S100A10 is a key plasminogen receptor of the extracellular cell surface that is overexpressed in many cancer cells. Typically, S100A10 is thought to be anchored to the plasma membrane via the phospholipid-binding sites of its binding partner, annexin A2. Here, using the potent and highly sequence-specific mechanism of RNA interference (RNAi), we have stably silenced the expression of the S100A10 gene in colorectal (CCL-222) cancer cells. We show that siRNA expression mediated by the pSUPER vector causes efficient, stable, and specific down-regulation of S100A10 gene expression. The siRNA-mediated down-regulation of S100A10 gene expression resulted in a major decrease in the appearance of extracellular S100A10 protein and correlated with a 45% loss of plasminogen binding, a 65% loss in cellular plasmin generation and a complete loss in plasminogendependent cellular invasiveness. We also observed that the CCL-222 cells do not express annexin A2 on their extracellular surface. Thus, the data show that annexin A2 is not required by S100A10 for its association with the plasma membrane, for its colocalization with uPAR, or for its binding and activation of plasminogen.

Neuroblastoma (NB) is derived from intrinsic migratory neural crest cells and has a high potential for distant metastasis. Growing evidence has implicated chemokine receptors, especially CXCR4, which normally control immune and inflammatory cell migration, as having important roles in tumor progression. In this study, we investigated the expression of CXCR4 in eight different NB cell lines and found that CXCR4 expression is dynamically regulated in NB and can be modulated by different tissue stromata. In addition, we demonstrate that IL-5 and IFN-gamma are released from stromal cells and act as differential mediators for CXCR4 expression. We also overexpressed CXCR4 in two NB cell lines, NUB-7 and SK-N-BE(2), and studied the role of CXCR4 in NB metastasis both in vitro and in vivo. In vitro transwell invasion assay showed that CXCR4 overexpression promoted NB cell migration preferentially toward a bone marrow stromal cell-conditioned medium. Using an in vivo xenograft model, CXCR4-overexpressing cells showed an increased incidence of metastasis, most notably bone marrow metastasis. Our studies reveal critical roles for CXCR4 in NB metastasis and provide insights into the regulatory mechanism of chemokine receptors in NB and the importance of the tissue microenvironment in modulating tumor cell behavior.

Purpose Low dose metronomic (LDM) chemotherapy, combined with VEGF signaling pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth in many adult preclinical cancer models. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, a VEGF receptor inhibitor, in three pediatric extracranial solid tumor mouse models. Experimental Design In vitro dose–response study of topotecan and pazopanib was conducted on several neuroblastoma, osteosarcoma, and rhabdomyosarcoma cell lines. In vivo antitumor efficacies of the LDM topotecan and pazopanib as single agents and in combination were tested on 4 subcutaneous xenograft models and on 2 neuroblastoma metastatic models. Circulating angiogenic factors such as circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP), and microvessel densities were used as surrogate biomarker markers of antiangiogenic activity. Results In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, combination of topotecan + pazopanib (TP + PZ) showed significant antitumor activity and significant enhancement in survival compared with the respective single agents in all models. Reductions in viable CEP and/or CEC levels and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. Pharmacokinetic studies of both drugs did not reveal any drug–drug interaction. Conclusion Metronomic administration of TP + PZ showed a statistically significant antitumor activity compared with respective single agents in pediatric tumor mouse models and represent a valid option as a maintenance therapy in aggressive pediatric solid tumors.

Neuroblastoma (NB) is the most deadly extra-cranial solid tumour in children necessitating an urgent need for effective and less toxic treatments. One reason for the lack of efficacious treatments may be the inability of existing drugs to target the tumour-initiating or cancer stem cell population responsible for sustaining tumour growth, metastases and relapse. Here, we describe a strategy to identify compounds that selectively target patient-derived cancer stem cell-like tumour-initiating cells (TICs) while sparing normal paediatric stem cells (skin-derived precursors, SKPs) and characterize two therapeutic candidates. DECA-14 and rapamycin were identified as NB TIC-selective agents. Both compounds induced TIC death at nanomolar concentrations in vitro, significantly reduced NB xenograft tumour weight in vivo, and dramatically decreased self-renewal or tumour-initiation capacity in treated tumours. These results demonstrate that differential drug sensitivities between TICs and normal paediatric stem cells can be exploited to identify novel, patient-specific and potentially less toxic therapies.

Purpose: Angiogenesis is increased in aggressive histology non^Hodgkin's lymphoma and may be a target with selective cyclooxygenase-2 inhibition and metronomic chemotherapy. Experimental Design: We assessed response, toxicity, and biomarkers of angiogenesis to lowdose cyclophosphamide (50 mg p.o. o.d.) and high-dose celecoxib (400 mg p.o. b.i.d.) in adult patients with relapsed or refractory aggressive non^Hodgkin's lymphoma in a multicenter phase II prospective study. Results: Thirty-two of 35 patients (median age, 62 years) are evaluable for response. Patients had primarily relapsed diffuse large B-cell lymphoma (63%) were heavily pretreated (median of three regimens) and high risk (79% international prognostic index, z2) and 34% were relapsed after autologous stem cell transplant.With a median follow-up of 8.4 months, the overall best response rate is 37% (2 complete clinical response/complete clinical response unconfirmed and 9 partial response), with 22% achieving stable disease. Median overall and progression-free survivals are14.4 and 4.7 months, respectively. The median response duration was 8.2 months.The most common toxicity was skin rash (40%); myelosuppression and gastrointestinal side effects were uncommon. Three patients developed deep vein thromboses and two heavily pretreated patients developed treatment-related acute myelogenous leukemia or myelodysplasia after 3.7 and 12 months of therapy. Circulating endothelial cells and their precursors declined and remained low in responders, whereas plasma vascular endothelial growth factor trended to decline in responding patients but increase in nonresponders.Trough celecoxib levels achieved targeted ''antiangiogenic'' levels. Conclusions: Low-dose cyclophosphamide and high-dose celecoxib is well tolerated and active in pretreated aggressive non^Hodgkin's lymphoma. Close surveillance for arterial and venous thrombotic events is recommended. The decline in circulating endothelial cells and their precursors suggests that this combination may be working by inhibiting angiogenesis but should be validated in a larger patient sample.

Neuroblastoma (NB) is one of the most common pediatric solid tumors originating from the neural crest lineage. Despite intensive treatment protocols including megatherapy with hematopoietic stem cell transplantation, the prognosis of NB patients remains poor. More effective therapeutics are required. High vascularity has been described as a feature of aggressive, widely disseminated NB. Our previous work demonstrated the overexpression of vascular endothelial growth factor (VEGF) in NB, and we showed that an anti-VEGF receptor (VEGFR-2) antibody could induce sustained NB tumor suppression and regression. Sunitinib is a kinase inhibitor targeting platelet-derived growth factor receptors and VEGFRs and, therefore, a promising antiangiogenic agent. In this study, we investigated the antitumor activity of sunitinib and its synergistic cytotoxicity with conventional (cyclophosphamide) and novel (rapamycin) therapies. Both NB cell lines and tumor-initiating cells from patient tumor samples were used in our in vitro and in vivo models for these drug testing. We show that sunitinib inhibits tumor cell proliferation and phosphorylation of VEGFRs. It also inhibits tumor growth, angiogenesis, and metastasis in tumor xenograft models. Low-dose sunitinib (20 mg/kg) demonstrates synergistic cytotoxicity with an mTOR inhibitor, rapamycin, which is more effective than the traditional chemotherapeutic drug, cyclophosphamide. These preclinical studies provide the evidence of antitumor activity of sunitinib both in the early stage of tumor formation and in the progressive metastatic disease. These studies also provide the framework for clinical trial of sunitinib, alone and in combination with conventional and novel therapies to increase efficacy and improve patient outcome in NB.

Annexin II heterotetramer (AIIt) is a Ca 2؉-and phospholipid-binding protein that consists of two copies of a p36 and p11 subunit. AIIt regulates the production and autoproteolysis of plasmin at the cell surface. In addition to its role as a key cellular protease, plasmin also plays a role in angiogenesis as the precursor for antiangiogenic proteins. Recently we demonstrated that the primary antiangiogenic plasmin fragment, called A 61 (Lys -Lys 468) was released from cultured cells. In the present study we report for the first time that AIIt possesses an intrinsic plasmin reductase activity. AIIt stimulated the reduction of the plasmin Cys 462-Cys 541 bond in a time-and concentration-dependent manner, which resulted in the release of A 61 from plasmin. Mutagenesis of p36 C334S and either p11 C61S or p11 C82S inactivated the plasmin reductase activity of the isolated subunits, suggesting that specific cysteinyl residues participated in the plasmin reductase activity of each subunit. Furthermore, we demonstrated that the loss of AIIt from the cell surface of HT1080 cells transduced with a retroviral vector encoding p11 antisense dramatically reduced the cellular production of A 61 from plasminogen. This is the first demonstration that AIIt regulates the cellular production of the antiangiogenic plasminogen fragment, A 61 .Angiostatin was originally identified in the urine of mice bearing Lewis lung carcinoma as a 38-kDa proteolytically derived fragment of plasminogen which encompassed the first four kringle domains (Lys 78 -Ala 440 ). Angiostatin was shown to be a potent antiangiogenic protein that inhibited the growth of human and murine carcinomas and also induced dormancy in their metastases. Angiostatin was also characterized as a specific antiangiogenic protein that blocked microvascular endothelial cell proliferation but not the proliferation of nonendothelial cells (1).It is now apparent that angiostatin is a member of a family of antiangiogenic plasminogen fragments (AAPFs).1 Physiologically relevant AAPFs include a 38-kDa AAPF isolated from the conditioned media of tumor-infiltrating macrophages (2) and AAPFs of 48, 42, and 50 kDa present in macrophage-conditioned media (3). Other AAPFs include a 50-kDa AAPF isolated from the conditioned media of human prostate carcinoma PC-3 cells (4, 5) and AAPFs of 66, 60, and 57 kDa detected in the conditioned media of HT1080 and Chinese hamster ovary cells (6). Because the carboxyl terminus of most of these AAPFs was not determined, the exact primary sequence of most of the AAPFs is not known. Two distinct pathways for the formation of AAPFs have been identified. First, certain proteinases can directly cleave plasminogen into AAPFs. These proteinases include metalloelastase, gelatinase B (matrix metalloproteinase-9), stromelysin-1 (matrix metalloproteinase-3), matrilysin (matrix metalloproteinase-7), cathepsin D, and prostate-specific antigen (7-11). The source of these proteinases may be tumor-infiltrating macrophages (2) or the cancer cells themselves. For example...

We enrolled 23 patients with relapsed follicular lymphoma (FL) in a prospective single-arm study of auto-SCT combined with in vivo rituximab graft purging and post transplant rituximab maintenance. Minimal residual disease was monitored with quantitative PCR testing. With a median follow-up of 74.2 months, neither median overall survival (OS) nor PFS has been reached. Here, 5-year OS and 5-year PFS are 78% (95% confidence interval (CI) 61-95%) and 59% (95% CI 38-80%), respectively. Time to progression (TTP) with the experimental regimen was significantly improved compared with TTP with the last prior treatment (Po0.001). Durable molecular remissions occurred in 11 of 13 assessable patients. PFS was significantly longer in patients who achieved a molecular remission by 3 months post-auto-SCT (P ¼ 0.001). Prolonged hypogammaglobulinemia occurred in most patients; however, no increase in major infections was observed.