Research ArticleExperimental Studies

Advanced Chemoembolization by Anti-angiogenic Calcium-Phosphate Ceramic Microspheres Targeting the Vascular Heterogeneity of Cancer Xenografts

MAKOTO EMOTO, HAJIME YOSHIHISA, KYOKO YANO, BATSUREN CHOIJAMTS, HITOSHI TSUGU, KATSURO TACHIBANA and MAMORU AIZAWA
Anticancer Research September 2015, 35 (9) 4757-4764;

Abstract

The purpose of the present study was to develop an advanced method of anti-angiogenic chemoembolization to target morphological vascular heterogeneity in tumors and further the therapeutic efficacy of cancer treatment. This new chemoembolization approach was designed using resorbable calcium-phosphate ceramic microspheres (CPMs), in a mixture of three different sizes, which were loaded with an anti-angiogenic agent to target the tumor vasculature in highly angiogenic solid tumors in humans in vivo. The human uterine carcinosarcoma cell line, FU-MMT-3, was used in this study because the tumor is highly aggressive and exhibits a poor response to radiotherapy and chemotherapeutic agents that are in current use. CPMs loaded with TNP-470, an anti-angiogenic agent, were injected into FU-MMT-3 xenografts in nude mice three times per week for 8 weeks. The treatment with TNP-470-loaded CPMs of three different diameters achieved a greater suppression of tumor growth in comparison to treatment with single-size TNP-470-loaded CPMs alone, and the control. Severe loss of body weight was not observed in any mice treated with any size of TNP-470-loaded CPMs. These results suggest that treatment with a mixture of differently-sized anti-angiogenic CPMs might be more effective than treatment with CPMs of a single size. This advanced chemoembolization method, which incorporated an anti-angiogenic agent to target the morphological vascular heterogeneity of tumors may contribute to effective treatment of locally advanced or recurrent solid tumors.

Uterine sarcomas are among the most aggressive uterine malignancies. Most uterine sarcomas are associated with a poor prognosis, with an estimated 2-year survival of <50%, even when discovered at an early stage. These tumors show a poor response to radiotherapy and chemotherapeutic agents (with substantial toxic effects) that are in current use (1). Previous studies in uterine sarcomas have apparently shown a higher expression of vascular endothelial growth factor (VEGF)-A and angiopoietin-2 genes, as well as a higher frequency of lymphovascular invasion and high microvascular density in comparison to other human uterine carcinomas (2).

In most solid tumors, the neovasculature developed through angiogenesis has an abnormal structure, with defects of the walls that make them more permeable than normal vessels. This vascular heterogeneity causes differences in the distribution patterns of perfusion and permeability (3). Thus, the tumor microvasculature possesses a high degree of heterogeneity in its structure and function. The tumor microenviroment, including the vasculature changes that occur according to tumor progression, results in irregular (unequal) distribution of hypoxic lesions and normoxic lesions within the tumor. This, in turn, leads to variations in the supply of oxygen and nutrients to the tumor due to the abnormal vascular network (4). These changes are remarkable in highly aggressive solid tumors, including uterine carcinosarcomas.

View this table:
Table I.

The preparation of the calcium-phosphate ceramic microspheres (CPMs) and their crystalline phases.

TNP-470 is a low-molecular-weight synthetic analog of fumagillin, a natural compound secreted by Aspergillus fumigatus (5), which inhibits angiogenesis via endothelial cell cycle arrest in the late G1 phase (6). Methionine aminopeptidase-2 has been identified as a molecular target of TNP-470 (7). The antitumor effect of TNP-470 has been shown in various human malignancies both in vitro and in vivo, including in our own studies (8-12). Our previous studies have also shown the efficacy of anti-angiogenic therapy using TNP-470 for highly aggressive human uterine carcinosarcoma both in vitro and in vivo (13-15).

In our previous study, we first designed a novel biomaterial composed of resorbable hollow ceramic microspheres loaded with TNP-470 to target the tumor vasculature and showed its usefulness and safety in a xenograft model of highly aggressive human uterine sarcoma xenograft model (16). As the next step for this new chemoembolization approach using ceramic microspheres, we designed a new form of anti-angiogenic microspheres as an advanced model to target (or measure) vascular heterogeneity in highly aggressive and highly angiogenic solid tumors. The results in this study suggest that the treatment with a mixture of differently-sized anti-angiogenic CPMs might be more effective than treatment with CPMs of a single size.

Materials and Methods

The preparation of calcium-phosphate ceramic microspheres with and without the agent TNP-470. The calcium-phosphate ceramic microspheres (CPMs) were synthesized by an ultrasonic spray-pyrolysis technique, as previously reported (17-20). Briefly, three types of starting solutions with a Ca/P ratio of 1.50 were prepared by mixing Ca(NO3)2, (NH4)2HPO4 and HNO3 (Table I). The upper- and lower-furnace temperatures were fixed at 850°C and 300°C, respectively. The above solution was sprayed into the heating zone of the furnace using an ultrasonic vibrator with a frequency of 2.4 MHz, and then the sprayed droplets were dried and pyrolyzed to form CPMs.

View this table:
Table II.

Ten treatment groups in vivo

The resulting CPMs were washed with pure water, and freeze-dried to prepare non-loaded CPMs for the in vitro and in vivo evaluations. TNP-470 was kindly donated by Takeda Chemical Industries (Osaka, Japan) suspended in a vehicle of 0.5% ethanol plus 5% gum arabic in saline. Its structure and characteristics have been previously described (14). TNP-470, was dissolved in ethanol to a concentration of 2,000 μg/cm3. CPMs loaded with TNP-470 were prepared by adding the CPMs (25 mg) into this ethanolic solution (1 cm3) and then freeze-drying it. Hereafter, we refer to the CPMs with and without TNP-470 on the basis of their concentrations of Ca2+ and PO43− ions as 1510(+) and 1510(−) (mol dm−3 Ca2+: 0.15, PO43−: 0.10); 6040(+) and 6040(−) (mol dm−3 Ca2+: 0.60, PPO43−: 0.40); and 9060(+) and 9060(−) (mol dm−3 Ca2+: 0.90, PO43−: 0.60), respectively. In addition, samples named Mix(+) and Mix(−) were prepared by mixing the three sizes of the above CPMs at the rate of 1:1:1 (w/w/w) with and without TNP-470, respectively.

The crystalline phases of the resulting sample powders were identified using an X-ray diffractometer (XRD). The phase composition was determined on the basis of the area of typical XRD diffraction lines: (221) for Hydroxyapatite (Ca10(PO4)6(OH)2; HAp) and (0210) for β-tricalcium phosphate (β-Ca3(PO4)2; β-TCP. The particle morphology was observed by scanning electron microscopy (SEM). The specific surface area (S) was measured by the BET (Brunauer, Emmet and Teller) method using N2 as an adsorption gas.

In vitro drug release. The release behavior of the drug from the resulting CPMs loaded with TNP-470 was examined as follows. The TNP-470-loaded microspheres (0.05 g) were immersed in physiological saline (1 cm3) at 37°C for 0.5, 1, 3, 5, 10, 24, 48, 72, and 120 h during shaking at the rate of 100 strokes/min. After the desired immersion periods, the released TNP-470 agent was separated by centrifuging the above suspension at 1,000 rpm (89.5 ×g) for 5 min. The amount of TNP-470 in the supernatant was determined by high-performance liquid chromatography (HPLC) (UV/VIS Detector SSC-5410; Senshu Scientific Co., Ltd., Tokyo, Japan). The column used was ODS-1251-N (4.6×250 mm), and the mobile phase was a solution of pure water/acetonitrile=30/70 (v/v).The wavelength of the HPLC detector was 217 nm in the UV region.

Figure 1.
Figure 1.

Scanning electron microscopy (SEM) images of calcium-phosphate ceramic microspheres (CPMs) with different particle sizes: a: 1510(−), b: 6040(−), and c: 9060(−).

Figure 2.
Figure 2.

The release profile of the drug from the CPMs loaded with TNP-470. 1510: Ca2+: 0.15, PO43−: 0.10 mol dm−3; 6040: Ca2+: 0.60, PO43−: 0.40 mol dm−3; and 9060 Ca2+: 0.90, PO43−: 0.60 mol dm−3; Mix: Ca2+: 0.15, PO43−: 0.10 mol dm−3+Ca2+: 0.60, PO43−: 0.40 mol dm−3+Ca2+: 0.90, PO43−: 0.60 mol dm−3.

The cell line and nude mice. A human uterine sarcoma cell line, FU-MMT-3, previously established by our laboratory from a patient with uterine carcinosarcoma, was used in the present study because this tumor is one of the most malignant neoplasms of the human solid tumors, and because it exhibits a poor response to radiotherapy and the chemotherapeutic agents that are in current use. FU-MMT-3 also exhibits a highly progressive activity both in vitro and in vivo. This cell line is chiefly composed of myogenic sarcoma cells; its immunophenotype, tumorigenicity, and cytogenetic characteristics have been reported (21). Female BALB/cA Jcl-nu athymic nude mice were obtained from Clea (Tokyo, Japan), and 5- to 6-week-old mice (body weight: 20 g) were used in the experiments. All animals were kept in isolation rooms at a controlled temperature and were caged in groups of five or fewer with ad libitum access to standard animal chow and water according to the instructions of the Institute of Experimental Animal Science, Fukuoka University Medical School. The in vivo experiments were performed in accordance with the Declaration of Helsinki and the World Medical Association, and were approved by the Institutional Animal Care and Use Committee of Fukuoka University (no. 0803222).

Injection of TNP-470-loaded microspheres. On the basis of the in vitro results, an in vivo evaluation was conducted using the microspheres prepared from the 1000 μg/ml TNP-470 solution. First, the mice were injected subcutaneously with 2×105 FU-MMT-3 cells in 0.2 ml Dulbecco's Modified Eagle's Medium in the right auxiliary region of the flank. Mice bearing resultant tumors measuring 5-10 mm in diameter on day 14 were randomly separated into 10 treatment groups as indicated in Table II.

1) injection of 1510(−) CPMs of 1.8 μm median diameter (25 mg); 2) injection of 6040(−) CPMs of 2.6 μm median diameter (25 mg); 3) injection of 9040(−) CPMs of 3.0 μm median diameter (25 mg); 4) injection of Mix(−) 1:1:1 mixture of 1510(−), 6040(−), 9040(−) CPMs; 5) injection of 1510(+) CPMs of 1.8 μm median diameter loaded with 1000-μg/ml TNP-470 suspended in physiological saline (0.4 cm3); 6) injection of 6040(+) CPMs of 2.6 μm median diameter loaded with 1000-μg/ml TNP-470 suspended in physiological saline (0.4 cm3); 7) Injection of 9040(+) CPMs of 3.0 μm in median diameter loaded with 1000-μg/ml TNP-470 suspended in physiological saline (0.4 cm3); 8) Injection of Mix(+) 1:1:1 mixture of 1510(+), 6040(+), 9040(+) CPMs with 1000-μg/ml TNP-470 suspended in physiological saline (0.4 cm3); 9) TNP-470 injection alone; and 10) non-treatment control (injection of 0.5% ethanol plus 5% gum arabic in saline) (n=4-5 in each group).

These therapies were administered three times per week for 8 weeks. TNP-470 alone for group 9 was injected subcutaneously at a dose of 30 mg/kg three times per week. CPMs for groups 1-8 (as described above) were injected subcutaneously at a dose of 200 μg three times per week. The mice were anesthetized with ether before the injection. The materials were then subcutaneously injected around the xenografts, under the guidance of a transdermal color Doppler ultrasound (SSD-4000; Aloka Ltd., Tokyo, Japan) with a 7.5-MHz curved array transducer (UST-987-7.5; Aloka Ltd.) while searching for the feeding arteries, as previously described (16). Briefly, a 30-gauge microinjection needle (0.25 mm in diameter) was advanced toward the feeding arteries into the bottom of the xenografts while monitoring the feeding arteries by color Doppler ultrasound. The solution was then slowly injected into these vessels. Where feeding arteries could not be fully visualized, the injection was made close to the vessels.

Figure 3.
Figure 3.

The antitumor effects of CPM treatment of FU-MMT-3 xenografts. Mix(+) treatment significantly suppressed tumor growth in comparison to 6040(+), and 9060(+).

Tumor growth was monitored by measuring the weekly volume twice, calculated as V=a×b2/2 (where a=length; b=width). Necropsies were performed on all mice soon after death during or after the course of therapies, and both the size and weight of the excised tumors were measured. The mean, SD, median, and SE of the tumor size during the different therapies and of the tumor weight after the completion of the therapies were calculated for each group.

Immunohistochemical staining. Hematoxylin and eosin (HE) staining was performed on all tumors in each FU-MMT-3 xenograft, which were resected from mice soon after death. Cryosections were used to determine the microvessels within each tumor and the sections were incubated with primary antibody to CD31 (dilution 1:100; PharMingen, San Diego, CA, USA), and these micorvessels were counted as described previously (15).

Statistical analysis. The in vivo data were expressed as the mean±SD. The Mann–Whitney U-test (non-parametric) was used to compare tumor growth, or tumor weight, between the three treatment groups and the control. These statistical analyses were performed using the StatView 5.0 for Macintosh software (SAS Institute, Inc., Cary, NC, USA). In each of the statistical tests, a p-value of less than 0.05 was considered to be statistically significant.

Results

The characterization of CPMs. The resulting CPMs were composed of β-TCP and ‘apatite’ biphases on the basis of XRD measurements. The Ca/P molar ratio, as determined by X-ray fluorescence spectrometry, was 1.49, which agreed well with the nominal composition of the starting solution. Thus, the ‘apatite’ presented in the CPMs may be a calcium-deficient hydroxyapatite (Ca-def HAp). Table I shows that the CPMs were composed of β-TCP and Ca-def HAp in the ranges of 54-74% and 26-46%, respectively. β-TCP and Ca-def HAp are known biodegradable ceramics.

The resulting CPMs were observed by SEM; typical micrographs are shown in Figure 1. These SEM micrographs indicated that the resulting powders were composed of spherical particles. The diameter of the particles decreased with decreasing concentration of the starting solution. The spherical particles may be formed via the following processes: (i) the removal of the solvent from the droplet surface, (ii) the formation of microcrystalline calcium phosphates, and (iii) the crystal growth of calcium phosphates. The resulting spherical particle diameters are dependent upon the droplet sizes of the starting solution. The droplet size may be reduced by decreasing the concentration of the starting solution, which in turn reduces the diameter of the spherical particles.

The release profile of TNP-470 from microspheres in vitro. Figure 2 shows the release profile of the drug from the CPMs loaded with TNP-470 over a period of 120 h. Ninety percent of the total TNP-470 from the CPMs was slowly released within 20 h. The amounts of TNP-470 released from the 1510(+) powder were the highest among examined sample powders.

Suppression of tumor growth and angiogenesis in vivo. The weekly changes in the mean tumor volume in FU-MMT-3 xenografts during the course of treatments are shown in Figure 3. Mix(+), 1510(+), and 9060(+) treatment significantly inhibited the growth of these FU-MMT-3 xenografts in comparison to controls (p<0.05, Mann–Whitney U-test). Mix(+) treatment significantly suppressed tumor growth in comparison to 6040(+), and 9060(+) (p<0.05, Mann–Whitney U-test).

Figure 4.
Figure 4.

The macroscopic findings on the surface and the histopathological findings of the FU-MMT-3 xenografts. A: Control; no treatment, B: Mix(+) treatment;. C-F: The histopathologic findings of FU-MMT-3 xenografts treated by Mix(+). Tumor microvessels with diameters of various sizes are seen in the control (A). The reduction of tumor microvessels is seen in the Mix(+) treatment in comparison to the control. Aggregates of CPMs can be seen on the tumor surface (arrow) (B). A feeding artery was entirely embolized by these CPMs (arrow). Lymphocytic infiltration is observed in the lumen and the surrounding fibrous tissues of the feeding arteries in Mix(+) treatment (400×) (C). Coagulative necrosis (asterisk) by chemoembolization is extensively seen following Mix(+) treatment (40×) (D). CPMs showing uniform basophilia, and aggregates of CPMs in tumor microvessels (E, F). CPMs are remarkably embolized in tumor microvessels of various sizes after Mix(+) treatment (F) (E: HE staining, ×200; F: immunohistochemical staining, ×400).

Severe loss of body weight was not observed in any mouse treated with any size of TNP-470-loaded CPMs.

Histopathological findings. Macroscopically, there was an apparent reduction of tumor microvessels on the surface in the tumor in comparison to the control (Figure 4A and B). Microscopically, the feeding arteries (arrow in Figure 4C) were observed to be entirely embolized by the CPMs. Lymphocytic infiltration was observed in the lumen and surrounding fibrous tissues of the feeding arteries; no injury caused by microspheres was found in the vessel walls (Figure 4C). Areas of tissue necrosis were extensively seen in all treatment groups, and especially in the Mix(+), and 1510(+) treated groups (Figure 4D). In all of the CPM treatment groups, aggregates of CPMs could be seen in the lumen of many microvessels within the tumor (Figure 4E and F).

Discussion

Drug delivery systems (DDS) can potentially be used for the active or passive targeting of tumor tissues. Passive targeting is based on the enhanced permeability and retention effect, which is based on the pathophysiological characteristics of solid tumor tissues, including their hypervascularity, incomplete vascular architecture, the secretion of vascular permeability factors stimulating extravasation within cancer tissue, and the absence of effective lymphatic drainage from tumors that impedes efficient clearance of macromolecules accumulated in solid tumor tissues (22). However, DDS using anti-angiogenic agents has not yet been fully-studied. Thus, anti-angiogenic DDS is expected to suppress tumor growth in two steps, not only by directly embolizing tumor vessels, but also by releasing an angiogenesis inhibitor from the microsphere carrier to the neighboring endothelial, stromal, and tumoral cells. Resorbable ceramic microparticles have been recently developed as a new embolic material. Excellent results have been reported regarding their use in microcatheter superselective embolization in rabbits and in the clinical therapy of 13 patients with meningioma (23, 24). Because the microparticles are spherical in shape, no complications such as hemorrhage or microcatheter clogging have occurred. These studies described their excellent biocompatibility and good visibility during injection control to produce the occlusion of the distal arteriocapillary bed (23, 24).

Many biomaterials are used as substitutes for human hard tissues such as bone or cartilage; these biomaterials have also recently been studied as carriers for drug delivery (25). CPMs were synthesized for clinical use in our previous studies (26-28). These biomaterials dissolve gradually without any cytotoxic or allergic reactions and elicit no immune response (29-32). Because of these characteristics, resorbable hollow CPMs were created in our laboratory, both for use as a drug carrier and as an embolization material (16). In our previous study, it was shown that treatment with CPMs alone significantly inhibited the growth of FU-MMT-3 xenografts in comparison to the control (16). It is suggested that a direct antitumor effect of this material by embolization occurred in vivo because histopathological investigations revealed that microspheres were remarkably embolized in the feeding arteries of the xenografts, as well as in many of the tumor microvessels. This strong embolization effect exhibited by CPMs alone is potentially useful for the treatment of cancer and some solid tumors.

Intra-tumoral vascular heterogeneity is an important feature of solid tumors (4) and needs to be considered when the therapeutic response to a targeted anti-angiogenic regimen is evaluated. Checkley et al. observed that in human PC-3 prostate adenocarcinoma xenografts that were treated with ZD6474, an anti-angiogenic agent, the core region in the xenograft tumor revealed a larger reduction in the pharmacokinetic parameter Ktrans (volume transfer coefficient) than the enhancing rims, suggesting a change in the distribution patterns of perfusion and permeability (3). An inadequate vascular supply and the subsequently induced hypoxia are the driving force of angiogenesis and increased vascular permeability (33). In the present study, in order to adapt our biomaterial for chemoembolization, the resorbable hollow CPMs were further refined (in our laboratory) to target vascular morphological heterogeneity. The in vitro results showed that the amounts of TNP-470 released from the 1510(+) powder were the highest among the examined sample powders. The 1510(-) powder consists of microspheres of the smallest diameter; therefore, the powder has the largest specific surface area (20 m2/g) in comparison to 6040(-) (14.4 m2/g) and 9060(-) (16.3 m2/g) powders. Thus, the 1510(-) powder with largest specific surface area can be loaded the most amounts of TNP-470 among examined CPMs. As the 1510 powder consists of microspheres of the smallest diameter, the 1510(+) solution would have the largest surface area of microspheres, thus this solution may include the highest amounts of TNP-470 in comparison to 6040(+), and 9040(+). As TNP-470 exhibited a two-step sustainable release in vitro from the internal space and external surface of the hollow CPMs, the smallest size of microspheres would be thought to be the most useful for drug carrier among these CPMs in vitro.

However, the in vivo results of the present study showed that the Mix(+) treatment tended to achieve a better suppression of tumor growth in FU-MMT-3 than the 1510(+) treatment. This result suggests that as the feeding arteries and tumor microvessels vary in diameter (they exhibit vascular morphological heterogeneity), these vessels might be more successfully embolized by different sizes of CPMs or their aggregates in the Mix(+) treatment. Additionally, the diameter of the CPMs was approximately 0.5-3 μm, and there was no evidence of blood injury, such as a remarkable hemorrhage or hematoma, in any of the treated mice. Thus, this new DDS using a mixture of sizes of CPMs might be a useful approach to chemoembolization in the treatment of many solid tumors, such as deep-seated cancer, including advanced or recurrent tumors in the abdomen, and urogenital and gynecological tumors that have already received radiotherapy. This advanced approach in chemoembolization to target (or measure) vascular morphological heterogeneity should be followed by clinical studies to assess its efficacy in the treatment of aggressive solid tumors.

Histopathologically, CPMs were remarkably embolized in the feeding arteries in the peripheral areas of the xenografts, as well as in many of the tumor microvessels in vivo. The destruction of tumor vessels and areas of coagulative necrosis were seen in all of the groups treated with CPMs, with and without TNP-470, in contrast to treatment with TNP-470 alone, suggesting a physical effect of embolization by the CPMs. The growth of the xenografts was significantly reduced in treatment with Mix(+) compared to treatment with Mix(−). The synergistic effect of the anti-angiogenic CPM delivery was shown in vivo. It is also suggested that TNP-470 is successfully released from the hollow CPMs after embolization at the tumor site. Thus, the physiochemical effects may be involved in a two-step inhibitory process of tumor angiogenesis.

In previous studies using TNP-470, the loss of body weight of mice was frequently observed (10-11, 34). In our present study, there were no remarkable side-effects, including severe loss of body weight, to any of the treatments using TNP-470-loaded CPMs, suggesting that TNP-470 may be more gently released than in treatment with TNP-470 alone (data not shown) (16). This result may support the safety of our new DDS as a chemoembolization approach using CPMs to target vascular heterogeneity in tumors.

In conclusion, this advanced chemoembolization method incorporating an anti-angiogenic agent to target morphological vascular heterogeneity may contribute to the effective treatment of locally advanced or recurrent solid tumors.

Acknowledgements

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 11671164).

Footnotes

  • Conflicts of Interest

    The Authors have no conflict of interest in regard to this study.

  • Received April 17, 2015.
  • Revision received May 21, 2015.
  • Accepted May 22, 2015.

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Advanced Chemoembolization by Anti-angiogenic Calcium-Phosphate Ceramic Microspheres Targeting the Vascular Heterogeneity of Cancer Xenografts
MAKOTO EMOTO, HAJIME YOSHIHISA, KYOKO YANO, BATSUREN CHOIJAMTS, HITOSHI TSUGU, KATSURO TACHIBANA, MAMORU AIZAWA
Anticancer Research Sep 2015, 35 (9) 4757-4764;
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