Biodistribution and Efficacy of Low Temperature-Sensitive Liposome Encapsulated Docetaxel Combined with Mild Hyperthermia in a Mouse Model of Prostate Cancer

Abstract

Purpose

Low temperature sensitive liposome (LTSL) encapsulated docetaxel were combined with mild hyperthermia (40–42°C) to investigate in vivo biodistribution and efficacy against a castrate resistant prostate cancer.

Method

Female athymic nude mice with human prostate PC-3 M-luciferase cells grown subcutaneously into the right hind leg were randomized into six groups: saline (+/− heat), free docetaxel (+/− heat), and LTSL docetaxel (+/− heat). Treatment (15 mg docetaxel/kg) was administered via tail vein once tumors reached a size of 200–300 mm³. Mice tumor volumes and body weights were recorded for up to 60 days. Docetaxel concentrations of harvested tumor and organ/tissue homogenates were determined by LC-MS. Histological evaluation (Mean vessel density, Ki67 proliferation, Caspase-3 apoptosis) of saline, free Docetaxel and LTSL docetaxel (+/− heat n = 3–5) was performed to determine molecular mechanism responsible for tumor cell killing.

Result

LTSL/heat resulted in significantly higher tumor docetaxel concentrations (4.7-fold greater compared to free docetaxel). Adding heat to LTSL Docetaxel or free docetaxel treatment resulted in significantly greater survival and growth delay compared to other treatments (p < 0.05). Differences in body weight between all Docetaxel treatments were not reduced by >10% and were not statistically different from each other. Molecular markers such as caspase-3 were upregulated, and Ki67 expression was significantly decreased in the chemo-hyperthermia group. Vessel density was similar post treatment, but the heated group had reduced vessel area, suggesting thermal enhancement in efficacy by reduction in functional perfusion.

Conclusion

This technique of hyperthermia sensitization and enhanced docetaxel delivery has potential for clinical translation for prostate cancer treatment.

INTRODUCTION

Prostate cancer is the most common tumor in the United States and the second leading cause of cancer death in American males (1). It is estimated that a man’s lifetime risk of prostate cancer is one in six. Over 95% of prostate cancers are adenocarcinomas, arising from epithelial cells within prostate gland (2). Thus, for decades, patients with prostate cancer limited to prostate gland underwent prostatectomy, essentially curing the patient but exposing the patient to the risk of surgery and its post-operative effects. However, for a significant proportion of men with aggressive prostate cancer, postoperative recurrence of the disease is a major concern. While recurrence rates depends on a variety of factors such as pre-operative PSA, Gleason score, tumor stage, and patient age, Cooperberg et al. estimated that 15% of all men undergoing prostatectomy will develop a recurrence for prostate cancer (3). Androgen deprivation therapy can be used to initially treat men with recurrent or metastatic cancer but these neoplastic cells typically become hormone resistant and are referred to as “castrate resistant” after 2 years of treatment (3,4). For these patients, chemotherapy is the only therapy that has been shown to improve survival (5). The 2011 National Comprehensive Cancer Network (NCCN) recommends docetaxel based regimen as first line chemotherapy against castrate resistant cancer and patients with metastatic disease that have failed androgen deprivation (6).

Docetaxel (DTX) is in the taxane class of chemotherapeutic drugs, acting mainly against highly proliferative cancer cells by binding to microtubules of the cells. While DTX has been shown to improve survival, it comes with significant side effects on patients. Side effects range from significant hematologic problems such as neutropenia, leukopenia, and thrombocytopenia to neurologic and respiratory problems (7). Therefore, the development of a synergistic treatment modality in combination with DTX is highly desirable for achieving enhanced efficacy and reduced side effects compared to conventional chemotherapy. While a clear DTX combination treatment is yet to emerge, researchers are eagerly searching for a clinically proven regimen. In this regard, a limited amount of studies has indicated that DTX action can be potentiated in the presence of heat. For instance, Mohamed et al. showed that hyperthermia modestly increase the mean tumor growth time in combination treatment with DTX in a fibrosarcoma cell line (8). This is an important finding, and a similar demonstration of hyperthermia sensitization of chemotherapy in a prostate specific cancer cell line may pave the way for eventual clinical studies. The benefits of DTX/heat additive regimen can be leveraged further by encapsulation into low temperature sensitive liposomes (LTSLs) as a means to enhance tumor delivery compared to free drug alone. LTSLs contain a lysolecithin lipid that rapidly melts and allow release of encapsulated doxorubicin upon being heated to mild hyperthermic temperatures (40–42°C) (9,10). This approach has been shown to result in significant reduction in tumor volume and enhance drug delivery in mouse and rabbit tumor models using water-soluble drugs (e.g., Doxorubicin) compared to conventional free drug or non-thermally sensitive liposome therapy (11–15). The ability of LTSL to induce release of water soluble DTX-prodrug has been investigated in healthy nude rats (16). Recently, a thermosensitive liposome containing DTX was also investigated for tumor inhibition in mice breast tumor (17). However, a comprehensive study that combines biodistribution, efficacy and mechanism of cell killing in a clinically relevant mouse tumor model is yet to be demonstrated. To address this, the objective of this study was 1) to determine the synergism between DTX and mild hyperthermia (40–42°C) therapy, 2) evaluate enhancement in tumor delivery of LTSL-DTX in combination with mild hyperthermia, and 3) assess histopathological features of tumors following DTX/hyperthermia treatment. To ease clinical translation, a GMP grade DTX-LTSL was obtained from Celsion Corp., USA. Celseon leads ThermoDox®, a proprietary heat-activated liposomal encapsulation of doxorubicin, currently in Phase III development for the treatment of primary liver cancer and Phase II development for the treatment of recurrent chest wall breast cancer. Data from this study suggest that LTSL encapsulation of DTX can similarly enhance tumor delivery in presence of hyperthermia, and achieves treatment efficacy equivalent to free docetaxel.

MATERIAL AND METHODS

Chemicals

DTX was obtained and reconstituted (20 mg/mL) according to the manufacturer’s recommendation (Sanofi Aventis, USA). Aliquots of the stock solution were diluted in 0.9% sterile saline to the final dosing concentration immediately prior to treatment. Phosphate buffer and 0.9% saline (PBS) were obtained from Sigma–Aldrich (Saint Louis, MO, USA). For histopathology, prolong Gold with DAPI mounting medium was obtained from Invitrogen (Carlsbad, CA, USA), and CD31 antibodies were obtained from BD Biosciences (CA, USA).

LTSL

A lyophilized GMP grade lyso-lecithin containing LTSL (lipid: DPPC, DSPG, MSPG and DSPE-mPEG2000; weight ratio of 83:8.3:8.3:8.3) was provided by Celsion Corp., USA through a Collaborative Research and Development Agreement with NIH. DTX was loaded passively into the LTSL bilayer at a lipid: drug ratio of 95:5. Prior to animal treatment, lyophilized LTSL were suspended in sterile water at a concentration of 1 mg DTX/mL, and size was measure by dynamic light scattering (~160 ± 6 nm). A detailed description of formulation stability over 12-month is shown in Table I.

Table I.

Lyso-Thermosentitive Liposome Stability Over 12 Months

Analysis	Months
	0	1	2	3	6	9	12
pH	7.15	7.13	7.13	7.10	7.08	7.09	7.06
Mean Vesicle size (nm)	97	103	105	106	107	110	115
CDrug %		100.09	99.82	99.74	99.47	99.12	98.95
Encapsulation %	89.4	89.2	89.3	89.0	88.7	88.6	88.4
Lipid concentration* (mg/mL)	DPPC	–	–	–	–	22.78	22.63
	DSPE-PEG2000	–	–	–	–	2.53	2.49
	MSPC	–	–	–	–	2.51	2.54
	DSPG	–	–	–	–	2.45	2.42

Animal Experiment

Animal and Tumor Cells

All animal-related procedures were approved and carried out under the guidelines of the National Institutes of Health (NIH) Animal Care and Use Committee. All drug delivery studies were performed in six- to 8-week-old female athymic nude mice (nu/nu NCR strain, body weight 20–25 g). Animals were kept as five/cage under specific-pathogen-free conditions with water and food ad-libitum. For tumor initiation, castrate resistant prostate adenocarcinoma PC-3 M-luc2, a luciferase expressing cell line (Caliper) was maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C with 5% CO₂.

Tumor Xenografts

The growth of PC3M-Luc 2 cells in athymic nude mice was established as previously described (18). PC3M-Luc 2 cells at 80–90% confluence were harvested, washed and diluted with sterile PBS buffer. 3–5 million cells were injected subcutaneously, in a total volume of 100 μl, into the rear thigh region of mouse leg using a 27 Ga needle. Mice were monitored and weighed daily for tumor growth by serial caliper measurement. Tumor volume was calculated using the formula (length × width²)/2, where length (a) is the largest dimension and width (b) the smallest dimension perpendicular to the length (a × b²)/2. Drug treatments were initiated when the tumor sizes reached 200–300 mm³.

Tumor Drug Delivery Study Design

For efficacy studies, nude mouse (n = 6–7) with PC3M-Luc 2 tumors, were randomized into six groups: saline (+/− heat), free DTX (+/− heat), and LTSL DTX (+/− heat). For biodistribution and histopathology, 4–5 mouse/group were randomized similarly. In all groups, 15 mg DTX/kg body weight was administered intravenously.

Hyperthermia Protocol

Hyperthermia treatment were performed using a water bath method as described previously (15,19). Briefly, mice were anesthetized with an i.p., injection of pentobarbital (60 mg/kg) with a 27 Ga needle (15). This dose of anesthesia often provided adequate immobilization for up to 1 h. If the tumor bearing mouse demonstrated signs of alertness, an additional dose at 1/3 (20 mg/kg) was injected subcutaneously or i.v. (20), depending on accessibility in the mouse holder/hyperthermia setup. Immediately after receiving a tail vein injection of treatment solution, the mice were positioned in specially designed holders that allow the isolated hind flank tumor to be placed in a water bath for 1 h. The water bath temperature was set to ~43°C; a temperature that was already been calibrated to give tumor temperatures of 41–42°C. Rise in body temperature during hyperthermia treatment was adequately addressed by placing a fan on top of the water bath which helped maintain the body temperature at ~37°C. Upon completion of hyperthermia treatment, mice were followed either for efficacy studies for 60 days or were euthanized for biodistribution study.

Biodistribution Studies

Biodistribution studies were performed in tumor bearing mice as follows. Saline, free DTX, and LTSL (15 mg DTX/kg, n = 5 (+/− heat)) was administered via tail vein once tumors reached a size of 300–400 mm³. Upon completion of hyperthermia for 1 h, cardiac perfusion was performed under isoflurane anesthesia by opening the chest cavity and giving a 10 mL intra-cardiac (i.c.) injection of PBS to clear the vasculature of drug/liposomes. Following confirmation of euthanasia, the tumors, and tumor adjacent muscle were collected from each treatment group; snap frozen and stored in −80°C for HPLC until DTX analysis. Similar protocols were also followed for non-hyperthermia group.

Docetaxel Analysis

All tissue DTX quantification were performed at Apredica in Watertown, MA by LC/MS/MS as given below.

Tissue Homogenization

Tissue samples at a concentration of 100 mg/mL were homogenized at 4°C in acetonitrile using Mini-Beadbeater-16 (Biospec Products Inc., OK, USA) at 3450 oscillations/min for three minutes in a 2 mL polypropylene screw-cap micro vials (Biospec Products Inc., OK, USA) using zirconia beads (1 mm diameter, Biospec Products Inc., OK, USA). Homogenized tissue samples were dried at room temperature under vacuum in a desiccator before use. This method of sample preparation didn’t impact DTX stability or quantification in quality control studies.

Sample Preparation

Previously homogenized dried samples were reconstituted with water and then protein and tissue were precipitated with three volumes of methanol containing internal standard. This was then centrifuged to remove precipitated protein and the supernatant was analyzed by LC/MS/MS. All tumor samples were compared to a calibration curve prepared in blank tissue homogenate (tumor, muscle and spleen) using DTX. A linear fit calibration curve was used, with a 1/X2 weighting factor applied to the data points. LLOQ was established as 41.2 ng/g.

LC/MS/MS Equipment and Conditions

Tumor samples were analyzed by LC/MS/MS using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 HPLC and a CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent). After separation on a C18 reverse phase HPLC column (Agilent, Waters, or equivalent) using an acetonitrile-water gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in MRM mode. The signal was optimized for each compound by ESI positive or negative ionization mode. An MS2 scan or a SIM scan was used to optimize the fragmenter voltage and a product ion analysis was used to identify the best fragment for analysis, and the collision energy was optimized using a product ion or MRM scan. An ionization ranking was assigned indicating the compound’s ease of ionization.

Histological and Immunohistochemical Staining and Analysis

Both frozen and paraffin tissues were step sectioned at 8-μm thickness. Tumors were divided into three (3) regions depending on the size of the tumor; with at least 1 mm separation between each region. Serial step sections from each region were evaluated using bright field and fluorescence microscopy for overall necrosis, blood vessel density, vessel size, apoptosis (caspase-3) and cell proliferation (Ki67). For histological analysis, sections were stained with H&E using standard methods. Tumors were classified as minimal, mild, moderate, and severe necrosis using a grading scheme defined by a veterinary pathologist at the National Cancer Institute. Caspase 3 and Ki67 assay to detect tumor apoptosis was performed as described previously (21). To determine blood vessel density and size, CD31 an endothelial cell marker was enumerated as follows. Briefly, frozen sections were thawed for 15 min, hydrated with PBS, blocked with a blocking buffer (Invitrogen) for 30 min, fixed in 4%PFA for 10 min. and were incubated for 45 min. with rhodamine red-X-conjugated antiCD31 antibody (rat anti mouse Mab, BD Biosciences). Fluorescently labeled tissues were mounted with mounting medium containing DAPI (Vector Laboratories) to visualize cell nuclei as previously described (2). Whole section digital histological scans were acquired with a 20× objective on a ScanScope CS (Aperio, Vista, CA) equipped with a color CCD camera and image processing software (ImageScope, Aperio). For Ki67 and TUNEL quantification, two to three random fields were obtained using a 40× objective lens from at least three tumors per mouse and at least five mice per group. The proliferation or apoptosis index was calculated as the percentage of total cells per field that were Ki67- or Caspase-positive, respectively, using the Aperio microscope morphometric analysis software package. Vessel density and size was determined by a NIH custom-made MATLAB software package.

Bioluminescent Imaging Study

The mice were imaged weekly during the treatment using a Xenogen Lumina bioluminescent imager to track tumor cell growth. Prior to each imaging, mice were injected with D-Luciferin sodium salt (Gold Bio Inc.) 60 mg/kg I.P. 5 min.. The region of interest (ROI) was defined over the contour of each individual tumor to include all photon emission from the entire tumor. All images were formatted with the same color-coded scale for visual assessment. Decreased signal intensity from luciferase expressing cancer cell was used to track and confirm the tumor growth/regression in the various treatment groups.

Statistical Analysis

The data of the tumor progression and regression, represented as the percent change from baseline for each individual animal, was analyzed by one-way ANOVA for statistical comparison. Treatment groups were compared for differences in mean survival, tumor DTX concentration, and cell cytotoxicity using analysis of variance (ANOVA) followed by Tukeys multiple comparison post-hoc tests. All analyses were performed using GraphPad Prism 5.0 (GraphPad Software Inc.). All p-values were two-sided, and a p-value less than 0.05 indicated statistical significance. Values are reported as mean ± SEM unless otherwise indicated.

RESULTS

Tumor Drug Delivery

Tumor DTX concentrations were 1.85 ± 0.4, 4.8 ± 1.4, 6.7 ± 2.8, 9.4 ± 1.8 μg DTX/g tissue for free DTX, DTX+heat, LTSL and LTSL+heat, respectively (Fig. 1; Table II). In the tumor adjacent muscle, the DTX concentration were 1.8 ± 1, 2.85 ± 0.5, 1.54 ± 0.2, 3.9 ± 1 for free DTX, DTX+heat, LTSL and LTSL+heat, respectively. LTSL+heat resulted in a significantly greater (~4.7-fold) tumor drug delivery compared to free DTX (p < 0.05, Tukeys) and ~1.5-fold greater delivery compared to LTSL alone. The drug delivery in tumor adjacent muscles for LTSL+heat group was 2–3 fold greater than the free DTX or LTSL treatments (p < 0.05, Tukeys test).

Fig. 1.

Docetaxel (DTX) detected in mouse prostate tumor, adjacent heated and contralateral unheated muscles following treatment either with free DTX, DTX+ Heat, LTSL or LTSL+ heat at a dose of 15 mg/kg DTX. Data are shown as mean DTX concentration with standard error of mean (n = 5). * p < 0.05.

Table II.

Bio-Distribution of Docetaxel in Tumor and Adjacent Muscle in Various Treatment Groups

Treatment group	Tumor (μg DTX/gram tissue)		Muscle Adjacent (μg DTX/gram tissue)
	Average	SEM	Average	SEM
Docetaxel	1.85	0.334477204	1.8	0.906228448
Docetaxel + heat (42°C)	4.775	1.771078414	2.85	0.588960949
LTSL	6.725	2.39488387	1.375	0.159295323
LTSL+ heat (42°C)	9.325	1.732546897	3.975	1.061779167

Tumor Growth Delay and Survival Rates in the Prostate Tumor Model

The median survival for the Saline, Saline+heat, LTSL, DTX, LTSL+heat, DTX+heat treated mice was 9, 12, 17, 17, 48.5, and 44.5 days (Fig. 2). Treatment of the prostate tumor bearing mice with either free DTX or LTSL in combination with mild hyperthermia significantly increased the median survival time by more than 30 days compared to the control group. Compared to free DXT+heat, LTSL treated demonstrated a slight increase in survival (~3 days). However, these two effective treatments were not significantly different (p > 0.05). Tumor growth delay was determined by computing the difference in the number of days required for tumor size to grow to a five-fold increase from initial treatment volume relative to saline control (Table III). In the hyperthermia group, mice treated with DTX with hyperthermia had a significantly longer growth delay of 34 days compared to matched saline. Also, DTX alone treatments were not different compared to matched saline. Similarly, the tumor volume of mice till first death showed a significant reduction in free DTX and LTSL plus heat treated mice (Fig. 3).

Fig. 2.

Median survival of prostate tumor bearing mouse following different treatment. Median survival for each treatment group is shown in the Kaplan-Meier survival curve. The percent survival in the hyperthermia group (DTX & LTSL+Heat) is significantly improved compared to control, * P < 0.05 for Free DTX and LTSL in combination compared to vs. Saline (± heat), LTSL and Free DTX (± heat).

Table III.

The Tumor Growth Delay of Each Treatment Group is Determined Compared to Saline Control

Group	Growth delay (Days)	SEM
Saline+heat	4	0.7
Docetaxel	14.7	5.1
LTSL	8	1.4
Docetaxel+heat (42 C)	34.2	8.4
LTSL+ heat (42°C)	36	7.9

Fig. 3.

Tumor volumes following treatment with saline (±heat), LTSL (±heat), and DTX (±heat). Volumes are reported till first death in each group.

Histopathological Analysis

For H&E analysis, tumors were graded on a scale of 1–4 for necrosis with 1 indicating minimal necrosis and 4 representing severe necrosis. DTX treatments combined with hyperthermia resulted in severe necrosis compared to untreated control as well as DTX or LTSL alone therapy (Figs. 4a and 5a–e, P < 0.05). Programed cell deaths assessed using a caspase 3 apoptotic analysis showed sequential activation and upregulation of caspases in the heated tumor (Figs. 4b and 5f–j, P < 0.05). Similarly, quantitative assessment of Ki-67 nuclear staining on paraffin-embedded tumor sections (expressed only in cycling cells) staining revealed significantly greater reduction in expression of proliferation markers in the heated tumor both for LTSL and DTX treated mice (Figs. 4c and 5k–o, P < 0.05). Finally, an assessment of DTX plus hyperthermia therapy on density and size of micro-vasculatures in tumor using CD31 analysis indicated similar micro-vessel density across all treatment groups (Figs. 4d and 5p–t). However, the size of vessels was significantly decreased in the free DTX group suggesting that the vessels were not functionally perfused at 72 h post treatment (Fig. 4e).

Fig. 4.

Histological and fluorescence analysis of prostate cancer tumors following treatment. (a) H&E analysis showing minimal necrosis in control group and severe necrosis in Docetaxel and LTSL in combination with heat; (b) Caspase-3 are highly upregulated in the heated tumor, and are significantly different compared to other treatments; (c) Quantitative assessment of Ki-67 staining on paraffin-embedded tumor sections suggesting significantly greater reduction in expression of proliferation markers in the heated tumor; (d–e) CD31 analysis showing similar vessel density but a significant decrease in perfusion as indicated by total vessel area for LTSL plus heat group. p < 0.05 (Tukey’s multiple comparison).

Fig. 5.

Histological and fluorescence analysis of prostate tumors following treatment. (a–e) H&E staining of tumor; (f–j) Caspase-3 apoptotic staining of tumors (viable = yellow/purple, clear/white = cellular death); (k–o) Ki-67 cell proliferation staining of tumors; (p–t) Fluorescence images of vessel distribution using CD31 marker (nuclei = blue and CD31 = red).

Bioluminescent Imaging Study

Although the tumor sizes were measured with calipers, periodic imaging with an in-vivo imaging system was conducted as a visual representation of tumor progression for up to 60 days. The tumor progression as shown at 7 days indicated significant differences in photon emission from tumor cells detected by the IVIS. Notably, the mice in the heat group demonstrated significant reduction in luciferase expression consistent with survival and histopathology data for both DTX and LTSL heated tumors (Fig. 6). These findings followed a similar pattern over 60 days of treatment.

Fig. 6.

Bio-luminescent imaging (BLI) of prostate cancer cell transfected with the luciferase gene. Significant reduction in luciferase expression in the LTSL or DTX+ heated tumor was noted at 7 and 14 days post treatment.

DISCUSSION

Prostate cancer can be treated with hormones, radical prostatectomy, radiation therapy, chemotherapy or combinations of them depending on the stage of the disease (22–27). The objective of this study was to understand the impact of hyperthermia in enhancing survival and chemotherapy distribution in prostate tumor bearing mice. Traditionally, mild hyperthermia between 40 and 45°C can increase tumor blood flow, oxygenation level, sensitivity to and uptake rates of chemotherapeutic drugs, and vessel permeability; all of which can potentially enhance drug accumulation in tumor tissues especially when combined with LTSLs (15,28–32). For example, we and others found that LTSL with a clinical magnetic resonance-guided high-intensity focused US (MR-HIFU) hyperthermia platform can enhance intratumoral doxorubicin distribution by 7-fold compared to free drug in a rabbit Vx2 tumor model (12,33,34). Such demonstration of drug delivery has mostly focused on delivery of water soluble drugs (e.g. Doxorubicin) (14,35), from LTSLs. In contrast to water soluble drugs, delivery of water insoluble hydrophobic drugs (e.g. DTX) using LTSL is challenging due to the poor entrapment and release of encapsulated content from liposomes in serum rich systemic circulation (36,37). In this study, a clinical grade LTSL encapsulated DTX that was characterized under GMP conditions was kindly provided by Celsion corporation to investigate hyperthermia-induced release of DTX from LTSLs in vivo. Data suggest that the biodistribution of DTX-LTSL in the mice prostate tumor following hyperthermia was significantly greater (~5 and 1.5 fold compared to free DTX and LTSL without heat, Fig. 1, (Table II). A possible mechanism is that hyperthermia reduced interstitial Fluid Pressure (38) and improved tumor perfusion (39) that then established a high intravascular drug concentration leading to greater drug coverage in the tumor. Further, the delivery of free DTX to tumor and muscle in presence of heat was ~2fold compared to DTX alone. Previously, Yeo et al. reported a DTX-prodrug encapsulated themonsensitive liposome with low accumulation in reticulo-endothelial system in nude rats (16). However, the pro-drug DTX-LTSL was not utilized for demonstration of drug delivery in tumor model. To address this limitation, we utilized a clinically relevant prostate cancer model to simulate human tumor conditions. Thus, we believe that the preclinical demonstration of a significantly greater DTX delivery in tumors compared to DTX alone in our study provides important insights and basis for translation of this technology in patient population.

A variety of combinatorial approaches have been investigated for enhancing DTX efficacy (40,41). For instance, DTX efficacy was shown to be markedly increased by co-administration with an analog of noscapine, a naturally occurring nontoxic plant alkaloid (42). Similarly, the combination of DTX with Imatinib mesylate, a platelet-derived growth factor receptor (PDGFR) inhibitor in men with androgen independent prostate cancer (AIPC) significantly decreases the PDGFR-expressing tumor burden in the bone marrow (43). In this study, we attempted to determine whether the action of DTX could be potentiated in the presence of hyperthermia. The fundamental basis of this hypothesis originated from previous studies where heat was shown to modify the cytotoxicity of many chemotherapeutic agents (e.g. cyclophosphamide, ifosfamide, 5-fluorodeoxyuridin and methotrexate) (44). A variety of mechanisms have been proposed including increased rate constants of alkylation, increased drug uptake and inhibition of repair of drug-induced lethal or sublethal damage. Further, these studies have shown that hyperthermia applied directly following drug administration was most effective (44). Most importantly, significant tumor size reduction and tumor growth delay in mice with fibrosacoma treated with free DTX in combination with heat (41.5°C) has been described (8,45). Recently, Zhang et al. reported a DTX-loaded thermosensitive liposomes composed of DPPC–DSPE-PEG2000–EPC–MSPC–DTX (molar ratio: 82:11:4:3:4) with greater tumor inhibition ratio of DTX-TL group than the unheated liposome (17). Thus, a similar translation of this idea using a GMP grade LTSL for achieving site selective drug delivery, and hyperthermic potentiation of prostate chemotherapy is a feasible idea. Our tumor volume as a function of time suggest that the cell killing of DTX in the encapsulated and free form was significantly improved in the presence of hyperthermia, and this resulted in a drastic increase in survival response compared to no heat groups (Figs. 2 and 3). Further, the molecular markers of apoptotic ell death such as caspase-3 genes, (46) were upregulated only on the heat treated groups (free DTX and LTSL) (Fig. 4b). Similarly, the nuclear expression of Ki-67, a marker expressed in proliferating cells that is required for maintaining cell proliferation (34) was significantly decreased (P < 0.05) in the heated tumor (Fig. 4c). This suggests that hyperthermia can serve as an adjuvant to chemotherapy of DTX in both free and encapsulated state. Interestingly, in mice model of prostate cancer the survival achievable between free and encapsulated form of DTX in presence of hyperthermia was similar. This provides a strong basis of future investigation of free DTX and hyperthermia combination in clinical trials. However, clinically free DTX is cleared fast, and thus an encapsulated carrier would provide much better control of drug delivery as demonstrated in our biodistribution studies. More detailed studies are needed with larger cohorts of animals especially in orthotopic models to verify these findings. Additional areas of future work can also focus on sequence, duration, and frequency of such treatments in orthotopic and xenograft mode especially in combination with applicators such as MR-HIFU, and electromagnetic deep heating devices. This will guide initial clinical trials with DTX alone, and their subsequent translation with DTX-LTSL. Finally, at single dose treatment of DTX-LTSL, we did not notice significant decrease in body weight in the treated animals compared to saline control (data not shown) suggesting that the tolerance to LTSL upon parenteral treatment was good in the mice model. We also tracked the animal tumors longitudinally using in vivo bioluminescence imaging to track tumor growth. To do so, mice were inoculated with human prostate cancer cells that were expressing luciferase genes. In many of the DTX-LTSL plus heated tumor, detectable signals in the inoculated region and other organs (liver, lung, kidney) was not demonstrated after 1–2 weeks and this continued for 60 days treatment period (Fig. 6). These findings suggest that an elevated temperature can improve drug delivery without drastically changing or enhancing the malignancy profile of solid tumors. Studies are underway to determine these mechanisms in more detail.

CONCLUSION

This study demonstrates that combining DTX encapsulated LTSLs with mild hyperthermia enhanced drug accumulation in the model prostate tumor and prolongs survival. DTX in combination with heat improves treatment efficacy. Preliminary evidence suggests that the mechanism of action is by an increase in apoptosis response. Bioluminescent emission after 2 weeks of treatment demonstrates no signs of re-growth in the heated regions. This combination approach has potential for clinical translation as a novel means to improve prostate cancer therapy.

ABBREVIATIONS

DLS

Dynamic light scattering

DPPC

Dipalmitoylphosphatidylcholine

DSPE-PEG

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000

DSPG

1,2-dioctadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol)

DTX

Docetaxel

H&E

Hematoxylin & Eosin

IVIS

In vivo imaging system

LC/MS

Liquid chromatography–mass spectrometry

LTSL

Low temperature sensitive liposomes

MSPC

1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine

PBS

Phosphate buffered Saline