Publications

Many cancers begin with the formation of a small nest of transformed cells that can remain dormant for years. Thrombospondin-1 (TSP-1) initially promotes dormancy by suppressing angiogenesis, a key early step in tumor progression. Over time, increases in drivers of angiogenesis predominate, and vascular cells, immune cells, and fibroblasts are recruited to the tumor mass forming a complex tissue, designated the tumor microenvironment. Numerous factors, including growth factors, chemokine/cytokine, and extracellular matrix, participate in the desmoplastic response that in many ways mimics wound healing. Vascular and lymphatic endothelial cells, and cancer-associated pericytes, fibroblasts, macrophages and immune cells are recruited to the tumor microenvironment, where multiple members of the TSP gene family promote their proliferation, migration and invasion. The TSPs also affect the immune signature of tumor tissue and the phenotype of tumor-associated macrophages. Consistent with these observations, expression of some TSPs has been established to correlate with poor outcomes in specific types of cancer.

Background: Anaplastic thyroid carcinoma (ATC) is a rapidly fatal cancer with a median survival of a few months. Enhanced therapeutic options for durable management of ATC will rely on an understanding of genetics and the role of the tumor microenvironment. The prognosis for patients with ATC has not improved despite more detailed scrutiny of underlying tumor genetics. Pericytes in the microenvironment play a key evasive role for thyroid carcinoma (TC) cells. Lenvatinib improves outcomes in patients with radioiodine-refractory well-differentiated TC. In addition to the unclear role of pericytes in ATC, the effect and mechanism of lenvatinib efficacy on ATC have not been sufficiently elucidated. Design: We assessed pericyte enrichment in ATC. We determined the effect of lenvatinib on ATC cell growth cocultured with pericytes and in a xenograft mouse model from human BRAFWT/V600E-ATC-derived cells coimplanted with pericytes. Results: ATC samples were significantly enriched in pericytes compared with normal thyroid samples. BRAFWT/V600E-ATC-derived cells were resistant to lenvatinib treatment shown by a lack of suppression of MAPK and Akt pathways. Moreover, lenvatinib increased CD47 protein (thrombospondin-1 [TSP-1] receptor) levels over time vs. vehicle. TSP-1 levels were downregulated upon lenvatinib at late vs. early time points. Critically, ATC cells, when cocultured with pericytes, showed increased sensitivity to this therapy and ultimately decreased number of cells. The coimplantation in vivo of ATC cells with pericytes increased ATC growth and did not downregulate TSP-1 in the microenvironment in vivo. Conclusions and Implications: Pericytes are enriched in ATC samples. Lenvatinib showed inhibitory effects on BRAFWT/V600E-ATC cells in the presence of pericytes. The presence of pericytes could be crucial for effective lenvatinib treatment in patients with ATC. Degree of pericyte abundance may be an attractive prognostic marker in assessing pharmacotherapeutic options. Effective durable management of ATC will rely on an understanding not only of genetics but also on the role of the tumor microenvironment.

Considerable progress has been made in our understanding of the process of angiogenesis in the context of normal and tumor tissue over the last fifty years. Angiogenesis, like most physiological processes, is carefully controlled by dynamic and opposing effects of positive factors, such as vascular endothelial growth factor (VEGF), and negative factors, such as thrombospondin-1. In most cases, the progression of a small mass of cancerous cells to a life-threatening tumor depends upon the initiation of angiogenesis and involves the dysregulation of the angiogenic balance. Whereas our newfound appreciation for the role of angiogenesis in cancer has opened up new avenues for treatment, the success of these treatments, which have focused almost exclusively on antagonizing the VEGF pathway, has been limited to date. It is anticipated that this situation will improve as more therapeutics that target other pathways are developed, more strategies for combination therapies are advanced, more detailed stratification of patient populations occurs, and a better understanding of resistance to anti-angiogenic therapy is gained.

OBJECTIVES: Tumor vasculature is structurally abnormal, with anatomical deformities, reduced pericyte coverage and low tissue perfusion. As a result of this vascular dysfunction, tumors are often hypoxic, which is associated with an aggressive tumor phenotype, and reduced delivery of therapeutic compounds to the tumor. We have previously shown that a peptide containing the thrombospondin-1 type I repeats (3TSR) specifically targets tumor vessels and induces vascular normalization in a mouse model of epithelial ovarian cancer (EOC). However, due to its small size, 3TSR is rapidly cleared from circulation. We now introduce a novel construct with the 3TSR peptide fused to the C-terminus of each of the two heavy chains of the Fc region of human IgG1 (Fc3TSR). We hypothesize that Fc3TSR will have greater anti-tumor activity in vitro and in vivo compared to the native compound.

METHODS: Fc3TSR was evaluated in vitro using proliferation and apoptosis assays to investigate differences in efficacy compared to native 3TSR. In light of the multivalency of Fc3TSR, we also investigate whether it induces greater clustering of its functional receptor, CD36. We also compare the compounds in vivo using an orthotopic, syngeneic mouse model of advanced stage EOC. The impact of the two compounds on changes to tumor vasculature morphology was also investigated.

RESULTS: Fc3TSR significantly decreased the viability and proliferative potential of EOC cells and endothelial cells in vitro compared to native 3TSR. High-resolution imaging followed by image correlation spectroscopy demonstrated enhanced clustering of the CD36 receptor in cells treated with Fc3TSR. This was associated with enhanced downstream signaling and greater in vitro and in vivo cellular responses. Fc3TSR induced greater vascular normalization and disease regression compared to native 3TSR in an orthotopic, syngeneic mouse model of advanced stage ovarian cancer.

CONCLUSION: The development of Fc3TSR which is greater in size, stable in circulation and enhances receptor activation compared to 3TSR, facilitates its translational potential as a therapy in the treatment of metastatic advanced stage ovarian cancer.

The thrombospondins (TSPs) are a family of multimeric extracellular matrix proteins that dynamically regulate cellular behavior and response to stimuli. In so doing, the TSPs directly and indirectly affect biological processes such as embryonic development, wound healing, immune response, angiogenesis, and cancer progression. Many of the direct effects of Thrombospondin 1 (TSP-1) result from the engagement of a wide range of cell surface receptors including syndecans, low density lipoprotein receptor-related protein 1 (LRP1), CD36, integrins, and CD47. Different or even opposing outcomes of TSP-1 actions in certain pathologic contexts may occur, depending on the structural/functional domain involved. To expedite response to external stimuli, these receptors, along with vascular endothelial growth factor receptor 2 (VEGFR2) and Src family kinases, are present in specific membrane microdomains, such as lipid rafts or tetraspanin-enriched microdomains. The molecular organization of these membrane microdomains and their constituents is modulated by TSP-1. In this review, we will describe how the presence of TSP-1 at the plasma membrane affects endothelial cell signal transduction and angiogenesis.

A basic requirement of tumorigenesis is the development of a vascular network to support the metabolic requirements of tumor growth and metastasis. Tumor vascular formation is regulated by a balance between promoters and inhibitors of angiogenesis. Typically, the pro-angiogenic environment created by the tumor is extremely aggressive, resulting in the rapid vessel formation with abnormal, dysfunctional morphology. The altered morphology and function of tumor blood and lymphatic vessels has numerous implications including poor perfusion, tissue hypoxia, and reduced therapy uptake. Targeting tumor angiogenesis as a therapeutic approach has been pursued in a host of different cancers. Although some preclinical success was seen, there has been a general lack of clinical success with traditional anti-angiogenic therapeutics as single agents. Typically, following anti-angiogenic therapy, there is remodeling of the tumor microenvironment and widespread tumor hypoxia, which is associated with development of therapy resistance. A more comprehensive understanding of the biology of tumor angiogenesis and insights into new clinical approaches, including combinations with immunotherapy, are needed to advance vascular targeting as a therapeutic area.

[This corrects the article DOI: 10.3389/fendo.2013.00189.].

An integral step in the development of solid tumors is the recruitment of blood vessels to fuel tumor growth. Antiangiogenic therapies can inhibit this process and control solid tumor growth. Thrombospondin-1 is an antiangiogenic protein possessing three type I repeats (3TSR) near the center of the protein and a CD47-binding peptide (CD47) in its C-terminus. Previously, we showed that treatment with recombinant 3TSR induces tumor regression, normalizes tumor vasculature, and improves uptake of chemotherapy drugs in an orthotopic, syngeneic mouse model of advanced stage epithelial ovarian cancer (EOC). While effective, this intervention required daily intraperitoneal injections. To circumvent this, here we employ adeno-associated virus (AAV) gene therapy vectors to express 3TSR alone or in combination with the CD47-binding peptide of TSP-1 and evaluate the impact on tumor development and survival in a mouse model of EOC. A single intraperitoneal injection of 1 × 1011 vg of AAV expressing 3TSR, CD47-binding peptide, or 3TSR + CD47 effectively suppressed primary tumor growth; however, only AAV-3TSR was able to inhibit development of secondary lesions at 90-days post-tumor implantation and significantly improve survival. Taken together, AAV-mediated expression of 3TSR appears safe and effective at inhibiting tumor development and represents a novel, less invasive approach for treating ovarian carcinoma.

PURPOSE: Intravenous delivery of oncolytic viruses often leads to tumor vascular shutdown, resulting in decreased tumor perfusion and elevated tumor hypoxia. We hypothesized that using 3TSR to normalize tumor vasculature prior to administration of an oncolytic Newcastle disease virus (NDV) would enhance virus delivery and trafficking of immunologic cell subsets to the tumor core, resulting in systemically enhanced immunotherapy and regression of advanced-stage epithelial ovarian cancer (EOC).

EXPERIMENTAL DESIGN: Using an orthotopic, syngeneic mouse model of advanced-stage EOC, we pretreated mice with 3TSR (4 mg/kg per day) alone or followed by combination with fusogenic NDV(F3aa) (1.0 × 108 plaque-forming units).

RESULTS: Treatment with 3TSR normalized tumor vasculature, enhanced blood perfusion of primary EOC tumors, and induced disease regression. Animals treated with combination therapy had the greatest reduction in primary tumor mass, ascites accumulation, and secondary lesions (50% of mice were completely devoid of peritoneal metastases). Combining 3TSR + NDV(F3aa) led to enhanced trafficking of immunologic cells into the primary tumor core.

CONCLUSIONS: We have shown, for the first time, that NDV, like other oncolytic viruses, is a potent mediator of acute vascular shutdown and that preventing this through vascular normalization can promote regression in a preclinical model of advanced-stage ovarian cancer. This challenges the current focus on induction of intravascular thrombosis as a requisite for successful oncolytic virotherapy.See related commentary by Bykov and Zamarin, p. 1446.

PURPOSE: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown.

EXPERIMENTAL DESIGN: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment.

RESULTS: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFβ1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFβ1 expression evoke gene-regulatory networks and pathways (TGFβ signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand-VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFβ1 axis reduces tumor cell growth and overcomes drug resistance.

CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFβ1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.