine (GIBCO; Grand Island, NY) at concentrations of 1%, 7%, and 10% wt/wt. Solutions were filtered with a sterile 0.22-µm syringe filter (Millex TM-GP; Millipore, Billerica, Mass) and stored at 4°C. RPMI medium was used as a solvent in all studies to maintain consistency between cell and animal experiments.

In Vitro Cytotoxicity
Cytotoxicity of Pluronic P85 solutions was determined on a rat colorectal carcinoma cell line (DHD/K12/TRb) in vitro (T.M.K.). This cell line, which originated from a 1,2-dimethylhydrazine-induced colon adenocarcinoma in BDIX/CrCrlBR rats, is a model of metastatic colon carcinoma that can be propagated both in vitro and in vivo (10,11). Cells from this line were donated by W. G. Pitt, PhD, Brigham Young University, Provo, Utah (original source, European Collection of Cell Cultures). The cells were grown in RPMI medium containing 10% fetal bovine serum (Hyclone, Logan, Utah) and 1% penicillin/streptomycin (GIBCO) and were passaged weekly (12). For cytotoxicity studies, cells were detached with trypsin–edetic acid (GIBCO), resuspended in RPMI medium, and plated into 96-well plates at 105 cells per milliliter. After 24 hours, the medium was replaced with 0%, 7%, or 10% Pluronic P85 solution, and the cells were placed in a covered water bath in an incubator at 37°C ± 0.25 or 43°C ± 0.25 (C24, New Brunswick Scientific, Edison, NJ) for 15, 30, or 60 minutes (four plates per time and temperature combination). The cells were washed and incubated overnight before mitochondrial enzyme activity was measured with the QIA127 colorimetric assay (Oncogene, Cambridge, Mass). Absorbance data were collected with a plate reader (ELx808; Bio-Tek, Winooski, Vt), and the results were determined by averaging the absorbance of 16 individual wells. Mitochondrial enzyme viability was then compared between cells treated with Pluronic P85 and control cells at each temperature.

Cell proliferation ability was further assessed with a clonogenic survival study. Cells were plated into six-well plates at 105 cells per milliliter, and, after 24 hours, cells were again treated with 1% or 7% Pluronic P85 solution at 37°C ± 0.25 or 43°C ± 0.25 for 30 or 60 minutes (three plates per time and temperature combination). The treated cells were washed, diluted to 700 cells per milliliter, and replated. After 9 days, cells were fixed in methanol and stained with May-Grunwald and Giemsa stains (Sigma-Aldrich, St Louis, Mo); stained colonies with more than 50 cells were then counted manually. Resulting colony-forming ability was determined by using the mean number of colonies in three plates. Results were assessed by comparing cells treated with Pluronic P85 against control cells at each temperature.

Tumor Inoculation
Tumors for use in in vivo studies were inoculated subcutaneously (B.D.W. and T.M.K.) into a total of 41 BDIX/CrCrlBR rats, with nine of 41 rats used for coagulation necrosis measurement (described below). On the day of inoculation, cells were trypsinized and resuspended in RPMI medium at a final concentration of 2 x 106 cells per milliliter. BDIX/CrCrlBR rats were obtained from an in-house rat colony (original source, Charles River Laboratories, Wilmington, Mass). The rats were anesthetized with 1% isoflurane and an O2 flow rate of 1 L/min (EZ150 Isoflurane Vaporizer; EZ Anesthesia, Palmer, Pa). Tumors were inoculated bilaterally on the back, approximately 1 cm distal to the scapula and 1 cm lateral to the midline, by means of subcutaneous injection of 50 µL of the cell suspension.

Tumor Treatment
Four weeks after tumor inoculation, rats were anesthetized, the tumor site was shaved and cleaned, and tumor size was measured with calipers (B.D.W. and T.M.K.). Rats were then randomly assigned to three groups. If tumors had not developed by this time or were too small to be treated, they were excluded from further study prior to treatment. In the first group (the control group), both tumors were treated with RF ablation only (seven rats, 14 tumors). In the second group (local Pluronic P85), the right tumor was treated with a local injection of 100 µL of 7% Pluronic P85 solution (mean dose, 28.1 mg per kilogram of body weight) into the center of the tumor, followed by RF ablation 15 minutes later (13 rats, 13 tumors), while the left tumor was treated with RF ablation alone. The time after intratumoral injection was chosen to give the liquid time to be absorbed into the surrounding tumor tissue and minimize leakage during the ablation. Only one tumor was treated to limit the confounding effects of systemic exposure to Pluronic P85 in the contralateral tumor. The third group of rats (systemic Pluronic P85) was given an intravenous injection of 100 µL of 1% Pluronic P85 (mean dose, 3.3 mg/kg), and both tumors were ablated after 75 minutes (eight rats, 15 tumors), a time chosen on the basis of preliminary unpublished results of using a range of times after injection. In all groups, monopolar RF was applied by using a 480-kHz RF generator (3E; Radionics, Burlington, Mass), a 21-gauge non–internally cooled electrode, and an abdominal grounding pad (B.D.W. and T.M.K.). Tumors were ablated with a typical power of 2–5 W to achieve an electrode temperature of 80°C for 2 minutes.

Tumor Assessment and Histologic Analysis
Rat weight and tumor sizes measured with calipers were recorded weekly for 2 weeks (B.D.W. and T.M.K.). Tumor volume was then calculated in cubic millimeters according to the approximation V = 1/2ab2, where a is the long and b is the short axis of the tumor in millimeters (13). All tumor volumes were normalized with respect to the pretreatment volume, and then tumors treated with Pluronic P85 were compared with control tumors. After 2 weeks, rats were euthanized, and tumors were excised and fixed in 10% buffered formalin for at least 24 hours. Fixed tissues were dehydrated, embedded in paraffin, and sliced into 5-µm slices, which were subsequently stained with hematoxylin-eosin or Masson trichrome stains.

Histologic slides were digitized by using a video microscopy system consisting of a light microscope (BX60; Olympus, Tokyo, Japan), a video camera (DXC-390; Sony, Tokyo, Japan), a position-encoded motorized stage (ProScan; Prior Scientific, Rockland, Mass), and software (Image-Pro with Scope-Pro; Media Cybernetics, Silver Springs, Md). Images of the Masson trichrome–stained slides with objective magnification of x4 were obtained by using the tiling function of Scope-Pro (14). For analysis, two independent observers (B.D.W. and A.A.E.) manually segmented and measured the visible areas of coagulation necrosis and viable tumor cells by using software (Image J; National Institutes of Health, Bethesda, Ma), confirming the identification of each region on hematoxylin-eosin– and Masson trichrome–stained slides in high magnification. Measurements from the two independent segmentations were averaged and used to determine coagulated area, viable tumor area, total histologic area, and percentage of the total extracted region showing coagulation necrosis. These areas were compared between groups treated with local or systemic Pluronic P85 and control groups. Viable tumor regions were those with cells similar to untreated cells and featuring the following: intact cell membranes, low degree of differentiation, high nuclear fraction, high pleomorphism, and abundance of mitotic figures. Slides in which the entire extracted region appeared to be necrotic at high magnification were designated as having no viable tumor present.

Coagulation Area Measurement
To measure the acute effects of Pluronic P85 administration on the size of ablation-induced coagulation, additional tumors were inoculated in nine rats (six tumors per group) and were treated as described above for the ablation-only, local Pluronic P85, and systemic Pluronic P85 groups. Rats were euthanized 24 hours after treatment, and 2-mm tumor slices were cut perpendicular to the ablation needle track and soaked with 2% 2,3,5-triphenyltetrazolium chloride (TTC) (BD Biosciences, San Jose, Calif) for 30 minutes to stain tissues with intact mitochondrial activity (7). Areas of the coagulated regions were then measured (B.D.W.).

Statistical Analysis
All data are presented as means ± standard errors of the mean unless otherwise noted. Statistical tests were performed by using a software package (S-Plus, version 7.0, 2005; Insightful, Seattle, Wash). Cytotoxicity data were normalized with respect to control data, and analysis was then performed separately at each temperature by using linear regression analysis with treatment group and time as independent variables. The two-sided Tukey-Kramer comparison was used when comparing groups. Clone formation was assessed by comparing treated cells versus control cells at each time point and temperature by using a two-sided unpaired t test. For the in vivo data, two-sided unpaired t tests were used when comparing tumor volumes, areas of viability, percentage coagulation necrosis, and coagulation area. To compare the proportions of treated tumors in each group that had no detectable viable tumor cells, the Fisher exact test was used. The P value considered to indicate significance for all tests was .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
In Vitro Effects of Pluronic P85
Cell viability as a function of time and Pluronic P85 exposure is shown for two temperatures in Figure 1. At 37°C, viability of cells exposed to 7% Pluronic P85 was comparable to that of cells without Pluronic P85 exposure, but cells with 10% Pluronic P85 showed a significant loss of viability (23% ± 5, P < .001). At 43°C, all test groups experienced significant toxicity because of the heat exposure. Cells treated with 7% and 10% Pluronic P85 were 22% ± 5 (P < .001) and 28% ± 5 (P < .001) less viable than control cells, respectively. Importantly, while 7% Pluronic P85 was not inherently toxic at 37°C, it substantially increased the toxicity of mild hyperthermia, even for exposures as short as 15 minutes.


Figure 1A
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  		Figure 1a: Viability of DHD/K12/TRb carcinoma cells treated with three Pluronic P85 concentrations at (a) 37°C and (b) 43°C. Each measurement is the mean of data in 16 wells ± standard error of the mean. * = Viability of treated cells significantly different from that of control cells.

 

Figure 1B
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  		Figure 1b: Viability of DHD/K12/TRb carcinoma cells treated with three Pluronic P85 concentrations at (a) 37°C and (b) 43°C. Each measurement is the mean of data in 16 wells ± standard error of the mean. * = Viability of treated cells significantly different from that of control cells.

 
Results of clonogenic measurement of cell proliferative activity confirmed the effects of Pluronic P85 in sensitizing the cells to heat exposure (Fig 2). After 60 minutes of exposure at 37°C, cells treated with 1% and 7% Pluronic P85 formed 33% ± 10 (P = .04) and 46% ± 10 (P = .02) fewer colonies than control cells, respectively, showing some loss of proliferative activity even without exposure to heat. However, cells treated with 1% and 7% Pluronic P85 at 43°C lost even greater fractions of their replicative ability: 92% ± 17 (P = .01) and 100% ± 17 (P = .008), respectively. The combination of Pluronic P85 and mild heat exposure inhibited colony formation substantially more than did either component alone.


Figure 2
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  		Figure 2: Bar graph shows proliferation ability of DHD/K12/TRb carcinoma cells treated with 1% or 7% Pluronic P85 versus that of control cells at 37°C and 43°C. Each measurement is the mean of data in three plates ± standard error of the mean. * = Treated cells significantly different from control cells at that temperature exposure.

 
Tumor Growth Suppression
Tumors had a pretreatment diameter of 8.9 mm ± 0.5, with no significant difference in size between groups. Seven days after treatment, tumor volume increased by 159% ± 17 in the control group (Fig 3). However, the volume of tumors treated with Pluronic P85 before ablation remained unchanged relative to the original tumor volume (local Pluronic P85, 3% ± 12; systemic Pluronic P85, 1% ± 23). By day 14, the volume of the ablation-only tumors was near the original tumor volume (16% ± 28), but in both Pluronic P85 pretreatment groups the tumor volume had decreased by more than half (local Pluronic P85, –55% ± 14; systemic Pluronic P85, –59% ± 14). At day 14, the differences between the control group and both Pluronic P85 pretreatment groups were significant (local Pluronic P85, P = .03; systemic Pluronic P85, P = .02).


Figure 3
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  		Figure 3: Graph shows progression of tumor volume for in vivo treatment groups over 14 days. Volume change is shown as a percentage of pretreatment tumor volume. Error bars = standard error of the mean, ** = volumes of tumors treated with local Pluronic P85 and tumors treated with systemic Pluronic P85 significantly different from volumes of tumors treated with ablation alone.

 
Histologic Analysis
Quantitative histologic analysis of explanted tumor tissue was used to assess the pathologic outcome of the tumors, and results of this analysis are summarized in the Table. Histologic findings from the three groups could be categorized into two types: slices containing no viable tumor and slices containing residual viable tumor nodes. The number of slices with no viable tumor present was similar in the ablation-only (four [29%] of 14) and local Pluronic P85 (three [23%] of 13, P = .65) groups and was higher but not significantly different in the systemic Pluronic P85 group (seven [47%] of 15, P = .26). Histologic slices containing viable tumor exhibited a similar pattern throughout the three groups, and a representative slice is shown in Figure 4. These slices were characterized by two main differentiable regions: coagulation necrosis and viable untreated tumor. In contrast, slices containing no residual viable tumor cells consisted of a single large ablated region, as shown in Figure 5.


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  		Summary of Results of Quantitative Histologic Analysis

 

Figure 4A
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  		Figure 4a: Representative Masson trichrome–stained slice from control group with residual tumor cells 14 days after RF ablation. (a) Overview slide shows treated region abutting large untreated tumor nodule. The locations of high-magnification images b and c are noted with black rectangles. (Original magnification, x40; scale bar = 2 mm.) (b) High-magnification image shows region of granulation tissue resolving into a fibrous scar, complete with collagen deposition and revacularization. The main cellular components are fibroblasts and macrophages. (Original magnification, x250; scale bar = 100 µm.) (c) High-magnification image of 4.6-mm-diameter tumor nodule shows viable poorly differentiated and pleomorphic tumor cells with large, irregular nuclei and darkly staining cytoplasm. The tumor cells were irregularly organized and interspersed within a collagenous matrix. (Original magnification, x250; scale bar = 100 µm.)

 

Figure 4B
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  		Figure 4b: Representative Masson trichrome–stained slice from control group with residual tumor cells 14 days after RF ablation. (a) Overview slide shows treated region abutting large untreated tumor nodule. The locations of high-magnification images b and c are noted with black rectangles. (Original magnification, x40; scale bar = 2 mm.) (b) High-magnification image shows region of granulation tissue resolving into a fibrous scar, complete with collagen deposition and revacularization. The main cellular components are fibroblasts and macrophages. (Original magnification, x250; scale bar = 100 µm.) (c) High-magnification image of 4.6-mm-diameter tumor nodule shows viable poorly differentiated and pleomorphic tumor cells with large, irregular nuclei and darkly staining cytoplasm. The tumor cells were irregularly organized and interspersed within a collagenous matrix. (Original magnification, x250; scale bar = 100 µm.)

 

Figure 4C
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  		Figure 4c: Representative Masson trichrome–stained slice from control group with residual tumor cells 14 days after RF ablation. (a) Overview slide shows treated region abutting large untreated tumor nodule. The locations of high-magnification images b and c are noted with black rectangles. (Original magnification, x40; scale bar = 2 mm.) (b) High-magnification image shows region of granulation tissue resolving into a fibrous scar, complete with collagen deposition and revacularization. The main cellular components are fibroblasts and macrophages. (Original magnification, x250; scale bar = 100 µm.) (c) High-magnification image of 4.6-mm-diameter tumor nodule shows viable poorly differentiated and pleomorphic tumor cells with large, irregular nuclei and darkly staining cytoplasm. The tumor cells were irregularly organized and interspersed within a collagenous matrix. (Original magnification, x250; scale bar = 100 µm.)

 

Figure 5A
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  		Figure 5a: Representative Masson trichrome–stained slice from systemic Pluronic P85 group with no residual viable tumor cells 14 days after systemic Pluronic P85 and RF ablation. (a) Overview slide shows circular coagulated region with central inflammatory core and injury resolution around the periphery. The locations of high-magnification images b and c are noted with black rectangles. (Original magnification, x40; scale bar = 2 mm.) (b) Collagenous granulation tissue, featuring prominent fibroblasts and endothelial cells, encircles the treated area. (Original magnification, x250; scale bar = 100 µm.) (c) Central coagulated region features cellular debris and many chronic inflammatory cells. Coagulation necrosis is less resolved and exhibits cellular debris and inflammatory infiltrate. (Original magnification, x250; scale bar = 100 µm.)

 

Figure 5B
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  		Figure 5b: Representative Masson trichrome–stained slice from systemic Pluronic P85 group with no residual viable tumor cells 14 days after systemic Pluronic P85 and RF ablation. (a) Overview slide shows circular coagulated region with central inflammatory core and injury resolution around the periphery. The locations of high-magnification images b and c are noted with black rectangles. (Original magnification, x40; scale bar = 2 mm.) (b) Collagenous granulation tissue, featuring prominent fibroblasts and endothelial cells, encircles the treated area. (Original magnification, x250; scale bar = 100 µm.) (c) Central coagulated region features cellular debris and many chronic inflammatory cells. Coagulation necrosis is less resolved and exhibits cellular debris and inflammatory infiltrate. (Original magnification, x250; scale bar = 100 µm.)

 

Figure 5C
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  		Figure 5c: Representative Masson trichrome–stained slice from systemic Pluronic P85 group with no residual viable tumor cells 14 days after systemic Pluronic P85 and RF ablation. (a) Overview slide shows circular coagulated region with central inflammatory core and injury resolution around the periphery. The locations of high-magnification images b and c are noted with black rectangles. (Original magnification, x40; scale bar = 2 mm.) (b) Collagenous granulation tissue, featuring prominent fibroblasts and endothelial cells, encircles the treated area. (Original magnification, x250; scale bar = 100 µm.) (c) Central coagulated region features cellular debris and many chronic inflammatory cells. Coagulation necrosis is less resolved and exhibits cellular debris and inflammatory infiltrate. (Original magnification, x250; scale bar = 100 µm.)

 
The size of the coagulated area at histologic examination on day 14 was largest in the control group, but the differences with local Pluronic P85 (P = .06) and systemic Pluronic P85 (P = .28) were not significant. Mean viable tumor area was highest in the ablation-only group and was lower in both Pluronic P85 groups, although this difference was significant only for the ablation-only versus the systemic Pluronic P85 group (P = .02). Additionally, the percentage of resected tissue scored as coagulated in the systemic Pluronic P85 group was significantly higher than that in the intratumoral Pluronic P85 group (P = .02) but was not significantly different from that in the control group (P = .12).

Effects on Coagulation Area
Areas of coagulation necrosis determined from TTC-stained slices 24 hours after treatment are shown in Figure 6. The coagulated areas in the ablation-only (0.30 cm2 ± 0.03) and systemic Pluronic P85 (0.25 cm2 ± 0.05) tumors were similar. However, coagulated area in the local Pluronic P85 group (0.17 cm2 ± 0.05) was reduced by 44% relative to that in the control group (P = .03).


Figure 6
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  		Figure 6: Bar graph shows areas of coagulation necrosis measured on TTC-stained tissue slices 24 hours after ablation. Each point is the mean of six measurements ± standard error of the mean. * = Results with a treatment were significantly different from those with ablation only.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
Recent technologic developments, such as internally cooled or multitined expandable electrodes (15), the use of imaging guidance and treatment assessment (16), and saline coadministration (17), have further increased the scope of tumors that can be treated with RF ablation. Nonetheless, peripheral cooling of tumor tissue near major blood vessels, the restricted size of energy deposition, and seeding of residual tumor around the needle electrode track have all been shown to be causes of incomplete treatments leading to local tumor regrowth, particularly in larger tumors (5,6).

Several investigators have reported the use of pharmacologic agents as adjuvants to RF ablation in an attempt to increase treatment volumes and improve clinical outcomes. Goldberg et al (7,8) have described the benefits of coadministration of liposomal doxorubicin through intratumoral or intravenous injection with ablation. Both methods resulted in increased tumor necrosis, but intravenous liposomes appeared more promising because they resulted in increased necrosis at the tumor periphery, where tumors are more likely to recur. In the only trial in patients to our knowledge, in 2002 (18), 10 patients given intravenous liposomal doxorubicin before ablation showed increased necrosis in perivascular and peripheral tumor sites 2–4 weeks after treatment. Another experimental study by Haaga et al (19) revealed that 5-fluorouracil–impregnated polyanhydride implants along with RF ablation led to improved tumor treatment response compared with RF alone in a rabbit liver cancer model. These results underscore the potential to maximize the effects of RF ablation by supplementing it with systemically or locally administered pharmacologic agents.

Pluronics are nonionic, triblock copolymers composed of blocks of ethylene oxide (EO) and propylene oxide (PO) with a generic structure of EOa-POb-EOa, where a and b are the number of repeats of each unit. These polymers are not inherently toxic at active concentrations and have been used to solubilize and deliver hydrophobic drugs within micelles (20,21). Despite their lack of inherent toxicity, Pluronics have been shown to sensitize cancer cells to chemotherapy (22) on the basis of several mechanisms, including reducing activity of cell detoxification systems (such as p-glycoprotein and glutathione sulfhydryl), depleting intracellular adenosine triphosphate concentration, reducing expression of antiapoptotic genes, and changing cell membrane fluidity (9,23). These effects occur at relatively low Pluronic concentrations below the critical micellar concentration, which is the concentration of polymer above which individual polymer molecules associate into micelles, or spherical supermolecular aggregates. For this reason, the cellular effects are believed to be due to unimers rather than micellar aggregates. Pluronic P85, with an EO repeat length of 26 and a PO repeat length of 40, is particularly suitable for inducing these effects because of its balance between relatively high hydrophobicity and intermediate critical micellar concentration (6.5 x 10–5 mol/L), which allows for a high concentration of unimers (24). Most recently, Pluronic P85 was found to increase the cytotoxicity of carboplatin in the cell line used in our study (12). The negligible systemic toxicity of Pluronic P85, combined with its wide range of biologic activity, make this surfactant an ideal thermosensitizer for use with RF ablation.

Our study examined the use of Pluronic P85 as a thermosensitizer prior to RF ablation treatment of experimental tumors. Although previous work has successfully supplemented RF ablation with various cytotoxic agents (7,8,18,19,25), to the best of our knowledge, our study is the first attempt to establish an ablation protocol that utilizes a nontoxic agent to increase cellular susceptibility to heat-related injury while minimizing systemic drug side effects. Experiments were performed to demonstrate the feasibility of this approach both in vitro in a cell culture environment and in vivo in an animal model. In vitro, cells were exposed to mild hyperthermia ranging from 15 to 60 minutes at 43°C to simulate heat doses that might be experienced by tumor cells at the outer periphery of the ablated region (26). This heat exposure combined with Pluronic P85 was shown to decrease both the mitochondrial enzyme activity (and thus viability) and proliferative ability of a rat colorectal carcinoma cell line. This finding is important because it represents a first step in developing compounds that can increase the toxicity of heat without resulting in substantial toxicity to normal cells.

In vivo, tumors treated with either intratumoral or systemic Pluronic P85 before ablation had reduced volumes compared with control tumors on days 7 and 14. The large increase in control tumor volume on day 7 may have resulted from inflammation and edema associated with the resolving ablated tissue. This inflammation and edema appeared to be reduced in tumors treated with Pluronic P85 and ablation. It is possible that Pluronic P85 has an effect on the extent of postablation inflammation, which would explain why tumors in groups treated with Pluronic P85 were smaller on day 7. By day 14, both Pluronic P85 treatment groups showed a substantially reduced tumor volume compared with the group treated with ablation alone, indicating that it may be possible to extend the thermosensitization effects of Pluronic P85 to tumor treatment strategies. One notable difference between the two pretreatment strategies was that tumors treated with intratumoral Pluronic P85 had a smaller percentage of necrosis on histologic slices than tumors treated with systemic Pluronic P85. Furthermore, the local Pluronic P85 group had the smallest area of coagulation necrosis at histologic examination. This finding is supported by the TTC assay, with which smaller areas of necrosis were seen in the local Pluronic P85 group 24 hours after ablation.

The explanation for the different coagulation sizes in this study is not clear. It is possible that the intratumoral injection of polymer interferes with tissue heating by either insulating the surrounding tumor or providing an alternate path for RF-induced current. In contrast, the size of the TTC-demarcated coagulated region after 24 hours was similar for the systemic Pluronic P85 and ablation-only groups. Thus, the difference in tumor volume between the ablation-only and systemic Pluronic P85 groups after 14 days is not likely to be explained by short-term changes in the coagulated region. These results suggest that Pluronic P85 does not immediately increase the size of the region destroyed by ablation but instead contributes to slow cellular processes that take place over a period of several days. Because the effects of Pluronic P85 on the inflammatory response to ablation are unknown, the best measurable outcome at histologic examination is the gross amount of viable tumor on day 14, which was least in the systemic Pluronic P85 group.

To our knowledge, our work is the first to demonstrate the thermosensitizing effects of Pluronic P85 both in vitro and in vivo. Although the exact mechanism is unknown, several previously established effects could be relevant. First, adenosine triphosphate depletion could decrease the ability of heat-damaged cells to recover from sublethal injury, like that occurring at the outer boundary of the ablated region. Second, altered gene expression, such as a decrease in antiapoptotic or heat shock proteins, could increase the number of affected cells undergoing apoptosis in the days following the treatment. Third, the copolymer may disrupt the cell membranes, leaving them more vulnerable to heat damage.

Our study had limitations. First, the results were based on effects after 2 weeks, but earlier histologic time points, such as 4 or 8 days, could provide more information on the exact mechanism of these effects. Second, these effects were established in only a single cell line and tumor model and may not be universally applicable. Future study in additional cell and tumor types could supplement these results. Third, no temperature mapping was performed in this study, which limits the conclusions that can be drawn about effects in specific temperature conditions. Experiments that correlate temperature exposure with cell death could provide much more detailed information about how and where Pluronic P85 exerts its effects.

Practical applications: Pluronic P85 increased the cytotoxic effects of hyperthermia on cancer cells, both in vitro when cells were exposed to mildly elevated temperatures (43°C) and in vivo in the context of RF ablation of experimental tumors. Our study results establish the potential for using a thermosensitizer, particularly Pluronic P85, to improve the therapeutic outcome of tumor RF ablation. Such knowledge can lead to more in-depth exploration of the effects and mechanisms of using a thermosensitizer and, ultimately, to improvement of the best available clinical care for minimally invasive tumor treatment.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 


   IMPLICATION FOR PATIENT CARE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
References
 


   FOOTNOTES
 

Abbreviations: RF = radiofrequency • RPMI = Roswell Park Memorial Institute • TTC = triphenyltetrazolium chloride

Author contributions: Guarantors of integrity of entire study, B.D.W., A.A.E.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, B.D.W., T.M.K., A.A.E.; experimental studies, B.D.W., T.M.K., A.A.E.; statistical analysis, B.D.W., T.M.K., A.A.E.; and manuscript editing, all authors

Authors stated no financial relationship to disclose.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 

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T. M. Krupka, D. Dremann, and A. A. Exner
Time and Dose Dependence of Pluronic Bioactivity in Hyperthermia-Induced Tumor Cell Death
Experimental Biology and Medicine, January 1, 2009; 234(1): 95 - 104.
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