Nanoparticulate Tetrac Inhibits Growth and Vascularity of Glioblastoma Xenografts

Abstract

Thyroid hormone as L-thyroxine (T₄) stimulates proliferation of glioma cells in vitro and medical induction of hypothyroidism slows clinical growth of glioblastoma multiforme (GBM). The proliferative action of T₄ on glioma cells is initiated nongenomically at a cell surface receptor for thyroid hormone on the extracellular domain of integrin αvβ3. Tetraiodothyroacetic acid (tetrac) is a thyroid hormone derivative that blocks T₄ action at αvβ3 and has anticancer and anti-angiogenic activity. Tetrac has been covalently bonded via a linker to a nanoparticle (Nanotetrac, Nano-diamino-tetrac, NDAT) that increases the potency of tetrac and broadens the anticancer properties of the drug. In the present studies of human GBM xenografts in immunodeficient mice, NDAT administered daily for 10 days subcutaneously as 1 mg tetrac equivalent/kg reduced tumor xenograft weight at animal sacrifice by 50%, compared to untreated control lesions (p < 0.01). Histopathological analysis of tumors revealed a 95% loss of the vascularity of treated tumors compared to controls at 10 days (p < 0.001), without intratumoral hemorrhage. Up to 80% of tumor cells were necrotic in various microscopic fields (p < 0.001 vs. control tumors), an effect attributable to devascularization. There was substantial evidence of apoptosis in other fields (p < 0.001 vs. control tumors). Induction of apoptosis in cancer cells is a well-described quality of NDAT. In summary, systemic NDAT has been shown to be effective by multiple mechanisms in treatment of GBM xenografts.

Fig. 1

Effects at 16 days after single injection of U87MG cancer cells along with various treatments. There were four animals per group and two tumors per animal, n = 8. Error bars represent ±SEM and statistical significance is compared to control, *p < 0.01. a Tumor weights for control (PBS), void nanoparticle (NP), tetrac (3 μg), tetrac (10 μg), NDAT (3 μg), and NDAT (10 μg). b Hemoglobin content for the same groups

Fig. 2

IVIS imaging of harvested tumors 16 days after single injections of U87MG-luc cancer cells along with various treatments; there were four animals per group and two tumors per animal. a Effects of control (PBS), void nanoparticle (NP), tetrac (3 μg), tetrac (10 μg), NDAT (3 μg), and NDAT (10 μg). The vertical luminescence color bar (right side) estimates viability, ranging from nonviable (blue) to fully viable (red). NDAT at 10 μg/implant approximates 10⁻⁷ M tissue tetrac equivalent drug concentration. b Quantitation of the tumor bioluminescent signal intensity in a. Error bars represent ±SEM compared to control, n = 8

Fig. 3

Effect of 10 days of daily s.c. NDAT (1 mg/kg) treatment on U87MG glioblastoma xenograft volume and weight. There were four animals per group and two tumors per animal, n = 8. Error bars represent ±SEM and statistical significance is compared to control. Three of the four control animals died on day 9, and their tumors’ volume and weight reflect 9 days of treatment. a Volumes of harvested tumors decreased by 60%, compared to controls. b There was a reduction in harvested tumor weight of 50% (*p < 0.01), compared to control lesions

Fig. 4

Histology showing effect of 10 days of daily s.c. NDAT treatment (1 mg/kg) on U87MG glioblastoma xenografts. a–c: Low power views of histologic sections of tumor xenografts harvested from control and NDAT-treated animals. a Sections from untreated animal, showing decreased cellularity in central area of tumor (small arrows), but not complete necrosis. b, c Sections from tumors of treated animals reveal extensive central necrosis involving 50% of tumor mass (b) or a smaller tumor (c) containing less than 30% viable cells. Small arrows show border of viable cells, and in b, part of the central necrotic area has been lost during processing of the tissue. Lower 3 panels (d–f) are high-power (400×) views of sections from tumors of NDAT-treated animals, showing cells undergoing necrosis (black arrows) and apoptosis (white arrows). Insets D′ and D″ show mitosis and apoptosis, respectively. Apoptotic cells have pyknotic nuclei and condensed eosinophilic cytoplasm and show separation from adjacent viable cells. In necrotic areas, there is no cellular morphology or nuclear staining

Fig. 5

Induction of necrosis by 10 days of daily s.c. NDAT treatment (1 mg/kg) in U87MG glioblastoma xenografts significantly increased necrosis and apoptosis in various fields. As a result, cell density was reduced. The vasculature essentially disappeared from xenografts of NDAT-treated animals. Data were collected from four animals per group and two tumors per animal, n = 8. Error bars represent ±SEM and statistical significance is compared to control, **p < 0.001

Fig. 6

Effect of 10 days of daily s.c. NDAT (1 mg/kg) treatment on U87MG glioblastoma xenografts. Data were collected from four animals per group and two tumors per animal, n = 8. Error bars represent ±SEM and statistical significance is compared to control, **p < 0.001. Quantitative representation of a histologic estimates of tumor area and b average number of mitotic figures/field examined