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Published online before print July 26, 2002, 10.1148/radiol.2243011421
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(Radiology 2002;224:823-829.)
© RSNA, 2002

Experimental Studies

Radio-frequency Ablation Increases Intratumoral Liposomal Doxorubicin Accumulation in a Rat Breast Tumor Model1

Wayne L. Monsky, MD, PhD, Jonathan B. Kruskal, MD, PhD, Anatoly N. Lukyanov, PhD, Geoffrey D. Girnun, PhD, Muneeb Ahmed, MD, G. Scott Gazelle, MD, PhD, Juan Carlos Huertas, MD, Keith E. Stuart, MD, Vladimir P. Torchilin, PhD, DSc and S. Nahum Goldberg, MD

1 From the Depts of Radiology (W.L.M., J.B.K., M.A., J.C.H., S.N.G.) and Medical Oncology (K.E.S.), Beth Israel Deaconess Med Ctr, Harvard Med School, 330 Brookline Ave, Boston, MA 02215; Dept of Pharmaceutical Sciences, Bouve College of Health Sciences, Northeastern Univ, Boston, Mass (A.N.L., V.P.T.); Dept of Cancer Biology, Dana-Farber Cancer Inst, Boston, Mass (G.D.G.); and Dept of Radiology, Massachusetts Gen Hosp, Boston, Mass (G.S.G.). Received Aug 23, 2001; revision requested Oct 11; final revision received Feb 27, 2002; accepted Mar 28. Supported in part by grants from Radionics, Burlington, Mass. Address correspondence to S.N.G. (e-mail: sgoldber@caregroup.harvard.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To determine whether intratumoral accumulation of liposomal doxorubicin or free unencapsulated doxorubicin is increased when combined with radio-frequency (RF) ablation.

MATERIALS AND METHODS: Two 1.2–1.5-cm R3230 mammary adenocarcinomas were grown within the mammary fat pads of 19 female Fischer rats. One tumor of each pair was treated with RF ablation (tip temperature, 70°C ± 2 [SD]; 120 mA ± 75) for 5 minutes, whereas the other tumor was a control. Intravenous liposomal doxorubicin (1 mg in 500 µL, n = 6) or intravenous free unencapsulated doxorubicin (n = 7) was administered immediately following RF ablation. Doxorubicin was extracted in acid alcohol from tumors 24 hours following RF ablation, and fluorescent spectrophotometry was used to quantify extracted doxorubicin. Comparisons of intratumoral doxorubicin accumulation in tumors treated with RF ablation and in untreated tumors were analyzed with parametric (paired Student t test) and nonparametric (Wilcoxon rank sum test) statistics. Findings at autoradiography with densitometry (six additional tumors) demonstrated the spatial distribution of the intratumoral accumulation of liposomal doxorubicin.

RESULTS: When RF ablation preceded administration of liposomal doxorubicin, mean intratumoral doxorubicin concentration was 5.6 µg/g ± 2.1 (range, 1.9–7.7 µg/g), whereas 1.0 µg/g ± 0.4 (range, 0.5–1.5 µg/g) was present in control tumors not treated with RF ablation (P < .05). Thus, there was a mean 7.1-fold ± 4.9 increase in intratumoral doxorubicin accumulation following RF ablation (range, 2.1–14.5-fold) compared with the amount without RF pretreatment (P < .05). Increased intratumoral accumulation was not seen in animals receiving free doxorubicin with (mean, 0.4 µg/g ± 0.1) or without (mean, 0.8 µg/g ± 0.4) RF pretreatment (P = .07). Autoradiographic findings demonstrated accumulation of liposomal doxorubicin in a peripheral rim of tumor adjacent to the zone of coagulation.

CONCLUSION: RF ablation augments the delivery of systemic antineoplastic agents such as liposomal doxorubicin.

© RSNA, 2002

Index terms: Animals • Chemotherapy • Hyperthermia • Liposomes • Neoplasms • Radiofrequency (RF) ablation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Radio-frequency (RF) ablation has been successfully used for an ever-increasing number of malignancies (1,2). In the majority of cases, RF ablation was performed in small, focal hepatocellular carcinomas and hepatic metastases (3–8). However, to date, the fundamental challenge that limits the wide-scale adoption of RF ablation for the treatment of larger liver tumors and other malignancies has been the inability to reliably create adequate large volumes of complete tumor destruction (9,10). Additionally, the local tumor recurrence observed in some cases suggests that residual microscopic foci of viable tumor can persist following thermal ablation (3–8,11).

It has been shown that there is nonuniform heating surrounding the RF electrodes, with a rapid exponential decrease in tissue temperatures as distance increases from the electrode (12). Increasing the amount of thermal energy deposited during ablation by either increasing the generator output or modifying the electrode has been successful in some cases. Coagulation diameters of 3.5–5.0 cm from a single RF application (13–17) have been achieved. However, the application of high-current energy has not been without increased patient risk from complications such as grounding pad burns (18). Thus, strategies to further optimize the volume of induced tumor coagulation are still required.

We have demonstrated a significant dose-dependent increase in tumor coagulation when we combined RF ablation with direct intratumoral injection of doxorubicin in small (<1.5-cm) rat breast tumors (19). However, direct injection strategies have been difficult to implement in larger tumors, since high interstitial pressures likely result in poor diffusion of chemical agents throughout the tumor (20). Thus, we have recently demonstrated synergy between RF ablation and an intravenously administered long circulating liposomal preparation of doxorubicin (Doxil; Alza Pharmaceuticals, Palo Alto, Calif) (21). Preliminary results confirm increased destruction of tumors treated with RF ablation and liposomal doxorubicin compared with that of tumors treated with RF ablation alone; an increase in coagulation diameter from 6.7 to 13.5 mm was achieved 48 hours following combined treatment (21). These results suggest a role for this type of adjuvant therapy with a combination of systemic chemotherapy and RF ablation. However, the mechanisms for this increased tumor destruction are incompletely understood.

Hyperthermia, and hence thermal ablation, has been proposed to increase vascular permeability and result in improved intratumoral delivery of cytotoxic agents (22). Thermal damage to tissues that is induced by using hyperthermia has been demonstrated to double maximum blood flow and, at higher temperatures, to cause vascular stasis (23). We hypothesized that RF tissue heating increases intratumoral drug delivery and, thus, augments intratumoral accumulation of liposomal doxorubicin and results in synergistic treatment effectiveness. The purpose of this study was to determine whether there was increased intratumoral accumulation of liposomal doxorubicin or free unencapsulated doxorubicin when either was administered following RF ablation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Model
Approval of the institutional animal care and use committee was obtained prior to the initiation of these studies. All experiments and procedures were performed in fully anesthetized animals. Anesthesia was induced by using an intraperitoneal injection of a mixture of 50 mg per kilogram of body weight of ketamine hydrochloride (Ketaject; Phoenix Pharmaceutical, St Joseph, Mo) and 5 mg/kg of xylazine hydrochloride (Bayer, Shawnee Mission, Kan). Booster anesthesia injections at 1/10 the dose were administered every 30–60 minutes as needed.

Experiments were performed (W.L.M., A.N.L., M.A., J.C.H.) by using a well-characterized established R3230 mammary adenocarcinoma cell line obtained from a laboratory (Ralph Weissleder, MD, PhD, Center for Molecular Imaging Research, Massachusetts General Hospital, Boston) (19,21,24,25). Fresh tumor (approximately 1 cm in diameter) was initially harvested from a live carrier. Within 1 hour of this tumor explantation, the tumor was homogenized with a tissue grinder (model 23; Kontes Glass, Vineland, NJ) by using an aseptic technique and was suspended in 7 mL of medium (RPMI 1640; INC Biomedicals, Aurora, Ill). In prior control experiments, we documented that this produces a concentration of approximately 1 x 107 tumor cells per 0.1 mL, with greater than 95% cellular viability. With direct visualization, an 18-gauge needle was used to slowly inject 0.2–0.3 mL of the tumor suspension into the mammary fat pad of 22 female Fischer 344 rats, the strain of animals from which this tumor was initially derived. All animals were acquired from a single vendor (Taconic, Germantown, NY) at 8–9 weeks of age (mean weight, 150 g ± 10).

One tumor was implanted within the mammary fat pad on each side of the abdomen in each animal, and a total of 44 tumors were implanted. Tumors were grown for 10–20 days until the desired size was achieved. Animals were monitored every 3–4 days to measure tumor growth. Solid 12–15-mm-diameter nonnecrotic (as determined by using ultrasonography) tumors were used for ablation studies.

Quantitation of Intratumoral Doxorubicin
A total of 19 rats with two tumors each (n = 38) were used for this initial experiment. One tumor of each pair was treated with RF ablation, whereas the other tumor was used as an internal control that was not treated with RF ablation. The tumor to be treated with RF ablation was randomly selected to minimize potential selection bias. RF ablation was administered at 500 KHz by using a monopolar 1-cm-tip electrode (Radionics, Burlington, Mass), according to a standardized protocol (mean tip temperature, 70°C ± 2 [SD]; 120 mA ± 75) for 5 minutes. Findings in previous studies have shown that with these parameters, coagulation of a 7-mm-diameter area, or approximately half the tumor diameter in this model, can be achieved (19,25). Thirteen of 19 rats received additional intravenous chemotherapy through direct femoral vein injection administered 20–30 minutes following RF application.

The remaining six rats did not receive chemotherapy because their 12 tumors were used as controls, and these tumors were treated with RF ablation alone (n = 6) or were not treated (n = 6). In six of the 13 rats receiving chemotherapy, 1 mg in a concentration of 500 µL (approximately 8 mg/kg of body weight) of intravenous methoxypolyethylene glycol–stabilized long circulating liposomal doxorubicin was administered. This dose was selected because findings in previous studies have shown that it increases coagulation to 13.5 mm at 48 hours following RF ablation with use of the previously mentioned parameters (19). In the other seven rats, an equivalent dose (1 mg in a concentration of 500 µL) of free unencapsulated doxorubicin (doxorubicin hydrochloride injection, Adriamycin PFS; Pharmacia & Upjohn, Kalamazoo, Mich) was injected.

Twenty-four hours following treatment, the 19 animals were sacrificed with an overdose (0.2 mL/kg) of pentobarbital sodium (Nembutal; Abbott Laboratories, North Chicago, Ill). This time point was selected because findings in prior studies demonstrated that maximum intrahepatic liposomal doxorubicin uptake occurred 24 hours after administration (26). The fluorescent properties of doxorubicin were used to quantify the amount of intratumoral doxorubicin. Tumors were harvested, weighed, and homogenized in acid alcohol (0.3-N hydrochloric acid, 50% alcohol), and extraction was continued for 24 hours at 5°C. As a control, the left lobe of the liver was also harvested from each rat, and similar doxorubicin extraction was performed. Doxorubicin extracted from the tumor or liver homogenate supernatant was quantitated by using fluorometry, with an excitation wavelength of 470 nm, and by measuring the intensity of emission at 590 nm (26).

Autoradiography
Autoradiography was performed in the three remaining rats to demonstrate the spatial distribution of the intratumoral accumulation of liposomal doxorubicin. A total of six tumors (two per animal) were used for this experiment, with one tumor in each animal being treated with RF ablation. In place of liposomal doxorubicin, 500 µL of tritiated 100-nm liposomes of equivalent size, molecular composition, and dose to 1 mg of liposomal doxorubicin, as discussed later, was administered via the tail vein 20–30 minutes following RF ablation. The six tumors were surgically removed from the rats 24 hours after therapy and were frozen at -80°C. Thirty-micrometer-thick tissue slices were prepared by using a microtome (Hacker Instruments, Fairfield, NJ) at -20°C. The slices were allowed to dry overnight at room temperature and were exposed to a phosphor imaging screen (Molecular Dynamics; Amersham Biosciences, Piscataway, NJ) for 7 days. Images of the specimens were quantified for both average and maximum densitometric intensity by using densitometry software (ImageQuant; Molecular Dynamics). Additional tissue sections were fixed in 10% formalin, and subsequently, they were stained with the hematoxylin-eosin stain for microscopic analysis and histopathologic correlation.

Preparation of Liposomes
All lipids were purchased from Avanti Polar Lipids, Alabaster, Ala. Liposomes exactly repeating the lipid composition of liposomal doxorubicin were prepared by extrusion. A lipid mixture of 6.38 mg of N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 19.16 mg of fully hydrogenated soy phosphatidylcholine, and 6.38 mg of cholesterol was prepared by using solutions of individual lipids in organic solvents. To label liposomes, 7.4 x 105 Bq (20 µCi) of cholesteryl(1-14C)-oleate (Nycomed Amersham, Piscataway, NJ) in a concentration of 2.1 x 109 Bq (56 mCi/mmol) was added to the lipid mixture. Organic solvents were removed in a vacuum. The lipid film obtained was dispersed in 2 mL of 10 mM (10 mmol/L) of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, or HEPES, and 140 mM (140 mmol/L) of sodium chloride at pH 7.4 (HEPES buffered saline; Sigma, St Louis, Mo). Liposomes were formed by extrusion of the crude lipid dispersion through 0.4-, 0.2-, and 0.1-µm polycarbonate filters at 50°C. The size of liposomes obtained was measured with dynamic light scattering by using a particle sizer (Coulter N4 Plus Submicron; Beckman Coulter, Miami, Fla). The majority of liposomes were 90–120 nm.

Statistical Analysis
All data are provided as mean ± SD. Comparisons of intratumoral doxorubicin accumulation in the tumors treated with RF ablation and in the nontreated tumors were analyzed with parametric (paired Student t test) and nonparametric (Wilcoxon rank sum test) statistics, as appropriate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
RF ablation combined with the intravenous administration of liposomal doxorubicin significantly increased the intratumoral accumulation of this chemotherapeutic agent (Table, Fig 1). All seven tumors treated with RF ablation in addition to liposomal doxorubicin had a higher intratumoral doxorubicin accumulation than any of the seven tumors treated with liposomal doxorubicin alone (P < .05, rank-sum nonparametric statistic). Specifically, a mean tissue concentration of 1.0 µg/g ± 0.4 (range, 0.5–1.5 µg/g) of doxorubicin was present in the pair-matched control tumors that were not treated with RF ablation, whereas a mean tissue concentration of 5.6 µg/g ± 2.1 (range, 1.9–7.7 µg/g) of doxorubicin was present in tumors treated with RF ablation prior to administration of liposomal doxorubicin. Thus, there was a mean 7.1 ± 4.9-fold increase in intratumoral doxorubicin accumulation following RF ablation (range, 2.1–14.5-fold increase) compared with that with administration of liposomal doxorubicin without pretreatment with RF ablation (Table, Fig 1).


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  		Tumoral and Hepatic Doxorubicin Accumulation Following RF Ablation and/or Doxorubicin Chemotherapy

 


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  		Figure 1. Graph shows doxorubicin accumulation according to treatment. Intratumoral doxorubicin accumulation is increased sevenfold in tumors treated with RF ablation immediately preceding intravenous administration of liposomal doxorubicin (Lipo Doxo) (1 mg doxorubicin) relative to tumors treated with RF ablation in the same animal in which liposomal doxorubicin was not previously administered. The absolute values of the two reported ratios are drawn to the numeric scale.

 
By comparison, tumors from animals treated with unencapsulated free doxorubicin had a mean doxorubicin accumulation of 0.4 µg/g ± 0.1 when they were treated with RF ablation and a mean doxorubicin accumulation of 0.8 µg/g ± 0.4 when they were not treated with RF ablation (P = .07 between these two groups, Fisher exact test). Although there was no significant difference in the amount of doxorubicin uptake by comparing liposomal doxorubicin and free doxorubicin administered in tumors that were not treated with RF ablation (P = .39), the difference in the amount of intratumoral uptake when comparing liposomal doxorubicin and free doxorubicin in tumors that were treated with RF ablation was significant (P < .001). The supernatant extracted from tumors that were not treated with doxorubicin had fluorescence equivalent to less than 0.1 µg/g, or essentially no activity.

Normal liver uptake of doxorubicin was 3.9 times higher when doxorubicin was administered as a liposomal preparation (mean, 5.8 µg/g ± 0.7) compared with that when free doxorubicin (mean, 1.5 µg/g ± 0.5) (P < .001) was administered (Table). Background intrinsic tissue fluorescence was equivalent to fluorescence generated by 0.08 µg/g of doxorubicin.

Autoradiographic findings demonstrated minimal heterogeneous accumulation of tritiated liposomes in control tumors that were not treated with RF ablation (Fig 2). However, in tumors treated with a combination of RF ablation and tritiated liposomes, there was a substantial increase in signal predominantly localized in a 1–2-mm-thick circumferential rim peripheral to the zone of coagulation (Fig 2). To a lesser extent, the labeled liposomes diffused into the adjacent "normal" tumor tissue and into the adjacent central coagulated tissue. Densitometry results confirmed that there was a 2.6-fold increase in average liposomal accumulation (from a mean of 3.0 intensity units ± 0.7 to a mean of 7.8 intensity units ± 3.7) inthe tumors treated with RF ablation compared with the pair-matched controls that were not treated with RF ablation (P < .05).



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  		Figure 2. Autoradiographs of tumors 24 hours following the intravenous administration of tritiated liposomes. (A) Image shows tumor that was not treated with RF ablation prior to administration. (B-D) Images show tumors that were treated with RF ablation immediately preceding administration of the liposomes. The borders of tumor A are delineated by open arrows. Note that the central zone in the tumor treated with RF ablation is without densitometric signal, and this zone corresponds to the coagulated zone with a predominantly peripheral rim of accumulated signal (solid arrows). Tumors A and B are an experimental pair from the same animal.

 
Likewise, the maximum intensity of liposomal accumulation was 2.1 times greater in the tumors treated with RF ablation (mean, 157.7 intensity units ± 25.2) compared with that in the pair-matched control tumors (mean, 75.3 intensity units ± 13.7; P < .01). Histologic analysis demonstrated a 1-mm hyperemic zone of intratumoral microvasculature peripheral to the zone of coagulation, which correlated to the outer portion of the zone in which the greatest liposome concentration was observed (Fig 3).



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  		Figure 3. Histologic specimen (hematoxylin-eosin stain; original magnification, x10) of tumor treated with RF ablation and liposomal doxorubicin demonstrates a thin rim of hyperemia (arrows) surrounding central coagulated tumor tissue (Coag.). Normal tumor tissue is seen peripheral to the zone of hyperemia.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
REFERENCES
 
Minimally invasive image-guided tumor ablation therapy, including RF, microwave, and laser, represents a new paradigm for the treatment of primary and metastatic neoplastic disease (9,10). Encouraging preliminary results have been obtained by using RF ablation for the treatment of small focal lesions (3–8). However, a key limitation of this technique has been incomplete treatment of larger lesions (ie, persistent, residual viable tumor with continued local growth). Researchers in a study of percutaneous ablation for hepatocellular carcinoma reported a local recurrence in 65% of lesions greater than 3.5 cm in diameter (4) and in 75% of tumors larger than 5 cm (8). For the percutaneous RF ablation treatment of colorectal metastases, local recurrence has been reported for 35%–89% of lesions larger than 2.5 cm (3–9). Thus, strategies to further optimize the volume of induced tumor coagulation are still required.

To overcome these limitations, previous investigators have studied the combination of RF therapy with adjuvants, such as percutaneous ethanol instillation (25), saline (27,28), and embolization (29), to effectively modulate either electrical conductivity or blood flow during the ablation to promote greater tissue heating. Another strategy, use of RF ablation combined with doxorubicin chemotherapy either directly injected into the tumor or administered intravenously in a liposomal vehicle, has also been shown to significantly increase the region of induced coagulation (21). Given the known antineoplastic effects of hyperthermia (ie, reversible cell damage induced at 42°C–48°C) and chemotherapeutic agents, such as doxorubicin, it is believed that the increased tumor destruction occurs within a sizable peripheral zone of elevated, but sublethal, temperatures that surround a region of heat-induced coagulation (30,31).

While both intratumoral injection and intravenous administration of liposomal doxorubicin have been shown to increase coagulation induced with RF ablation, the mechanisms responsible for this synergy are not completely understood and may have important implications for clinical practice. In this study, a marked increase in intratumoral doxorubicin accumulation was demonstrated when RF ablation was combined with intravenous administration of liposomal doxorubicin when compared with that of controls, but no increase was observed when unencapsulated doxorubicin was administered. This sevenfold increase in intratumoral doxorubicin accumulation is likely to be at least partially responsible for prior observations of increased coagulation in this tumor model when liposomal doxorubicin was administered as an adjuvant to RF ablation therapy (21). In addition, given that thermal damage induced with RF ablation can promote highly concentrated pharmaceutical delivery to well-defined areas, this process of selective intratumoral deposition may potentially allow a reduction of overall chemotherapeutic dosage and, thereby, reduce systemic toxicity while delivery of high doses to the tumor target is maintained.

Hyperthermia, and hence thermal ablation, has been proposed to increase vascular permeability (22), and, thus, it should result in improved intratumoral delivery of cytotoxic agents. With normal conditions, the intratumoral delivery of systemic chemotherapeutic agents is limited (31). Although tumor microvasculature is known to be "hyperpermeable," spatial and temporal heterogeneity of transvascular transport may limit the delivery of therapeutic agents, such as doxorubicin, in relatively hypopermeable regions of the tumor. This limitation, in turn, results in less than optimum therapeutic effectiveness (32). Prior investigators (33) have demonstrated enhanced antitumoral activity of liposomal doxorubicin in tumors that infiltrate the liver and spleen. It has been hypothesized that liposomes are taken up by the reticuloendothelial system and that free doxorubicin is subsequently released to liver metastases or to the systemic circulation to enter extrahepatic tumors (34). This is consistent with our findings and those of prior studies of the liver in which a fourfold greater uptake was noted for liposomal doxorubicin compared with that of free doxorubicin (35). However, the activity against extrahepatic (subcutaneous) tumors has been reported to be inferior to that of free doxorubicin at milligram-equivalent doses (36). Findings in our study suggest that augmenting liposomal doxorubicin delivery and increasing its accumulation in subcutaneous tumors by combining it with RF ablation could potentially expand the clinical use of chemotherapeutic agents that have previously been shown to lack effectiveness caused by an inability to achieve sufficient intratumoral drug concentrations.

Our observation that RF ablation does not increase doxorubicin accumulation when it is administered in a nonliposomal preparation suggests that increased doxorubicin accumulation when liposomal doxorubicin is administered is not entirely caused by passive diffusion of the agent through the RF treatment zone. Were such a mechanism responsible, increased accumulation of the low-molecular-weight free doxorubicin would have been observed. Rather, this result supports the contention that the liposome delivery vector used in our study further enhances local drug accumulation in the RF-heated tumor (30,37).

Given that the methoxypolyethylene glycol–coated liposomal doxorubicin preparation has increased circulation time (30,35), increased antitumoral activity compared with that of free doxorubicin is likely caused in part by increased drug delivery to the zone of thermal damage induced by RF ablation over an extended therapeutic window (26,37). It is also possible that the increase in doxorubicin is caused by better drug retention from entrapment of the liposomes. One plausible hypothesis would describe active phagocytosis of the liposomes by an increased number of macrophages that are recruited to the tumor as part of the acute inflammatory response to thermal injury induced by RF ablation.

The significant enhancement of liposomally delivered doxorubicin accumulation, following RF ablation, suggests that this synergy is, at least in part, caused by hyperthermia-related increased microvascular permeability and transvascular transport, perhaps as a result of endothelial injury (22,23). Tumors have regions of hypopermeable microvessels, where the transvascular channels are limited in number and or size, that limit the extravasation of larger (100.0 nm to 1.2 mm) antineoplastic agents, such as liposomes and gene therapy vectors (32). Lower-temperature hyperthermia that does not cause coagulation in the peripheral zone surrounding the thermally mediated coagulation induced by RF ablation may behave as other permeability-causing agents and may open interendothelial gaps and, thus, augment microvascular permeability (32).

Although findings of this study establish that there is increased tumoral delivery of doxorubicin when liposomal doxorubicin is combined with RF ablation, this mechanism alone is unlikely to be the only reason for improved tumor destruction. In prior studies, Goldberg et al (21) have demonstrated that the combination of RF ablation and administration of liposomes found in liposomal doxorubicin also increases tumor destruction greater than RF ablation alone, but the extent of destruction is less than that caused by a combination of liposomal doxorubicin and RF ablation. To account for these findings, increased free radical generation was implicated as a potential mechanism for the synergistic effects of the empty liposomes on RF ablation efficiency (38,39). Other researchers (40) have postulated that transient damage to cellular homeostatic mechanisms, such as the multidrug resistance membrane protein, permits increased cellular or nuclear membrane permeability to and retention of doxorubicin. Thus, synergy between RF ablation and liposomal doxorubicin is likely multifactorial.

Findings of our autoradiographic studies demonstrate that the spatial distribution of liposomal deposition is heterogeneous and, specifically, that labeled liposomes accumulate in the periphery of the zone treated with RF ablation. This pattern of distribution with absent central liposome accumulation is in accord with findings in prior studies that have demonstrated absent perfusion of the coagulated zone (11), a finding that would limit the delivery of intravenously administered agents to that portion of the tumor. Analysis of this pattern can help clarify some of the differences in accumulation with free doxorubicin in tumors that have been treated with RF ablation and in those that have not been treated with RF ablation. The trend toward a lower doxorubicin concentration observed in tumors treated with RF ablation versus those that are not treated with ablation when free doxorubicin is injected can be attributed to a reduction in tumor volume that is perfused by and subject to passive diffusion or extravasation of intravascular doxorubicin.

The autoradiographic findings also help to clarify why doxorubicin, when administered as a liposomal preparation, is also likely to be complementary to RF ablation. The majority of the liposomal doxorubicin concentrated in a zone immediately peripheral to the area coagulated by RF heating alone. As such, this corresponds to the region in which we have previously observed increased coagulation at 48 hours when RF ablation was combined with administration of liposomal doxorubicin. Given that this area also correlates to the area of an inflammatory reaction that has also been observed in both tumor and normal tissue (11), it is plausible that further investigation will demonstrate that a combination of RF ablation and liposomal doxorubicin therapy will help increase the surgical margin of ablated tissue surrounding a lesion without the need for additional ablation (41).

Additionally, the patchy penetration of some of the liposomes into the zone of coagulation suggests that there are residual patent vessels in areas of the tumor that have been traditionally classified as being completely destroyed. Such patent vessels have been previously demonstrated to protect and harbor clusters of residual viable tumor (27). Given the known heterogeneity of tumor heating observed in clinical practice (3–8), it is likely that the infiltration of chemotherapy into the coagulated focus will result in further decreases in local residual tumor, which may potentially increase the rate of complete local ablation.

Observed differences in net increased accumulation when comparing findings at autoradiography with those at spectrophotometry (two- to fourfold versus a sevenfold increase, respectively) can be attributed to the qualitative nature of the autoradiography in which densitometry is performed on thin, representative tumor sections. This contrasts with our quantitative study in which spectrophotometry was used to measure total doxorubicin accumulation throughout the entire tumor. Alternatively, differences in formulation of the tritiated liposomes and liposomal doxorubicin may affect net accumulation and detection.

Researchers in future investigations will focus on optimizing the synergy between RF ablation and administration of liposomal doxorubicin, will specifically assess RF temperature and liposomal doxorubicin dose escalations, and will determine the optimal timing between RF ablation and liposomal doxorubicin administration. Additionally, several investigators (42) are actively developing strategies to increase intratumoral drug delivery by heating thermally labile liposomes. Furthermore, other chemotherapeutic agents may also exhibit synergism with RF ablation and may warrant further investigation.

Practical application: When RF ablation is combined with systemic therapies, some of the inherent limitations of each treatment are overcome. The result is increased coagulation for a given RF application and increased intratumoral accumulation of the chemotherapeutic agent. Findings in this study support the adjuvant use of minimally invasive RF therapy to augment the delivery of liposomal encapsulated antineoplastic agents and, thus, to improve the overall effectiveness of both treatments. This approach may likely find use in clinical trials for the treatment of focal liver, breast, and other neoplasms.


   FOOTNOTES
 
Abbreviation: RF = radio frequency

Author contributions: Guarantor of integrity of entire study, S.N.G.; study concepts, S.N.G., W.L.M., G.D.G., J.B.K., V.P.T.; study design, S.N.G., K.E.S., W.L.M., J.B.K., G.D.G., V.P.T.; literature research, S.N.G., G.D.G., W.L.M., V.P.T.; experimental studies, W.L.M., S.N.G., J.C.H., M.A., A.N.L.; data acquisition, W.L.M., S.N.G., A.N.L., M.A., J.C.H.; data analysis/interpretation, S.N.G., G.S.G., M.A., A.N.L., J.B.K.; statistical analysis, S.N.G., G.S.G.; manuscript preparation, W.L.M., S.N.G., V.P.T.; manuscript definition of intellectual content, W.L.M., S.N.G., G.D.G., G.S.G., J.B.K.; manuscript editing, S.N.G., G.D.G., M.A., G.S.G., K.E.S., J.B.K., W.L.M., V.P.T.; manuscript revision/review and final version approval, all authors.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 

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