Molecular Cancer Therapeutics
Large Molecule Therapeutics
SapC–DOPS Nanovesicles as Targeted Therapy for Lung Cancer Shuli Zhao1, Zhengtao Chu2,3, Victor M. Blanco2, Yunzhong Nie4, Yayi Hou4, and Xiaoyang Qi2,3
Abstract Lung cancer is the deadliest type of cancer for both men and women. In this study, we evaluate the in vitro and in vivo efficacy of a biotherapeutic agent composed of a lysosomal protein (Saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS), which can be assembled into nanovesicles (SapC–DOPS) with selective antitumor activity. SapC–DOPS targets phosphatidylserine, an anionic phospholipid preferentially exposed in the surface of cancer cells and tumor-associated vasculature. Because binding of SapC to phosphatidylserine is favored at acidic pHs, and the latter characterizes the milieu of many solid tumors, we tested the effect of pH on the binding capacity of SapC–DOPS to lung tumor cells. Results showed that
SapC–DOPS binding to cancer cells was more pronounced at low pH. Viability assays on a panel of human lung tumor cells showed that SapC–DOPS cytotoxicity was positively correlated with cell surface phosphatidylserine levels, whereas mitochondrial membrane potential measurements were consistent with apoptosis-related cell death. Using a fluorescence tracking method in live mice, we show that SapC–DOPS specifically targets human lung cancer xenografts, and that systemic therapy with SapC–DOPS induces tumor apoptosis and significantly inhibits tumor growth. These results suggest that SapC–DOPS nanovesicles are a promising treatment option for lung cancer. Mol
Introduction
drugs, they provide improved biocompatibility, pharmacokinetics, biodistribution, and toxicologic profile. Despite promising results in preclinical models of lung cancer (6) and many other cancer types (7), only a few nontargeted formulations (i.e., relying on passive tumor accumulation through the enhanced permeability and retention effect) have been approved for cancer treatment by regulatory agencies (8, 9). Clinical trials are under way to evaluate novel liposomal formulations of the chemotherapy drugs paclitaxel (LEP-ETU; ref. 10) and cisplatin (Lipoplatin; ref. 11) in patients with lung cancer. Liposomes can deliver large drug payloads but, as numerous clinical studies have shown, this does not always translate into enhanced therapeutic efficacy; results so far show that liposomal drug delivery offers reduced toxicity, but only modest survival benefits, compared with free drug administration (12, 13). In this study we report a novel targeted biotherapeutic agent, saposin C-dioleoylphosphatidylserine (SapC–DOPS) proteoliposomes, with selective antineoplastic activity on lung cancer cells both in vitro and in vivo. SapC is a small glycoprotein present in all tissues that acts as biologic activator of lysosomal enzymes, such as acid b-glucosidase, acid sphingomyelinase, and acid b-galactosylceramide, which degrade fatty acid substrates resulting in the production of ceramide, a key intermediate in the synthesis of sphingolipids, and a well-known apoptosis inducer (14, 15). SapC has a strong affinity for negatively charged phospholipids such as phosphatidylserine (PS), to which it binds more avidly at acidic pH (16–18). Because the expression of PS is relatively abundant in the surface of tumor cells and tumor-associated vasculatures (19, 20), but not in normal cells, and in view of the characteristically acidic tumor microenvironment that results from glycolytic conversion of glucose into lactate, we hypothesized that SapC–DOPS nanovesicles could be an effective antitumor agent. Previous work from our group showed that SapC–DOPS targets a variety of cancer cells in vitro and in vivo, and has anticancer
Lung cancer is the most common and the deadliest type of cancer worldwide, accounting for 1.8 million new cases and 1.6 million deaths in 2012 (1). Cigarette smoking is the single leading cause of lung cancer, followed by environmental and occupational exposure to pollutants such as radon, arsenic, asbestos, Bis(chloromethyl) ether, chromium, nickel, vinyl chloride, and polycyclic aromatic compounds (2–4). Startlingly, a direct causal relationship between atmospheric particulate matter (air pollution) and lung and other cancers was recently established. As reported by the International Agency for Research on Cancer, more than half of lung cancer–related deaths caused by air pollutants worldwide occurred in China and other East Asian countries (5). Standard treatment options for lung cancer (chemotherapy, radiation, and surgery) have several undesirable side effects that affect the quality of life of the cancer patient. Liposomal formulations as vehicles for drug delivery are the subject of intense research. Compared with nonencapsulated, free
1 State Key Laboratory of Reproductive Medicine, Central Laboratory of Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China. 2Division of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio. 3Division of Human Genetics, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio. 4Immunology and Reproductive Biology Laboratory, Medical School and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, Jiangsu, China.
Corresponding Author: Xiaoyang Qi, Division of Hematology and Oncology, Department of Internal Medicine, College of Medicine, University of Cincinnati, The Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH 45267. Phone: 513-558-4025; Fax: 513-558-6703; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-14-0661 2015 American Association for Cancer Research.
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effects in animal models of human solid tumors (21–26). In this study, we evaluate the tumor-targeting and cytotoxic potential of SapC–DOPS on human lung cancer cells, and assess its effectiveness in suppressing lung carcinoma growth in vivo. To this end, we first describe the pH dependence of SapC–DOPS cell binding and assess its cytotoxicity in a panel of lung cancer cells. The induction of apoptosis in cultured lung cancer cells is confirmed through DNA fragmentation analysis. Next, we use in vivo fluorescence imaging to assess SapC–DOPS tumorselective targeting in allogeneic lung tumor mouse models. Finally, the therapeutic efficacy of SapC–DOPS against tumor growth is studied in nude mice bearing human lung cancer xenografts. Our results show that SapC–DOPS effectively targets lung cancer cells in vitro and in vivo, and possesses antitumor activity in a preclinical model of human lung cancer.
Materials and Methods Cell culture The human lung carcinoma A549 and mouse Lewis lung carcinoma (LLC) cell lines were from American Type Culture Collection (ATCC; 1998 and 2001). The human lung carcinoma cell lines SPC-A-1, H1299, NCI-H661, SK-MES-1, NCIH460, and 801-D were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences from 1999 to 2010. Cells were routinely tested for mycoplasma contamination. No authentication of the cell lines was done by the authors. Human cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 mg/ mL streptomycin and 100 U/mL penicillin. LLC cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 100 mg/mL streptomycin, and 100 U/mL penicillin. Culture media and FBS were purchased from Life Technologies. All cell lines were cultured at 37 C in a humidified 5% CO2 atmosphere. Preparation of SapC–DOPS nanovesicles Dioleoylphosphatidylserine (DOPS; Avanti Polar Lipids Inc.) was dried under a stream of N2 gas, combined with recombinant SapC protein (27) in citrate/phosphate buffer (pH 5.0), and bath sonicated in PBS as described in detail previously (21). To fluorescently label SapC–DOPS nanovesicles, an aliquot of CellVue Maroon (CVM; Molecular Targeting Technologies Inc.) in ethanol was dried together with DOPS before SapC addition and bath sonication. Free CVM was eliminated from this preparation by filtration through a Sephadex G25 column (PD-10; Amersham Pharmacia Biotech). For in vivo treatment, a lyophilized formulation was used: dry DOPS was suspended in 80% tert-butanol. SapC and sucrose (10 mg/mL) were dissolved in water. DOPS suspension and SapC plus sucrose (1:0.6, vol:vol) was lyophilized in a freeze dryer (VirTis Unitop 1000L linked to a Freezemobile 25XL; The VIRTIS). The stable powder cake was resuspended in saline solution to form SapC–DOPS nanovesicles. Vesicles were monitored with a submicron particle size analyzer (Coulter Model N4 Plus). The molar ratio of SapC: DOPS was 1:7. SapC–DOPS binding in vitro To evaluate the influence of pH on the binding of SapC–DOPS to tumor cells, SapC–DOPS–CVM (containing 50 mg/mL SapC) or free, equimolar CVM were dissolved in complete RPMI-1640
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medium with different pH values (adjusted by addition of NaOH or HCl). A549 cells were collected by trypsinization, washed twice with PBS, and incubated in media adjusted to pH 5.5, 6.6, 7.5, or 8.5 for 2 hours at 37 C. The cells were then washed twice with PBS and analyzed by flow cytometry (FACSCanto II; BD Biosciences), using excitation at 633 nm and a 760 nm band-pass filter to detect CVM emission. Viability assays The colorimetric Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories) was used to monitor the cytotoxic effects of SapC–DOPS. Lung carcinoma cells were plated at a density of 5,000 cells per well in 96-well plates in triplicate. After 12 hours in culture, cells were treated with SapC–DOPS at final SapC concentrations of 0 (control), 0.5, 1, 2, 4, 8, 16, 32, 64, or 128 mg/mL. After 72 hours, 10 mL CCK-8 was added to each well. Optical density (OD) values were obtained from absorbance readings at 450 nm. Viability was calculated as the percentage of viable cells compared with control using the formula: growth inhibition (%) ¼ (ODC ODT)/ODC 100, where ODC and ODT are the OD values of the control and treated samples, respectively. IC50 was calculated by linear interpolation using the formula: IC50 ¼ [(50 A)/(B A)] (D C) þ C, where A is the first point on the curve, expressed as percent inhibition, that is less than 50%; B is the first point on the curve, expressed as percent inhibition, that is greater than or equal to 50%; C is the concentration of inhibitor that gives A% inhibition; and D is the concentration of inhibitor that gives B% inhibition (28). Phosphatidylserine levels, DNA fragmentation, and mitochondrial membrane potential measurements Membrane phosphatidylserine (PS) levels were measured using Annexin V-FITC (Invitrogen) according to the manufacturer's instructions. In brief, cells were trypsinized, washed with PBS, and resuspended at 2,500,000 cells/mL in Annexin V binding buffer. Aliquots of cells (0.2 mL/tube) were incubated with 10 mL of Annexin V-FITC for 15 minutes at room temperature in the dark. Then, 800 mL of binding buffer was added to each tube. Samples were kept on ice and analyzed by flow cytometry within 1 hour. Analysis of DNA fragmentation and mitochondrial membrane potential (Dcm) measurements were carried out in A549 cells cultured at a density of 5 105 cells per well in 6-well plates. Twenty-four hours after plating, cultures were treated with SapC–DOPS (containing 0, 10, 20, or 40 mg/mL of SapC) for 48 hours. To identify apoptotic cells by DNA fragmentation analysis, cells were harvested, washed with PBS, and fixed in 70% ice-cold ethanol overnight. Fixed cells were incubated with 20 U/mL RNase I and 50 mg/mL propidium iodide (PI) for 30 minutes. PI signal was quantified by flow cytometry and cells in the sub-G0/G1 phase of the cell-cycle were identified as apoptotic. Changes in Dcm were monitored using the lipophilic cationic probe 5,50 ,6,60 -tetrachloro-1,10 ,3,30 - tetraethyl-benzimidazolcarbocyanine iodide (JC-1). After treatment for 48 hours, the cells were trypsinized, washed with PBS, and incubated with JC-1 as per the manufacturer's instructions at 37 C in the dark for 30 minutes. Dcm was measured by flow cytometry by exciting with a 488 nm laser line and measuring emitted fluorescence with the FL1 (green) and FL2 (red) channels.
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Targeted Lung Cancer Therapy with SapC–DOPS
housed in specific pathogen-free conditions at Drum Tower Hospital Animal Center, Nanjing, China with controlled temperature (21 2 C), humidity (55 5%), and light (12 hour light/dark cycle). Water was available to mice ad libitum. After the mice had acclimated for 2 weeks, 2 106 A549 cells in 0.1 mL PBS were injected subcutaneously into their dorsal right flanks. Mice were monitored for solid tumor growth for approximately 10 days, and when the tumor volume reached 100 mm3, they were randomly assigned to 5 groups (n ¼ 6/group). The mice then received intravenous injections (0.2 mL) every other day for 5 weeks as follows: group 1 (control, normal saline), group 2 (cisplatin 4 mg/kg), group 3 (highdose, SapC–DOPS 11.8 mg/kg), group 4 (medium-dose, SapC– DOPS 5.9 mg/kg), and group 5 (low-dose, SapC–DOPS 2.9 mg/kg). Tumor dimensions were measured every other day during treatment. After the treatment regimen was completed, the mice were sacrificed and the tumors were removed, measured, and weighed. Tumor volume (in mm3) was calculated using the formula: L W2 0.5. The inhibition of tumor growth was calculated as % inhibition ¼ [1 (average tumor weight for each treatment group/average tumor weight for the control group)] 100%. Apoptosis in paraffin-embedded tumor samples was evaluated using a TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) assay (Boster Bio). Statistical analysis A t test analysis or two-way ANOVA/Tukey test was used to determine statistical significance for experiments with two or more groups, respectively. Analyses were done with SPSS 12.0. Data are presented as the arithmetic mean SEM.
Figure 1. SapC–DOPS binding to tumor cells is enhanced by acidic pH. A, flow cytometry analysis of A549 cells incubated with CMV (medium pH ¼ 7.5; purple-filled trace) or SapC–DOPS–CVM in media of various pH: 5.5 (green); 6.5 (pink); 7.5 (blue); 8.5 (orange). B, quantification of data from experiments like the one shown in A. , P < 0.05; , P < 0.0.1; , P < 0.001 with respect to control (one-way ANOVA followed by Tukey–Kramer multiple-comparison test); n ¼ 3.
In vivo imaging of tumor targeting by SapC–DOPS–CVM All procedures involving animals were approved by the Ethics Committee for Animal Research of Nanjing University. Murine lung tumor cells (Lewis lung cells, LLC; 5 105 cells) were injected into 4 week-old BALB/c female nude mice subcutaneously or into FVB male mice through the tail vein. Sham animals received a PBS injection. At day 10, 200 mL SapC–DOPS–CMV, DOPS–CVM, CVM, or PBS in normal saline were intravenously injected into tumor-bearing mice. Two to 150 hours later, mice were anesthetized to effect with 2% isoflurane and imaged with an IVIS 200 imaging system (PerkinElmer). In vivo SapC–DOPS treatment Healthy female BALB/c nude mice (5–6 weeks old) were obtained from Vital River Lab Animal Co., Ltd., Beijing Laboratory Animal Research Center (Beijing, China), and were
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Figure 2. Half-maximal cytotoxic dose of SapC–DOPS as a function of cell surface membrane phosphatidylserine levels in lung tumor cells. MTT assays were performed to determine SapC–DOPS cytotoxicity in cultured human lung cancer cells. The concentration of SapC–DOPS required to reduce cell viability by 50% is significantly lower in cells with high PS exposure, suggesting that increased PS levels enhance SapC–DOPS antitumor activity. Cell lines were grouped on the basis of their surface PS expression levels, as denoted by Annexin V-FITC binding (% signal increase over unstained controls): SPC-A-1, 28.9%; H1299, 48.4%; NCIH661, 51.1% (low PS group); A549, 195.8%; SK-MES-1, 336.8%; NCI-H460, 108.9; 801-D, 81.9% (high PS group). , P < 0.05.
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Results Acidic pH enhances the binding of SapC–DOPS to tumor cells Acidosis, resulting from high glycolytic activity and impaired perfusion, is a prominent feature in the microenvironment of solid tumors. Because the binding of SapC to anionic phospholipids such as PS is favored by low pH, we tested whether a weakly acidic environment could promote the binding and internalization of SapC–DOPS in lung cancer cells in vitro. For these experiments, we chose A549 lung adenocarcinoma cells, which in addition to expressing high levels of surface PS (see next section), are among the best characterized and most widely used lung tumor cell lines. A549 cells were incubated with SapC–DOPS–CMV nanovesicles in culture media with different pH values (5.5, 6.6, 7.5, or 8.5), at 37 C for 2 hours. Cellbound CVM fluorescence intensity was then assessed by flow cytometry (Ex 633 nm/Em 760 nm). The results depicted in Fig. 1A and quantified in Fig. 1B show that SapC–DOPS–CMV targeting was inversely correlated with pH, i.e., binding was higher as media pH decreased. SapC–DOPS has cytotoxic activity against lung cancer cells To evaluate the cytotoxic effects of SapC–DOPS on lung tumor cells, viability assays were performed in vitro in a panel of human lung cancer cell lines. Cells were incubated with SapC–DOPS at concentrations ranging from 0 to 128 mg/mL and 72 hours later, the effects on cell growth were measured using the CCK-8 assay. Figure 2 shows the calculated IC50 values plotted as a function of cell surface membrane PS levels (low PS vs. high PS;
cutoff ¼ 52%), as assessed with annexinV-FITC staining. Similar to previous observations in glioblastoma (23) and pancreatic cancer cell lines (24), the selectivity of SapC–DOPS toward PS was evidenced by a significant, inverse correlation between IC50 values and surface PS in these lung cancer cell lines. SapC–DOPS induces apoptosis in lung tumor cells To assess whether the loss in cell viability caused by SapC– DOPS was associated with the induction of apoptosis, two relevant apoptosis parameters, namely mitochondrial membrane potential (Dcm) and DNA fragmentation, were evaluated by flow cytometry in A549 cells. Dcm was assessed with the fluorescent dye JC-1. When living cells are incubated with JC-1, the dye is incorporated into the membranes of healthy, polarized mitochondria, forming aggregates that emit red (590 nm) fluorescence upon excitation with a 488 nm laser. During early apoptosis, the collapse of Dcm induces dissociation and release of JC-1 monomers to the cytosol, causing a fluorescence emission shift to green (529 nm) fluorescence. Treatment of A549 cells with SapC–DOPS (0, 10, 20, or 40 mg/mL) for 48 hours induced a dose-dependent shift from red to green fluorescence, reflecting a collapse of Dcm consistent with apoptotic cell death (Fig. 3A). In parallel experiments, we analyzed the effects of SapC–DOPS on cell-cycle progression and apoptotic cell death by quantifying the amount of DNA-bound PI representing DNA content. As DNA is fragmented in apoptotic cells, PI fluorescence intensity is lower than in intact cells in the G0–G1 cycle, giving raise to the "sub G0/G1" phase. As shown in Fig. 3B, SapC–DOPS incubation
Figure 3. SapC–DOPS induces apoptosis in human lung adenocarcinoma A549 cells. Cultured A549 cells were treated with SapC–DOPS (0, 10, 20, and 40 mg/mL) for 48 hours. A, SapC–DOPS causes dose-dependent Dcm depolarization, as assessed by quantification of P2-gated populations. B, dose-dependent DNA fragmentation induced by SapC–DOPS. The percentage of cells with fragmented DNA is represented by the Sub G0/G1 (M1) population. The figures are representative profiles of at least three experiments. C, quantification of results from experiments like those shown in A (P2 events; n ¼ 4) and B (M1 events; n ¼ 3). , P < 0.01; , P < 0.001.
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Figure 4. SapC–DOPS targets lung tumor cells in vivo. Nude mice bearing subcutaneous LLC cell allografts were imaged 24 hours after SapC–DOPS–CVM, DOPS-CVM, CVM, or PBS administration via intraperitoneal (A) or tail vein (B) injection. C, a lung tumor–bearing mouse (LLC) and a sham-injected, control mouse were intravenously injected with SapC–DOPS–CVM and imaged after 2 hours (in vivo) and 150 hours (ex vivo).
caused a dose-dependent increase in the percentage of cells in sub G0/G1 phase (M1). Figure 3C shows summary data for these experiments. Taken together, the dose-dependent loss of Dcm and the DNA fragmentation observed in these experiments demonstrate that SapC–DOPS induces apoptosis in A549 lung cancer cells. SapC–DOPS targets lung tumors in vivo The tumor-targeting capability of SapC–DOPS was tested in mouse allogeneic tumor models generated through subcutaneous implantation or systemic injection of LLC cells into female BALB/c nude mice or male FVB mice, respectively. Ten days after tumor implantation, animals were injected into the peritoneal cavity or via tail vein with SapC–DOPS–CVM or DOPS-CVM nanovesicles, CVM alone, or PBS; in vivo imaging was performed 24 hours later. As shown in Fig. 4A (intraperitoneal injections) and Fig 4B (intravenous injections), tumor-specific fluorescence in subcutaneous xenografts was detected only in SapC–DOPS– CVM–injected mice. SapC–DOPS' tumor-targeting capability was also assessed in mice harboring allogeneic lung tumors produced by systemic injection of LLC cells. Figure 4C shows a LLC-tumor-bearing mouse and a sham (PBS-injected) mouse imaged 2 and 150 hours after intravenous injection with SapC– DOPS–CVM. As in the prior model, SapC–DOPS–CVM fluorescence localized specifically to the tumor. In vivo antitumor efficacy of SapC–DOPS nanovesicles The anticancer efficacy of SapC–DOPS against human lung cancer cells was evaluated in nude mice bearing subcutaneous A549 lung tumors. About 10 days after implantation (when
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tumors reached 100 mm3), animals (6 per group) were treated intravenously with SapC–DOPS, PBS, or the chemotherapy drug cisplatin, and tumor growth was monitored over time. Quantification of tumor burden revealed that SapC–DOPS treatment at all three different doses tested reduced tumor weight by approximately 50% at day 35 (Fig. 5). Cisplatin, an alkylating chemotherapy drug used here as a positive control for tumor growth inhibition (29), was also effective in reducing tumor growth. Consistent with our observations in lung tumor cells in vitro, TUNEL staining confirmed the presence of apoptotic cells in SapC–DOPS–treated subcutaneous A549 lung tumors (Fig. 5C).
Discussion Lung cancer is the most common and the deadliest form of cancer worldwide. Because most lung tumors are diagnosed only when they reach advanced stages, standard treatments, namely surgical resection, radio- and chemotherapy, are mostly ineffective. Hence, recent efforts, including the development of immunotherapy agents, reflect the urgent need for more effective, specific therapeutic approaches (30–33). The present study shows that SapC–DOPS, a liposomal biotherapeutic agent composed of a lysosomal hydrolase coactivator (SapC) and a phospholipid (DOPS), induces apoptosis in lung tumor cells in vitro and delays the growth of lung cancer xenografts in vivo. Unlike typical anticancer liposomal formulations, SapC– DOPS possesses intrinsic tumor-targeting and tumoricidal activities, which result from the combined properties of both its lipid phase and its targeting protein. In this sense, SapC– DOPS is not merely a platform for chemotherapy drug delivery,
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Figure 5. Effects of SapC–DOPS on the growth of human lung tumor xenografts. After tumor establishment (day 0), mice were intravenously injected with saline (control), SapC–DOPS (high, medium, or low dose), or cisplatin every 2 days for 5 weeks. A, subcutaneous A549 tumor volume measurements. B, tumor weight at sacrifice. By 5 weeks after treatment onset, all experimental regimens significantly reduced tumor growth. C, apoptosis detection by TUNEL staining in subcutaneous A549 tumors. Original magnification, 400. Bar graph, quantification of apoptotic cells (averaged from 10 microscopic fields per treatment). , P < 0.05; , P < 0.01; , P < 0.001.
but an anticancer drug in itself. While multiple modifications are being assayed to overcome some common drawbacks of targeted liposomal formulations, such as limited cell internalization, incomplete drug release and lysosomal degradation (7), SapC–DOPS readily targets exposed PS residues on cancer cells' membranes and exploits the cell's own lysosomal machinery to induce tumor toxicity. Importantly, a key feature of liposomes, i.e., the ability to be endowed with enhanced targeting and cytotoxic capabilities by functionalization with different ligands and drugs, is also shared by SapC–DOPS. The basis of SapC–DOPS anticancer actions resides in the affinity of SapC toward PS, a membrane phospholipid which is sequestered in the inner side of the plasma membrane in healthy cells, but is aberrantly exposed on the surface of many different (nonapoptotic) tumor cells (19, 20). This peculiarity has prompted the development of a number of PS-targeting, antibody-based imaging and therapeutic probes, with recent studies using liposomal formulations showing promising results (34, 35). Upon tumor cell binding, SapC–DOPS cytotoxic activity is thought to arise from lysosomal destabilization and leakage, and/or activation of lysosomal fatty acid hydrolases and ceramide accumulation leading to caspase-mediated apoptosis (16, 21, 22, 36).
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In this study, we show that SapC–DOPS exerts cytotoxic actions against a variety of human lung carcinoma cells in vitro, and that higher levels of exposed PS correlate with higher killing efficacy by SapC–DOPS (i.e., lower IC50). We reported similar results in glioblastoma (23) and pancreatic cancer (24) cell lines. Conversely, SapC–DOPS' lack of toxicity toward untransformed human cells in vitro has been demonstrated in mammary (MCF-10A) cells, fibroblasts (21), and pancreatic ductal epithelial cells (24). The binding of SapC to PS is pH dependent, with an estimated pKa of 5.3 (17, 18). The present results show that SapC–DOPS binding to A549 human lung cancer cells in media with different pHs (5.5 to 8.5) was highest at the most acidic pH value tested. Because acidosis, secondary to hypoperfusion, hypoxia and augmented glycolytic lactate production, is a common feature of the microenvironment of solid tumors, SapC–DOPS may be particularly effective to counteract hypoxia-driven malignant progression and to treat tumors that show resistance to antiangiogenic therapies (23). The present data also indicate that treatment of A549 lung cancer cells with SapC–DOPS induces a dose-dependent decrease in Dcm paralleled by fragmentation of cellular DNA (sub G0/G1 peak), consistent with apoptotic cell death.
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Using fluorescently labeled nanovesicles, we show that systemically injected SapC–DOPS specifically targets subcutaneously implanted mouse lung tumor allografts, as well as mouse pulmonary tumors in vivo. Finally, the therapeutic efficacy of SapC– DOPS was evidenced by its ability to significantly reduce the growth of xenografted human lung tumors. As shown by TUNEL staining, and consistent with the reported effects of SapC–DOPS on lung tumor cells in vitro, this effect can be attributed, at least in part, to apoptosis induction. Taken together, the present results suggest that SapC–DOPS is a promising compound with potential clinical applicability for the diagnosis and treatment of lung tumors. Aberrant PS exposure is a common aspect of different cancer types, and we have shown that SapC–DOPS exerts both in vitro and in vivo antitumor actions in mouse models of neuroblastoma (21), glioblastoma (23), squamous cell carcinoma (25), pancreatic cancer (24), and metastatic brain tumors (26). Unlike chemotherapy drugs, SapC–DOPS nanovesicles are well tolerated and nontoxic in preclinical cancer models (21). In conclusion, our in vivo and in vitro experiments suggest that SapC–DOPS nanovesicles are a promising treatment option for lung cancer, worthy of further clinical development.
Authors' Contributions Conception and design: Y. Hou, X. Qi Development of methodology: S. Zhao, Z. Chu, Y. Nie, X. Qi Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Zhao, Z. Chu, X. Qi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V.M. Blanco, Y. Nie, X. Qi Writing, review, and/or revision of the manuscript: V.M. Blanco, X. Qi Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Zhao, Z. Chu Study supervision: Y. Hou, X. Qi
Acknowledgments The authors thank Dr. S. Abu-Baker and A. Stevens for the editing help.
Grant Support This work was supported in part by New Drug State Key Project grant number 009ZX09102-205 (to X. Qi), 1R01CA158372 (to X. Qi), and Research Funds/ Hematology-Oncology Programmatic Support from University of Cincinnati College of Medicine (to X. Qi). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Disclosure of Potential Conflicts of Interest X. Qi is a consultant/advisory board member for Bexion Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.
Received August 7, 2014; revised October 30, 2014; accepted November 12, 2014; published online February 10, 2015.
References 1. Stewart B, Wild C. World Cancer Report 2014. Lyon, France: International Agency for Research on Cancer. World Health Organization; 2014. 2. Field RW, Withers BL. Occupational and environmental causes of lung cancer. Clin Chest Med 2012;33:681–703. 3. Fucic A, Gamulin M, Ferencic Z, Rokotov DS, Katic J, Bartonova A, et al. Lung cancer and environmental chemical exposure: a review of our current state of knowledge with reference to the role of hormones and hormone receptors as an increased risk factor for developing lung cancer in man. Toxicol Pathol 2010;38:849–55. 4. Hubaux R, Becker-Santos D, Enfield K, Lam S, Lam W, Martinez V. Arsenic, asbestos and radon: emerging players in lung tumorigenesis. Environ Health 2012;11:89. 5. Kurt Straif AC, Jonathan Samet, eds. Scientific Publication No. 161: Air Pollution and Cancer. Lyon, France: IARC Scientific Publications; 2013. 6. Ahmad I, Longenecker M, Samuel J, Allen TM. Antibody-targeted delivery of doxorubicin entrapped in sterically stabilized liposomes can eradicate lung cancer in mice. Cancer Res 1993;53:1484–8. 7. Sawant R, Torchilin V. Challenges in development of targeted liposomal therapeutics. AAPS J 2012;14:303–15. 8. Maeda H. Macromolecular therapeutics in cancer treatment: the EPR effect and beyond. J Control Release 2012;164:138–44. 9. Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev 2013;65:36–48. 10. Slingerland M, Guchelaar H-J, Rosing H, Scheulen ME, van Warmerdam LJ, Beijnen JH, et al. Bioequivalence of Liposome-Entrapped Paclitaxel EasyTo-Use (LEP-ETU) formulation and paclitaxel in polyethoxylated castor oil: a randomized, two-period crossover study in patients with advanced cancer. Clin Ther 2013;35:1946–54. 11. Stathopoulos GP, Antoniou D, Dimitroulis J, Stathopoulos J, Marosis K, Michalopoulou P. Comparison of liposomal cisplatin versus cisplatin in non-squamous cell non-small-cell lung cancer. Cancer Chemother Pharmacol 2011;68:945–50. 12. Chang H-I, Yeh M-K. Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy. Int J Nanomedicine 2012;7:49.
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13. Slingerland M, Guchelaar H-J, Gelderblom H. Liposomal drug formulations in cancer therapy: 15 years along the road. Drug Discov Today 2012;17:160–6. 14. Henry B, Moller C, Dimanche-Boitrel MT, Gulbins E, Becker KA. Targeting the ceramide system in cancer. Cancer Lett 2013;332:286–94. 15. Mullen TD, Obeid LM. Ceramide and apoptosis: exploring the enigmatic connections between sphingolipid metabolism and programmed cell death. Anticancer Agents Med Chem 2012;12:340–63. 16. Vaccaro AM, Ciaffoni F, Tatti M, Salvioli R, Barca A, Tognozzi D, et al. pH-dependent conformational properties of saposins and their interactions with phospholipid membranes. J Biol Chem 1995;270: 30576–80. 17. de Alba E, Weiler S, Tjandra N. Solution structure of human saposin C: pHdependent interaction with phospholipid vesicles. Biochemistry 2003;42: 14729–40. 18. Liu A, Qi X. Molecular dynamics simulation of saposin C-membrane binding. Open Struct Biol J 2008;2:21–30. 19. Ran S, Thorpe PE. Phosphatidylserine is a marker of tumor vasculature and a potential target for cancer imaging and therapy. Int J Radiat Oncol Biol Phys 2002;54:1479–84. 20. Utsugi T, Schroit AJ, Connor J, Bucana CD, Fidler IJ. Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res 1991;51:3062–6. 21. Qi X, Chu Z, Mahller YY, Stringer KF, Witte DP, Cripe TP. Cancer-selective targeting and cytotoxicity by liposomal-coupled lysosomal saposin C protein. Clin Cancer Res 2009;15:5840–51. 22. Kaimal V, Chu Z, Mahller YY, Papahadjopoulos-Sternberg B, Cripe TP, Holland SK, et al. Saposin C coupled lipid nanovesicles enable cancerselective optical and magnetic resonance imaging. Mol Imaging Biol 2011;13:886–97. 23. Wojton J, Chu Z, Mathsyaraja H, Meisen WH, Denton N, Kwon CH, et al. Systemic delivery of SapC-DOPS has antiangiogenic and antitumor effects against glioblastoma. Mol Ther 2013;21:1517–25. 24. Chu Z, Abu-Baker S, Palascak MB, Ahmad SA, Franco RS, Qi X. Targeting and cytotoxicity of SapC-DOPS nanovesicles in pancreatic cancer. PLoS ONE 2013;8:e75507.
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Downloaded from mct.aacrjournals.org on February 23, 2015. © 2015 American Association for Cancer Research.
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Zhao et al.
25. Abu-Baker S CZ, Stevens AM, Li J, Qi X. Cytotoxicity and selectivity in skin cancer by SapC-DOPS nanovesicles. J Cancer Ther 2012;3:321–6. 26. Blanco VM, Chu Z, Vallabhapurapu SD, Sulaiman MK, Kendler A, Rixe O, et al. Phosphatidylserine-selective targeting and anticancer effects of SapCDOPS nanovesicles on brain tumors. Oncotarget 2014;5:7105–18. 27. Qi X, Leonova T, Grabowski GA. Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase. J Biol Chem 1994;269:16746–53. 28. Mahata S, Maru S, Shukla S, Pandey A, Mugesh G, Das BC, et al. Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells. BMC Complement Altern Med 2012;12:15. 29. Yamori T, Sato S, Chikazawa H, Kadota T. Anti-tumor efficacy of paclitaxel against human lung cancer xenografts. Jpn J Cancer Res 1997;88:1205–10. 30. Ramlogan-Steel CA, Steel JC, Morris JC. Lung cancer vaccines: current status and future prospects. Transl Lung Cancer Res 2013;3:46–52. 31. Morrison BJ, Steel JC, Morris JC. Immunotherapy in lung cancer: the potential of cancer stem cells in future therapies. Future Oncol 2013;9:623–5.
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32. Spiro SG, Silvestri GA. One hundred years of lung cancer. Am J Respir Crit Care Med 2005;172:523–9. 33. Huber RM, Reck M, Thomas M. Current status and future strategies for multimodality treatment of unresectable stage III non-small-cell lung cancer. Eur Respir J 2013;42:1119–33. 34. Zhang L, Zhao D. Liposomal encapsulation enhances in vivo near infrared imaging of exposed phosphatidylserine in a mouse glioma model. Molecules 2013;18:14613–28. 35. Zhou H, Stafford JH, Hallac RR, Zhang L, Huang G, Mason RP, et al. Phosphatidylserine-targeted molecular imaging of tumor vasculature by magnetic resonance imaging. J Biomed Nanotechnol 2014;10: 846–55. 36. Ditaranto-Desimone K, Saito M, Tekirian T, Saito M, Berg M, Dubowchik G, et al. Neuronal endosomal/lysosomal membrane destabilization activates caspases and induces abnormal accumulation of the lipid secondary messenger ceramide. Brain Res Bull 2003;59: 523–31.
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SapC−DOPS Nanovesicles as Targeted Therapy for Lung Cancer Shuli Zhao, Zhengtao Chu, Victor M. Blanco, et al. Mol Cancer Ther 2015;14:491-498.
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Large Molecule Therapeutics
SapC–DOPS Nanovesicles as Targeted Therapy for Lung Cancer Shuli Zhao1, Zhengtao Chu2,3, Victor M. Blanco2, Yunzhong Nie4, Yayi Hou4, and Xiaoyang Qi2,3
Abstract Lung cancer is the deadliest type of cancer for both men and women. In this study, we evaluate the in vitro and in vivo efficacy of a biotherapeutic agent composed of a lysosomal protein (Saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS), which can be assembled into nanovesicles (SapC–DOPS) with selective antitumor activity. SapC–DOPS targets phosphatidylserine, an anionic phospholipid preferentially exposed in the surface of cancer cells and tumor-associated vasculature. Because binding of SapC to phosphatidylserine is favored at acidic pHs, and the latter characterizes the milieu of many solid tumors, we tested the effect of pH on the binding capacity of SapC–DOPS to lung tumor cells. Results showed that
SapC–DOPS binding to cancer cells was more pronounced at low pH. Viability assays on a panel of human lung tumor cells showed that SapC–DOPS cytotoxicity was positively correlated with cell surface phosphatidylserine levels, whereas mitochondrial membrane potential measurements were consistent with apoptosis-related cell death. Using a fluorescence tracking method in live mice, we show that SapC–DOPS specifically targets human lung cancer xenografts, and that systemic therapy with SapC–DOPS induces tumor apoptosis and significantly inhibits tumor growth. These results suggest that SapC–DOPS nanovesicles are a promising treatment option for lung cancer. Mol
Introduction
drugs, they provide improved biocompatibility, pharmacokinetics, biodistribution, and toxicologic profile. Despite promising results in preclinical models of lung cancer (6) and many other cancer types (7), only a few nontargeted formulations (i.e., relying on passive tumor accumulation through the enhanced permeability and retention effect) have been approved for cancer treatment by regulatory agencies (8, 9). Clinical trials are under way to evaluate novel liposomal formulations of the chemotherapy drugs paclitaxel (LEP-ETU; ref. 10) and cisplatin (Lipoplatin; ref. 11) in patients with lung cancer. Liposomes can deliver large drug payloads but, as numerous clinical studies have shown, this does not always translate into enhanced therapeutic efficacy; results so far show that liposomal drug delivery offers reduced toxicity, but only modest survival benefits, compared with free drug administration (12, 13). In this study we report a novel targeted biotherapeutic agent, saposin C-dioleoylphosphatidylserine (SapC–DOPS) proteoliposomes, with selective antineoplastic activity on lung cancer cells both in vitro and in vivo. SapC is a small glycoprotein present in all tissues that acts as biologic activator of lysosomal enzymes, such as acid b-glucosidase, acid sphingomyelinase, and acid b-galactosylceramide, which degrade fatty acid substrates resulting in the production of ceramide, a key intermediate in the synthesis of sphingolipids, and a well-known apoptosis inducer (14, 15). SapC has a strong affinity for negatively charged phospholipids such as phosphatidylserine (PS), to which it binds more avidly at acidic pH (16–18). Because the expression of PS is relatively abundant in the surface of tumor cells and tumor-associated vasculatures (19, 20), but not in normal cells, and in view of the characteristically acidic tumor microenvironment that results from glycolytic conversion of glucose into lactate, we hypothesized that SapC–DOPS nanovesicles could be an effective antitumor agent. Previous work from our group showed that SapC–DOPS targets a variety of cancer cells in vitro and in vivo, and has anticancer
Lung cancer is the most common and the deadliest type of cancer worldwide, accounting for 1.8 million new cases and 1.6 million deaths in 2012 (1). Cigarette smoking is the single leading cause of lung cancer, followed by environmental and occupational exposure to pollutants such as radon, arsenic, asbestos, Bis(chloromethyl) ether, chromium, nickel, vinyl chloride, and polycyclic aromatic compounds (2–4). Startlingly, a direct causal relationship between atmospheric particulate matter (air pollution) and lung and other cancers was recently established. As reported by the International Agency for Research on Cancer, more than half of lung cancer–related deaths caused by air pollutants worldwide occurred in China and other East Asian countries (5). Standard treatment options for lung cancer (chemotherapy, radiation, and surgery) have several undesirable side effects that affect the quality of life of the cancer patient. Liposomal formulations as vehicles for drug delivery are the subject of intense research. Compared with nonencapsulated, free
1 State Key Laboratory of Reproductive Medicine, Central Laboratory of Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu, China. 2Division of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio. 3Division of Human Genetics, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio. 4Immunology and Reproductive Biology Laboratory, Medical School and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, Jiangsu, China.
Corresponding Author: Xiaoyang Qi, Division of Hematology and Oncology, Department of Internal Medicine, College of Medicine, University of Cincinnati, The Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH 45267. Phone: 513-558-4025; Fax: 513-558-6703; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-14-0661 2015 American Association for Cancer Research.
Cancer Ther; 14(2); 491–8. 2015 AACR.
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effects in animal models of human solid tumors (21–26). In this study, we evaluate the tumor-targeting and cytotoxic potential of SapC–DOPS on human lung cancer cells, and assess its effectiveness in suppressing lung carcinoma growth in vivo. To this end, we first describe the pH dependence of SapC–DOPS cell binding and assess its cytotoxicity in a panel of lung cancer cells. The induction of apoptosis in cultured lung cancer cells is confirmed through DNA fragmentation analysis. Next, we use in vivo fluorescence imaging to assess SapC–DOPS tumorselective targeting in allogeneic lung tumor mouse models. Finally, the therapeutic efficacy of SapC–DOPS against tumor growth is studied in nude mice bearing human lung cancer xenografts. Our results show that SapC–DOPS effectively targets lung cancer cells in vitro and in vivo, and possesses antitumor activity in a preclinical model of human lung cancer.
Materials and Methods Cell culture The human lung carcinoma A549 and mouse Lewis lung carcinoma (LLC) cell lines were from American Type Culture Collection (ATCC; 1998 and 2001). The human lung carcinoma cell lines SPC-A-1, H1299, NCI-H661, SK-MES-1, NCIH460, and 801-D were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences from 1999 to 2010. Cells were routinely tested for mycoplasma contamination. No authentication of the cell lines was done by the authors. Human cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 mg/ mL streptomycin and 100 U/mL penicillin. LLC cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 100 mg/mL streptomycin, and 100 U/mL penicillin. Culture media and FBS were purchased from Life Technologies. All cell lines were cultured at 37 C in a humidified 5% CO2 atmosphere. Preparation of SapC–DOPS nanovesicles Dioleoylphosphatidylserine (DOPS; Avanti Polar Lipids Inc.) was dried under a stream of N2 gas, combined with recombinant SapC protein (27) in citrate/phosphate buffer (pH 5.0), and bath sonicated in PBS as described in detail previously (21). To fluorescently label SapC–DOPS nanovesicles, an aliquot of CellVue Maroon (CVM; Molecular Targeting Technologies Inc.) in ethanol was dried together with DOPS before SapC addition and bath sonication. Free CVM was eliminated from this preparation by filtration through a Sephadex G25 column (PD-10; Amersham Pharmacia Biotech). For in vivo treatment, a lyophilized formulation was used: dry DOPS was suspended in 80% tert-butanol. SapC and sucrose (10 mg/mL) were dissolved in water. DOPS suspension and SapC plus sucrose (1:0.6, vol:vol) was lyophilized in a freeze dryer (VirTis Unitop 1000L linked to a Freezemobile 25XL; The VIRTIS). The stable powder cake was resuspended in saline solution to form SapC–DOPS nanovesicles. Vesicles were monitored with a submicron particle size analyzer (Coulter Model N4 Plus). The molar ratio of SapC: DOPS was 1:7. SapC–DOPS binding in vitro To evaluate the influence of pH on the binding of SapC–DOPS to tumor cells, SapC–DOPS–CVM (containing 50 mg/mL SapC) or free, equimolar CVM were dissolved in complete RPMI-1640
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medium with different pH values (adjusted by addition of NaOH or HCl). A549 cells were collected by trypsinization, washed twice with PBS, and incubated in media adjusted to pH 5.5, 6.6, 7.5, or 8.5 for 2 hours at 37 C. The cells were then washed twice with PBS and analyzed by flow cytometry (FACSCanto II; BD Biosciences), using excitation at 633 nm and a 760 nm band-pass filter to detect CVM emission. Viability assays The colorimetric Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories) was used to monitor the cytotoxic effects of SapC–DOPS. Lung carcinoma cells were plated at a density of 5,000 cells per well in 96-well plates in triplicate. After 12 hours in culture, cells were treated with SapC–DOPS at final SapC concentrations of 0 (control), 0.5, 1, 2, 4, 8, 16, 32, 64, or 128 mg/mL. After 72 hours, 10 mL CCK-8 was added to each well. Optical density (OD) values were obtained from absorbance readings at 450 nm. Viability was calculated as the percentage of viable cells compared with control using the formula: growth inhibition (%) ¼ (ODC ODT)/ODC 100, where ODC and ODT are the OD values of the control and treated samples, respectively. IC50 was calculated by linear interpolation using the formula: IC50 ¼ [(50 A)/(B A)] (D C) þ C, where A is the first point on the curve, expressed as percent inhibition, that is less than 50%; B is the first point on the curve, expressed as percent inhibition, that is greater than or equal to 50%; C is the concentration of inhibitor that gives A% inhibition; and D is the concentration of inhibitor that gives B% inhibition (28). Phosphatidylserine levels, DNA fragmentation, and mitochondrial membrane potential measurements Membrane phosphatidylserine (PS) levels were measured using Annexin V-FITC (Invitrogen) according to the manufacturer's instructions. In brief, cells were trypsinized, washed with PBS, and resuspended at 2,500,000 cells/mL in Annexin V binding buffer. Aliquots of cells (0.2 mL/tube) were incubated with 10 mL of Annexin V-FITC for 15 minutes at room temperature in the dark. Then, 800 mL of binding buffer was added to each tube. Samples were kept on ice and analyzed by flow cytometry within 1 hour. Analysis of DNA fragmentation and mitochondrial membrane potential (Dcm) measurements were carried out in A549 cells cultured at a density of 5 105 cells per well in 6-well plates. Twenty-four hours after plating, cultures were treated with SapC–DOPS (containing 0, 10, 20, or 40 mg/mL of SapC) for 48 hours. To identify apoptotic cells by DNA fragmentation analysis, cells were harvested, washed with PBS, and fixed in 70% ice-cold ethanol overnight. Fixed cells were incubated with 20 U/mL RNase I and 50 mg/mL propidium iodide (PI) for 30 minutes. PI signal was quantified by flow cytometry and cells in the sub-G0/G1 phase of the cell-cycle were identified as apoptotic. Changes in Dcm were monitored using the lipophilic cationic probe 5,50 ,6,60 -tetrachloro-1,10 ,3,30 - tetraethyl-benzimidazolcarbocyanine iodide (JC-1). After treatment for 48 hours, the cells were trypsinized, washed with PBS, and incubated with JC-1 as per the manufacturer's instructions at 37 C in the dark for 30 minutes. Dcm was measured by flow cytometry by exciting with a 488 nm laser line and measuring emitted fluorescence with the FL1 (green) and FL2 (red) channels.
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Targeted Lung Cancer Therapy with SapC–DOPS
housed in specific pathogen-free conditions at Drum Tower Hospital Animal Center, Nanjing, China with controlled temperature (21 2 C), humidity (55 5%), and light (12 hour light/dark cycle). Water was available to mice ad libitum. After the mice had acclimated for 2 weeks, 2 106 A549 cells in 0.1 mL PBS were injected subcutaneously into their dorsal right flanks. Mice were monitored for solid tumor growth for approximately 10 days, and when the tumor volume reached 100 mm3, they were randomly assigned to 5 groups (n ¼ 6/group). The mice then received intravenous injections (0.2 mL) every other day for 5 weeks as follows: group 1 (control, normal saline), group 2 (cisplatin 4 mg/kg), group 3 (highdose, SapC–DOPS 11.8 mg/kg), group 4 (medium-dose, SapC– DOPS 5.9 mg/kg), and group 5 (low-dose, SapC–DOPS 2.9 mg/kg). Tumor dimensions were measured every other day during treatment. After the treatment regimen was completed, the mice were sacrificed and the tumors were removed, measured, and weighed. Tumor volume (in mm3) was calculated using the formula: L W2 0.5. The inhibition of tumor growth was calculated as % inhibition ¼ [1 (average tumor weight for each treatment group/average tumor weight for the control group)] 100%. Apoptosis in paraffin-embedded tumor samples was evaluated using a TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) assay (Boster Bio). Statistical analysis A t test analysis or two-way ANOVA/Tukey test was used to determine statistical significance for experiments with two or more groups, respectively. Analyses were done with SPSS 12.0. Data are presented as the arithmetic mean SEM.
Figure 1. SapC–DOPS binding to tumor cells is enhanced by acidic pH. A, flow cytometry analysis of A549 cells incubated with CMV (medium pH ¼ 7.5; purple-filled trace) or SapC–DOPS–CVM in media of various pH: 5.5 (green); 6.5 (pink); 7.5 (blue); 8.5 (orange). B, quantification of data from experiments like the one shown in A. , P < 0.05; , P < 0.0.1; , P < 0.001 with respect to control (one-way ANOVA followed by Tukey–Kramer multiple-comparison test); n ¼ 3.
In vivo imaging of tumor targeting by SapC–DOPS–CVM All procedures involving animals were approved by the Ethics Committee for Animal Research of Nanjing University. Murine lung tumor cells (Lewis lung cells, LLC; 5 105 cells) were injected into 4 week-old BALB/c female nude mice subcutaneously or into FVB male mice through the tail vein. Sham animals received a PBS injection. At day 10, 200 mL SapC–DOPS–CMV, DOPS–CVM, CVM, or PBS in normal saline were intravenously injected into tumor-bearing mice. Two to 150 hours later, mice were anesthetized to effect with 2% isoflurane and imaged with an IVIS 200 imaging system (PerkinElmer). In vivo SapC–DOPS treatment Healthy female BALB/c nude mice (5–6 weeks old) were obtained from Vital River Lab Animal Co., Ltd., Beijing Laboratory Animal Research Center (Beijing, China), and were
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Figure 2. Half-maximal cytotoxic dose of SapC–DOPS as a function of cell surface membrane phosphatidylserine levels in lung tumor cells. MTT assays were performed to determine SapC–DOPS cytotoxicity in cultured human lung cancer cells. The concentration of SapC–DOPS required to reduce cell viability by 50% is significantly lower in cells with high PS exposure, suggesting that increased PS levels enhance SapC–DOPS antitumor activity. Cell lines were grouped on the basis of their surface PS expression levels, as denoted by Annexin V-FITC binding (% signal increase over unstained controls): SPC-A-1, 28.9%; H1299, 48.4%; NCIH661, 51.1% (low PS group); A549, 195.8%; SK-MES-1, 336.8%; NCI-H460, 108.9; 801-D, 81.9% (high PS group). , P < 0.05.
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Results Acidic pH enhances the binding of SapC–DOPS to tumor cells Acidosis, resulting from high glycolytic activity and impaired perfusion, is a prominent feature in the microenvironment of solid tumors. Because the binding of SapC to anionic phospholipids such as PS is favored by low pH, we tested whether a weakly acidic environment could promote the binding and internalization of SapC–DOPS in lung cancer cells in vitro. For these experiments, we chose A549 lung adenocarcinoma cells, which in addition to expressing high levels of surface PS (see next section), are among the best characterized and most widely used lung tumor cell lines. A549 cells were incubated with SapC–DOPS–CMV nanovesicles in culture media with different pH values (5.5, 6.6, 7.5, or 8.5), at 37 C for 2 hours. Cellbound CVM fluorescence intensity was then assessed by flow cytometry (Ex 633 nm/Em 760 nm). The results depicted in Fig. 1A and quantified in Fig. 1B show that SapC–DOPS–CMV targeting was inversely correlated with pH, i.e., binding was higher as media pH decreased. SapC–DOPS has cytotoxic activity against lung cancer cells To evaluate the cytotoxic effects of SapC–DOPS on lung tumor cells, viability assays were performed in vitro in a panel of human lung cancer cell lines. Cells were incubated with SapC–DOPS at concentrations ranging from 0 to 128 mg/mL and 72 hours later, the effects on cell growth were measured using the CCK-8 assay. Figure 2 shows the calculated IC50 values plotted as a function of cell surface membrane PS levels (low PS vs. high PS;
cutoff ¼ 52%), as assessed with annexinV-FITC staining. Similar to previous observations in glioblastoma (23) and pancreatic cancer cell lines (24), the selectivity of SapC–DOPS toward PS was evidenced by a significant, inverse correlation between IC50 values and surface PS in these lung cancer cell lines. SapC–DOPS induces apoptosis in lung tumor cells To assess whether the loss in cell viability caused by SapC– DOPS was associated with the induction of apoptosis, two relevant apoptosis parameters, namely mitochondrial membrane potential (Dcm) and DNA fragmentation, were evaluated by flow cytometry in A549 cells. Dcm was assessed with the fluorescent dye JC-1. When living cells are incubated with JC-1, the dye is incorporated into the membranes of healthy, polarized mitochondria, forming aggregates that emit red (590 nm) fluorescence upon excitation with a 488 nm laser. During early apoptosis, the collapse of Dcm induces dissociation and release of JC-1 monomers to the cytosol, causing a fluorescence emission shift to green (529 nm) fluorescence. Treatment of A549 cells with SapC–DOPS (0, 10, 20, or 40 mg/mL) for 48 hours induced a dose-dependent shift from red to green fluorescence, reflecting a collapse of Dcm consistent with apoptotic cell death (Fig. 3A). In parallel experiments, we analyzed the effects of SapC–DOPS on cell-cycle progression and apoptotic cell death by quantifying the amount of DNA-bound PI representing DNA content. As DNA is fragmented in apoptotic cells, PI fluorescence intensity is lower than in intact cells in the G0–G1 cycle, giving raise to the "sub G0/G1" phase. As shown in Fig. 3B, SapC–DOPS incubation
Figure 3. SapC–DOPS induces apoptosis in human lung adenocarcinoma A549 cells. Cultured A549 cells were treated with SapC–DOPS (0, 10, 20, and 40 mg/mL) for 48 hours. A, SapC–DOPS causes dose-dependent Dcm depolarization, as assessed by quantification of P2-gated populations. B, dose-dependent DNA fragmentation induced by SapC–DOPS. The percentage of cells with fragmented DNA is represented by the Sub G0/G1 (M1) population. The figures are representative profiles of at least three experiments. C, quantification of results from experiments like those shown in A (P2 events; n ¼ 4) and B (M1 events; n ¼ 3). , P < 0.01; , P < 0.001.
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Figure 4. SapC–DOPS targets lung tumor cells in vivo. Nude mice bearing subcutaneous LLC cell allografts were imaged 24 hours after SapC–DOPS–CVM, DOPS-CVM, CVM, or PBS administration via intraperitoneal (A) or tail vein (B) injection. C, a lung tumor–bearing mouse (LLC) and a sham-injected, control mouse were intravenously injected with SapC–DOPS–CVM and imaged after 2 hours (in vivo) and 150 hours (ex vivo).
caused a dose-dependent increase in the percentage of cells in sub G0/G1 phase (M1). Figure 3C shows summary data for these experiments. Taken together, the dose-dependent loss of Dcm and the DNA fragmentation observed in these experiments demonstrate that SapC–DOPS induces apoptosis in A549 lung cancer cells. SapC–DOPS targets lung tumors in vivo The tumor-targeting capability of SapC–DOPS was tested in mouse allogeneic tumor models generated through subcutaneous implantation or systemic injection of LLC cells into female BALB/c nude mice or male FVB mice, respectively. Ten days after tumor implantation, animals were injected into the peritoneal cavity or via tail vein with SapC–DOPS–CVM or DOPS-CVM nanovesicles, CVM alone, or PBS; in vivo imaging was performed 24 hours later. As shown in Fig. 4A (intraperitoneal injections) and Fig 4B (intravenous injections), tumor-specific fluorescence in subcutaneous xenografts was detected only in SapC–DOPS– CVM–injected mice. SapC–DOPS' tumor-targeting capability was also assessed in mice harboring allogeneic lung tumors produced by systemic injection of LLC cells. Figure 4C shows a LLC-tumor-bearing mouse and a sham (PBS-injected) mouse imaged 2 and 150 hours after intravenous injection with SapC– DOPS–CVM. As in the prior model, SapC–DOPS–CVM fluorescence localized specifically to the tumor. In vivo antitumor efficacy of SapC–DOPS nanovesicles The anticancer efficacy of SapC–DOPS against human lung cancer cells was evaluated in nude mice bearing subcutaneous A549 lung tumors. About 10 days after implantation (when
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tumors reached 100 mm3), animals (6 per group) were treated intravenously with SapC–DOPS, PBS, or the chemotherapy drug cisplatin, and tumor growth was monitored over time. Quantification of tumor burden revealed that SapC–DOPS treatment at all three different doses tested reduced tumor weight by approximately 50% at day 35 (Fig. 5). Cisplatin, an alkylating chemotherapy drug used here as a positive control for tumor growth inhibition (29), was also effective in reducing tumor growth. Consistent with our observations in lung tumor cells in vitro, TUNEL staining confirmed the presence of apoptotic cells in SapC–DOPS–treated subcutaneous A549 lung tumors (Fig. 5C).
Discussion Lung cancer is the most common and the deadliest form of cancer worldwide. Because most lung tumors are diagnosed only when they reach advanced stages, standard treatments, namely surgical resection, radio- and chemotherapy, are mostly ineffective. Hence, recent efforts, including the development of immunotherapy agents, reflect the urgent need for more effective, specific therapeutic approaches (30–33). The present study shows that SapC–DOPS, a liposomal biotherapeutic agent composed of a lysosomal hydrolase coactivator (SapC) and a phospholipid (DOPS), induces apoptosis in lung tumor cells in vitro and delays the growth of lung cancer xenografts in vivo. Unlike typical anticancer liposomal formulations, SapC– DOPS possesses intrinsic tumor-targeting and tumoricidal activities, which result from the combined properties of both its lipid phase and its targeting protein. In this sense, SapC– DOPS is not merely a platform for chemotherapy drug delivery,
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Figure 5. Effects of SapC–DOPS on the growth of human lung tumor xenografts. After tumor establishment (day 0), mice were intravenously injected with saline (control), SapC–DOPS (high, medium, or low dose), or cisplatin every 2 days for 5 weeks. A, subcutaneous A549 tumor volume measurements. B, tumor weight at sacrifice. By 5 weeks after treatment onset, all experimental regimens significantly reduced tumor growth. C, apoptosis detection by TUNEL staining in subcutaneous A549 tumors. Original magnification, 400. Bar graph, quantification of apoptotic cells (averaged from 10 microscopic fields per treatment). , P < 0.05; , P < 0.01; , P < 0.001.
but an anticancer drug in itself. While multiple modifications are being assayed to overcome some common drawbacks of targeted liposomal formulations, such as limited cell internalization, incomplete drug release and lysosomal degradation (7), SapC–DOPS readily targets exposed PS residues on cancer cells' membranes and exploits the cell's own lysosomal machinery to induce tumor toxicity. Importantly, a key feature of liposomes, i.e., the ability to be endowed with enhanced targeting and cytotoxic capabilities by functionalization with different ligands and drugs, is also shared by SapC–DOPS. The basis of SapC–DOPS anticancer actions resides in the affinity of SapC toward PS, a membrane phospholipid which is sequestered in the inner side of the plasma membrane in healthy cells, but is aberrantly exposed on the surface of many different (nonapoptotic) tumor cells (19, 20). This peculiarity has prompted the development of a number of PS-targeting, antibody-based imaging and therapeutic probes, with recent studies using liposomal formulations showing promising results (34, 35). Upon tumor cell binding, SapC–DOPS cytotoxic activity is thought to arise from lysosomal destabilization and leakage, and/or activation of lysosomal fatty acid hydrolases and ceramide accumulation leading to caspase-mediated apoptosis (16, 21, 22, 36).
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In this study, we show that SapC–DOPS exerts cytotoxic actions against a variety of human lung carcinoma cells in vitro, and that higher levels of exposed PS correlate with higher killing efficacy by SapC–DOPS (i.e., lower IC50). We reported similar results in glioblastoma (23) and pancreatic cancer (24) cell lines. Conversely, SapC–DOPS' lack of toxicity toward untransformed human cells in vitro has been demonstrated in mammary (MCF-10A) cells, fibroblasts (21), and pancreatic ductal epithelial cells (24). The binding of SapC to PS is pH dependent, with an estimated pKa of 5.3 (17, 18). The present results show that SapC–DOPS binding to A549 human lung cancer cells in media with different pHs (5.5 to 8.5) was highest at the most acidic pH value tested. Because acidosis, secondary to hypoperfusion, hypoxia and augmented glycolytic lactate production, is a common feature of the microenvironment of solid tumors, SapC–DOPS may be particularly effective to counteract hypoxia-driven malignant progression and to treat tumors that show resistance to antiangiogenic therapies (23). The present data also indicate that treatment of A549 lung cancer cells with SapC–DOPS induces a dose-dependent decrease in Dcm paralleled by fragmentation of cellular DNA (sub G0/G1 peak), consistent with apoptotic cell death.
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Targeted Lung Cancer Therapy with SapC–DOPS
Using fluorescently labeled nanovesicles, we show that systemically injected SapC–DOPS specifically targets subcutaneously implanted mouse lung tumor allografts, as well as mouse pulmonary tumors in vivo. Finally, the therapeutic efficacy of SapC– DOPS was evidenced by its ability to significantly reduce the growth of xenografted human lung tumors. As shown by TUNEL staining, and consistent with the reported effects of SapC–DOPS on lung tumor cells in vitro, this effect can be attributed, at least in part, to apoptosis induction. Taken together, the present results suggest that SapC–DOPS is a promising compound with potential clinical applicability for the diagnosis and treatment of lung tumors. Aberrant PS exposure is a common aspect of different cancer types, and we have shown that SapC–DOPS exerts both in vitro and in vivo antitumor actions in mouse models of neuroblastoma (21), glioblastoma (23), squamous cell carcinoma (25), pancreatic cancer (24), and metastatic brain tumors (26). Unlike chemotherapy drugs, SapC–DOPS nanovesicles are well tolerated and nontoxic in preclinical cancer models (21). In conclusion, our in vivo and in vitro experiments suggest that SapC–DOPS nanovesicles are a promising treatment option for lung cancer, worthy of further clinical development.
Authors' Contributions Conception and design: Y. Hou, X. Qi Development of methodology: S. Zhao, Z. Chu, Y. Nie, X. Qi Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Zhao, Z. Chu, X. Qi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V.M. Blanco, Y. Nie, X. Qi Writing, review, and/or revision of the manuscript: V.M. Blanco, X. Qi Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Zhao, Z. Chu Study supervision: Y. Hou, X. Qi
Acknowledgments The authors thank Dr. S. Abu-Baker and A. Stevens for the editing help.
Grant Support This work was supported in part by New Drug State Key Project grant number 009ZX09102-205 (to X. Qi), 1R01CA158372 (to X. Qi), and Research Funds/ Hematology-Oncology Programmatic Support from University of Cincinnati College of Medicine (to X. Qi). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Disclosure of Potential Conflicts of Interest X. Qi is a consultant/advisory board member for Bexion Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.
Received August 7, 2014; revised October 30, 2014; accepted November 12, 2014; published online February 10, 2015.
References 1. Stewart B, Wild C. World Cancer Report 2014. Lyon, France: International Agency for Research on Cancer. World Health Organization; 2014. 2. Field RW, Withers BL. Occupational and environmental causes of lung cancer. Clin Chest Med 2012;33:681–703. 3. Fucic A, Gamulin M, Ferencic Z, Rokotov DS, Katic J, Bartonova A, et al. Lung cancer and environmental chemical exposure: a review of our current state of knowledge with reference to the role of hormones and hormone receptors as an increased risk factor for developing lung cancer in man. Toxicol Pathol 2010;38:849–55. 4. Hubaux R, Becker-Santos D, Enfield K, Lam S, Lam W, Martinez V. Arsenic, asbestos and radon: emerging players in lung tumorigenesis. Environ Health 2012;11:89. 5. Kurt Straif AC, Jonathan Samet, eds. Scientific Publication No. 161: Air Pollution and Cancer. Lyon, France: IARC Scientific Publications; 2013. 6. Ahmad I, Longenecker M, Samuel J, Allen TM. Antibody-targeted delivery of doxorubicin entrapped in sterically stabilized liposomes can eradicate lung cancer in mice. Cancer Res 1993;53:1484–8. 7. Sawant R, Torchilin V. Challenges in development of targeted liposomal therapeutics. AAPS J 2012;14:303–15. 8. Maeda H. Macromolecular therapeutics in cancer treatment: the EPR effect and beyond. J Control Release 2012;164:138–44. 9. Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev 2013;65:36–48. 10. Slingerland M, Guchelaar H-J, Rosing H, Scheulen ME, van Warmerdam LJ, Beijnen JH, et al. Bioequivalence of Liposome-Entrapped Paclitaxel EasyTo-Use (LEP-ETU) formulation and paclitaxel in polyethoxylated castor oil: a randomized, two-period crossover study in patients with advanced cancer. Clin Ther 2013;35:1946–54. 11. Stathopoulos GP, Antoniou D, Dimitroulis J, Stathopoulos J, Marosis K, Michalopoulou P. Comparison of liposomal cisplatin versus cisplatin in non-squamous cell non-small-cell lung cancer. Cancer Chemother Pharmacol 2011;68:945–50. 12. Chang H-I, Yeh M-K. Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy. Int J Nanomedicine 2012;7:49.
www.aacrjournals.org
13. Slingerland M, Guchelaar H-J, Gelderblom H. Liposomal drug formulations in cancer therapy: 15 years along the road. Drug Discov Today 2012;17:160–6. 14. Henry B, Moller C, Dimanche-Boitrel MT, Gulbins E, Becker KA. Targeting the ceramide system in cancer. Cancer Lett 2013;332:286–94. 15. Mullen TD, Obeid LM. Ceramide and apoptosis: exploring the enigmatic connections between sphingolipid metabolism and programmed cell death. Anticancer Agents Med Chem 2012;12:340–63. 16. Vaccaro AM, Ciaffoni F, Tatti M, Salvioli R, Barca A, Tognozzi D, et al. pH-dependent conformational properties of saposins and their interactions with phospholipid membranes. J Biol Chem 1995;270: 30576–80. 17. de Alba E, Weiler S, Tjandra N. Solution structure of human saposin C: pHdependent interaction with phospholipid vesicles. Biochemistry 2003;42: 14729–40. 18. Liu A, Qi X. Molecular dynamics simulation of saposin C-membrane binding. Open Struct Biol J 2008;2:21–30. 19. Ran S, Thorpe PE. Phosphatidylserine is a marker of tumor vasculature and a potential target for cancer imaging and therapy. Int J Radiat Oncol Biol Phys 2002;54:1479–84. 20. Utsugi T, Schroit AJ, Connor J, Bucana CD, Fidler IJ. Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res 1991;51:3062–6. 21. Qi X, Chu Z, Mahller YY, Stringer KF, Witte DP, Cripe TP. Cancer-selective targeting and cytotoxicity by liposomal-coupled lysosomal saposin C protein. Clin Cancer Res 2009;15:5840–51. 22. Kaimal V, Chu Z, Mahller YY, Papahadjopoulos-Sternberg B, Cripe TP, Holland SK, et al. Saposin C coupled lipid nanovesicles enable cancerselective optical and magnetic resonance imaging. Mol Imaging Biol 2011;13:886–97. 23. Wojton J, Chu Z, Mathsyaraja H, Meisen WH, Denton N, Kwon CH, et al. Systemic delivery of SapC-DOPS has antiangiogenic and antitumor effects against glioblastoma. Mol Ther 2013;21:1517–25. 24. Chu Z, Abu-Baker S, Palascak MB, Ahmad SA, Franco RS, Qi X. Targeting and cytotoxicity of SapC-DOPS nanovesicles in pancreatic cancer. PLoS ONE 2013;8:e75507.
Mol Cancer Ther; 14(2) February 2015
Downloaded from mct.aacrjournals.org on February 23, 2015. © 2015 American Association for Cancer Research.
497
Zhao et al.
25. Abu-Baker S CZ, Stevens AM, Li J, Qi X. Cytotoxicity and selectivity in skin cancer by SapC-DOPS nanovesicles. J Cancer Ther 2012;3:321–6. 26. Blanco VM, Chu Z, Vallabhapurapu SD, Sulaiman MK, Kendler A, Rixe O, et al. Phosphatidylserine-selective targeting and anticancer effects of SapCDOPS nanovesicles on brain tumors. Oncotarget 2014;5:7105–18. 27. Qi X, Leonova T, Grabowski GA. Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase. J Biol Chem 1994;269:16746–53. 28. Mahata S, Maru S, Shukla S, Pandey A, Mugesh G, Das BC, et al. Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells. BMC Complement Altern Med 2012;12:15. 29. Yamori T, Sato S, Chikazawa H, Kadota T. Anti-tumor efficacy of paclitaxel against human lung cancer xenografts. Jpn J Cancer Res 1997;88:1205–10. 30. Ramlogan-Steel CA, Steel JC, Morris JC. Lung cancer vaccines: current status and future prospects. Transl Lung Cancer Res 2013;3:46–52. 31. Morrison BJ, Steel JC, Morris JC. Immunotherapy in lung cancer: the potential of cancer stem cells in future therapies. Future Oncol 2013;9:623–5.
498 Mol Cancer Ther; 14(2) February 2015
32. Spiro SG, Silvestri GA. One hundred years of lung cancer. Am J Respir Crit Care Med 2005;172:523–9. 33. Huber RM, Reck M, Thomas M. Current status and future strategies for multimodality treatment of unresectable stage III non-small-cell lung cancer. Eur Respir J 2013;42:1119–33. 34. Zhang L, Zhao D. Liposomal encapsulation enhances in vivo near infrared imaging of exposed phosphatidylserine in a mouse glioma model. Molecules 2013;18:14613–28. 35. Zhou H, Stafford JH, Hallac RR, Zhang L, Huang G, Mason RP, et al. Phosphatidylserine-targeted molecular imaging of tumor vasculature by magnetic resonance imaging. J Biomed Nanotechnol 2014;10: 846–55. 36. Ditaranto-Desimone K, Saito M, Tekirian T, Saito M, Berg M, Dubowchik G, et al. Neuronal endosomal/lysosomal membrane destabilization activates caspases and induces abnormal accumulation of the lipid secondary messenger ceramide. Brain Res Bull 2003;59: 523–31.
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SapC−DOPS Nanovesicles as Targeted Therapy for Lung Cancer Shuli Zhao, Zhengtao Chu, Victor M. Blanco, et al. Mol Cancer Ther 2015;14:491-498.
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