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Supplementary Figure 3 from Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

Supplementary Figure 1 from Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

Supplementary Figure 4 from Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

Supplementary Figure 2 from Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

Supplementary Methods from Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

Supplementary Figure Legends 1-4 from Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

Supplementary Figures 1-6 from Differential Effects of VEGFR-1 and VEGFR-2 Inhibition on Tumor Metastases Based on Host Organ Environment

Supplementary Figure Legends 1-6 from Differential Effects of VEGFR-1 and VEGFR-2 Inhibition on Tumor Metastases Based on Host Organ Environment

Numerous preclinical studies have demonstrated that angiogenesis inhibitors can increase the efficacy of radiotherapy (RT). We sought to examine the safety and efficacy of bevacizumab (BV) and RT in soft tissue sarcomas and explore biomarkers to help determine the treatment response.Patients with ≥5 cm, intermediate- or high-grade soft tissue sarcomas at significant risk of local recurrence received neoadjuvant BV alone followed by BV plus RT before surgical resection. Correlative science studies included analysis of the serial blood and tumor samples and serial perfusion computed tomography scans.The 20 patients had a median tumor size of 8.25 cm, with 13 extremity, 1 trunk, and 6 retroperitoneal/pelvis tumors. The neoadjuvant treatment was well tolerated, with only 4 patients having Grade 3 toxicities (hypertension, liver function test elevation). BV plus RT resulted in ≥80% pathologic necrosis in 9 (45%) of 20 tumors, more than double the historical rate seen with RT alone. Three patients had a complete pathologic response. The median microvessel density decreased 53% after BV alone (p.05). After combination therapy, the median tumor cell proliferation decreased by 73%, apoptosis increased 10.4-fold, and the blood flow, blood volume, and permeability surface area decreased by 62-72% (p.05). Analysis of gene expression microarrays of untreated tumors identified a 24-gene signature for treatment response. The microvessel density and circulating progenitor cells at baseline and the reduction in microvessel density and plasma soluble c-KIT with BV therapy also correlated with a good pathologic response (p.05). After a median follow-up of 20 months, only 1 patient had developed local recurrence.The results from the present exploratory study indicated that BV increases the efficacy of RT against soft tissue sarcomas and might reduce the incidence of local recurrence. Thus, this regimen warrants additional investigation. Gene expression profiles and other tissue and circulating biomarkers showed promising correlations with treatment response.

Tumors induce new blood vessel growth primarily from host organ microvascular endothelial cells (EC), and microvasculature differs significantly between the lung and liver. Vascular endothelial growth factor (VEGF or VEGF-A) promotion of tumor angiogenesis is thought to be mediated primarily by VEGF receptor-2 (VEGFR-2). In this study, VEGFR-2 antibody (DC101) inhibited growth of RenCa renal cell carcinoma lung metastases by 26%, whereas VEGFR-1 antibody (MF-1) had no effect. However, VEGFR-2 neutralization had no effect on RenCa liver metastases, whereas VEGFR-1 neutralization decreased RenCa liver metastases by 31%. For CT26 colon carcinoma liver metastases, inhibition of both VEGFR-1 and VEGFR-2 was required to induce growth delay. VEGFR-1 or VEGFR-2 inhibition decreased tumor burden not by preventing the establishment of micrometastases but rather by preventing vascularization and growth of micrometastases by 55% and 43%, respectively. VEGF induced greater phosphorylation of VEGFR-2 in lung ECs and of VEGFR-1 in liver ECs. EC proliferation, migration, and capillary tube formation in vitro were suppressed more by VEGFR-2 inhibition for lung EC and more by VEGFR-1 inhibition for liver EC. Collectively, our results indicate that liver metastases are more reliant on VEGFR-1 than lung metastases to mediate angiogenesis due to differential activity of VEGFRs on liver EC versus lung EC. Thus, therapies inhibiting specific VEGFRs should consider the targeted sites of metastatic disease. Cancer Res; 70(21); 8357–67. ©2010 AACR.

Herein we describe a pathogenic role for the Pseudomonas aeruginosa type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which P. aeruginosa wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of P. aeruginosa pneumonia-induced lung endothelial barrier disruption.

Background: Numerous linkage studies have been performed in pedigrees of Autism Spectrum Disorders, and these studies point to diverse loci and etiologies of autism in different pedigrees. The underlying pattern may be identified by an integrative approach, especially since ASD is a complex disorder manifested through many loci. Method: Autism spectrum disorder (ASD) was studied through two different and independent genome-scale measurement modalities. We analyzed the results of copy number variation in autism and triangulated these with linkage studies. Results: Consistently across both genome-scale measurements, the same two molecular themes emerged: immune/chemokine pathways and developmental pathways. Conclusion: Linkage studies in aggregate do indeed share a thematic consistency, one which structural analyses recapitulate with high significance. These results also show for the first time that genomic profiling of pathways using a recombination distance metric can capture pathways that are consistent with those obtained from copy number variations (CNV).

Metastases to any organ site require angiogenesis for tumor expansion. Tumor angiogenesis is restrained by a variety of endogenous inhibitors including thrombospondin 1 (TSP1). The principal anti-angiogenic activity of TSP1 resides in a domain containing 3 TSP1 repeats (3TSR), and TSP1 cleavage is regulated, in part, by the metalloproteinase ADAMTS1. Here we examine the role of TSP1 and ADAMTS1 in controlling metastatic disease in the liver and lung. The growth of CT26 colon carcinoma cells and RenCa renal carcinoma cells over-expressing TSP1 was inhibited in the liver but not in the lung. B16F10 melanoma liver metastases demonstrated accelerated growth in Tsp1-null mice compared to controls, while B16F10 lung metastases grew similarly in Tsp1-null mice and controls. We compared cleavage of recombinant TSP1 by liver and lung lysates, and found that liver lysate cleaved TSP1 much more efficiently that lung lysate. This cleavage activity could be blocked with neutralizing antibody or RNA interference directed at ADAMTS1, and cleavage activity was significantly abrogated when liver lysates from Adamts1-null mice were used. Finally to confirm that lack of TSP1 cleavage resulted in ineffective anti-angiogenic function in the lung, we generated CT26 expressing colon cancer cells stably secreting only the 3TSR domain. 3TSR secretion from CT26 cells inhibited both CT26 liver and lung metastases. Collectively, these data indicate that the anti-angiogenic activity of TSP1 is differentially regulated by ADAMTS1 in the liver and lung, and emphasize the notion of variations in the regulation of angiogenesis in different host organ environments.

Purpose Sunitinib (SU) is a multitargeted receptor tyrosine kinase inhibitor of the vascular endothelial growth factor and platelet-derived growth factor receptors. The present study examined SU and radiotherapy (RT) in a genetically engineered mouse model of soft tissue sarcoma (STS). Methods and Materials Primary extremity STSs were generated in genetically engineered mice. The mice were randomized to treatment with SU, RT (10 Gy × 2), or both (SU+RT). Changes in the tumor vasculature before and after treatment were assessed in vivo using fluorescence-mediated tomography. The control and treated tumors were harvested and extensively analyzed. Results The mean fluorescence in the tumors was not decreased by RT but decreased 38–44% in tumors treated with SU or SU+RT. The control tumors grew to a mean of 1378 mm[superscript 3] after 12 days. SU alone or RT alone delayed tumor growth by 56% and 41%, respectively, but maximal growth inhibition (71%) was observed with the combination therapy. SU target effects were confirmed by loss of target receptor phosphorylation and alterations in SU-related gene expression. Cancer cell proliferation was decreased and apoptosis increased in the SU and RT groups, with a synergistic effect on apoptosis observed in the SU+RT group. RT had a minimal effect on the tumor microvessel density and endothelial cell-specific apoptosis, but SU alone or SU+RT decreased the microvessel density by >66% and induced significant endothelial cell apoptosis. Conclusion SU inhibited STS growth by effects on both cancer cells and tumor vasculature. SU also augmented the efficacy of RT, suggesting that this combination strategy could improve local control of STS., National Cancer Institute (U.S.) (Grant U54 CA119349), National Cancer Institute (U.S.) (Grant P30-CA14051)

Purpose: Thrombospondin-1 (Tsp1), endostatin, and tumstatin are extracellular matrix–associated proteins that inhibit angiogenesis. We examined the mechanisms by which tumor cells may bypass the antiangiogenic effects of these endogenous regulators. Experimental Design: CT26 colon and RenCa renal carcinoma cells were stably transfected with Tsp1, endostatin, or tumstatin cDNA. Subcutaneous and metastatic tumor growth in syngeneic mice was analyzed. Expression of proangiogenic factors in resulting tumors was measured by quantitative real-time PCR. The combination of Tsp1 and vascular endothelial growth factor (VEGF) receptor-2 inhibition was also examined. Results: There was significant suppression of angiogenesis in flank tumors and liver metastases formed from cells overexpressing Tsp1, endostatin, or tumstatin. However, all tumors ultimately escaped angiogenesis inhibition. The combination of all three angiogenesis inhibitors had no additive effect beyond overexpression of a single inhibitor. Using quantitative real-time PCR, we found that VEGF and platelet-derived growth factor (PDGF)-A levels were routinely up-regulated at least 5-fold in all CT26 tumors overexpressing any antiangiogenic protein, and there were variable increases in angiopoietin 2 (Ang2), basic fibroblast growth factor, and PDGF-B. In contrast, RenCa tumors, which have high baseline levels of VEGF and PDGF-B, relied on basic fibroblast growth factor, Ang1, and PDGF-A up-regulation to counteract Tsp1 overexpression. Growth of CT26 cells with Tsp1 overexpression was suppressed when anti–VEGFR-2 treatment was added. Conclusions: Cancer cells with overexpression of three different endogenous angiogenesis inhibitor eventually escape angiogenesis inhibition by up-regulation of various proangiogenic factors. Tsp1, endostatin, and tumstatin may be functionally redundant in this system. These endogenous angiogenesis inhibitors are likely best used in combination with the blockade of proangiogenic pathways or with traditional chemotherapy or radiation therapy.

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Metastases to any organ site require angiogenesis for tumor expansion. Tumor angiogenesis is restrained by a variety of endogenous inhibitors including thrombospondin 1 (TSP1). The principal anti-angiogenic activity of TSP1 resides in a domain containing 3 TSP1 repeats (3TSR), and TSP1 cleavage is regulated, in part, by the metalloproteinase ADAMTS1. Methods and Results: Wild-type B16F10 melanoma cells formed increased liver metastases in Tsp1-null mice compared to controls but equivalent lung metastases in Tsp1-null mice and controls. Stable cell lines of B16F10, CT26 colon carcinoma, and RenCa renal carcinoma overexpressing full-length TSP1 or 3TSR were generated. Metastases from CT26 and RenCa cell lines over-expressing TSP1 were reduced in liver but not in lung. To determine why TSP1 blocked metastatic tumor growth in the liver but not the lung, we examined cleavage of recombinant TSP1 protein by liver and lung lysates and found that liver lysate cleaved TSP1 into its [3TSR][1] domain much more efficiently that lung lysate. TSP1-mediated inhibition of CT26 lung metastases was restored when only the 3TSR domain was over-expressed rather than full-length TSP1. Liver lysate cleavage activity was significantly abrogated when ADAMTS1 activity was (1) neutralized using ADAMTS-1 antibody, (2) knocked down by lentiviral shRNA, or (3) absent in Adamts1-null liver lysate. Livers metastases generated from B16F10 cells over-expressing TSP1 in Adamts1-haploinsufficient mice demonstrated accelerated growth compared to growth in wild-type mice. Conclusions: TSP1 inhibits the growth of liver metastases but not lung metastases secondary to differential cleavage of TSP1 into anti-angiogenic fragments by ADAMTS1. This study emphasizes the importance of host organ environment in the regulation of tumor angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2325. [1]: /lookup/external-ref?link_type=PDB&access_num=3TSR&atom=%2Fcanres%2F70%2F8_Supplement%2F2325.atom