Thrombospondins in the tumor microenvironment.

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.

The Tyrosine Kinase Inhibitor Lenvatinib Inhibits Anaplastic Thyroid Carcinoma Growth by Targeting Pericytes in the Tumor Microenvironment.

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.

Counter regulation of tumor angiogenesis by vascular endothelial growth factor and thrombospondin-1.

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.

Addition of an Fc-IgG induces receptor clustering and increases the in vitro efficacy and in vivo anti-tumor properties of the thrombospondin-1 type I repeats (3TSR) in a mouse model of advanced stage ovarian cancer.

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.

Normalizing Tumor Vasculature to Reduce Hypoxia, Enhance Perfusion, and Optimize Therapy Uptake.

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.

Endothelial Cell Behavior Is Determined by Receptor Clustering Induced by Thrombospondin-1.

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.

Corrigendum: Thrombospondin-1 Silencing Down-Regulates Integrin Expression Levels in Human Anaplastic Thyroid Cancer Cells With BRAFV600E: New Insights in the Host Tissue Adaptation and Homeostasis of Tumor Microenvironment.

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

Readmission to hospital following laparoscopic cholecystectomy: a meta-analysis.

BACKGROUND: Laparoscopic cholecystectomy (LC) is one of the most commonly performed surgical procedures. Despite this, patterns of readmission following LC are not well defined. This meta-analysis aimed to determine rates and predictors of readmission. METHODS: An ethically approved International Prospective Register of Systematic Reviews (PROSPERO)-registered meta-analysis was undertaken searching PubMed, Scopus, Web of Science and Cochrane Library databases from January 2013-June 2018 adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Published literature potentially suitable for data analysis was graded using methodological index for non-randomised studies (MINORS) criteria; papers scoring ≥ 16/24 for comparative and ≥ 10/16 for non-comparative studies were included. A meta-analysis of potential risk factors was performed by computing the odds ratio using Mantel-Haenszel method and fixed-effects model with 95% confidence intervals. RESULTS: Three thousand and eight hundred thirty-two articles were reduced to 44 studies qualifying for a final analysis of 1,573,715 laparoscopic cholecystectomies from 25 countries. Overall readmission rate was 3.3% (range: 0.0-11.7%); 52,628 readmissions out of 1,573,715 LCs. Surgical complications accounted for 76% of reported reasons for readmission, predominantly bile duct complications (33%), wound infection (17%) and nausea and vomiting (9%). Pain (15%) and cardiorespiratory complications (8%) account for the remainder. Obesity, single port LC and day case LC were not associated with increased rates. CONCLUSIONS: Pain, nausea and vomiting and surgical complications, particularly bile duct obstruction are the most common causes for readmission. Intra-operative cholangiography may reduce readmission rates. Causes for readmission were inconsistently reported throughout. The mean readmission rate of 3.3% may act as a quality benchmark for improving LC, and clearer reporting of reasons for readmission are required to advance care.

AAV-mediated expression of 3TSR inhibits tumor and metastatic lesion development and extends survival in a murine model of epithelial ovarian carcinoma.

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.

Meta-analysis of unplanned readmission to hospital post-appendectomy: an opportunity for a new benchmark.

BACKGROUND: Appendicitis is the most common cause of acute abdominal pain requiring surgical intervention. While many studies report readmission, a meta-analysis of readmission post-appendectomy has not been published. This meta-analysis was undertaken to determine rates and predictors of hospital readmission following appendectomy and to potentially provide a metric benchmark. METHODS: An ethically approved PROSPERO-registered (ID CRD42017069040) meta-analysis following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, using databases PubMed and Scopus, was undertaken for studies published between January 2012 and June 2017. Articles relating to outcomes and readmissions after appendectomy were identified. Those scoring >15 for comparative studies and >10 for non-comparative studies, using Methodological Index for Non-Randomized Studies criteria were included in the final analysis. The odds ratios (OR) using random-effects, Mantel-Haenszel method with 95% confidence intervals (CI), were computed for each risk factor with RevMan5. RESULTS: A total of 1757 articles reviewed were reduced to 45 qualifying studies for a final analysis of 836 921 appendectomies. 4.3% (range 0.0-14.4%) of patients were readmitted within 30 days. Significant preoperative patient factors for increased readmission were diabetes mellitus (OR 1.93, CI 1.63-2.28, P < 0.00001), complicated appendicitis (OR 3.6, CI 2.43-5.34, P < 0.00001) and open surgical technique (OR 1.39, CI 1.08-1.79, P < 0.00001). Increased readmission was not associated with gender, obesity or paediatric versus general surgeons or centres. CONCLUSION: This meta-analysis identified that readmission is not uncommon post-appendectomy, occurring in one in 25 cases. The mean readmission rate of 4.3% may act as a quality benchmark for improving emergency surgical care. Targeting high-risk groups with diabetes or complicated appendicitis and increasing use of laparoscopic technique may help reduce readmission rates.

Combining Vascular Normalization with an Oncolytic Virus Enhances Immunotherapy in a Preclinical Model of Advanced-Stage Ovarian Cancer.

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.

Pericytes Elicit Resistance to Vemurafenib and Sorafenib Therapy in Thyroid Carcinoma via the TSP-1/TGFß1 Axis.

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 TGFß1, 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 TGFß1 expression evoke gene-regulatory networks and pathways (TGFß 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/TGFß1 axis reduces tumor cell growth and overcomes drug resistance. CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFß1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

Integration of pro- and anti-angiogenic signals by endothelial cells.

Angiogenesis or neovascularization is a complex multi-step physiological process that occurs throughout life both in normal tissues and in disease. It is tightly regulated by the balance between pro-angiogenic and anti-angiogenic factors. The angiogenic switch has been identified as the key step during tumor progression in which the balance between pro-angiogenic and anti-angiogenic factors leans toward pro-angiogenic stimuli promoting the progression of tumors from dormancy to dysplasia and ultimately malignancy. This event can be described as either the outcome of a genetic event occurring in cancer cells themselves, or the positive and negative cross-talk between tumor-associated endothelial cells and other cellular components of the tumor microenvironment. In recent years, the mechanisms underlying the angiogenic switch have been extensively investigated in particular to identify therapeutic targets that can lead to development of effective therapies. In this review, we will discuss the current findings on the regulatory pathways in endothelial cells that are involved in the angiogenic switch with an emphasis on the role of anti-angiogenic protein, thrombospondin-1 (TSP-1) and pro-angiogenic factor, vascular endothelial growth factor (VEGF).

Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations.

KRIT1 mutations are the most common cause of cerebral cavernous malformation (CCM). Acute Krit1 gene inactivation in mouse brain microvascular endothelial cells (BMECs) changes expression of multiple genes involved in vascular development. These changes include suppression of Thbs1, which encodes thrombospondin1 (TSP1) and has been ascribed to KLF2- and KLF4-mediated repression of Thbs1 In vitro reconstitution of TSP1 with either full-length TSP1 or 3TSR, an anti-angiogenic TSP1 fragment, suppresses heightened vascular endothelial growth factor signaling and preserves BMEC tight junctions. Furthermore, administration of 3TSR prevents the development of lesions in a mouse model of CCM1 (Krit1ECKO ) as judged by histology and quantitative micro-computed tomography. Conversely, reduced TSP1 expression contributes to the pathogenesis of CCM, because inactivation of one or two copies of Thbs1 exacerbated CCM formation. Thus, loss of Krit1 function disables an angiogenic checkpoint to enable CCM formation. These results suggest that 3TSR, or other angiogenesis inhibitors, can be repurposed for TSP1 replacement therapy for CCMs.

Fluvastatin inhibits intimal hyperplasia in wild-type but not Thbs1-null mice.

BACKGROUND: Thrombospondin-1 (TSP-1) is functionally important to intimal hyperplasia (IH) development. Statin drugs have beneficial pleiotropic effects, including reduced IH; however, the effect of statins on IH in a TSP-1-independent setting is unknown. HYPOTHESIS: Statins will be less effective in attenuating IH after vascular injury in TSP-1-null (Thbs1-/-) mice compared with wild-type (WT) mice. MATERIALS AND METHODS: Carotid artery ligation was performed on WT and Thbs1-/- mice. Each strain was divided into two groups: no statin control or standard chow containing fluvastatin (10 or 40 mg/kg/d). After 28 d, analysis included morphometric analysis and real-time quantitative reverse transcription polymerase chain reaction on the arteries and enzyme-linked immunosorbent assay on plasma (TSP-1 WT, TSP-2 WT, and Thbs1-/-). Comparisons were made by analysis of variance, with P < 0.05 considered significant. RESULTS: In no statin controls, WT mice had more IH than Thbs1-/- mice (0.46 ± 0.09 versus 0.15 ± 0.04). Fluvastatin reduced IH in the WT (0.46 ± 0.09 versus 0.23 ± 0.06), but not in Thbs1-/- groups (0.15 ± 0.04 versus 0.22 ± 0.07). No difference in IH existed between Thbs1-/- no statin controls and fluvastatin WT and Thbs1-/- groups. Statin dose did not affect IH. TSP-1 plasma levels were increased in fluvastatin WT. TSP-2 levels were decreased in fluvastatin WT and elevated in fluvastatin Thbs1-/-. Fluvastatin had no effect on tissue Thbs1 or Thbs2 gene expression. CONCLUSIONS: TSP-1 is necessary for robust IH after arterial injury. Because fluvastatin had no effect on IH in Thbs1-/-, the data suggest that the statin effect on IH may be largely TSP-1 dependent. Both statins and the presence of TSP-1 affect TSP-1 and TSP-2 plasma levels.

Thrombospondin-1 in a Murine Model of Colorectal Carcinogenesis.

Colorectal Cancer (CRC) is one of the late complications observed in patients suffering from inflammatory bowel diseases (IBD). Carcinogenesis is promoted by persistent chronic inflammation occurring in IBD. Understanding the mechanisms involved is essential in order to ameliorate inflammation and prevent CRC. Thrombospondin 1 (TSP-1) is a multidomain glycoprotein with important roles in angiogenesis. The effects of TSP-1 in colonic tumor formation and growth were analyzed in a model of inflammation-induced carcinogenesis. WT and TSP-1 deficient mice (TSP-1-/-) of the C57BL/6 strain received a single injection of azoxymethane (AOM) and multiple cycles of dextran sodium sulfate (DSS) to induce chronic inflammation-related cancers. Proliferation and angiogenesis were histologically analyzed in tumors. The intestinal transcriptome was also analyzed using a gene microarray approach. When the area containing tumors was compared with the entire colonic area of each mouse, the tumor burden was decreased in AOM/DSS-treated TSP-1-/- versus wild type (WT) mice. However, these lesions displayed more angiogenesis and proliferation rates when compared with the WT tumors. AOM-DSS treatment of TSP-1-/- mice resulted in significant deregulation of genes involved in transcription, canonical Wnt signaling, transport, defense response, regulation of epithelial cell proliferation and metabolism. Microarray analyses of these tumors showed down-regulation of 18 microRNAs in TSP-1-/- tumors. These results contribute new insights on the controversial role of TSP-1 in cancer and offer a better understanding of the genetics and pathogenesis of CRC.

Thrombospondin-1-Based Antiangiogenic Therapy.

Ocular angiogenesis is one of the underlying causes of blindness and vision impairment and may occur in a spectrum of disorders, including diabetic retinopathy, neovascular age-related macular degeneration, retinal artery or vein occlusion, and retinopathy of prematurity. As such, strategies to inhibit angiogenesis by suppressing vascular endothelial growth factor activity have proven to be effective in the clinic for the treatment of eye diseases. A complementary approach would be to increase the level of naturally occurring inhibitors of angiogenesis, such as thrombospondin (TSP)-1. This article summarizes the development of TSP-1-based inhibitors of angiogenesis.