Letter pubs.acs.org/NanoLett
Inhibition of Mammary Tumor Growth Using Lysyl OxidaseTargeting Nanoparticles to Modify Extracellular Matrix Mathumai Kanapathipillai,†,⊥ Akiko Mammoto,‡,⊥ Tadanori Mammoto,‡ Joo H. Kang,† Elisabeth Jiang,‡ Kaustabh Ghosh,‡,∥ Netanel Korin,† Ashley Gibbs,‡ Robert Mannix,‡ and Donald E. Ingber*,†,‡,§ †
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States Vascular Biology Program, Departments of Pathology & Surgery, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, United States § School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138 United States ‡
S Supporting Information *
ABSTRACT: A cancer nanotherapeutic has been developed that targets the extracellular matrix (ECM)-modifying enzyme lysyl oxidase (LOX) and alters the ECM structure. Poly(D,Llactide-co-glycolide) nanoparticles (∼220 nm) coated with a LOX inhibitory antibody bind to ECM and suppress mammary cancer cell growth and invasion in vitro as well as tumor expansion in vivo, with greater efficiency than soluble anti-LOX antibody. This nanomaterials approach opens a new path for treating cancer with higher efficacy and decreased side effects. KEYWORDS: Cancer, nanoparticle, lysyl oxidase, extracellular matrix, mechanics
C
Lysyl oxidase (LOX) is an interesting target candidate because it is an ECM-associated enzyme found in elevated levels in the tumor microenvironment that increases ECM stiffness and promotes malignancy by catalyzing cross-linking of collagen and elastin.13−17 Importantly, mammary cancer progression can be slowed, and metastasis suppressed, by inhibiting LOX cross-linking activity using systemic injection of high doses of chemical inhibitors or soluble antibodies.9,18−21 But chemical inhibitors of LOX and soluble antibodies must be used in high doses to induce these effects, and hence, administration of these agents can be limited by significant side effects (e.g., lathyrism, immune reaction).22,23 Thus, in the present study, we set out to design a nanocarrier-based delivery system to efficiently target and concentrate LOX inhibitory antibody within the tumor ECM. The goal was to develop improved LOX-modifying agents that could produce tumor suppression at lower administered doses, thereby minimizing systemic side effects and improving therapeutic efficacy. First, nanoparticles were formed from amphiphilic poly(D,Llactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-bPEG-COOH) copolymer that is widely used for tissueengineering applications and has been shown to be safe in vivo,24−26 using a simple solvent-displacement method followed by self-assembly in aqueous solution (Figure 1). The surface of the nanoparticles was then covalently modified with a LOXinhibiting antibody (LOXAb) using carbodiimide chemistry27,28
ancer therapies are often limited in their effectiveness due to dose-limiting systemic side effects. Nanoparticle targeting approaches that selectively concentrate drug delivery to tumor sites offer a potential way to overcome this obstacle; however, most cancer nanotherapeutics are currently designed to leverage nonspecific enhanced permeability and retention (EPR) effects, and so these drugs also accumulate in other organs.1−3 Alterative delivery approaches have been developed to target drugs or nanocarriers bearing active agents to specific molecules on the cancer cell surface; this increases the efficiency of drug delivery to tumor sites, thereby increasing therapeutic index and reducing immunogenicity and other side effects.3−9 But long-term efficacy can be limited by down regulation of expression of these cell surface molecules.4,5 Another less explored option for increasing cancer drug efficacy is to target drugs to the extracellular matrix (ECM) that surrounds the tumor cells to selectively concentrate drug at these sites, thereby enhancing drug retention and increasing its half-life in the tumor microenvironment.6,7 An advantage of this approach is that the ECM is not surrounded by a plasma membrane, and hence, nanoparticles have essentially unrestricted access to these sites. However, in the present study, we explore another possible advantage of this approach. Because changes in the structure and mechanics of the tumor ECM also actively contribute to tumor formation and cancer progression,8−10 and recombination of epithelial cancers with embryonic ECM can inhibit tumor proliferation and induce differentiation,11,12 it might be possible to suppress cancer growth using nanocarriers to target drugs that alter physical properties of the ECM that normally help to drive tumor progression. © 2012 American Chemical Society
Received: March 29, 2012 Revised: April 23, 2012 Published: May 4, 2012 3213
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appropriate to support passive targeting to tumors, while the LOXAb coating provides an added active ECM targeting and retention capability.3,29,30 As increasing LOX activity has been shown to promote tumor cell growth and metastasis in vivo, we first performed in vitro cell growth and migration assays to confirm the inhibitory effects of LOXAbNPs. 4T1 mouse mammary tumor cells incubated with 50 μg LOXAbNP/mL (which corresponds to 0.5 μg LOXAb/mL) showed significant inhibition of cell growth, whereas soluble antibody had no effect on proliferation when added at the same effective concentration (Figure 3A). Similar
Figure 1. Schematic representation of the LOXAbNP formulation. (A) PLGA-PEG nanoparticles were produced by simple solvent displacement and subsequent self-assembly in aqueous solution. (B) LOXAb was covalently conjugated to the surface of the nanoparticles using carbodiimide chemistry.
to create LOX-targeting nanoparticles (LOXAbNPs). Covalent modification was confirmed by fluorescence-activated cell sorting (FACS) (Figure 2A), and quantification of the efficiency of
Figure 3. Inhibitory effect of LOXAbNPs on mammary 4T1 tumor cells. (A) 4T1 tumor cell growth and (B) migration in a transwell matrigel invasion assay in vitro. The conditions used are control (50 μg NP/mL) and LOXAbNP (50 μg LOXAbNP/mL = 0.5 μg LOXAb/mL = LOXAbNP and 0.5 μg LOXAb/mL). Note that LOXAbNPs inhibited tumor cell growth and migration compared to an equivalent dose of soluble LOXAb (*p < 0.01; **p < 0.001).
findings were also obtained when the LOXAbNPs were tested in an in vitro tumor cell migration assay (Figure 3B). Importantly, when LOXAbNP was added at the same dose of soluble LOXAb, the nanoparticles were three- to four-times more effective at suppressing these tumor cell behaviors in vitro (Figures 3A,B). This increased therapeutic efficacy of LOXAbNP may be due to preferential binding of the nanoparticles to the LOX enzyme, retention of the larger particles in the tumor microenvironment for longer times, and/or extended release of the LOXAb due to hydrolysis of the NP polymer matrix. All of these factors would lead to an increase in half-life and function of the antibody compared to soluble LOXAb. We then used an orthotopic mammary 4T1 tumor model to explore whether this increase in therapeutic index obtained in vitro with the LOXAbNPs can be leveraged to achieve targeted therapeutic delivery and more effective tumor growth suppression in vivo compared to systemic delivery of soluble LOXAb. Intravenous injection of LOXAbNPs (5 mg/kg) twice a week in mice bearing 4T1 tumors implanted within the mammary fat pad resulted in significant inhibition of tumor growth compared to
Figure 2. characterization of LOXAbNP size and morphology. (A) FACS results confirming the conjugation of TRITC-labeled LOXAb to the surface of the PLGA-PEG-NPs that were labeled with fluorescent coumarin (left, NP alone; right, NPs conjugated to LOXAb). (B) A histogram showing the results of DLS measurements, which depict the size range of the nanoparticles; inset showing transmission electron micrograph of the nanoparticles (scale bar, 500 nm).
conjugation using protein analysis revealed nanoparticle coating concentrations of approximately 1 μg LOXAb/100 μg of polymer nanoparticles. The coated nanoparticles appeared round and displayed at an average size of ∼220 nm, as determined using dynamic light scattering (DLS) and transmission electron microscopy (TEM) (Figure 2B). The particle size is in a range 3214
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untreated tumor-bearing mice (Figure 4A,B), whereas administration of an equal amount of soluble LOXAb alone showed no significant effect on the tumor expansion. Moreover, the LOXAbNP formulation was able to suppress cancer growth in mice using 25to 30-fold lower doses than that previously reported to be required to obtain similar tumor inhibition using soluble LOXAb.9,18,19,23 These findings suggest that use of optimized nanoparticle formulations of the LOXAb could significantly reduce doses required to produce clinical effects, and hence reduce systemic side effects, in patients with cancer and other LOX associated pathologies, including fibrotic diseases19 in the future. Based on past studies that showed LOX increases ECM stiffness by increasing collagen cross-linking during tumor progression,9,10,16 we then investigated whether the tumor growth inhibition produced by the LOXAbNP correlates with its effects on the tumor ECM. Frozen sections of tumor tissues removed from mice were decellularized31,32 and stained with picrosirius red to label collagen, and collagen fiber structural organization was analyzed using birefringence microscopy.33,34 These studies revealed that tumors treated with LOXAbNPs exhibited lower birefringent retardance and fewer thick fibers relative to untreated tumors or tumors treated with soluble LOXAb (Figure 5A−C). Thus, the differential effects of LOXAbNP and soluble LOXAb on mammary tumor expansion correlate directly with their relative abilities to inhibit collagen cross-linking and suppress ECM fiber assembly in vivo. The ability of LOXAbNPs to preferentially target and concentrate within the tumor ECM also could offer a novel approach to image tumor structure, which is important because collagen cross-linking and related increases in ECM stiffness appear to be causally involved in cancer progression and metastasis.9,35 In fact, fluorescence spectroscopic analysis of tumor sections from animals treated with LOXAbNPs labeled with the fluorescent dye Coumarin 6 revealed preferential NP accumulation in the treated tumor tissue (Figure S1, Supporting Information). Moreover, preliminary in vivo biodistribution studies revealed that coating of NPs with LOXAb resulted in an approximate
Figure 4. LOXAbNPs inhibit tumor growth in vivo. (A) Growth of 4T1 mammary tumors implanted orthotopically in the mammary fat pad of mice that were either untreated (□) or treated with soluble LOXAb (○, 50 μg/kg = LOXAb), NP alone (▲, 5 mg/kg), low-dose LOXAbNP (◆, 5 mg/kg = 50 μg/kg), or high-dose LOXAbNP (■, 5 mg/kg = 100 μg/kg) twice per week (n = 7). LOXAbNP treated tumors are significantly smaller than nontreated tumors or tumors treated with either soluble LOXAb or NP alone (*p < 0.05). (B) Representative photographs of untreated tumors and tumors treated with LOXAbNP (5 mg/kg = 100 μg/kg). Treatments began 3 days after tumor implantation and were administered for 12 days twice per week. Note that treatment with LOXAbNP produced significant inhibition of tumor growth, while an equal amount of soluble LOXAb had no effect.
Figure 5. Effect of LOX inhibition on tumor ECM organization and thickness. (A) Birefringence images of picosirius red stained collagen fibers in decellularized frozen sections of tumors from untreated mice or mice treated with NP alone (5 mg/kg), soluble LOXAb (50 μg/kg), or LOXAbNP (5 mg/kg =50 μg/kg LOXAb). (B) Retardance and (C) fiber area measurements obtained from birefringence microscopic analysis of the picrosiriusstained specimens. Note that the LOXAbNP treatment resulted in significant decreases in retardance and fiber area compared to tumor tissues that were either untreated and treated with soluble LOXAb alone (*p < 0.01; **p < 0.001). 3215
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doubling in the amount of NP retained within tumors compared to bare NPs (Figure S2, Supporting Information). LOXAbNPs concentrated more in tumor tissue than spleen or kidney, although high levels of accumulation were observed in liver and lung as well. It is important to note that a related lysyl oxidase-like enzyme (LOXL2) is also known to play a major role in cancer metastasis.18,19 Thus, even greater anticancer effects might be obtained in the future by coating our nanoparticles with inhibitory antibodies directed against both LOX and LOXL2. Regardless of the antibody coating, our nanoparticles will require further optimization (e.g., alterations of size, shape, PEG length, and polymer molecular weight and composition) to render long circulation and less immunogenicity3,36 as well as to control degradation and minimize toxicity,29 before they could be developed as effective therapeutic agents for LOX-related pathologies. In summary, we have demonstrated that LOXAbNPs exhibit enhanced therapeutic effects on tumor growth inhibition in vitro and in a mouse xenograft model in vivo, when compared to soluble LOXAb. Most importantly, the LOXAbNPs were effective at less than one-fiftieth the dose of the soluble antibody when used in vitro, and they also displayed a high therapeutic index in vivo. In addition, the antitumor effects of the LOXAbNPs correlated with their ability to inhibit collagen cross-linking in vivo, and our studies confirmed that these tumor-targeting nanoparticles can incorporate imaging agents for visualization of the tumor microenvironment. Thus, this nanomaterial-based approach for targeting LOX enzymes offers a unique approach to modulate and analyze the physical properties of the ECM, which plays a central role in disease development and represents a potential target for inducing cancer reversal.37 Importantly, in addition to having direct therapeutic implications for treatment of cancer, LOXAbNPs also should prove useful for analysis and manipulation and potential treatment of fibrosis, inflammation, various developmental abnormalities, and virtually all other forms of disease that are associated with abnormal ECM remodeling.
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ASSOCIATED CONTENT
* Supporting Information S
Detailed experimental procedures and supporting experiments are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] Present Address ∥
Department of Bioengineering, University of California, Riverside Riverside, California 92521
Author Contributions ⊥
These authors contributed equally
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the DOD innovator award (W81XWH-08-1-0659, to D.E.I.), ABTA (to A.M.), and the Wyss Institute for Biologically Inspired Engineering at Harvard University. 3216
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