Journal of Controlled Release 160 (2012) 239–244

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Combination radiofrequency (RF) ablation and IV liposomal heat shock proteinsuppression: Reduced tumor growth and increased animal endpoint survival in asmall animal tumor model☆

Wei Yang a,c, Muneeb Ahmed a,⁎, Beenish Tasawwar a, Tatynana Levchenko d, Rupa R. Sawant d,Vladimir Torchilin d, S. Nahum Goldberg a,b

a Laboratory forMinimally Invasive Tumor Therapies, Department of Radiology, Beth Israel DeaconessMedical Center/HarvardMedical School, 330 Brookline Ave, Boston,MA02215, United Statesb Division of Image-guided Therapy and Interventional Oncology, Department of Radiology, Hadassah Hebrew University Medical Center, Jerusalem, Israelc Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Ultrasound, Peking University School of Oncology, Beijing Cancer Hospital & Institute,Beijing 100142, Chinad Department of Pharmaceutical Sciences and Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA 02115, United States

☆ Supported by grants from the National Cancer InHealth, Bethesda, MD (R01CA133114, R01 CA100041U54CA151881-01). WY is a recipient of the NationalChina, Commission No. 81101745.⁎ Corresponding author at: Department of Radiology

coness Medical Center, 1 Deaconess Road, Boston, MA 0fax: +1 617 754 2545.

E-mail address: mahmed@bidmc.harvard.edu (M. Ah

0168-3659/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.jconrel.2011.12.031

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 12 September 2011Accepted 21 December 2011Available online 30 December 2011

Keywords:RadiofrequencyTumor ablationHeat shock proteinLiposomal quercetinLiposomal doxorubicin

Background: To investigate the effect of IV liposomal quercetin (a known down-regulator of heat shock pro-teins) alone and with liposomal doxorubicin on tumor growth and end-point survival when combined withradiofrequency (RF) tumor ablation in a rat tumor model.Methods: Solitary subcutaneous R3230 mammary adenocarcinoma tumors (1.3–1.5 cm) were implanted in48 female Fischer rats. Initially, 32 tumors (n=8, each group) were randomized into four experimentalgroups: (a) conventional monopolar RF alone (70 °C for 5 min), (b) IV liposomal quercetin alone (1 mg/kg), (c) IV liposomal quercetin followed 24 hr later with RF, and (d) no treatment. Next, 16 additional tumorswere randomized into two groups (n=8, each) that received a combined RF and liposomal doxorubicin(15 min post-RF, 8 mg/kg) either with or without liposomal quercetin. Kaplan-Meier survival analysis wasperformed using a tumor diameter of 3.0 cm as the defined survival endpoint.

Results: Differences in endpoint survival and tumor doubling time among the groups were highly significant(Pb0.001). Endpoint survivals were 12.5±2.2 days for the control group, 16.6±2.9 days for tumors treatedwith RF alone, 15.5±2.1 days for tumors treated with liposomal quercetin alone, and 22.0±3.9 days withcombined RF and quercetin. Additionally, combination quercetin/RF/doxorubicin therapy resulted in the lon-gest survival (48.3±20.4 days), followed by RF/doxorubicin (29.9±3.8 days).Conclusions: IV liposomal quercetin in combination with RF ablation reduces tumor growth rates and im-proves animal endpoint survival. Further increases in endpoint survival can be seen by adding an additionalanti-tumor adjuvant agent liposomal doxorubicin. This suggests that targeting several post-ablation process-es with multi-drug nanotherapies can increase overall ablation efficacy.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Minimally-invasive, image-guided radiofrequency (RF) ablation isincreasingly used to treat focal tumors in a range of organs, and hasbeen incorporated into treatment paradigms for focal tumors of theliver, lung, bone, and kidney [1]. However, additional work is requiredto overcome difficulties in achieving a complete ablative margin for

stitute, National Institutes of5, and 2R01 HL55519, CCNENatural Science Foundation of

, WCC 308-B, Beth Israel Dea-2215. Tel.: +1 617 754 2674;

med).

rights reserved.

larger tumors, where residual, untreated tumor leads to local tumorprogression [2–5]. Most often, tumor cells survive ablation becauseof the biophysical limitations of the procedure, such as poor thermalconductivity coupled with perfusion-mediated tissue cooling, thatprevent uniform heating of the entire tumor volume to a temperaturesufficient for inducing coagulation necrosis (50–60 °C), especially inthe peripheral area of ablation [5–7]. Therefore, strategies that can in-crease of the extent of RF tumor destruction are desired.

One such successful strategy has been to target residual viablecells with adjuvant chemotherapy or radiation to improve complete-ness of RF ablation [7,8]. The rationale of this combined approach is toincrease tumor destruction occurring within the sizable peripheralzone of sub-lethal, temperatures (i.e., largely reversible cell damageinduced by mildly elevating tissue temperatures to 41–45 °C) sur-rounding the heat-induced coagulation [7]. Indeed, several studies

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have reported increased tumor destruction with RF ablation and ad-juvant IV liposome-encapsulated chemotherapeutics, such as doxoru-bicin and/or paclitaxel in part due to RF inducing a potentiallycontrolled increase intratumoral nanodrug accumulation [9-11]. Sev-eral additional mechanisms accounting for the increased tumor de-struction of combination therapy have been identified, includingamplification of cell stress and apoptosis by the nanodrugs most nota-bly in the rim of periablational tissue exposed to sublethal hyperther-mia [12]. Interestingly, in these experiments a rim of increased heat-shock protein (HSP) production was also observed immediatelyperipheral to the zone of apoptosis in a concentric ring of still-viabletumor surrounding the ablation zone. As these HSPs have known cellu-lar protective effects against apoptosis [13-15], the presence of a rim ofincreasedHSP expression beyond the ablation zonewith co-localizationto persistently viable tumor suggests that protective HSP effects may beinhibiting additional gains in tumor destruction at the ablation margin.

Based upon this observation, most recently, RF ablation has beencombined with adjuvant IV liposome-encapsulated quercetin, a flava-noid with known suppressive effects on the synthesis of heat shockproteins in a variety of cell lines [16]. Indeed, the rationale for thiscombined paradigm was to use image-guided RF to control the re-lease of a nanodrug that is particularly active against its unwanted ef-fects at the periphery of the thermal ablation within a given tumortarget. In one study, RF ablation sufficiently increased liposomal quer-cetin concentrations in the tumor to increase apoptosis, reduce HSPexpression, preferentially in the periablational rim, and increasedoverall tumor destruction compared to RF alone at 24 h [17]. Evengreater gains observed for triple therapy combining RF ablationwith both adjuvant liposomal doxorubicin and liposomal quercetin[17]. Yet, short-term mechanistic studies alone are often insufficientto predict the actual effect of different liposomal compounds on thecritical endpoint outcome of survival [11]. Accordingly, the purposeof this study is to investigate the effect of IV liposomal quercetinand/or liposomal doxorubicin when combined with RF tumor abla-tion on tumor growth and end-point survival in a rat tumor modelas a necessary step in evaluating the potential utility of this regimenprior to consideration of clinical trials.

2. Materials and methods

2.1. Experimental overview

Approval of the Institutional Animal Care and Use Committee wasobtained prior to the start of this study. The study was performed intwo phases to systematically investigate the potential synergistic ef-fects of RF ablation with adjuvant liposomal quercetin alone andthen in combination with liposomal doxorubicin. A total of 48 tumorsin 48 animals were used. In sections below, references to “quercetin”and “doxorubicin” imply liposomal encapsulation as no free drug(either quercetin or doxorubicin) was administered in this study.

2.2. Phase I. Effect of liposomal quercetin and RF ablation on tumorgrowth rates and animal endpoint survival

To investigate the endpoint survival and tumor growth for animalsfrom different treatment strategies, 32 tumors/animals were random-ized to receive one of four therapeutic regimens, including standard-ized RF ablation (conventional, monopolar 1 cm tip; applied for5 min; with the tip temperature titrated to 70±1 °C) and/or IV lipo-somal quercetin (0.5 ml/0.29 mg). These groups included (n=8, eachgroup): (a) RF alone, (b) IV liposomal quercetin alone, (c) IV liposo-mal quercetin followed 24 hr later with RF, and (d) no treatment.The survival endpoint was a maximum tumor diameter of 30 mm orsurvival of 90 days, whichever was reached first. Secondary endpointswere the rate of tumor growth and local control (i.e., no visible tumoron the chest wall).

2.3. Phase II. Effect of adding liposomal doxorubicin to RF/liposomalquercetin on end point survival

This experiment was performed to determine whether the liposo-mal doxorubicin would affect the survival results of combinationtreatment with RF and liposomal quercetin. Sixteen additionaltumors were randomized into two groups (n=8, each group) that re-ceived either triple therapy with combined RF, liposomal quercetin(24 hrs pre-RF, 0.5 ml/0.29 mg), and liposomal doxorubicin (15 minpost-RF, 8 mg/kg) or RF with liposomal doxorubicin alone.

2.4. Animal model

For all experiments and procedures, anesthesia was induced withintraperitoneal injection of a mixture of ketamine (50 mg/kg,Ketaject; Phoenix Pharmaceutical, St. Joseph, MO) and xylazine(5 mg/kg, Bayer, Shawnee Mission, KS). Animals were sacrificedwith an overdose (0.2 mL/kg) of pentobarbital sodium (Nembutal;Abbott Laboratories, North Chicago, IL).

Experiments were performed with a well-characterized estab-lished R3230 mammary adenocarcinoma cell line used in multipleprior studies [10,18,19]. In total, 48 female Fisher rats (120±20 g;13–14 weeks old, Taconic Farms, Germantown, NY) with R3230 tu-mors were used in this study. Fresh tumor was harvested from alive carrier and homogenized in a tissue grinder (Model 23; KontesGlass, Vineland, NJ) and suspended in Roswell Park Memorial Institu-tion (RPMI) 1640 medium (Biomedicals, Aurora, IL). One tumor wasimplanted into each animal by slowly injecting 0.3–0.4 mL of tumorsuspension into the mammary fat pad of each animal via an18-gauge needle. Animals were monitored every 2–3 days to measuretumor growth. 1.3–1.5 cm solid non-necrotic tumors (determined onultrasonography by an absence of more than 3 separate cystic spaces>0.5 mm in diameter within the entire tumor) were used for thisstudy, randomized to different treatment arms. Tumors were mea-sured in a longitudinal and transverse diameter with a mechanicalcaliper (W.Y., B.T.). Accuracy of the final measurement was verifiedby the senior author, who was blinded to treatment group) every2–3 days until they reached 30 mm in largest diameter, at whichpoint the animals were sacrificed. This surrogate for endpoint survivalwas selected because of requirements of the institutional animalcommittee, which mandated sacrifice at this point on the basis of an-imal size and tumor burden as dictated by the U.S. Department of Ag-riculture Animal Welfare Act (31–32). Any animal with tumorsweighing more than 10% of its body weight, corresponding to atumor diameter of 30 mm in this model, was considered moribundand underwent mandatory sacrifice. For all measurements, anesthe-sia was induced, as described previously, to permit accurate measure-ments. The largest diameter measured at every time point wasrecorded and plotted.

2.5. RF application

Conventional monopolar RFA was applied by using a 500-kHz RFAgenerator (model 3E; Radionics, Burlington, Mass). To complete theRF circuit, the animal was placed on a standardized metallic ground-ing pad (Radionics). Contact was ensured by shaving the animal'sback and by liberally applying electrolytic contact gel. Initially, the1-cm tip of a 21-gauge electrically insulated electrode (SMK elec-trode; Radionics) was placed at the center of the tumor. RF was ap-plied for 5 min with the generator output titrated to maintain adesignated tip temperature (70±2°C, mean 90.1±21.6 mA, range48–156 mA). This standardized method of RFA application has beendemonstrated previously to provide reproducible coagulation vol-umes with use of this conventional RFA system [18,19].

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2.6. Preparation and administration of adjuvant intravenous liposomalagents

Quercetin-loaded liposomes were prepared such that the lipidcomposition in these liposomes was identical to Doxil, in a mannersimilar to prior reports [12]. Briefly, 0.29 mg of quercetin (1 mg/mLsolution in methanol) was added to hydrogenated soyphosphatidylcholine, cholesterol and polyethyleneglycolphosphatidylethanolamine (PEG2000-PE) (57.25:37.57:5.18 mol%,respectively) solutions in chloroform, and a lipid film was formed ina round bottom flask by solvent removal on a rotary evaporator. Thelipid film was then rehydrated with 1 mL of phosphate buffered sa-line, pH 7.4 and the preparation was probe-sonicated with a SonicDismembrator (Model 100, Fisher Scientific, Pittsburgh, PA) at apower output of 7 watts for 30 min. To remove any titanium particleswhich may have been shed from the tip of the probe during sonica-tion, the sample was centrifuged for 10 min at 2000 rpm. Liposomeswere loaded with 5 mol% quercetin (not to saturation), and the lipo-somal loading efficiency of quercetin was 100% (as noted above,0.29 mg of quercetin was loaded in each administered dose). The li-posomal size was 115 nm±43 nm. The zeta-potential was −34.7±5.5 mV. The liposomal formulation was tested and stable for1 week at 4 °C in phosphate buffered saline (PBS) at a pH of 7.4 andfor 24 hrs at 37 °C in PBS with 10% fetal bovine serum. When pre-pared for the experiment, all formulations were administered within24 h of preparation. Finally, the polydispersity coefficient was 0.315.For Phase II, a commercially-available preparation of liposomal doxo-rubicin (Doxil; ALZA Pharmaceuticals, Palo Alto, CA) was used.

Liposomal quercetin (0.5 ml) was injected slowly (for 30 s via a27-gauge needle) into the tail vein 24 hr pre-RF ablation in the com-bined therapy group; in order to provide maximal drug uptake andpotential HSP suppression prior to the thermal ablation. Liposomaldoxorubicin (1 mg; of a 2 mg/ml preparation) was injected separatelywith the same approach (0.5 ml IV per animal), but at a later timepoint, 15 min post-RF. This timing of IV liposomal doxorubicin admin-istration was selected based upon prior studies demonstrating maxi-mum tumor coagulation and intratumoral drug accumulation withthis paradigm [10,19].

2.7. Statistical analysis

SPSS 13.0 software (SPSS Inc., Chicago, IL) was used for statisticalanalysis in this study. All data were provided as mean plus or minusSD. The P value is less than 0.05 was considered as significant. Onthe basis of studies that demonstrated exponential tumor growthpatterns, tumor growth rates and doubling times were also calculat-ed by means of regression analysis of an exponential model. TheKaplan–Meier method and log-rank test were used for endpoint sur-vival analysis. Given the absence of censoring of our data, one-wayanalysis of variance was then performed on the survival endpointsfor each animal for the comparisons reported. Pairwise t tests(pb0.05; two-tailed test) based on the least square means were

Table 1Effect of RF Ablation and/or adjuvant liposomal chemotherapy (quercetin and/or doxorubic

Treatment No. of tumors Mean endpoint survival (d) Median en

Control 8 12.5±2.2 11.5RF alone 8 16.6±2.9 17Quercetin alone 8 15.5±2.1 15.5Quercetin-RF 8 22.0±3.9 22.5RF-doxorubicin 8 29.9±3.8 31Quercetin-RF-Doxorubicin 8 48.3±20.4 45

Note — Monopolar RF was applied for 5 min by using a 1-cm tip electrode, with the tempea Refers to b in the formula in results, tumor growth rates.

subsequently performed only if the overall P values were significant.We then used two way analysis of variance to determine the contri-butions of RF ablation and intravenous injection of liposomal quer-cetin to endpoint survival. One-way and two way analyses ofvariance were also performed to determine the contribution of RFablation and intravenous liposomal quercetin to the parameter oftumor doubling time.

3. Results

3.1. Endpoint survival

The mean endpoint survival (i.e., the time from treatment for thetumor to reach 30 mm in diameter) in the control group was 12.5±2.2 days. Mean endpoint survival for the groups treated with eitherIV liposomal quercetin alone or RF ablation alone was similar, butwas significantly improved when compared with the control (notreatment) group. Endpoint survival was 16.6±2.9 days for tumorstreated with RF ablation alone and 15.5±2.1 days for tumors treatedwith IV liposomal quercetin alone (Pb0.001 compared with control).Significantly greater endpoint survival of 22.0±3.9 days wasobtained for tumors treated with combined RF ablation and IV liposo-mal quercetin (Pb0 .001 compared with other groups) (Table 1,Fig. 1).

When liposomal doxorubicin was added to RF/quercetin (i.e., tri-ple therapy), survival increased to 48.3±20.4 days, compared to29.9±3.8 days for RF with only liposomal doxorubicin (Pb0.01).Pair-wise comparisons indicated that tumor growth rates and meanendpoint survival with the different treatments protocols (i.e. singleagent, combination of two treatments, and combination of threetreatments) were all significantly different (Pb0.005, all compari-sons). Although, animals that received combined RF/doxorubicin sur-vived longer than did animals that received combined RF/quercetin(Pb0.005), triple therapy (quercetin/RF/doxorubicin) showed thegreatest survival benefit. The maximum endpoint survival in thisstudy was limited to 90 days and was obtained for one animal inthe combined treatment group of triple therapies.

3.2. Tumor growth rate

Tumor growth rate curves were calculated as exponential func-tions to the formula y=aebx, where a=1.3–1.5, initial tumor size,according to study design. All curves had R2 values ranging from0.90 to 0.99 (Table1). The value representing tumor growth rate (b)varied from 0.035 to 0.009 (Table1). This corresponded to doublingtimes ranging from 19.7±3.2 days (untreated controls) to81.6±6.8 days in animals treated with triple therapy (i.e.,quercetin/RF/doxorubicin) (Fig. 2). Results of analysis of variancedemonstrated significant differences in tumor doubling times forthe combined treatments compared over no or single treatment(either RF, quercetin, or doxorubicin alone), and the longest tumordoubling times with triple therapy.

in) on endpoint survival and tumor doubling time.

dpoint survival (d) Exponential growth ratea Tumor doubling time (d) R2

0.035 19.7±3.2 0.900.028 24.3±1.9 0.970.032 21.3±2.8 0.940.024 29.2±1.4 0.990.017 39.8±2.0 0.980.009 81.6±6.8 0.91

rature titrated to 70 °C. Endpoint survival is defined as 30 mm in diameter.

Fig. 1. Kaplan-Meier analysis of animal endpoint survival following treatment with RFand/or intravenous liposomal quercetin (Qu) and/or liposomal doxorubicin (Doxil). An-imal endpoint survival was defined as tumor diameter greater than 30 mm, or survivalto 90 days, whichever came first (the graph was truncated at 60 days, as the final oneanimal had complete local control/tumor regression, and survived beyond 90 days).Mean survival for animals that received RF and single agent therapy (quercetin-RF orRF-doxorubicin) was greater than RF alone (16.6±2.9 days), quercetin alone (15.5±2.1 days), or the control group (12.5±2.2 days; Pb0.05, all comparisons). Greatest end-point survival was observed with triple therapy, quercetin-RF-doxorubicin (mean end-point survival of 48.3±20.4 days, pb0.005 for all comparisons).

A B

C D

E F

Fig. 3. Gross tumor local control and regression in animals treated with combined RFablation and IV liposomal quercetin and doxorubicin (A–D), and tumor had no treat-ment (E–F). (A) The initial tumor on day 0 (prior to any treatment) measures14×13 mm. (B) At day 14, there were signs of tumor necrosis. (C) At day 35, tumorsize decreased to 10×11 mm with necrosis. (D) At day 90, no evidence of tumor canbe seen, a fact verified by autopsy. (E) The control, untreated tumor on day 0 (priorto any treatment) measures 13×12 mm. (F) The tumor quickly grew up and increasedto 3 cm in size (and was sacrificed) at day 14.

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3.3. Local control

Local tumor control was achieved only once, in 1 (12.5%) of the8 animals that received triple therapy. In this particular animal,tumor size decreased progressively after treatment and completelycured until the animal was sacrificed at 90 days, when no viabletumor was identified at histopathologic examination. No localtumor control was seen in any other groups. Thus, a significant differ-ence was seen in local control achieved in the group of rats that re-ceived the triple therapy or not (Pb0.01 for all comparisons) (Fig. 3).

4. Discussion

Exposure to low, non-lethal levels of hyperthermia results in re-versible cellular injury and upregulation of protective cellular path-ways [20]. One such pathway, mediated by the heat shock protein

Fig. 2. Tumor growth following treatment with RF ablation and/or intravenous liposomalquercetin (Qu) and/or liposomal doxorubicin (Doxil). Themean tumordoubling time for an-imals that received RF ablation and liposomal quercetin was greater than RF alone (24.3±1.9 days, pb0.05), liposomal quercetin alone (21.3±2.8 days, pb0.05) or the controlgroup (19.7±3.2 days; Pb0.005). Triple therapy (quercetin-RF-doxorubicin) showed thegreatest survival benefit, with a mean tumor doubling time of 81.6±6.8 days. This was sig-nificantly greater than quercetin-RF (29.2±1.4 days; pb0.001) or RF-doxorubicin (39.8±2.0 days, pb0.001).

family and the HSP70 protein in particular, has known protective ef-fects in the face of cellular injury from exposure to low level hyper-thermia and chemotherapeutic agents [21]. Along these lines,several studies have demonstrated increased HSP70 expression in pa-tient serum and in the ablative zone up to 5 days after RF ablation[22–24]. Similarly, elevated HSP70 levels were also detected in biopsyspecimens obtained 24 hr after RF ablation of human hepatocellularcarcinoma [25].

Recently, markedly increased HSP70 expression after RF ablationhas been localized to the transitional zone at the ablation marginwhere the greatest exposure to sub-lethal hyperthermia occurs [12].Immunohistochemical characterization in subcutaneous rat breast tu-mors using co-localized staining for both cleaved caspase 3 (a markerof apoptosis) and HSP70 demonstrated a rim of apoptosis at the abla-tive margin, immediately surrounded by a rim of HSP70 that corre-sponds to partially-injured, viable tumor cells [12]. Most recently,Yang et al. have endeavored to exploit this observation further intoa factor that can improve nanoparticulate mediated cancer therapyby attempting to target the HSP70 cellular stress pathway to increaseoverall tumor coagulation through combining RF ablation with lipo-some encapsulated quercetin, a compound with known inhibitory ef-fects on HSP expression [17]. In their report, combination therapyresulted in reduced HSP70 expression in the ablative margin withcorrelative increases in both apoptosis and tumor coagulation. This

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correlated to an early (4 h) reduction in HSP70 expression and in-creased apoptosis for combination quercetin-RF compared to RFalone. However, whether gains in apoptosis and tumor destruction(with reductions in HSP70 expression) translate into correlative in-creases in animal endpoint survival and reduction in tumor growthhas not been determined.

In this study, combining RF ablation with adjuvant liposome en-capsulated quercetin resulted in reduced tumor growth (as evidenceby tumor doubling time) and increased animal endpoint survival overindividual treatments (either RF or liposomal quercetin alone). Thiscorrelates well with the increased tumor coagulation and immuno-histochemistry staining (increased apoptosis and reduced HSP70 ex-pression) observed in prior studies. Quercetin acts through severalmechanisms, including phosphorylation of AMP-activated protein ki-nase to down-regulate HSP70 expression [26], interfering with trans-location [27], reducing the availability of heat shock transcriptionfactor, suppressing initiation and elongation of the HSP70 mRNA[28], and separately increasing the activity of caspase 3 [26]. This af-fects both of the pathways (cleaved caspase 3 and HSP70) that wereinterrogated by Yang et al., and likely contributes to gains observedhere in animal endpoint survival. Interestingly, liposomal quercetinalso provided a survival benefit over control, untreated tumors (andsimilar to RF alone), suggesting a separate and independent suscepti-bility of this tumor model to quercetin. The effects of quercetin ontumor cell growth have been separately demonstrated in in vitrostudies [16,29], and in addition, liposomal quercetin has beenshown to slow tumor growth in several separate murine xenografttumor models [30].

A number of experimental and clinical studies have demonstratedthe benefits of combining RF ablation with IV liposomal doxorubicin,including RF-induced local increases of intratumoral drug accumula-tion, leading to increased tumor coagulation and animal endpointsurvival (as is also confirmed in our results) [7,10,31]. In our study,RF and liposomal doxorubicin resulted in longer animal survivaltimes (mean 29.9 days) compared to RF and liposomal quercetin(mean 22 days), despite the similar tumor coagulation observed inprior studies (13.2 mm vs. 13.1 mm for RF with liposomal doxorubi-cin or liposomal quercetin, respectively) [17]. This discordance un-derscores the need to carefully address the most relevant endpoints,such as tumor coagulation and endpoint survival, and that delayedevaluation of tumor coagulation must be viewed as primary, and sep-arate, outcome measure. Thus, while early evaluation of coagulationdiameter can identify early temporal differences between groups,the actual time it takes for a full effect to take place may vary.Hence, the determination of a full dose response curve likely needsto be tailored for each agent, and may make comparisons betweendifferent drugs even more difficult. Similarly, differences betweenintratumoral drug accumulation and coagulation demonstrate thatidentifying the drug concentration required for a threshold effect re-quires continued investigation. Finally, while tumor coagulation re-flects local improvements in tumor destruction, the systemic impactof adjuvant therapy may be under-represented. For example, extra-cellular HSP expression upregulates the immune-mediated responseto tumor cells [32–35], and therefore, gains in tumor coagulationfrom intracellular HSP suppression does not account for systemicanti-tumor effects, and may not translate directly to improvementsin animal endpoint survival.

Immunohistochemical characterization in small animal tumorstreated with combination RF and liposomal doxorubicin has alsoshown increased HSP70 expression in the periablational zone withcompared to RF alone, and has formed the basis for recent studies inwhich Yang et al. reported that introducing liposomal quercetin intothe treatment paradigm could result in HSP70 suppression with tripletherapy, resulting in the largest tumor coagulation compared to sin-gle or dual therapy regimens (RF with or without a single adjuvant li-posomal chemotherapy agent). Our results support these findings,

with significantly longer animal endpoint survival compared toother treatment arms. Potential mechanisms of synergy for tripletherapy include improved therapeutic index and efficacy (quercetinmay reverse doxorubicin cell resistance in hypoxic conditions [36])and targeting separate pathways of cellular stress (quercetin predom-inantly through anti-HSP effects, while adjuvant doxorubicin hasbeen shown to both increase apoptosis and HSP expression [12]). In-terestingly, Yang et al. reported only partial HSP70 suppression withthe addition of liposomal quercetin to the RF and liposomal doxorubi-cin, possibly reflecting limitations with intratumoral spatial deliveryor temporal effects of HSP70 suppression. This suggests that refine-ment of the treatment paradigm (for example, by increasing liposomeloading of quercetin or using a multi-dosing approach) could result ineven greater gains in animal endpoint survival. Finally, in a separatestudy, Yang et al. have also previously combined RF and liposomaldoxorubicin with adjuvant liposomal paclitaxel, an agent with pre-dominant pro-apoptotic effects but with some secondary anti-HSP ac-tivity [11]. In the same rat breast tumor model, combination RF/doxorubicin/paclitaxel therapy resulted in a mean of 56.8 days of sur-vival, compared to the maximum of 48.3 days observed here withcombination RF/doxorubicin/quercetin. This suggests both that multi-ple drug regimens have the potential to increase overall tumor de-struction and that continued investigation into the optimal selectionof drug(s) is required.

Clearly, HSP70 has known cellular protective effects in the settingof low-level hyperthermic exposure that one may wish to eliminatewhen attempting to treat a tumor. Fortunately, as these results andseveral other studies demonstrate, HSP70 is locally increased whencells are exposed to sublethal hyperthermia immediately surroundingthe RF ablation zone, and this focal effect can be selectively sup-pressed with targeted local delivery of inhibitors/antagonists includ-ing quercetjn [17]. Yet, a word of caution and a call for additionalstudy is warranted prior to rapid clinical adoption. For example, theHSP70 family of proteins has other extensive roles in protein foldingand transport, molecular chaperone activities, and immune-relatedantigen presentation [37,38], such that downregulation of HSP activ-ity may have unintended and even unwanted side effects on tumorgrowth. Given the ubiquitous nature of HSP70, it is also theoreticallypossible that systemic HSP70 suppression from systemic administra-tion of specific antagonists could have unrelated secondary effects onnon-target tissues. However, in our current paradigm in which a sin-gle dosing regimen is used with nanoparticle encapsulated platforms,the non-target systemic exposure is likely minimal. Finally, quercetinitself has separate non-HSP-based effects (for example, anti-oxidative, anti-hypoxic, and anti-angiogenic effects [39–42]) suchthat manipulation of these pathways may also alter the cellular mi-croenvironment and tissue response at least in part through non-HSP mechanisms. The degree to which these potential secondaryeffects from targeted and localized HSP70 suppression occur, andtheir ultimate clinical significance, is currently unclear, but will hope-fully be more fully elucidated in the near future.

There are several additional limitations of this study worth men-tioning. Although this well-characterized R3230 tumor model toallow comparison to other studies of RF ablation and liposomal che-motherapy performed in this model, careful interpretation and appli-cation of the results is required. For example, the differencesidentified between quercetin and doxorubicin-based regimens likelyreflect in part underlying tumor susceptibility to each of these agentsfor this particular model, and so further study will be required inother models to determine which tumor types will respond to RFcombinedwith either or both liposomal agents. We have also selecteda commercially available pegylated stealth liposomal doxorubicinagent and prepared similarly constructed quercetin-containing lipo-somes. Prior studies have demonstrated good success with thesepreparations, such as increased tumor destruction and intratumoraldrug accumulation while reducing tumor growth rates [7]. As it has

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been previously shown that the empty liposome itself contributes toincreases in observed tumor destruction, the continued use of this de-livery vehicle allows a standardized comparison of the effects of dif-ferent chemotherapy agents. However, additional characterizationand optimization of the delivery vehicle and timing of the agent ad-ministration in future studies may improve in vivo drug delivery.For example, administering liposome-encapsulated quercetin at a dif-ferent time point or dosage (or multiple doses) may potentially resultin even greater HSP70 suppression, and therefore, further gains intumor destruction and animal endpoint survival. Similarly, we alsoacknowledge that other approaches, such as the incorporation ofthermosensitive liposomal preparations into our combination thera-py algorithm may yield different, and perhaps improved, results[43,44]. However, given the timing (4–12 hrs) of anti-HSP and pro-apoptosis effects that have been previously observed, it is far fromclear that an additional early burst of drug release (0–1 h), both spa-tially and temporally, will definitely increase tumor destruction com-pared to a slower, more prolonged drug release. In particular, theparadigm of tissue heating (both timing and variances in temperatureranges in the treatment zone) vary significantly between focal high-temperature RF tumor ablation and the low-temperature convention-al hyperthermia that has been combined with thermosensitiveliposomes in existing literature — such that, it is likely too early to as-sume that thermosensitive liposomal preparations will definitely be“better” than non-temperature sensitive agents. Clearly, further in-vestigation into identifying the delivery vehicle characteristics andadministration paradigm is still required.

In conclusion, combining RF ablation with liposome-encapsulatedquercetin to modulate heat shock reaction can reduce tumor growthand increased animal endpoint survival. Thus, our studies furthersupports the paradigm of using image-guided ablation to selectivelycontrol delivery of a nanodrug that is particularly active against itsunwanted effects at the periphery of the thermal ablation zone.Even further gains can be achieved by combining RF ablation withmultiple adjuvant agents that target separate underlying cellularstress pathways. Continued investigation into single and multi-drugcombinations as adjuvants to ablation therapy, including optimiza-tion of drug delivery vehicles and dosing regimens, may yield contin-ued gains in tumor destruction.

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