Gadolinium metallofullerenol nanoparticles inhibit cancer metastasis through matrix metalloproteinase inhibition: imprisoning instead of poisoning cancer cells

Article Outline

• Abstract
• Graphical Abstract
• Methods
  • Preparation of f-NPs
  • Physicochemical characterization and stability evaluation of f-NPs
  • Cell culture
  • Determination of MMP in macrophage or cancer cell–macrophage co-cultured system
  • RNA extraction and qPCR analysis
  • Western blotting
  • Matrigel invasion assay
  • Animal
    • Tissue invasion model
    • Blood vessel metastasis model
  • Bioluminescence imaging
  • Histological examination
  • Statistical analysis
• Results
  • Characterization and stability study of the metallofullerenol nanoparticles
  • f-NPs inhibited the production of MMPs and cancer cell invasion in vitro
  • f-NPs significantly inhibited tumor metastasis in a tissue invasion model along with MMP inhibition
  • f-NPs isolated cancer cells within a dense fibrous cage
  • f-NPs significantly inhibited the establishment of tumor foci in lung in a bloodstream transfer model
• Discussion
• Appendix A. Supplementary data
• References
• Copyright

Abstract 

The purpose of this work is to study the antimetastasis activity of gadolinium metallofullerenol nanoparticles (f-NPs) in malignant and invasive human breast cancer models. We demonstrated that f-NPs inhibited the production of matrix metalloproteinase (MMP) enzymes and further interfered with the invasiveness of cancer cells in tissue culture condition. In the tissue invasion animal model, the invasive primary tumor treated with f-NPs showed significantly less metastasis to the ectopic site along with the decreased MMP expression. In the same animal model, we observed the formation of a fibrous cage that may serve as a physical barrier capable of cancer tissue encapsulation that cuts the communication between cancer- and tumor-associated macrophages, which produce MMP enzymes. In another animal model, the blood transfer model, f-NPs potently suppressed the establishment of tumor foci in lung. Based on these data, we conclude that f-NPs have antimetastasis effects and speculate that utilization of f-NPs may provide a new strategy for the treatment of tumor metastasis.

From the Clinical Editor

In this study utilizing metallofullerenol nanoparticles, the authors demonstrate antimetastasis effects and speculate that utilization of these nanoparticles may provide a new strategy in metastatic tumor therapy.

Graphical Abstract 

Key words: Nanomedicine, Metallofullerenol nanoparticles, Cancer metastasis, Matrix metalloproteinase, Fibrous cage

Cancer nanotechnology has become an innovative trend with immense potential for the safe and efficacious clinical translation of chemotherapeutics to the clinic.¹, ², ³, ⁴ Engineered nanoparticles (NPs), often themselves pharmaceutically active,⁵ provide for a robust platform that shows numerous advantages for cancer therapy compared to the conventional chemotherapeutics.¹ An exciting field within nanotechnology involves the investigation of carbon nanomaterials for biomedical applications in cancer treatment, including carbon nanotubes,⁶, ⁷ fullerenes, and their derivatives.⁵, ⁸ Gd@C₈₂(OH)_{x (x = 20–24)} endohedral metallofullerenol nanoparticles (f-NPs) were recently developed for biomedical applications involving imaging⁹, ¹⁰, ¹¹ and cancer therapy.⁵, ¹² Our laboratory has reported that several mechanisms including regulatory effects on immune response⁵, ¹³ may become involved in the anticancer outcome of this highly biocompatible nanoparticle.¹², ¹⁴, ¹⁵ In our previous study we utilized high-throughput gene array analysis to identify and distinguish the targeted genes that are responsive to f-NPs treatment.¹² This technique revealed that the matrix metalloproteinase (MMP) family showed strong inhibitory responsiveness to the f-NPs treatment.¹² We therefore asked if f-NPs can be utilized in a disease scenario in which MMPs have an important role, and therefore if the suppressed MMP could lead to a therapeutic outcome. One example is tumor metastasis in which MMP enzymes degrade all kinds of extracellular matrix (ECM) proteins and facilitate tumor invasiveness.¹⁶ Tumor metastasis, which remains a tremendous hurdle and a major contributor to more than 90% of cancer deaths,¹⁷, ¹⁸ comprises a multitude of steps, with the initiating stage consisting of degradation of the ECM.¹⁶ For a tumor to invade and intravasate into blood vessels, it must first break free from its own immediate confines, which consist of a fibrin-¹⁹ and collagen-dense matrix.²⁰ To accomplish this, the tumor relies on tumor-associated macrophages (TAMs) capable of secretion of proteases, mainly MMPs, that break down the ECM in the peripheral space of tumors, allowing for the escape.²¹ The objective of this study was to study the interactions between metallofullerenols and components of the tumor microenvironment specifically through regulation of secreted cytokines (e.g., MMP) that facilitate cancer invasion, and to examine the antimetastasis effects arising from here.

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Methods 

Preparation of f-NPs 

Gadolinium metallofullerene (Gd@C₈₂) was synthesized by an arc-discharge method using composite rods consisting of Gd₂O₃ (purity >99.99%; Sigma-Aldrich, St Louis, Missouri) and graphite (purity >99.99%, Beijing Chemical Co., Beijing, China) in a 450-torr He gas atmosphere as we described elsewhere.⁵, ⁹, ¹², ²², ²³, ²⁴ A graphite rod filled with an atomic ratio of Gd/Ni/C = 1:1:100 was used as the anode for arc burning. The raw soot was collected and extracted in N,N-dimethylformamide (Beijing Chemical Co. Nishitama, Tokyo, Kyoto, Japan) at 170°C for 12 hours. The N,N-dimethylformamide solution was then transferred into water-free toluene. Gd@C₈₂ was subsequently separated by a two-step high-performance liquid chromatography (LC908-C60; Japan Analytical Industry Co., Nishitama, Tokyo, Japan) equipped with a 5PBB column (Nacalai Co., Kyoto, Japan) followed by Buckyprep column (Nacalai Co.). To synthesize metallofullerenol, we mixed Gd@C₈₂ solution with 50% NaOH water solution containing 0.1 mL 40% tetrabutylammonium hydroxide (Sigma-Aldrich). After stirring for 12 hours at room temperature (22--28°C), the precipitate was carefully collected. To remove excess tetrabutylammonium hydroxide and NaOH, the precipitate was repeatedly washed by methanol and water. Subsequently, the precipitate was dissolved in water with continuous stirring for 24 hours. To obtain Gd@C₈₂(OH)₂₂, the concentrated Gd@C₈₂(OH)_x aqueous solution was purified by a Sephadex G-25 column chromatography with an eluent of distilled water. The molecular weight was determined by elemental analysis, matrix-assisted laser desorption/ionization–time-of-flight–mass spectrometry (AutoFlex; Bruker Co., Bremen, Germany) and x-ray photoemission spectroscopy (Beijing Synchrotron Radiation Facility) as we described elsewhere.²⁴

Physicochemical characterization and stability evaluation of f-NPs 

The morphology of the f-NPs was characterized by transmission electron microscopy (TEM, CM120; FEI, Hillsboro, Oregon). Particle size and zeta potential were measured in pure water, saline, and saline supplemented with 1% mouse serum by ZetaSizer Nano (Malvern Instruments, Worcestershire, United Kingdom). All the measurements were performed at a particle concentration of 100 μg/mL.

To evaluate the stability of f-NPs, transparency check, dynamic light scattering (DLS), and inductively coupled plasma mass spectroscopy (ICP-MS) analysis were performed. f-NPs were suspended in saline with or without 1% mouse serum at 100 μg/mL and sonicated for 10 minutes before the experiment. Particle size kinetics measurements were conducted using DLS from 0 to 6 days. Photographs of the f-NPs suspension were taken at days 0 and 6. To determine whether Gd³⁺ ions could be released from the carbon cage, a dialysis experiment was performed. The dialysis bag (1000-Da cutoff size) was filled with 5 mL 100 μg/mL f-NPs suspension, then dialyzed against 200 mL saline with or without serum. The Gd concentrations in the dialysis bags were measured at days 0 and 6 by ICP-MS analysis.

Cell culture 

MDA-MB-231 and MDA-MB-231-luc (constitutively expressing luciferase) cancer cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, Frederick, Maryland) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine. To generate a MDA-MBA-231 cell line constitutively expressing green fluorescent protein (GFP), 1 × 10⁵ cancer cells were transduced with lentivirus in a six-well tissue culture plate. The virus- containing medium was removed after 16 hours and the cultures replenished with fresh DMEM. Cells were allowed to proliferate to 1 × 10⁶ cells. The GFP cancer cells were used within five generations after transduction. U937 macrophages were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Determination of MMP in macrophage or cancer cell–macrophage co-cultured system 

MDA-MB-231 cancer cells were seeded into an upper inserted chamber that was supported by a permeable membrane with 3-μm pores. U937 macrophage cells were seeded into the lower chamber of the transwell in 2 mL complete DMEM. Cancer cells and macrophages were exposed to f-NPs at indicated concentrations for 96 hours. In parallel, U937 cells were solely cultured in the same medium in the presence of f-NPs at indicated concentrations for 96 hours. RNA samples were collected for quantitative polymerase chain reaction (qPCR) analysis. The total protein was collected for the western blotting experiment.

RNA extraction and qPCR analysis 

To identify the amount of MMP-2 and MMP-9 messenger RNA (mRNA), cell and tumor samples were collected for qPCR analysis. Total RNA was extracted using the commercially available kit (NucleoSpin RNA II; Macherey-Nagel, Bethlehem, Pennsylvania) according to the manufacturer's protocol. qPCR analysis was performed using the iQ 5 multicolor qPCR detection system (Bio-Rad Laboratories, Hercules, California). The sequence-specific primer pairs are as follow. For tumor tissue samples, MMP-9-up: 5′-TGAATCAGCTGGCTTTTGTG-3′; MMP-9-down: 5′-GTGGATAGCTCGGTGGTGTT-3′; MMP-2-up: 5′- GGTCTCGATGGTGTTCTGGT-3′; MMP-2-down: 5′-GTCGCCCCTAAAACAGACAA-3′. For cell samples, MMP-9-up: 5′- GCCATTCACGTCGTCCTTAT-3′; MMP-9-down: 5′-TTGACAGCGACAAGAAGTGG-3′; MMP-2-up: 5′-ATGACAGCTGCACCACTGAG-3′; MMP-2-down: 5′-ATTTGTTGCCCAGGAAAGTG-3′. Results were analyzed using the ABI Prism SDS 2.0 software (Applied Biosystems, Foster City, California). Both tested genes were compared with β-actin for normalization.

Western blotting 

To examine the MMP-2 and MMP-9 expression in cells or tumor tissues, the samples were washed in phosphate buffered saline and lysed in a lysis buffer containing protease inhibitor. The protein content in the extraction was determined by the Bradford method. Eighty micrograms of total protein were electrophoresed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking using 5% milk, the membranes were incubated with 1:1000 dilution of primary monoclonal antibody to MMP-9 and MMP-2 (Santa Cruz Biotechnology, Santa Cruz, California) at 4°C overnight, respectively. The membranes were overlaid with secondary antibody (1:1000 dilution) before the addition of the horseradish peroxidase–conjugated streptavidin-biotin complex. The proteins were detected using ECL reagent (Invitrogen, Carlsbad, California) according to the manufacturer's instructions.

Matrigel invasion assay 

MDA-MB-231-GFP and U937 cells were suspended to a final concentration of 1 × 10⁵ cells and 2 × 10⁴ cells in 500 μL serum-free DMEM in the upper insert that had been precoated with Matrigel (BD Science, Sparks, Maryland) and treated with f-NPs at various concentrations. We also included phorbol-12-myristate-13-acetate (PMA)²⁵ (0.1 nmol/mL), which is capable of enhancing the level of MMPs and caffeine²⁶ (150 nmol/mL) capable of suppressing MMPs in this assay. We also included small-molecule MMP inhibitors, CID 10667540 (MMP-2/MMP-9 inhibitor, 100 nM) and CP 471474 (broad-spectrum MMP inhibitor to MMP-1/-2/-3/-9/-13, 100 nM), in this assay. Twenty-four hours after various treatments, fluorescence intensity of GFP-expressing cancer cells that migrated through the Matrigel was detected. The nontreated cells were regarded as 100% in the invasion index calculation.

Animal 

Athymic BALB/c nu/nu female mice (∼16 g) were maintained under specific pathogen-free conditions. All animal experiments were performed under protocol approved by the Animal Care and Use Committee.

Tissue invasion model 

The tumor cell suspension (0.2 mL, 1 × 10⁷ cells/mL) was injected subcutaneously into mice. Thirty days after injection, the mice were killed and the subcutaneous tumors were divided into small pieces (3–4 mm³). The small tumor tissues were then subcutaneously inoculated into the left inguinal region of mice using a trochar needle. Ten days after implantation, the mice were randomly divided into two groups (n = 6) and received daily intraperitoneal (i.p.) injections of f-NPs saline solution (2.5 μmol/kg) or saline alone for 6 weeks. Tumor metastasis was examined by bioluminescence imaging (BLI) at the end of treatments.

Blood vessel metastasis model 

To see the effect of f-NPs in the situation in which a small amount of invasive cancer cells have already escaped from the primary site, 0.1 mL cancer cell suspension containing 1 × 10⁶ cells was directly injected through tail vein. Seven days after injection, the mice were daily dosed with f-NPs at 2.5 μmol/kg or saline alone for 6 weeks by i.p. administration. Tumor metastases in lung were monitored weekly by BLI and magnetic resonance imaging (MRI). At the end of the experiment, positron emission tomography imaging was performed.

Bioluminescence imaging 

To visualize the tumor metastasis, anesthetized tumor-bearing mice were injected intraperitoneally with 75 mg/kg d-luciferin (Caliper Life Sciences, Hopkinton, Massachusetts). The images were acquired using an IVIS Imaging System (Xenogen, Caliper Life Sciences). Acquisition time was 2 minutes. Analyses were performed with LIVINGIMAGE software (Caliper Life Sciences) by measuring photon flux in the region of interest. Note that the lower parts of the mouse body required covering so as to achieve adequate contrast for imaging at the ectopic site.

Histological examination 

In a tissue invasion model, the primary tumor tissue together with major organs (heart, lung, spleen, liver, kidney, brain, bone, and muscle) were quickly collected and fixed for histology analysis. Parts of the tumor tissues were used for hematoxylin-eosin and Van Gieson (VG) staining. In a bloodstream transfer model, the lung tissues as well as normal tissues such as liver and kidney were collected for histology analysis. The sections were examined by light microscopy.

Statistical analysis 

All of the results were calculated as mean ± SD. The statistical significance of the differences between groups was analyzed by Student's t-test as well as analysis of variance using Excel software (Microsoft, Seattle, Washington) at a significance level of 0.05.

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Results 

Characterization and stability study of the metallofullerenol nanoparticles 

The engineered f-NPs were developed to possess a viruslike nanostructure functionalized with a certain number of hydroxyl groups on the surface. The morphology of the NPs was found to be spherical/ellipsoidal in nature, with a primary size of ∼100 nm, as verified using TEM (Figure 1, A). The size of f-NPs was corroborated using DLS, with the f-NPs showing a single peak size distribution in physiological medium, all the while exhibiting a negative zeta potential (Figure 1, B).

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• Figure 1. 

  Synthesis and characterization of Gd@C₈₂(OH)₂₂ nanoparticles (f-NPs). (A) The hydroxyl-functionalized Gd@C₈₂ nanoparticles were purified by Sephadex G-25 column chromatography with an eluent of distilled water. TEM image shows the morphology of f-NPs. Scale bar, 50 nm. (B) Size distributions, average sizes (first value in parentheses), and zeta potentials of f-NPs (second value in parentheses) in different physiological aqueous solutions were analyzed. (C) Stability of f-NPs in physiological solution was determined by transparency check, DLS, and ICP-MS analysis. f-NPs were suspended in saline with or without mouse serum (w/w, 1%) at 100 μg/mL and sonicated for 10 minutes before the measurement. Particle size measurements were conducted using DLS from 0 to 6 days. Photographs of f-NPs in saline were taken at 0 and 6 days. To determine whether Gd³⁺ ions could be released from the carbon cage, dialysis experiments were performed. The dialysis bags contained 5 mL f-NPs suspension at particle dose of 100 μg/mL, which was dialyzed against 200 mL saline with or without serum. Gd content in dialysis bags were measured at 0 and 6 days by ICP-MS.

Transparency check, DLS, and ICP-MS analysis were used as three parallel methods to evaluate the stability of f-NPs in physiological conditions. Figure 1, C shows that f-NPs maintained optical transparency and remained in the ∼100- and ∼130-nm size range during the 6-day kinetics study in saline with and without mouse serum, respectively. Using a dialysis experiment, it was possible to determine whether the Gd³⁺ ions could be released during the incubation. The Gd concentration in the dialysis bag at day 0 was used as the reference value to calculate particle stability at a later time point. In agreement with the DLS data, ICP-MS measurement indicated that there was no significant Gd³⁺ ions release within 6 days, an indication of the intrinsic stability of f-NPs in these solutions (Figure 1, C).

f-NPs inhibited the production of MMPs and cancer cell invasion in vitro 

To test whether TAMs-mediated production of MMP was influenced by f-NPs treatment, we examined the effect of f-NPs in macrophages and in a cancer cell–macrophage co-culture system. MMP-2 and MMP-9 are examined, because many ECM components (e.g., collagens and gelatin) could be digested by these MMP molecules.²⁷ The amounts of MMP-9 and MMP-2 mRNA in TAMs following administration of f-NPs were measured, and the results are shown in Figure 2, A. The presence of the f-NPs in increasing doses decreased MMP-9 and MMP-2 mRNA expression in human U937 TAMs-like cells (Figure 2, A). Significant decreases in both MMP-9 and MMP-2 were observed by f-NPs treatment at 0.04 μmol/mL. The dose-dependent decreasing trends of MMP-9 and MMP-2 expression were confirmed by western blotting (Figure 2, A, insert). Please note that non-metallofullerenol [C₆₀(OH)₂₂] and GdCl₃ have no effects on inhibiting MMPs expression (Supplementary Figure S1, available online at http://www.nanomedjournal.com). The levels of these MMPs were then examined in an experiment involving co-culture of U937 cells with MDA-MB-231 cancer cells, so as to evaluate the effect of a potential cross-talk between cancer cells and macrophages. As can be seen in Figure 2, B, the f-NPs had a dramatic effect on MMP-9 expression, bringing about an enormous and sustained decrease at 0.04 μmol/mL. As is evident, the cancer cells co-incubated with the TAMs were found to have an inhibitory effect on the decrease of MMP-2 expression, highlighting the link that exists between TAMs and cancer cells. Although MMP-2 was not as sensitive as MMP-9 to the f-NPs treatment, the NPs were capable of inducing a ∼40% decrease in MMP-2 expression at 1 μmol/mL, whereas for MMP-9, a decrease of 90% occurred at a dose of only 0.04 μmol/mL (Figure 2, B). These results were also confirmed by western blotting (Figure 2, B, insert).

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• Figure 2. 

  Study of the effects of f-NPs on MMP regulation and cancer cell invasion in vitro. (A) qPCR (histogram) and western blotting (insert) were used to study the level of MMP-9 and MMP-2 in U937 cells treated with f-NPs at various concentrations. ^⁎P < 0.05, compared to control. (B) qPCR (histogram) and western blotting (insert) were used to determine MMP-9 and MMP-2 levels in MDA-MB-231/U937 co-cultured system in the presence of f-NPs at indicated concentrations. ^⁎P < 0.05, compared to control. (C) To study the effect of f-NPs on cancer cell invasion, the GFP-labeled cancer cells that were able to pass through the Matrigel-coated membranes toward serum-containing medium (chemoattractant) were detected. PMA-, caffeine-, and small-molecule MMP inhibitors (CP 471474, CID 10667540)–treated cells were included in the assay. Nontreated cells were considered as 100% in the calculation of the invasion index. ^⁎P < 0.05, significantly lower compared to control; #P < 0.05, significantly higher compared to non-treated cells.

To escape from the primary tumor site, invasive cancer cells require the assistance of MMP enzymes capable of degrading collagen components in the ECM. We are therefore interested in whether the MMP inhibition could be linked to a decreased cancer invasive potential in the presence of f-NPs in vitro. To answer this question, a Matrigel invasion experiment was used to study the effects of f-NPs. The data showed that PMA (which is able to increase MMP production²⁵) significantly promoted cell invasion; however, f-NPs and caffeine (which is able to decrease MMP production²⁶) potently inhibited the invasion of MDA-MBA-231 cells in this assay (Figure 2, C). To further strengthen our conclusion, we also assayed the effect of small-molecule MMP inhibitors (CP 471474 and CID 10667540). In agreement with the effect of f-NPs, these MMP inhibitors also showed a significant inhibition of cancer cell migration (Figure 2, C).

f-NPs significantly inhibited tumor metastasis in a tissue invasion model along with MMP inhibition 

To test whether f-NPs are able to inhibit MMP in vivo, including the capability of inhibition on tumor metastasis, we subsequently examined the effects of f-NPs in an MDA-MB-231 xenograft mouse model where lung metastasis frequently occurrs.²⁸, ²⁹ To visualize tumor metastasis in nude mice, MDA-MB-231-luc cells that express a luciferase gene were utilized for the ease of localization via BLI. Ten days after tumor implantation in the groin (primary tumor), the animals received daily intraperitoneal injections of either the f-NPs at 2.5 μmol/kg or saline, for a sustained duration of 6 weeks. Consistent with our previous findings,⁵, ⁸, ¹⁵ the f-NPs–treated animals showed significant tumor inhibition of the primary tumor (which was covered by black paper) compared to saline control. Following examination of tumor metastasis, tumor foci were only observed in the lungs and lymph of mice receiving saline injections 6 weeks after implantation, but not in animals treated with f-NPs (Figure 3, A). To quantify the ability of f-NPs to inhibit the metastatic potential of tumors, the inhibition rate of tumor metastasis (R_M) was defined by the following equation:

where I_S represents the intensity of BLI signal in the saline group, I_NP the intensity in the same area of f-NPs–treated mice, and BG is BLI background. The f-NPs treatment significantly inhibited tumor metastasis in the ectopic site and resulted in an average R_M of ∼78% (Figure 3, B). Note that no abnormal behavior, body weight loss, and histological abnormalities were observed during the 6-week treatment.

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• Figure 3. 

  The f-NPs potently inhibited tumor metastasis in the tissue invasion animal model. (A) Representative bioluminescence images (BLI) of tumor foci obtained from control (left) and the f-NPs–treated animals (right) after the course of treatment. (B) Quantification of the bioluminescence intensity of tumor foci in the lungs of animals after the course of treatment. f-NPs treatment significantly inhibited tumor metastasis with an R_M = 78% in the tissue invasion model. ^⁎P < 0.05, compared with saline control. (C) The levels of MMP-9 and MMP-2 in the primary tumor tissues treated with f-NPs or saline were determined by qPCR (histogram) and western blotting (insert). ^⁎P < 0.05, compared to control.

In the tissue culture condition, we have shown that the level of MMP was remarkably inhibited in the presence of f-NPs (Figure 2). In light of these findings, the question remains as to whether MMP inhibition has a role in the therapeutic outcome of f-NPs in vivo. The qPCR results demonstrated a >90% and >60% decrease in MMP-9 and MMP-2 in tumor tissues, respectively, as compared to the control. This result was further bolstered by western blotting (Figure 3, C, insert).

f-NPs isolated cancer cells within a dense fibrous cage 

Interestingly, histological examination of primary subcutaneous tumor using VG staining demonstrated a thick fibrous layer on the tumor surface in the f-NPs–treated mice but not in saline-treated mice, the results of which are shown in Figure 4. This dense connective tissue at the tumor boundary is easily discernible given the presence of VG staining, which allows for staining of collagen. Saline control tumors possessed a natural tumor ECM composed of fibrin and type I collagen. The capsule was homogeneous in size throughout, and had an average thickness of ∼60 μm (Figure 4, A). In f-NPs–treated animals, the fibrous layer surrounding the surface of the primary tumor was ∼450 μm thick (Figure 4, B). A similar fibrous cage was also found in another breast cancer model, MCF-7, showing layers with a thickness of ∼32 μm and ∼578 μm for saline (Figure 4, C) and f-NPs (Figure 4, D) treatments, respectively. Note that no remarkable deposition of fibrous tissue was observed in other major organs. A closer examination of the tumor tissues from saline- and f-NPs–treated animals was conducted using TEM (Supplementary Figure S2). Saline-treated tumor tissue did not show the presence of collagen fibers under TEM. Contrastingly, collagen fibers can be easily found in tumor tissue treated with f-NPs.

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• Figure 4. 

  Photomicrographs of histological slides of excised tumors using Van Gieson staining to differentiate collagen fibers at tumor site in control and f-NPs–treated animals, respectively. (A) The excised tumors of the control mice and (B) of the f-NPs–treated mice of MDA-MB-231 human breast cancer model. (C) The excised tumors of the control mice and (D) of the f-NPs–treated mice of another human breast cancer model, MCF-7. The dotted line represents the boundary between the tumor and its fibrous capsule. The average thicknesses of fibrous capsules on the tumors in the f-NPs–treated group were 7–18 times larger than those of the control depending on tumor type.

f-NPs significantly inhibited the establishment of tumor foci in lung in a bloodstream transfer model 

The prognosis and survival rate greatly depend on the stage of the cancer and the extent of cancer spread.³⁰ We have demonstrated that f-NPs potently suppress tumor metastasis in a tissue invasion model, but because f-NPs cannot completely eliminate tumor invasion, there is a need for the data in the bloodstream transfer model, in which the cancer cells were directly injected into bloodstream. These data can help determine whether f-NPs would be able to inhibit the establishment of tumor foci and subsequent tumor growth if a small amount of invasive cancer cells have already escaped from the primary site. As can be seen in Figure 5, A, tumor signal was detected in the lungs ∼21 days after cancer cell injection in the saline group. By contrast, no cancer signal could be detected in mice with f-NPs treatment at this time point, and tumors did not appear in f-NPs group until day 42. Again, R_M was calculated at the end point of experimentation using BLI readout, the results of which can be seen in Figure 5, B. The treatment using f-NPs significantly suppressed the establishment of tumor foci in lungs compared to saline control, with an average R_M of 88%. These findings were further corroborated by ¹⁸F-fluorodeoxyglucose positron emission tomography (Supplementary Figure S3) and MRI results (Supplementary Figure S4). These diverse imaging data provide extra resolution by which f-NPs inhibition of tumor metastasis could be monitored in detail. Histological examination of the lungs of animals that received the various treatments further highlight the efficacy of f-NPs treatment. The saline control showed the presence of numerous tumor foci, as evidenced by the clusters of large, irregularly shaped tumor cells with darkly staining nuclei. In contrast, the lungs of animals receiving f-NPs showed minimal signs of tumor presence (Figure 5, C).

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• Figure 5. 

  f-NPs potently inhibited the establishment of tumor foci in the blood transfer model of MDA-MB-231-luc cancer mice. (A) MDA-MB-231-luc cells were harvested and resuspended at a concentration of 1 × 10⁷ cells/mL in saline. A 0.1-mL cell suspension was injected into the tail vein of the nude mice. Seven days after cancer cell injection, the mice received daily intraperitoneal doses of the f-NPs at 2.5 μmol/kg for a duration of 6 weeks. Saline was used as control. Tumor metastases in lung were monitored weekly by BLI. (B) Quantification of the BLI intensity of tumor foci in the lungs of animals after different treatments. f-NPs treatment significantly inhibited tumor metastasis with an R_M = 88% in the bloodstream transfer model. ^⁎P < 0.05, compared to control. (C) Histological examination of the lungs with different treatments was performed. Arrows indicate the tumor foci in a saline-treated animal. Higher magnification images of animal lung are shown in the lower panel.

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Discussion 

In this study we demonstrated that f-NPs potently suppressed MMPs at mRNA and protein levels in macrophage and a cancer cell–macrophage co-culture system. We further demonstrated that f-NPs caused a suppression of cancer cell invasion through Matrigel. In the tissue invasion animal model, we demonstrated that the treatment using f-NPs potently prevented metastasis and restricted tumor invasion mainly through an MMP-inhibitory process. Interestingly, f-NPs were able to induce a thick and dense fibrous cage. This may be in large part due to the NP treatment's ability to reduce the expression of MMPs and resulting accumulation of fibrous components. In an animal model simulating bloodstream metastasis, f-NPs treatment was also capable of preventing tumor foci establishment in lung. The present findings may suggest a new strategy in cancer therapeutics using engineered NP, namely imprisoning instead of poisoning cancer cells.

Production of MMPs at tumor sites has been shown to be upregulated in several human tumors, and if left rampant and unchecked, degrades the matrix surrounding the tumor, allowing for tumor invasion into surrounding tissues and the circulation.³¹ The downregulation of MMP expression in the tumor microenvironment is known as a major contributing factor in the decreased metastatic potential of tumors.³¹ It has been demonstrated that small-molecule inhibitors of MMPs, including synthetic and natural compounds–based MMP inhibitors, would be useful to prevent tumor metastasis.³² However, many MMP inhibitors eventually fail in the clinical trial because of unexpected side effects and toxicity (e.g., musculoskeletal syndrome), poor pharmacokinetic profile, low bioavailability, etc.³² These disappointing results highlight the need for a new type of MMP inhibitor such as a pharmaceutically active NP, which shows more effectiveness, high bioavailability, and low toxicity.

Another interesting finding in this work is the establishment of fibrous encapsulation that was mainly composed of collagen components (Figure 4). Although we do not have a full understanding of how the fibrous cage was formed, it is worthwhile to consider the important role of the decreased MMP level that is responsible for ECM degradation and further excessive collagen deposition. The lack of MMP expression allowed for thickening of the connective tissue boundary around the tumor, a direct product of the body's natural inflammatory response. This point is in agreement with previous findings³³ and has been experimentally proved by an in vitro collagen degradation assay (Supplementary Figure S5). However, in the presence of abundant MMPs, levels like those found in tumors of control mice, the fibrous layer would have undergone degradation that facilitates tumor invasion. Aside from the fibrous cage in the MDA-MB-231 breast cancer, a similar fibrous composition was also found in another breast cancer, the MCF-7 cancer model (Figure 4, C and D). This implies that the novel phenomenon can be applied to several different tumor types.

The thick collagen boundary surrounding the tumor surface may have several important roles in preventing metastasis. First, the fibrous layer separating the invasive cancer cells from normal tissue may act as a barrier that prevented cross-talk and signaling between the cancer cells and TAMs, thereby preventing production of the MMP enzymes. Several novel therapeutic strategies are currently being explored to exploit the link between macrophages, tumors, and metastasis. As an example, depletion of macrophages was able to reduce lung metastases in mouse models of polyoma middle T–induced mammary cancer.³⁴ Concomitantly, if the phenotype of TAMs can be reversed, macrophages can be used to restrain tumor growth rather than promote it.³⁵ Second, the thickened capsule that developed around the tumor over the course of f-NPs treatment physically encased the tumor within a matrix that may prevent cancer cells from escaping.³⁶, ³⁷ This possibility is also supported by a previous finding that expansion of tumor cells within a three-dimensional matrix of type I collagen or cross-linked fibrin was restricted in the absence of MMP.³⁷ With a low level of proteolysis, tumor cells that were embedded in ECM matrices were trapped in a compact, spherical configuration and failed in the cytoskeletal reorganization that is necessary for tumor growth and invasion.³⁷ Understanding of the antimetastasis activity of f-NPs may lead to a new approach in cancer metastasis management—namely, solid-tumor encapsulation using engineered NPs instead of direct cell killing as has been largely involved in conventional chemotherapeutics. Indeed, more in-depth mechanistic studies are needed to fully understand the function of this thick collagen layer, including how the layer is formed. This may involve the sophisticated regulatory mechanisms in which f-NPs treatment could influence cytokine and growth factor release (e.g., TGF-β1), alter proliferation and differentiation in fibroblasts, and decrease matrix degradation (e.g., MMP inhibition), etc.

Compared to existing chemicals capable of MMP inhibition, the f-NPs exhibited a safe and efficient nanotherapeutic platform for cancer metastasis management. These NPs hold great potential for antimetastatic treatment because of several properties. First, upon circulation in the bloodstream, f-NPs could preferentially accumulate in the stroma of the tumor surface, which may be due to a result of the enhanced permeability and retention effect.³⁸ Their particle phase may result in their being phagocytosed by immune cells both in the bloodstream (e.g., monocytes) and partially by resident TAMs at tumor sites, where they can exert immunoregulatory effects, such as affecting cytokine (e.g., MMP) secretion. Second, the f-NPs potently inhibited a series of key factors that triggered tumor metastasis,¹² including MMPs and angiogenesis factors.¹² The multiple bioactivities of the f-NPs may contribute to the therapeutic outcome via a combined and/or synergistic effect.^5,12, ¹³, ¹⁴, ¹⁵ Third, the nontoxic nature of the f-NPs,⁵, ¹² such as lack of toxicity to normal organs (Supplementary Figure S6) allows patients to receive continuous and long-term treatment, which is usually impossible for most anticancer agents including many existing MMP inhibitors.³²

In summary, we demonstrated a novel approach using Gd@C₈₂(OH)₂₂ metallofullerenol nanoparticles to inhibit tumor metastasis. Rather than direct cell killing, the NPs inhibited tumor metastasis mainly through an MMP-inhibitory process. The formation of a thick fibrous cage may serve as a “prison” capable of confining the invasive tumor cells in their primary site (Figure 6). We anticipate that these findings will result in a new approach in the management of tumor metastasis, namely, imprisoning instead of poisoning cancer cells.

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• Figure 6. 

  Schematic presentation of possible antimetastasis mechanism of f-NPs. Rather than direct cell killing, the metallofullerenol nanoparticles inhibited tumor metastases mainly through a MMP inhibition process. In the control group (left panel), TAMs-secreted MMP enzymes are able to efficiently degrade the fibrous matrices surrounding tumors and facilitate their invasiveness. In the nanoparticle-treated group (right panel), f-NPs decreased the production of MMP, subsequently reducing fibrous matrix degradation. The thick fibrous cage may therefore serve as a “prison” that tightly confines the invasive cancer cells within the primary site.

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Appendix A. Supplementary data 

The following is the Supplementary data to this article.

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