Cancer Therapy: Preclinical Noninvasive Radiofrequency Field Destruction of Pancreatic Adenocarcinoma Xenografts Treated with Targeted Gold Nanoparticles

Purpose: Pancreatic carcinoma is one of the deadliest cancers with few effective treatments. Gold nanoparticles (AuNP) are potentially therapeutic because of the safety demonstrated thus far and their physiochemical characteristics. We used the astounding heating rates of AuNPs in nonionizing radiofrequency (RF) radiation to investigate human pancreatic xenograft destruction in a murine model. Experimental Design: Weekly, Panc-1 and Capan-1 human pancreatic carcinoma xenografts in immunocompromised mice were exposed to an RF field 36 hours after treatment (intraperitoneal) with cetuximabor PAM4 antibody–conjugated AuNPs, respectively. Tumor sizes were measured weekly, whereas necrosis and cleaved caspase-3 were investigated with hematoxylin–eosin staining and immunofluorescence, respectively. In addition, AuNP internalization and cytotoxicity were investigated in vitrowith confocal microscopy and flow cytometry, respectively. Results: Panc-1 cells demonstrated increased apoptosis with decreased viability after treatment with cetuximab-conjugated AuNPs and RF field exposure (P1⁄4 0.00005). Differences in xenograft volumes were observed within 2 weeks of initiating therapy. Cetuximaband PAM4-conjugated AuNPs demonstrated RF field–induced destruction of Panc-1 and Capan-1 pancreatic carcinoma xenografts after 6 weeks of weekly treatment (P 1⁄4 0.004 and P 1⁄4 0.035, respectively). There was no evidence of injury to murine organs. Cleaved caspase-3 and necrosis were both increased in treated tumors. Conclusions: This study demonstrates a potentially novel cancer therapy by noninvasively inducing intracellular hyperthermia with targeted AuNPs in an RF field. While the therapy is dependent on the specificity of the targeting antibody, normal tissues were without toxicity despite systemic therapy and whole-body RF field exposure. Clin Cancer Res; 16(23); 5712–21. 2010 AACR. Despite decades of research in the biology and treatment of pancreatic carcinoma, it remains one of the deadliest and least curable forms of cancer (1, 2). Targeted therapy against antigens overexpressed in pancreatic cancer, such as epidermal growth factor receptor (EGFR-1), has only been minimally successful despite its use in other antigen overexpressing cancers (3). Clearly, novel therapeutic approaches to treat this disease that kills more than 95% of diagnosed patients are needed (4). Previous reports have demonstrated that metal nanoparticles induce hyperthermic cytotoxicity in vitro by exposing the nanoparticles to one of a few forms of nonionizing radiation, specifically near-infrared (NIR) and radiofrequency (RF; refs. 5–8). Furthermore, tumor necrosis has been demonstrated by directly injecting nanoparticles into rodent and rabbit syngeneic cancer implants that subsequently underwent noninvasive RF field exposure (9, 10). Importantly, normal tissues tolerate hyperthermia at higher temperatures and for longer periods of time than malignant tissues, portending an opportunistic thermal cancer treatment (11). The previous experimental models suffer from multiple challenges. First, NIR radiation does not penetrate deeply into tissue, limiting its use to superficial malignancies (12–14). Second, if a direct intratumoral injection of nanoparticles were required, then it would necessitate that the tumor be visualized by traditional imaging and an invasive procedure be required to actually inject the nanoparticles. Furthermore, direct injection is problematic because nanoparticles will diffuse through malignant and surrounding normal tissue, increasing the likelihood of damage to normal cells. Multiple nanoparticles (6, 8, 15) such as gold, silver, and semiconducting Authors' Affiliations: Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center; Department of Biomedical Engineering, Rice University; Department of Veterinary Medicine and Surgery, The University of Texas M. D. Anderson Cancer Center; and Department of Mechanical Engineering and Materials Science, Rice University, Houston, Texas Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Steven A. Curley, Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, 1400 Holcombe Blvd, Unit 444, Houston, TX 77030. Phone: 713-794-4957; Fax: 713-745-5235. E-mail: [email protected] doi: 10.1158/1078-0432.CCR-10-2055 2010 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 16(23) December 1, 2010 5712 Research. on May 5, 2017. © 2010 American Association for Cancer clincancerres.aacrjournals.org Downloaded from nanoparticles are candidates for hyperthermic treatment, but gold has the most immediate potential for use in human patients and seems to have a favorable safety profile (5, 16, 17). On the basis of previous in vitro work (8), we hypothesized that systemic delivery of antibody targeted gold nanoparticles (AuNPs) would induce hyperthermic cytotoxicity after RF field exposure in human pancreatic carcinoma xenografts without injury to normal tissues. Antibodies to 2 distinct human antigens (EGFR-1 and MUC-1) were utilized to deliver 2 AuNPs of different sizes to 2 unique human pancreatic xenografts. Although EGFR-1 is a problematic therapeutic target due to its diverse constitutive expression in normal tissues, PAM4 is a human antibody to MUC-1 that is specific to pancreatic carcinoma (18). The components were chosen such that the constructs had similar sizes that could lead to increased tumor internalization rates (19). The primary aim was to show human pancreatic cancer xenograft destruction. Materials and Methods Cell culture, antibodies, fluorophores, and gold nanoparticles Two human pancreatic carcinoma cell lines, Panc-1 and Capan-1, were acquired from the American Type Culture Collection (ATCC) confirmed by the Characterized Cell Line Core Service (M. D. Anderson Cancer Center; December 2009), and maintained according to ATCC’s cell media recommendations in standard conditions (37 C, 5%CO2). All experiments utilized standard cell culture coated dishes and equipment (BD Biosciences; Corning Inc.). Cetuximab (C225; Bristol-Myers Squibb), a chimeric monoclonal IgG1 antibody against human EGFR-1 was conjugated to spherical 10-nm AuNPs (Ted Pella, Inc.) via a linker. PAM4 (Immunomedics, Inc.), a human monoclonal antibody against a mucin glycoprotein, MUC-1, was directly conjugated to 20-nm AuNPs (Ted Pella, Inc.) via a thiol–gold bond described later. All fluorophores or fluorophore conjugates were used as directed by the manufacturer (Invitrogen Corp.). AuNP constructs and characterization C225 was conjugated via covalent hydrazide-thiol heterobifunctional linker (Sensopath Technologies, Inc.) from a previously published protocol with slight modifications based on glycosolation of the Fc region (20). Briefly, a solution of 10-nm AuNPs were twice washed in a borate buffer solution at pH8. A total of 450mg ofC225with linker was slowly added to a 1,000 mg of AuNP solution. It was placed on a continuous mixer and incubated at room temperature for 4 hours. Next, the conjugate was concentrated 15-fold in a 50,000 molecular weight centrifugation filter unit (Millipore Corp.) at 3,800 g. Because PAM4 does not have the same glycosolation status in the Fc region as C225, it was directly conjugated to slightly larger AuNPs without a linker via a thiol– gold interaction. First, PAM4 IgG was washed in borate buffer (pH 8) twice and resuspended at a concentration of 2 mg/mL. A monovalent, single-molecule IgG (Fc þ Fab with reactive sulfur groups on the heavy chain formerly of the disulfide bond) was created by reducing the interchain disulfide bond at the hinge region with 3 molar excess of tris(2-carboxyethyl)phosphine, which is a relatively gentle, but very specific, nonsulfur-containing disulfide bond reducing agent that does not reduce heavy-light intrachain disulfide bonds or internal disulfide bonds at the antigen binding site at these concentrations (21). Subsequently, 450 mg of ‘‘activated’’ PAM4 was mixed with 1,000 mg of 20-nm AuNP in borate buffer and mixed for 4 hours in the dark at room temperature to permit the reduced sulfur moieties to directly form thiol–gold bonds on the AuNPs. The construct was twice washed with borate buffer, concentrated 15-fold in a 50,000 molecular weight centrifugation filter unit (Millipore Corp.) at 3,800 g. A small shift in the peak plasmonic absorbance of the AuNPs (NS1; Applied NanoFluorescence) was indicative of a nonaggregated conjugation state after challenge with equivolume of 10% sodium chloride (21). Dynamic light scattering (DLS; Horiba, Ltd.) determined the average hydrodynamic diameter of the constructs (500 measurements per sample in triplicate). Target protein expression in Panc-1 and Capan-1 cells with immunoprecipitation The cellmembrane expressionof EGFR-1 andMUC-1was confirmed byWestern blot analysis. Briefly, cell pellets were lysed with cold radioimmunoprecipitation assay buffer and centrifuged at 13,000 rpm for 30 minutes. The protein extracts (50 mg per lane) were electrophoresed on Bis-Tris protein gel, transferred to a polyvinylidene difluoridemembrane, and sequentially incubated in 5% dry milk and primary antibodies (BD Biosciences and Immunomedics, Inc.). Next, the membranes were incubated with secondary anti-human IgG antibodies (H þ L chains; Jackson ImmunoResearch, West Grove, PA). Images were acquired by a high-resolution photoscanner (CanoScan 4400F; Translational Relevance Nanoparticle-mediated hyperthermic therapy offers a treatment that potentially will simultaneously have less adverse effects than systemic chemotherapy and a more direct action on pancreatic cancer cells. Gold colloids have a long history of minimal adverse effects, whereas nonionizing radiation is known to be safe. However, because AuNPs heat in nonionizing radiation, there is the potential to noninvasively target or direct the hyperthermic effect. Pancreatic cancer, one of the deadliest cancers, has multiple targeted therapies in development. As we have shown here, the proper combination of a targeting antibody conjugated with AuNPs results in effective tumor destruction with minimal toxicity. Nanoparticle and RF Field–Induced Tumor Destruction www.aacrjournals.org Clin Cancer Res; 16(23) December 1, 2010 5713 Research. on May 5, 2017. © 2010 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Canon, Inc.) after the bands were detected with a peroxide solution (GenDEPOT, Inc.). Confocal imaging of antibody-conjugated AuNPs in vitro Confocal images were prepared by growing Panc-1 cells on standard 1.5 glass cover slips, which were treated with AlexaFluor (AF) 647–labeled C225 (the primary antibody delivered to living cells), 10-nm AuNP alone, the labeled C225-AuNP–conjugated construct, or neither for 3 hours. Cell membranes were stained with 5 mg/mL of wheat germ agglutinin conjugated to AF 594 (Invitrogen Corp.) by incubating the cover slip for 10 minutes at 37 C. After phosphate buffered saline (PBS) washings, the cells were fixed with 4% paraformaldehyde and permeabilized with cold 100% methanol. Blocking was performed for 1 hour with blocking solution [3% bovine serum albumin (BSA) þ 1% animal serum in PBS). Cells were washed and stained with AF 488–labeled secondary antibody against the heavy and light chains of human IgG (Invitrogen Corp.). After washing, DNA was stained with 40,6-diamidino-2-phenylindole dihydrochloride (DAPI; 300 nmol/L) for 5 minutes. Cover slips were mounted (Dako Denmark A/S, Glostrup, Denmark), dried, sealed, and stored in the dark at 4 C until analyzed (Olympus DSU with Orca II ER camera; Olympus America Inc., Center Valley, PA). In a separate experiment, Panc-1 cells were treated with AF 647–labeled C225 at the same concentration with or without AuNPs to determine the percentage of cells in vitro with measurable amounts of primary fluorophore–labeled C225-AuNP. Cells were identified in the usual manner by an automated cytometer while the fluorescence of AF 647 was simultaneously measured for individual cells (Cellometer Vision; Nexcelom Bioscience, LLC). A fluorescence threshold of 7 arbitrary units was chosen to separate positive from negative populations, based on the control sample. For each group, 1,800 cells were counted and a histogram was created for each group with the same binning (Sigma Plot Version 11; Systat Software Inc.). TEM imaging in vitro Cell pellets were fixed with a 3% glutaraldehyde/ 2% paraformaldehyde solution in 0.1 mol/L of cacodylate buffer at pH 7.4. Samples were washed with 0.1% cacodylate-buffered tannic acid, treated with 1% buffered osmium tetroxide, and stained with 1% uranyl acetate. The samples were ethanol dehydrated and embedded in LX-112 medium. After polymerization, the samples were cut with a Leica Ultracut microtome (Leica), double stained with uranyl acetate/lead citrate in a Leica EM stainer, and imaged with a JEM 1010 TEM (Jeol USA, Inc.) at an accelerating voltage of 80 kV. Images were acquired with AMT Imaging System (Advanced Microscopy Techniques Corp.). Determination of gold concentration with inductively coupled plasma atomic emission spectrometry Cell pellets, tumor specimens, and organ specimens (liver, spleen, kidneys, and lung) were sectioned, weighed (cells were counted), and gently washed with PBS. Samples were partially digestedwith 2mLof certified 30%H2O2 and evaporated. Next, 5 mL of aqua regia (1 part of nitric acid combined with 3 parts of hydrochloric acid by volume, in a fume hood) was slowly added to each sample as the temperature was slowly raised to 130 C in order to completely digest the samples. After 3 hours, the samples were passively cooled, and diluted with 18-MW water to a final volume of 10 mL. The gold concentration of each sample was determined by inductively coupled plasma atomic emission spectrometry (ICP) according tomanufacturer’s recommendations (iCAP 6500; Thermo Fisher Scientific). Nanoparticle heating in an RF field Triplicate solutions of various concentrations of AuNPs alone or conjugated to antibodies were placed in the RF field in a transparent, 1.5-mL quartz cuvette directly on a copper ground plate and exposed to a high-voltage RF field at 600-W generator power for 1minute (13.56MHz, 10-cm air gap; ThermMed, LLC, Inc.). Temperatures were recorded every 30 seconds with an IR camera (FLIR SC 6000; FLIR Systems, Inc.). Panc-1 in vitro RF field exposure and cytotoxicity after C225-AuNP treatment Panc-1 cells were plated in quadruplicate at a concentration of 1 10 cells/mL in 60-mm dishes and incubated overnight. Negative controls remained untreated, whereas the others were treated with C225 alone or C225-AuNP. Separate groups (n1⁄4 4 each) underwent RF field exposure at 600 W for 10 minutes. Bulk media temperature remained between 36 Cand 41 C, asmeasured by an IR camera (FLIR SC 6000). Viability was measured with flow cytometry (LSRII; BD Biosciences) 36 hours after exposure. Briefly, cell media (i.e., dying cells that were floating) was collected and the adherent remaining cells were releasedwith trypsin. The trypsin was then neutralized with cell media and the cells were collected. Each sample was washed and stained without fixation or permeabilization. After a final wash, 50,000 cellular events were acquired for each sample and analyzed with FlowJo 8.8.6 (TreeStar, Inc.). After gating the single cell population, Annexin V, a protein that binds to membrane protein phosphatidylserine, positive cells were considered apoptotic; whereas propidium iodide (PI), a chemical that fluoresces when bound to DNA, positive cells were considered necrotic. Annexin V and PI doublepositive cells were considered dead cells, whereas doublenegative cells were characterized as viable. Panc-1 and Capan-1 pancreatic xenografts AuNP treatment and RF field exposure Nude balb/c mice (NCI Mouse Repository) were subcutaneously injected with 3 10 Panc-1 or Capan-1 cells to the right flank (n 1⁄4 20 each cell line). After the tumors were palpable (approximately 3 weeks), mice within each cancer cell type were randomly assigned to 1 of 4 groups. The mice with Panc-1 xenografts were treated/exposed to no treatment, C225-AuNP without RF exposure, RF expoGlazer et al. Clin Cancer Res; 16(23) December 1, 2010 Clinical Cancer Research 5714 Research. on May 5, 2017. © 2010 American Association for Cancer clincancerres.aacrjournals.org Downloaded from sure alone, or C225-AuNP treatment plus RF exposure. The mice were treated with C225-AuNP at 10 mg/kg by gold weight injected intraperitoneally to prevent AuNP collection at the site of an extremity (i.e., tail vein). RF exposure consisted of 600-W generator power for 10 minutes with an air gap of 10 cm. All antibody-AuNP treatments occurred weekly, whereas all RF exposures occurred 36 hours after treatments on a weekly basis. Tumors were measured 48 hours after RF exposure each week, whereas C225-AuNP treatment and RF exposure began after week 1. Mice with Capan-1 tumors were randomly assigned to 1 of 4 groups as well (n 1⁄4 5 each group). Those groups were untreated control, PAM4-AuNP treatment only, unconjugated AuNP treatment plus RF field exposure (600-W generator power, 10-minute duration, and 10cm air gap), and PAM4-AuNP treatment weekly with the same RF exposure weekly. Unconjugated AuNP and PAM4AuNP were also treated intraperitoneally at 10 mg/kg by gold weight for each. During the experiment, all mice were kept in accordance with an Institutional Animal Care and Use Committee approvedprotocol. To safely expose the animals toRF fields, their tails, ears, and paws were completely grounded to avoid excess current in the extremities that would result in electrothermal injuries. This was accomplished by placing micedirectly ona large grounded copperplate andattaching conducting copper tape (3M) to the extremities and plate. Mice were sedated with ketamine 0.1 mg/g and xylazine 0.01 mg/g intraperitoneally prior to each RF field exposure and monitored thereafter. The temperature of each mousewas continuouslymeasuredwith an IR camera (FLIR SC6000), andatno timedid the temperature exceed41.5 C. For the firstminute of every RF field exposure, a cuvettewith 100mg/mLof 20-nmAuNPswas placedwithin 1.5 cmof the tumor. This served as the control to confirm heating of AuNPs in the RF field. After 1 minute, the RF field generator was briefly turned off (<2 seconds) to remove the cuvette, as it approached boiling temperatures soon thereafter. Tumor volumes (width squared length)weremeasured weekly with electronic calipers. After 6 weeks of treatment, the animals were euthanized and selected organs (liver, spleen, kidney, lung, and tumor) were harvested for gold biodistribution and histopathologic evaluation. For evaluation of normal organ and tumor-specific injury, specimens were prepared for histologic procedures, embedded in paraffin, and sectioned at 5 mm. The sectionswere stained with hematoxylin and eosin stain (H&E) and examined by lightmicroscopy. Injury was assessed by grade (grade 1: rare <10%; grade 2: mild, 10%–20%; grade 3: moderate, 20%– 50%; and grade 4: severe, >50%) by an expert in comparative mammalian pathology (A.N.H.). Confocal immunofluorescent microscopy in vivo Tumor sections on standard glass slides were deparaffinized by heating to 60 C for 1 hour. They were then dewaxed and rehydrated by sequential washing for 5 minutes in xylene (3 ) followed by decreasing concentrations of ethanol in water. Sections were then placed in boiling antigen retrieval buffer (citrate based, pH 1⁄4 6, 0.5% Tween-20). This was heated in amicrowave at boiling temperatures for an additional 1 minute. Next, the sections were microwaved in the buffer for 15 minutes at 30% power. Sections were then washed with PBS and placed in blocking solution for 1.5 hours (1% BSA and 2% fetal bovine serum in PBS). Sections were stained with an antibody to cleaved caspase-3 (Cell Signaling Technology, Danvers, MA) diluted 1:250 in blocking solution for 1 hour at room temperature. Sections were thrice washed with PBS and a secondary antibody conjugated to AF 488 was applied to each (1:300) for 1 hour at room temperature. Sections were thrice washed in PBS, stained with DAPI (300 nmol/L for 10minutes), and subjected to a final washing in PBS. Fluorescent mounting media was applied (Dako North America, Inc.) and the sections were sealed with a 1.5 glass cover slip. Statistical methods Results are means standard errors of the mean unless otherwise noted. Statistical significance, a, was set to P 1⁄4 0.05. Two-tailed Student’s t test compared differences in means between groups, whereas multiple-way analysis of variation analyzed tumor volumes (SPSS Version 16.0, SPSS Inc.). Data were plotted with Sigma Plot Version 11 (Systat Software Inc.).