Taturo Udagawa

Targeting angiogenesis-dependent calcified neoplasms using combined polymer therapeutics.

Background There is an immense clinical need for novel therapeutics for the treatment of angiogenesis-dependent calcified neoplasms such as osteosarcomas and bone metastases. We developed a new therapeutic strategy to target bone metastases and calcified neoplasms using combined polymer-bound angiogenesis inhibitors. Using an advanced “living polymerization” technique, the reversible addition-fragmentation chain transfer (RAFT), we conjugated the aminobisphosphonate alendronate (ALN), and the potent anti-angiogenic agent TNP-470 with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer through a Glycine-Glycine-Proline-Norleucine linker, cleaved by cathepsin K, a cysteine protease overexpressed at resorption sites in bone tissues. In this approach, dual targeting is achieved. Passive accumulation is possible due to the increase in molecular weight following polymer conjugation of the drugs, thus extravasating from the tumor leaky vessels and not from normal healthy vessels. Active targeting to the calcified tissues is achieved by ALNs affinity to bone mineral. Methods and Finding The anti-angiogenic and antitumor potency of HPMA copolymer-ALN-TNP-470 conjugate was evaluated both in vitro and in vivo. We show that free and conjugated ALN-TNP-470 have synergistic anti-angiogenic and antitumor activity by inhibiting proliferation, migration and capillary-like tube formation of endothelial and human osteosarcoma cells in vitro. Evaluation of anti-angiogenic, antitumor activity and body distribution of HPMA copolymer-ALN-TNP-470 conjugate was performed on severe combined immunodeficiency (SCID) male mice inoculated with mCherry-labeled MG-63-Ras human osteosarcoma and by modified Miles permeability assay. Our targeted bi-specific conjugate reduced VEGF-induced vascular hyperpermeability by 92% and remarkably inhibited osteosarcoma growth in mice by 96%. Conclusions This is the first report to describe a new concept of a narrowly-dispersed combined polymer therapeutic designed to target both tumor and endothelial compartments of bone metastases and calcified neoplasms at a single administration. This new approach of co-delivery of two synergistic drugs may have clinical utility as a potential therapy for angiogenesis-dependent cancers such as osteosarcoma and bone metastases.

The antiangiogenic agents TNP-470 and 2-methoxyestradiol inhibit the growth of angiosarcoma in mice

BACKGROUND Endothelial malignancies, such as angiosarcoma and hemangioendothelioma, are often resistant to chemotherapy and surgery, and may result in death. Improved means of therapy are needed for these disorders. OBJECTIVE We wanted to determine whether angiosarcoma can be treated with angiogenesis inhibitors in mice. METHODS Mice were inoculated with a cell line that gives rise to angiosarcoma and were treated with the angiogenesis inhibitors 2-methoxyestradiol and TNP-470. Response to therapy was monitored by measurement of tumors. RESULTS TNP-470 caused an 84% reduction in tumor size, and 2-methoxyestradiol caused a 68% reduction in tumor size. CONCLUSION Angiogenesis inhibitors are highly effective in treatment of angiosarcoma in mice. Clinical trials of these agents in humans with angiosarcoma and hemangioendothelioma are warranted.

Matrigel cytometry: A novel method for quantifying angiogenesis in vivo

Many of the current in vivo methods to evaluate angiogenesis are poorly quantifiable. Recently, the Matrigel plug assay has become the method of choice in many studies involving in vivo testing for angiogenesis. When known angiogenic factors are mixed with Matrigel and injected subcutaneously into mice, endothelial cells migrate into the gel plug. These endothelial cells form vessel-like structures, a process that mimics the formation of capillary networks. Here, we present a modification of the traditional Matrigel assay with improved method to quantify the amount of endothelial cells that incorporate into the plug. The removed plugs were subjected to a mild protease treatment, yielding intact cells. The liberated cells were then stained using an endothelial cell-specific markers, and counted by flow cytometry. This novel combination of FACS analysis with the traditional Matrigel assay improves the ability to quantify in vivo angiogenesis, and for the first time enables to determine the number of migrating and proliferating endothelial cells which reflects the angiogenesis rate.

Bone marrow is a reservoir for proangiogenic myelomonocytic cells but not endothelial cells in spontaneous tumors

The hypothesis that bone marrow-derived, circulating endothelial cells incorporate into tumor blood vessels is unresolved. We have measured the numbers of bone marrow-derived versus resident endothelial cells in spontaneous prostate cancers during different stages of tumor progression and in age-matched normal prostates. Bone marrow-derived endothelial cells were rare in dysplasia and in well differentiated cancers representing between 0 and 0.04% of the total tumor mass. Instead, approximately 99% of all tumor-associated bone marrow-derived cells were CD45(+) hematopoietic cells, including GR-1(+), F4-80(+), and CD11b(+) myeloid cells. Similar to peripheral blood mononuclear cells, these tumor-associated myeloid cells expressed matrix metalloproteinases (MMPs), consistent with their proposed catalytic role during tumor angiogenesis. Furthermore, freshly isolated CD11b(+) cells stimulated tumor endothelial cell cord formation by 10-fold in an in vitro angiogenesis assay. The bone marrow is, therefore, a reservoir for cells that augment tumor angiogenesis, but the tumor endothelium is derived primarily from the local environment.

Chronic Suppression of Angiogenesis following Radiation Exposure Is Independent of Hematopoietic Reconstitution

Radiation can potentially suppress neovascularization by inhibiting the incorporation of hematopoietic precursors as well as damaging mature endothelial cells. The purpose of these studies was to quantify the effect of radiation on angiogenesis and to examine the relationship between bone marrow reconstitution and neovascularization. Immune competent, severe combined immunodeficient, RAG1-deficient, and green fluorescence protein transgenic mice in the C57 genetic background, as well as the highly angiogenic 129S1/SvlmJ strain of mice, underwent whole-body or localized exposure to radiation. The hematopoietic systems in the irradiated recipients were restored by bone marrow transfer. Hematopoietic reconstitution was assessed by doing complete blood counts. Angiogenesis was induced in the mouse cornea using 80 ng of purified basic fibroblast growth factor, and the neovascular response was quantified using a slit lamp biomicroscope. Following whole-body exposure and bone marrow transplantation, the hematopoietic system was successfully reconstituted over time, but the corneal angiogenic response was permanently and significantly blunted up to 66%. Localized exposure of the eyes to radiation suppressed corneal angiogenesis comparably to whole-body exposure. Whole-body irradiation with ocular shielding induced bone marrow suppression but did not inhibit corneal neovascularization. In mice exposed to radiation before tumor implantation, the reduced local angiogenic response correlated with significantly reduced growth of tumor cells in vivo. These results indicate that bone marrow suppression does not suppress neovascularization in the mouse cornea and that although hematopoietic stem cells can readily reconstitute peripheral blood, they do not restore a local radiation-induced deficit in neovascular response.

Thalidomide and Analogs

Thalidomide was developed in 1958 by Chemie Grunenthal as an oral sedative. Subsequently, McBride (1) and Lenz (2) described the association between maternal usage of thalidomide and fetal limb defects. The effect of thalidomide as a teratogen was experimentally confirmed in a number of species (3–8). By the time thalidomide was withdrawn from the market in 1962, thousands of babies worldwide were estimated to have been born with thalidomide-related defects. As a consequence, the mechanism by which thalidomide causes birth defects has been intensively investigated. Nevertheless, the mechanism has remained elusive (9).

X-linked dominant growth suppression of transplanted tumors in C57BL/6J-scid mice.

Tumor susceptibility, angiogenesis, and immune response differ between mouse strains. We, therefore, examined the growth rates of tumor xenografts in three genetically isolated strains of severe combined immunodeficient mice (C.B-17, C57BL/6J, and C3H). Tumors grew at significantly reduced rates in the C57BL/6J-scid strain. Engrafting bone marrow from the C57BL/6J-scid strain onto C.B-17-scid mice did not transfer the slow-growing tumor phenotype to the recipient mice; this counters the supposition that the slow-growing tumor phenotype is caused by a greater immune response to the xenograft in the C57BL/6J-scid strain. To establish the inheritance pattern of the slow-growing tumor phenotype, we reciprocally crossed C.B-17-scid mice and C57BL/6J-scid mice. Tumor growth was suppressed in all of the F1 progeny except the male mice derived from the cross between C.B-17-scid female and C57BL/6J-scid male mice. The F1 male mice that received the X chromosome from the C.B-17 strain displayed a fast-growing tumor phenotype. These results confirm that there are significant strain differences in capacity to support the growth of tumor xenografts. In addition, these results reveal the existence of a dominant allele involved in host suppression of tumor growth on the X chromosome of C57BL/6J mice.

Abstract 5235: Uncovering tumor-host molecular and cellular interactions involved in tumor dormancy using microRNAs

There is an increasing interest in elucidating the mechanisms of the earlier stages in tumor progression, the point at which a dormant tumor acquires the ability to grow and metastasize. Although the tumor dormancy phenomenon has important implications for early detection and treatment of cancer, its biology and genetic characteristics are poorly understood. microRNAs (miRs), small non-coding RNA molecules which regulate gene expression at the post transcriptional level, are emerging as key regulators in physiological and pathological processes, including tumorigenesis. We hypothesized that specific miRs may also play a key role in the regulation of tumor dormancy and the angiogenic switch. We therefore established a pair of cell lines that generate dormant avascular and fast-growing angiogenic osteosarcomas in SCID mice (Saos-2-D and Saos-2-E respectively). Although these cells share similar growth rate in vitro, when inoculated into mice, Saos-2-E cells establish vascularized and palpable tumors within one month, while Saos-2-D cells remain avascular and non-palpable for a year. Following our hypothesis, miR array of Saos-2-D and Saos-2-E cells, was performed. Several miRs that were differentially-expressed in the dormant versus fast-growing-tumor-forming cells were selected for further evaluation. Out of those miRs, miR200c and miR93 (representing either a mild or a substantial difference in the expression profile respectively) reverted cells forming fast-growing tumors to a dormant non-angiogenic phenotype, both in vitro and in vivo. Preliminary in vitro analysis of human brain, breast and prostate cancer cell lines revealed a similar phenotypic effect following overexpression of those miRs. We therefore propose that miR200c and miR93 play a role in the switch from dormancy. Further analysis on the target genes of those miRs is required in order to fully elucidate their role in tumor dormancy. Uncovering new molecular targets involved in the tumor dormancy phenomenon could provide important tools for novel dormancy-directed tumor therapy strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5235. doi:1538-7445.AM2012-5235