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Inhibition of mTOR signaling using the rapalog everolimus is an FDA-approved targeted therapy for patients with lung and gastroenteropancreatic neuroendocrine tumors (NET). However, patients eventually progress on treatment, highlighting the need for additional therapies. We focused on pancreatic NETs (pNET) and reasoned that treatment of these tumors upon progression on rapalog therapy, with an mTOR kinase inhibitor (mTORKi), such as CC-223, could overcome a number of resistance mechanisms in tumors and delay cardiac carcinoid disease. We performed preclinical studies using human pNET cells in vitro and injected them subcutaneously or orthotopically to determine tumor progression and cardiac function in mice treated with either rapamycin alone or switched to CC-223 upon progression. Detailed signaling and RNA sequencing analyses were performed on tumors that were sensitive or progressed on mTOR treatment. Approximately 57% of mice bearing pNET tumors that progressed on rapalog therapy showed a significant decrease in tumor volume upon a switch to CC-223. Moreover, mice treated with an mTORKi exhibited decreased cardiac dilation and thickening of heart valves than those treated with placebo or rapamycin alone. In conclusion, in the majority of pNETs that progress on rapalogs, it is possible to reduce disease progression using an mTORKi, such as CC-223. Moreover, CC-223 had an additional transient cardiac benefit on valvular fibrosis compared with placebo- or rapalog-treated mice. These results provide the preclinical rationale to further develop mTORKi clinically upon progression on rapalog therapy and to further test their long-term cardioprotective benefit in those NET patients prone to carcinoid syndrome. Mol Cancer Ther; 16(11); 2432–41. ©2017 AACR.

Table S1. CC-223-treated mice tend to develop less histopathologic lesions of the heart valves than either rapamycin- or placebo-treated mice. Figure S1. MLN0128 inhibited mTOR signaling in human pNET cell lines in a dose-dependent manner. Figure S2. Mean tumor volumes of rapamycin, CC-223 responders and non-responders over time. Figure S3. Tumor burden, DDC and/or serum serotonin levels in nude mice either orthotopically or subcutaneously injected with BON-luci cells and treated with placebo, rapamycin or CC-223.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

Purpose: Saposin C is a multifunctional protein known to activate lysosomal enzymes and induce membrane fusion in an acidic environment. Excessive accumulation of lipid-coupled saposin C in lysosomes is cytotoxic. Because neoplasms generate an acidic microenvironment, caused by leakage of lysosomal enzymes and hypoxia, we hypothesized that saposin C may be an effective anticancer agent. We investigated the antitumor efficacy and systemic biodistribution of nanovesicles comprised of saposin C coupled with dioleoylphosphatidylserine in preclinical cancer models.Experimental Design: Neuroblastoma, malignant peripheral nerve sheath tumor and, breast cancer cells were treated with saposin C–dioleoylphosphatidylserine nanovesicles and assessed for cell viability, ceramide elevation, caspase activation, and apoptosis. Fluorescently labeled saposin C–dioleoylphosphatidylserine was i.v. injected to determine in vivo tumor-targeting specificity. Antitumor activity and toxicity profile of saposin C–dioleoylphosphatidylserine were evaluated in xenograft models.Results: Saposin C–dioleoylphosphatidylserine nanovesicles, with a mean diameter of ∼190 nm, showed specific tumor-targeting activity shown through in vivo imaging. Following i.v. administration, saposin C–dioleoylphosphatidylserine nanovesicles preferentially accumulated in tumor vessels and cells in tumor-bearing mice. Saposin C–dioleoylphosphatidylserine induced apoptosis in multiple cancer cell types while sparing normal cells and tissues. The mechanism of saposin C–dioleoylphosphatidylserine induction of apoptosis was determined to be in part through elevation of intracellular ceramides, followed by caspase activation. In in vivo models, saposin C–dioleoylphosphatidylserine nanovesicles significantly inhibited growth of preclinical xenografts of neuroblastoma and malignant peripheral nerve sheath tumor. I.v. dosing of saposin C–dioleoylphosphatidylserine showed no toxic effects in nontumor tissues.Conclusions: Saposin C–dioleoylphosphatidylserine nanovesicles offer promise as a novel, nontoxic, cancer-targeted, antitumor agent for treating a broad range of cancers. (Clin Cancer Res 2009;15(18):5840–51)

Supplementary Data from Cancer-Selective Targeting and Cytotoxicity by Liposomal-Coupled Lysosomal Saposin C Protein

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs’ recapitulation of human tumors., Patient-derived xenograft models (PDX) have been extensively used to study the molecular and clinical features of cancers. Here the authors present a cohort of 536 PDX models from 25 cancers, as well as their genomic and evolutionary profiles and their suitability for clinical trials.

Background: Triple-Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with few targeted therapies, limiting patients to harsh systemic treatments and resulting in the lowest 5-year survival rate of all breast cancer subtypes. This survival rate plummets after metastasis to another site, making it crucial to identify new therapeutic options for TNBC patients to prevent recurrence and metastasis. The small molecule therapy birinapant, a secondary mitochondrial activator of caspases (SMAC) mimetic, represents a potential new avenue for TNBC treatment. Birinapant initiates apoptotic signaling through targeting of Inhibitor of Apoptosis (IAP) proteins in the Tumor Necrosis Factor Receptor (TNFR) extrinsic apoptosis signaling pathway, leading to the formation of a death complex, caspase activity, and apoptosis. We have found that approximately 25% of TNBC patient-derived xenograft (PDX) and xenograft-derived organoid (PDxO) models are exquisitely sensitive to birinapant treatment. However, the majority of our TNBC patient-derived lines remain resistant through unknown means. We seek to understand the parameters for birinapant response and the mechanism of inherent resistance in TNBC using our patient-derived TNBC models. Approach and Results: Preliminary Western blot experiments revealed that treatment of birinapant-sensitive and resistant PDX tumors in vivo and PDxOs in vitro with birinapant causes degradation of cIAP1 and cIAP2, but only causes apoptosis in birinapant-sensitive lines. Therefore, there may be a disruption of necessary downstream apoptotic signaling in the birinapant-resistant lines. Through follow-up Western blot experiments we determined that at baseline, birinapant-resistant PDxOs do not lack the TNFR extrinsic apoptosis proteins required for apoptosis signaling after IAP degradation, nor is there an abundance of these proteins in the birinapant-sensitive PDxOs. Furthermore, to determine if TNFR extrinsic apoptosis signaling is impaired in birinapant-resistant PDxOs, we treated birinapant-resistant and -sensitive PDxOs with the TNF-a cytokine, which is known to enhance extrinsic apoptosis signaling in the presence of birinapant. Treatment with TNF-a and birinapant did not induce apoptosis in the birinapant-resistant PDxOs, suggesting that the extrinsic apoptosis signaling pathway may be the culprit of resistance in these patient-derived lines. Discussion: Follow-up experiments will address whether an inability to execute extrinsic apoptosis through other death receptor pathways explains birinapant resistance in these models. This work will allow us to uncover what predisposes TNBC patients to be resistant or sensitive to birinapant, and determine how this may be harnessed therapeutically to improve outcomes for TNBC patients. This project was funded in part with federal funds under HHSN261201500003I and U54CA224076. Citation Format: Elisabeth A. Brown, Chieh-Hsiang Yang, Emilio Cortes-Sanchez, Zhengtao Chu, Jeevan Govindharajulu, Ralph Parchment, James Doroshow, Apurva Srivastava, Alana Welm. Determinants of birinapant efficacy in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 393.

Phosphatidylserine (PS) is often externalized in viable pancreatic cancer cells and is therapeutically targetable using PS-selective drugs. One of the first-line treatments for advanced pancreatic cancer disease, gemcitabine (GEM), provides only marginal benefit to patients. We therefore investigated the therapeutic benefits of combining GEM and the PS-targeting drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), for treating pancreatic ductal adenocarcinoma (PDAC). Using cell-cycle analyses and a cell surface PS-based sorting method in vitro, we observed an increase in surface PS as cells progress through the cell cycle from G1 to G2/M. We also observed that GEM treatment preferentially targets G1 phase cells that have low surface PS, resulting in an increased median surface PS level of PDAC cells. Inversely, SapC-DOPS preferentially targets high surface PS cells that are predominantly in the G2/M phase. Finally, combination therapy in subcutaneous and orthotopic PDAC tumors in vivo with SapC-DOPS and GEM or Abraxane (Abr)/GEM (one of the current standards of care) significantly inhibits tumor growth and increases survival compared with individual treatments. Our studies confirm a surface PS and cell cycle-based enhancement of cancer cytotoxicity following SapC-DOPS treatment in combination with GEM or Abr/GEM. Thus, PDAC patients treated with Abr/GEM may benefit from concurrent administration of SapC-DOPS.

Inhibition of mTOR signaling using the rapalog everolimus is an FDA-approved targeted therapy for patients with lung and gastroenteropancreatic neuroendocrine tumors (NET). However, patients eventually progress on treatment, highlighting the need for additional therapies. We focused on pancreatic NETs (pNET) and reasoned that treatment of these tumors upon progression on rapalog therapy, with an mTOR kinase inhibitor (mTORKi), such as CC-223, could overcome a number of resistance mechanisms in tumors and delay cardiac carcinoid disease. We performed preclinical studies using human pNET cells in vitro and injected them subcutaneously or orthotopically to determine tumor progression and cardiac function in mice treated with either rapamycin alone or switched to CC-223 upon progression. Detailed signaling and RNA sequencing analyses were performed on tumors that were sensitive or progressed on mTOR treatment. Approximately 57% of mice bearing pNET tumors that progressed on rapalog therapy showed a significant decrease in tumor volume upon a switch to CC-223. Moreover, mice treated with an mTORKi exhibited decreased cardiac dilation and thickening of heart valves than those treated with placebo or rapamycin alone. In conclusion, in the majority of pNETs that progress on rapalogs, it is possible to reduce disease progression using an mTORKi, such as CC-223. Moreover, CC-223 had an additional transient cardiac benefit on valvular fibrosis compared with placebo- or rapalog-treated mice. These results provide the preclinical rationale to further develop mTORKi clinically upon progression on rapalog therapy and to further test their long-term cardioprotective benefit in those NET patients prone to carcinoid syndrome. Mol Cancer Ther; 16(11); 2432–41. ©2017 AACR.

// Robert F. Koncar 1 , Zhengtao Chu 1 , Lindsey E. Romick-Rosendale 2 , Susanne I. Wells 2 , Timothy A. Chan 3 , Xiaoyang Qi 1 , El Mustapha Bahassi 1 1 Department of Internal Medicine, Division of Hematology/Oncology, University of Cincinnati, Cincinnati, OH, USA 2 Division of Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA 3 Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Correspondence to: El Mustapha Bahassi, email: bhassiem@uc.edu Keywords: glioma, IDH1, temozolomide, PLK1, checkpoint adaptation Received: September 09, 2016 Accepted: January 04, 2017 Published: February 02, 2017 ABSTRACT Despite multimodal therapy with radiation and the DNA alkylating agent temozolomide (TMZ), malignant gliomas remain incurable. Up to 90% of grades II-III gliomas contain a single mutant isocitrate dehydrogenase 1 ( IDH1 ) allele. IDH1 mutant-mediated transformation is associated with TMZ resistance; however, there is no clinically available means of sensitizing IDH1 mutant tumors to TMZ. In this study we sought to identify a targetable mechanism of TMZ resistance in IDH1 mutant tumors to enhance TMZ efficacy. IDH1 mutant astrocytes rapidly bypassed the G2 checkpoint with unrepaired DNA damage following TMZ treatment. Checkpoint adaptation was accompanied by PLK1 activation and IDH1 mutant astrocytes were more sensitive to treatment with BI2536 and TMZ in combination (

// Victor M. Blanco 1 , Zhengtao Chu 1, 2 , Kathleen LaSance 3 , Brian D. Gray 4 , Koon Yan Pak 4 , Therese Rider 5 , Kenneth D. Greis 5 , Xiaoyang Qi 1, 2 1 Division of Hematology-Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA 2 Division of Human Genetics, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA 3 Department of Radiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA 4 Molecular Targeting Technologies, Inc., West Chester, Pennsylvania 19380, USA 5 Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA Correspondence to: Xiaoyang Qi, email: xiaoyang.qi@uc.edu Keywords: glioblastoma, liposome, PET, optical imaging, SapC-DOPS Abbreviations: SapC, Saposin C; DOPS, dioleylphosphatidylserine; PS, phosphatidylserine Received: June 25, 2015 Accepted: March 28, 2016 Published: April 16, 2016 ABSTRACT Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

Background: Predicting the risk of relapse in patients with non-metastatic breast cancer is important for medical decisions and patient counseling. For patients who receive neoadjuvant chemotherapy, pathologic response and especially pathologic complete response (pCR) has been associated with risk of relapse; however this association is imperfect. Our prior work in patient-derived xenograft (PDX) models indicated that tumor engraftment in mice correlated with risk of recurrence. To understand further the prognostic utility of PDX, we designed a prospective clinical trial to determine the correlation between PDX generation with residual disease following neoadjuvant chemotherapy and relapse. Preliminary data are presented. Methods: Women with newly diagnosed non-metastatic hormone receptor low-positive (HR-low, ER and/or PR ≤ 10%) or negative breast cancer planned to receive systemic chemotherapy prior to definitive surgery were eligible. Tumor tissue at diagnosis was orthotopically implanted in NOD/SCID mice. The primary objective of the study is to correlate the ability of a tumor to engraft in mice with pathologic responses and clinical outcomes. Results: Between 12/2016 and 2/2020, 58 patients enrolled (triple negative breast cancer (TNBC), n=37; HR-low or negative/Her2+, n=11; or HR-low/Her2-, n=8; mixed, n=1). PDXs were successfully established from 16 patients. Patients uniformly received intensive preoperative chemotherapy per standard of care. Among the 12 patients whose tumors engrafted in mice (PDX(+)) and underwent surgery, the pCR rate was 41.7%. In the subgroup of patients with postoperative follow up >6 months (n=9), 3 patients had achieved a pCR, 1 of whom recurred; and 6 patients had residual disease, 3 of whom recurred (overall relapse rate 44.4%). All patients who relapsed (TNBC n=3; HR-low/Her2- n=1), experienced a very early relapse ( Among the 38 patients whose tumors did not engraft (PDX(-)) and underwent surgery, the overall pCR rate was 60.6%. In the subgroup of patients with postoperative follow up >6 months (n=30), 18 patients had achieved a pCR of whom none recurred, and 12 had residual disease of whom 1 patient (with TNBC) had locoregional recurrence 35.1 months after her definitive surgery (relapse rate 3.3%). She underwent repeat surgery followed by radiation therapy and 5.5 months after her recurrence, she remains disease-free. Achievement of pCR was not statistically different between the PDX(+) and PDX(-) group. In the entire cohort, the presence of residual disease was associated with high risk of relapse (22.2%) as compared to the achievement of pCR (4.8%, p = 0.16). However, successful tumor engraftment was associated not only with a higher risk of relapse (44.4% vs 3.3%, p = 0.0068, odds ratio 20.45), but also an early and exceptionally aggressive relapse. Conclusion. Our functional studies not only identify a patient population with a high risk of relapse with greater precision than the achievement of pCR, but also identify patients whose relapse has a particularly aggressive natural history. We established models that recapitulate this aggressive disease and in-depth genomic studies are underway. These studies will lead us to better patient stratification on the basis of risk of relapse and novel therapeutic strategies focused on patients with exceptionally aggressive breast cancers that our current diagnostic methods cannot identify. Citation Format: Christos Vaklavas, Zhengtao Chu, Rachel E Factor, Alana L Welm, Bryan E Welm, Cindy B Matsen. Towards personalized medicine - patient-derived breast tumor grafts as predictors of relapse and response to therapy. Preliminary results [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-01.

// Harold W. Davis 1 , Subrahmanya D. Vallabhapurapu 1 , Zhengtao Chu 1 , Swarajya L. Vallabhapurapu 1 , Robert S. Franco 1 , Michelle Mierzwa 2 , William Kassing 2 , William L. Barrett 2 and Xiaoyang Qi 1, 3 1 Division of Hematology/Oncology, Translational Research Laboratory, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA 2 Department of Radiation Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA 3 Division of Human Genetics, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Correspondence to: Xiaoyang Qi, email: xiaoyang.qi@uc.edu Keywords: radiotherapy-induced resistance; surface phosphatidylserine-selective; cancer cell death; SapC-DOPS nanovesicles; combined-treatment enhancement Received: August 15, 2018 Accepted: December 29, 2018 Published: January 25, 2019 ABSTRACT Normal living cells exhibit phosphatidylserine (PS) primarily within the intracellular leaflet of the plasma membrane. In contrast, viable cancer cells have high levels of PS on the external surface, and exhibit a broad range of surface PS, even within specific types of cancer. Agents that target surface PS have recently been developed to treat tumors and are expected to be more effective with higher surface PS levels. In this context, we examined whether surface PS is increased with irradiation. In vitro irradiation of cancer cell lines selected surviving cells that had higher surface PS in a dose- and time-dependent manner. This was more pronounced if surface PS was initially in the lower range for cancer cells. Radiation also increased the surface PS of tumor cells in subcutaneous xenografts in nude mice. We found an inverse relationship between steady state surface PS level of cancer cell lines and their sensitivity to radiation-induced cell death. In addition, serial irradiation, which selected surviving cells with higher surface PS, also increased resistance to radiation and to some chemotherapeutic drugs, suggesting a PS-dependent mechanism for development of resistance to therapy. On the other hand, fractionated radiation enhanced the effect of a novel anti-cancer, PS-targeting drug, SapC-DOPS, in some cancer cell lines. Our data suggest that we can group cancer cells into cells with low surface PS, which are sensitive to radiation, and high surface PS, which are sensitive to SapC-DOPS. Combination of these interventions may provide a potential new combination therapy.

Published version. Source at http://doi.org/10.18632/oncotarget.7955. License CC BY 3.0. Alternatively spliced Tissue Factor (asTF) is a secreted form of Tissue Factor (TF), the trigger of blood coagulation whose expression levels are heightened in several forms of solid cancer, including pancreatic ductal adenocarcinoma (PDAC). asTF binds to β1 integrins on PDAC cells, whereby it promotes tumor growth, metastatic spread, and monocyte recruitment to the stroma. In this study, we determined if targeting asTF in PDAC would significantly impact tumor progression. We here report that a novel inhibitory anti-asTF monoclonal antibody curtails experimental PDAC progression. Moreover, we show that tumor-derived asTF is able to promote PDAC primary growth and spread during early as well as later stages of the disease. This raises the likelihood that asTF may comprise a viable target in early- and late-stage PDAC. In addition, we show that TF expressed by host cells plays a significant role in PDAC spread. Together, our data demonstrate that targeting asTF in PDAC is a novel strategy to stem PDAC progression and spread.

Pancreatic cancer remains one of the most intractable cancers, with a dismal prognosis reflected by a 5-year survival of ~6%. Since early disease symptoms are undefined and specific biomarkers are lacking, about 80% of patients present with advanced, inoperable tumors that represent a daunting challenge. Despite many clinical trials, no single chemotherapy agent has been reliably associated with objective response rates above 10% or median survival longer than 5 to 7 months. Although combination chemotherapy regimens have in recent years provided some improvement, overall survival (8-11 months) remains very poor. There is therefore a critical need for novel therapies that can improve outcomes for pancreatic cancer patients. Here, we present a summary of the current therapies used in the management of advanced pancreatic cancer and review novel therapeutic strategies that target tumor biomarkers. We also describe our recent research using phosphatidylserine-targeted saposin C–coupled dioleoylphosphatidylserine nanovesicles for imaging and therapy of pancreatic cancer.

// Victor M. Blanco 1 , Zhengtao Chu 1,2 , Subrahmanya D. Vallabhapurapu 1 , Mahaboob K. Sulaiman 1 , Ady Kendler 3 , Olivier Rixe 4 , Ronald E. Warnick 5 , Robert S. Franco 1 and Xiaoyang Qi 1,2 1 Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 2 Division of Human Genetics, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 3 Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 4 Division of Hematology/Oncology, Georgia Regents University, GRU Cancer Center, Augusta, Georgia 5 Department of Neurosurgery, University of Cincinnati Brain Tumor Center, and Mayfield Clinic, Cincinnati, Ohio Correspondence: Xiaoyang Qi, email: // Keywords : Glioblastoma; brain metastasis; SapC-DOPS; imaging; cancer therapy Received : June 2, 2014 Accepted: July 13, 2014 Published: July 14, 2014 Abstract Brain tumors, either primary (e.g., glioblastoma multiforme) or secondary (metastatic), remain among the most intractable and fatal of all cancers. We have shown that nanovesicles consisting of Saposin C (SapC) and dioleylphosphatidylserine (DOPS) are able to effectively target and kill cancer cells both in vitro and in vivo . These actions are a consequence of the affinity of SapC-DOPS for phosphatidylserine, an acidic phospholipid abundantly present in the outer membrane of a variety of tumor cells and tumor-associated vasculature. In this study, we first characterize SapC-DOPS bioavailability and antitumor effects on human glioblastoma xenografts, and confirm SapC-DOPS specificity towards phosphatidylserine by showing that glioblastoma targeting is abrogated after in vivo exposure to lactadherin, which binds phosphatidylserine with high affinity. Second, we demonstrate that SapC-DOPS selectively targets brain metastases-forming cancer cells both in vitro , in co-cultures with human astrocytes, and in vivo , in mouse models of brain metastases derived from human breast or lung cancer cells. Third, we demonstrate that SapC-DOPS nanovesicles have cytotoxic activity against metastatic breast cancer cells in vitro , and prolong the survival of mice harboring brain metastases. Taken together, these results support the potential of SapC-DOPS for the diagnosis and therapy of primary and metastatic brain tumors.

BRAIN TUMORS ARE associated with extremely poor prognosis in both adult and pediatric patients with a 5-year survival rate of only 30% (1). It is estimated that nearly 70,000 new cases of brain tumor were diagnosed in the US in 2013 (1). In particular, glioblastoma multiforme (GBM) is a highly aggressive and drug-resistant form of primary brain tumor (2–4). Despite the use of radiation and chemotherapy, the median survival for GBM patients is less than 15 months (5,6). Novel imaging methods that can help characterize brain tumors, aid in the selection of an effective therapy, and monitor the therapeutic response in these patients could greatly improve their survival rate and quality of life. Vesicles formulated with saposin C and dioleylphosphatidylserine (SapC-DOPS) have been shown to selectively target tumors, but not normal tissues (7–12). Formulating SapC-DOPS vesicles with an MRI visible beacon would allow noninvasive tracking of the uptake of this tumor specific agent. The high resolution, lack of ionizing radiation, excellent tissue penetration, and soft tissue contrast available with MRI provides distinct advantages over other molecular imaging approaches, such as nuclear or optical imaging. Traditional anatomical MRI and nonspecific contrast agents can localize a tumor mass and assess the abnormal vasculature feeding the tumor, but molecular imaging with a specifically targeted contrast agent could provide biochemical characterization of the disease, which could guide the selection of an effective treatment or allow early detection of the therapeutic effect. Saposins are small nonenzymatic glycoproteins present in all normal tissues that act as biological activators of lysosomal enzymes (13). The functional organization of SapC consists of a membrane fusogenic domain and a region for activation of lysosomal enzymes (14,15). SapC associates with lipid membranes by embedding into membrane leaflets. Each helical peptide of SapC at the amino and carboxyl ends of the molecule is essential for insertion into lipid bilayers (16). SapC preferentially interacts with unsaturated, negatively charged phospholipids, such as phosphatidylserine (PS) (15,16). PS is present in all cells at relatively high amounts, approximately 2–10% of total cellular lipid (17). However, in normal cells, PS is only present on the intracellular aspect of the cellular membrane and not on the cell surface. In contradistinction, PS is relatively abundant on the surface of tumor cells and on tumor vasculature (18,19). Furthermore, the exposure level of PS on the cancer cell surface can be modulated by the metastatic potential (20) and at early stages of cell death (21,22), providing a metric of overall prognosis and therapeutic effect. Elevated PS levels have been observed in cultured brain tumor cells and tumors in vivo by flow cytometry using annexin A5 and immunohistochemical analysis with PS-specific binding proteins or anti-PS monoclonal antibodies (8,12). Specifically, GBM tumors express PS on the cell surface allowing fluorescent SapC-DOPS vesicles to selectively target these cells in vitro and in vivo (12). In vivo fluorescent imaging of SapC-DOPS vesicles has shown preferential tumor uptake and complete clearance from normal tissues by 24 h postinjection (9). Tumor selective imaging of SapC-DOPS vesicles by means of MRI has been previously reported by encapsulating iron oxide particles inside the vesicles (9). In this study, we explored the use of a paramagnetic gadolinium chelate for noninvasively tracking SapC-DOPS vesicles. Gadolinium based MRI contrast agents are widely used clinically to detect lesions and image blood vessels. At the present time, there are nine different gadolinium based contrast agents approved by the U.S. Food and Drug Administration (FDA) for clinical use (23). On the other hand, no iron oxide agents are currently commercially available for MRI (24). A superparamagnetic iron oxide nanoparticle agent called Ferumoxytol has been used for MRI (25,26), but it is only FDA approved for the treatment of anemia (27). In addition, gadolinium agents are often preferred because they generate an increase in the MRI signal, compared with iron oxide agents that typically generate a decrease in the MRI signal (28). The aim of this research was to investigate paramagnetic SapC-DOPS vesicles as a targeted contrast agent for imaging GBM cells in vitro and in vivo in the mouse brain.

Saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles are a nanotherapeutic which effectively target and destroy cancer cells. Here, we explore the systemic use of SapC-DOPS in several models of brain cancer, including glioblastoma multiforme (GBM), and the molecular mechanism behind its tumor-selective targeting specificity. Using two validated spontaneous brain tumor models, we demonstrate the ability of SapC-DOPS to selectively and effectively cross the blood–brain tumor barrier (BBTB) to target brain tumors in vivo and reveal the targeting to be contingent on the exposure of the anionic phospholipid phosphatidylserine (PtdSer). Increased cell surface expression of PtdSer levels was found to correlate with SapC-DOPS–induced killing efficacy, and tumor targeting in vivo was inhibited by blocking PtdSer exposed on cells. Apart from cancer cell killing, SapC-DOPS also exerted a strong antiangiogenic activity in vitro and in vivo. Interestingly, unlike traditional chemotherapy, hypoxic cells were sensitized to SapC-DOPS–mediated killing. This study emphasizes the importance of PtdSer exposure for SapC-DOPS targeting and supports the further development of SapC-DOPS as a novel antitumor and antiangiogenic agent for brain tumors.

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF-overexpressing high-grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell-derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

e16209Background: Phosphatidylserine (PS) is an anionic phospholipid primarily localized on the inner plasma membrane of healthy cells. In contrast, PS is externally expressed on cancer cells and t...

Background High toxicity, morbidity and secondary malignancy render chemotherapy of neuroblastoma inefficient, prompting the search for novel compounds. Nanovesicles offer great promise in imaging and treatment of cancer. SapC-DOPS, a stable nanovesicle formed from the lysosomal protein saposin C and dioleoylphosphatidylserine possess strong affinity for abundantly exposed surface phosphatidylserine on cancer cells. Here, we show that SapC-DOPS effectively targets and suppresses neuroblastoma growth and elucidate the molecular mechanism of SapC-DOPS action in neuroblastoma in vitro. Methods In vivo targeting of neuroblastoma was assessed in xenograft mice injected intravenously with fluorescently-labeled SapC-DOPS. Xenografted tumors were also used to demonstrate its therapeutic efficacy. Apoptosis induction in vivo was evaluated in tumor sections using the TUNEL assay. The mechanisms underlying the induction of apoptosis by SapC-DOPS were addressed through measurements of cell viability, mitochondrial membrane potential (ΔΨM), flow cytometric DNA fragmentation assays and by immunoblot analysis of second mitochondria-derived activator of caspases (Smac), Bax, Cytochrome c (Cyto c) and Caspase-3 in the cytosol or in mitochondrial fractions of cultured neuroblastoma cells. Results SapC-DOPS showed specific targeting and prevented the growth of human neuroblastoma xenografts in mice. In neuroblastoma cells in vitro, apoptosis occurred via a series of steps that included: (1) loss of ΔΨM and increased mitochondrial superoxide formation; (2) cytosolic release of Smac, Cyto c, AIF; and (3) mitochondrial translocation and polymerization of Bax. ShRNA-mediated Smac knockdown and V5 peptide-mediated Bax inhibition decreased cytosolic Smac and Cyto c release along with caspase activation and abrogated apoptosis, indicating that Smac and Bax are critical mediators of SapC-DOPS action. Similarly, pretreatment with the mitochondria-stabilizing agent bongkrekic acid decreased apoptosis indicating that loss of ΔΨM is critical for SapC-DOPS activity. Apoptosis induction was not critically dependent on reactive oxygen species (ROS) production and Cyclophilin D, since pretreatment with N-acetyl cysteine and cyclosporine A, respectively, did not prevent Smac or Cyto c release. Conclusions Taken together, our results indicate that SapC-DOPS acts through a mitochondria-mediated pathway accompanied by an early release of Smac and Bax. Specific tumor-targeting capacity and anticancer efficacy of SapC-DOPS supports its potential as a dual imaging and therapeutic agent in neuroblastoma therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0336-y) contains supplementary material, which is available to authorized users.

Acidic phospholipids and saposins associations are involved in the degradation process of glycosphingolipids/sphingolipids in late endosomes/lysosomes. In this report, we showed the colocalization of saposin C and lysobisphosphatidic acid (LBPA) in human fibroblasts by using cytoimmunofluorescence analysis. This colocalization pattern was not seen with other saposins. Large numbers of saposins A, B, and D illustrated the staining patterns that differ from LBPA. In addition, ingested anti-LBPA antibody altered the location of saposin C in human wild-type fibroblasts. In vitro assays demonstrated that saposin C at nM concentrations induced membrane fusion of LBPA containing phospholipid vesicles. Under the same condition, other saposins had no fusion induction on these vesicles. These results suggested a specific interaction between saposin C and LBPA. Total saposin-deficient fibroblasts showed a massive accumulation of multivesicular bodies (MVBs) by electron microscopic analysis. No significant increase of MVBs was found in saposins A and B deficient cells. Interestingly, the accumulated MVBs were significantly reduced by loading saposin C alone into the total saposin-deficient cells. Therefore, we propose that saposin C-LBPA interaction plays a role in the regulation of MVB formation in cells.

Saposin C, a sphingolipid activator protein with fusogenic activity, interacts specifically with the membrane containing negatively charged, unsaturated phospholipids. The kinetics and mechanism of saposin C-induced membrane fusion were previously investigated using acidic phospholipid liposomes. A hypothetic clip-on model for such a fusion process was illustrated by the ionic binding between saposin C and lipids, as well as the inter-saposin C hydrophobic interaction. Here, we report the location of the fusogenic domain in a linear sequence at the amino-terminal half of saposin C. This domain consisted of the first and second helical sequences. Selected positively charged lysines in the fusogenic domain were mutated to study the roles of basic residues in the saposin C-induced vesicle fusion. Based on the results, Lys13 and Lys17 were critical for the fusogenic activity, but had no effect on the enzymatic activation of acid beta-glucosidase (GCase). These results clearly indicate the segregation of the fusion and activation function into two different regions of saposin C. Interestingly, all the Lys mutant saposin Cs anchored on the acidic phospholipid membrane. Our data suggest that saposin C's fusogenic and activation functions have different requirements for the orientation and insertion manners of helical peptides in membranes.

Lung cancer is the deadliest type of cancer for both men and women. In this study, we evaluate the in vitro and in vivo efficacy of a biotherapeutic agent composed of a lysosomal protein (Saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS), which can be assembled into nanovesicles (SapC–DOPS) with selective antitumor activity. SapC–DOPS targets phosphatidylserine, an anionic phospholipid preferentially exposed in the surface of cancer cells and tumor-associated vasculature. Because binding of SapC to phosphatidylserine is favored at acidic pHs, and the latter characterizes the milieu of many solid tumors, we tested the effect of pH on the binding capacity of SapC–DOPS to lung tumor cells. Results showed that SapC–DOPS binding to cancer cells was more pronounced at low pH. Viability assays on a panel of human lung tumor cells showed that SapC–DOPS cytotoxicity was positively correlated with cell surface phosphatidylserine levels, whereas mitochondrial membrane potential measurements were consistent with apoptosis-related cell death. Using a fluorescence tracking method in live mice, we show that SapC–DOPS specifically targets human lung cancer xenografts, and that systemic therapy with SapC–DOPS induces tumor apoptosis and significantly inhibits tumor growth. These results suggest that SapC–DOPS nanovesicles are a promising treatment option for lung cancer. Mol Cancer Ther; 14(2); 491–8. ©2015 AACR.

SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

To investigate paramagnetic saposin C and dioleylphosphatidylserine (SapC-DOPS) vesicles as a targeted contrast agent for imaging phosphatidylserine (PS) expressed by glioblastoma multiforme (GBM) tumors.Gd-DTPA-BSA/SapC-DOPS vesicles were formulated, and the vesicle diameter and relaxivity were measured. Targeting of Gd-DTPA-BSA/SapC-DOPS vesicles to tumor cells in vitro and in vivo was compared with nontargeted paramagnetic vesicles (lacking SapC). Mice with GBM brain tumors were imaged at 3, 10, 20, and 24 h postinjection to measure the relaxation rate (R1) in the tumor and the normal brain.The mean diameter of vesicles was 175 nm, and the relaxivity at 7 Tesla was 3.32 (s*mM)(-1) relative to the gadolinium concentration. Gd-DTPA-BSA/SapC-DOPS vesicles targeted cultured cancer cells, leading to an increased R1 and gadolinium level in the cells. In vivo, Gd-DTPA-BSA/SapC-DOPS vesicles produced a 9% increase in the R1 of GBM brain tumors in mice 10 h postinjection, but only minimal changes (1.2% increase) in the normal brain. Nontargeted paramagnetic vesicles yielded minimal change in the tumor R1 at 10 h postinjection (1.3%).These experiments demonstrate that Gd-DTPA-BSA/SapC-DOPS vesicles can selectively target implanted brain tumors in vivo, providing noninvasive mapping of the cancer biomarker PS.

Only a small number of promising drugs target pancreatic cancer, which is the fourth leading cause of cancer deaths with a 5-year survival of less than 5%. Our goal is to develop a new biotherapeutic agent in which a lysosomal protein (saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS) are assembled into nanovesicles (SapC-DOPS) for treating pancreatic cancer. A distinguishing feature of SapC-DOPS nanovesicles is their high affinity for phosphatidylserine (PS) rich microdomains, which are abnormally exposed on the membrane surface of human pancreatic tumor cells. To evaluate the role of external cell PS, in vitro assays were used to correlate PS exposure and the cytotoxic effect of SapC-DOPS in human tumor and nontumorigenic pancreatic cells. Next, pancreatic tumor xenografts (orthotopic and subcutaneous models) were used for tumor targeting and therapeutic efficacy studies with systemic SapC-DOPS treatment. We observed that the nanovesicles selectively killed human pancreatic cancer cells in vitro by inducing apoptotic death, whereas untransformed cells remained unaffected. This in vitro cytotoxic effect correlated to the surface exposure level of PS on the tumor cells. Using xenografts, animals treated with SapC-DOPS showed clear survival benefits and their tumors shrank or disappeared. Furthermore, using a double-tracking method in live mice, we showed that the nanovesicles were specifically targeted to orthotopically-implanted, bioluminescent pancreatic tumors. These data suggest that the acidic phospholipid PS is a biomarker for pancreatic cancer that can be effectively targeted for therapy utilizing cancer-selective SapC-DOPS nanovesicles. This study provides convincing evidence in support of developing a new therapeutic approach to pancreatic cancer.

Squamous cell carcinoma (SCC) and melanoma are malignant human cancers of the skin with an annual mortality that exceeds 10,000 cases every year in the USA alone. In this study, the lysosomal protein saposin C (SapC) and the phospholipid dioloylphosphatidylserine (DOPS) were assembled into cancer-selective nanovesicles (SapC-DOPS) and successfully tested using several in vitro and in vivo skin cancer models. Using MTT assay that measures the percentage of cell death, SapC-DOPS cytotoxic effect on three skin tumor cell lines (squamous cell carcinoma, SK-MEL-28, and MeWo) was compared to two normal nontumorigenic skin cells lines, normal immortalized keratinocyte (NIK) and human fibroblast cell (HFC). We observed that the nanovesicles selectively killed the skin cancer cells by inducing apoptotic cell death whereas untransformed skin cancer cells remained unaffected. Using subcutaneous skin tumor xenografts, animals treated with SapC-DOPS by subcutaneous injection showed a 79.4% by volume tumor reduced compared to the control after 4 days of treatment. We observed that the nanovesicles killed skin cancer cells by inducing apoptotic cell death compared to the control as revealed by TUNEL staining of xenograft tumor sections.

Rheumatoid arthritis is a chronic inflammatory disease affecting approximately 1% of the population and is characterized by cartilage and bone destruction ultimately leading to loss of joint function. Early detection and intervention of disease provides the best hope for successful treatment and preservation of joint mobility and function. Reliable and non-invasive techniques that accurately measure arthritic disease onset and progression are lacking. We recently developed a novel agent, SapC-DOPS, which is composed of the membrane-associated lysosomal protein saposin C (SapC) incorporated into 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipid nanovesicles. SapC-DOPS has a high fusogenic affinity for phosphatidylserine-enriched microdomains on surfaces of target cell membranes. Incorporation of a far-red fluorophore, CellVue Maroon (CVM), into the nanovesicles allows for in vivo non-invasive visualization of the agent in targeted tissue. Given that phosphatidylserine is present only on the inner leaflet of healthy plasma membranes but is “flipped” to the outer leaflet upon cell damage, we hypothesized that SapC-DOPS would target tissue damage associated with inflammatory arthritis due to local surface-exposure of phosphatidylserine. Optical imaging with SapC-DOPS-CVM in two distinct models of arthritis, serum-transfer arthritis (e.g., K/BxN) and collagen-induced arthritis (CIA) revealed robust SapC-DOPS-CVM specific localization to arthritic paws and joints in live animals. Importantly, intensity of localized fluorescent signal correlated with macroscopic arthritic disease severity and increased with disease progression. Flow cytometry of cells extracted from arthritic joints demonstrated that SapC-DOPS-CVM localized to an average of 7–8% of total joint cells and primarily to CD11b+Gr-1+ cells. Results from the current studies strongly support the application of SapC-DOPS-CVM for advanced clinical and research applications including: detecting early arthritis onset, assessing disease progression real-time in live subjects, and providing novel information regarding cell types that may mediate arthritis progression within joints.

Nanovesicles composed of the phospholipid dioleylphosphatidylserine (DOPS) and a fusogenic protein, saposin C (SapC), selectively target and induce apoptotic cell death in a variety of human cancer cells in vitro and in vivo. We tested whether such tumor-homing nanovesicles are capable of delivering fluorescent probes and magnetic resonance (MR) contrast agents to cancerous tissue to aid in earlier detection and improve visualization.SapC-DOPS nanovesicles labeled with either a far-red fluorescent probe (CellVue® Maroon, CVM) or conjugated with a dextran coated MR contrast agent, ultrasmall superparamagnetic iron oxide (USPIO), were systemically administrated into xenografts for tumor detection using optical and MR imaging systems.SapC-DOPS nanovesicles were effectively detected in vivo in tumor-bearing animals using both optical and MR imaging techniques, thereby demonstrating the cancer-selective properties of these nanovesicles.SapC-DOPS nanovesicles offer promise as a new and robust theranostic agent for broad cancer-selective detection, visualization, and potential therapy.

INTRODUCTION: Glioblastoma Multiforme (GBM) is the most aggressive form of primary brain tumor with a median survival of

Saposin C is one of four small lipid-binding proteins that derive from a single precursor protein, named prosaposin (PSAP). PSAP has several neuronal effects, including neurite outgrowth stimulation, neuron preservation, and nerve regeneration enhancement. A minimal domain required for PSAP's neurotrophic function is located in the amino-terminal half of saposin C. Genetic defects of the PSAP gene in humans and mice lead to a complex lysosomal storage disease. The skin fibroblasts from PSAP- and saposin C-deficient patients have a massive accumulation of multivesicular bodies (MVBs). Incorporation of exogenous saposin C-containing liposomes into the cultured PSAP-/- cells reduced the accumulated MVBs to normal levels. Internalized saposin C was localized to late endosomes and lysosomes. MVBs are crucial for maintaining the cellular homeostasis required for neuronal development and growth. PSAP-/- mice have a short life span (30 days) and central nervous system (CNS) neuronal degeneration. Similar to PSAP-/- fibroblasts, excessive MVBs accumulated in CNS neurons and brain tissues of PSAP-null mice. Cultured cortical and hippocampal neurons from PSAP-/- mice had poor survival and displayed a neurite degenerative pattern. Delivery of saposin C ex vivo into cultured neurons and in vivo into brain neuronal cells in mice across the blood-brain barrier was accomplished with intravenously administered dioleoylphosphatidylserine (DOPS) liposomes. These studies may yield a new therapeutic approach for neuron protection, preservation, and regeneration.

To investigate the relationship between major histocompatibility complex class I chain-related A(MICA) gene and systemic lupus erythematosus (SLE).The alleles and frequencies of exons 4 and 5 of MICA gene were determined in 70 cases of SLE and 152 controls of Yunnan Hans by STR genotyping, polymerase chain reaction, single strand conformation polymorphism and bidirection DNA sequencing.Five alleles of exon 5 and 10 alleles of exon 4 were found in this study. The frequency of each allele was determined in patients and controls. There was no significant difference between the two groups in exons 4 and 5 of MICA gene.Exons 4 and 5 of MICA were not related to SLE in Yunnan Hans.

Pancreatic ductal adenocarcinoma (PDAC) is marked by high expression of Tissue Factor (TF). Alternatively spliced TF (asTF) is a secreted TF form that signals non-proteolytically via β1/β3 integrins. asTF is abundant in PDAC lesions and its constitutive overexpression promotes tumor growth, metastatic spread, monocyte recruitment, and elevated procoagulant potential of PDAC cells and cell-derived microparticles. In this study, we determined if 1) delayed-onset overexpression of asTF yields a phenotype distinct from that obtained with constitutive overexpression; 2) asTF contributes to PDAC cell migration; and 3) monoclonal antibody-based targeting of tumor-derived asTF slows PDAC progression and/or asTF release into circulation in an orthotopic setting. Nude mice were orthotopically transplanted with human PDAC cells harboring a doxycycline (Dox)-inducible asTF transgene system (line Pt45P1/asTFi); mice received Dox (2µg/mL) in sucrose at day 1 (“Dox”), day 25 (“late Dox”), or sucrose alone (“no Dox”), and tumor progression was monitored in vivo over 7 weeks via SapC-DOPS imaging. Migration of Pt45P1/asTFi cells toward serum was assessed using laminin-coated transwell inserts. Plasma of PDAC tumor-bearing mice was assayed for asTF using a custom ELISA. Pt45P1 cells were resuspended in PBS containing anti-asTF rabbit monoclonal antibody Rb1 and implanted in a) nude mice; b) SCID mice, and c) SCID mice with TF levels reduced by 99% (SCID/TF-low) and tumor progression was monitored as above over 5 weeks. Paraffin-embedded tumor specimens were assessed for vessel density (isolectin B4 and/or anti-CD31 staining) and monocyte/macrophage infiltration (anti-F4/80 and/or anti-CD206 staining). “Late Dox” mice developed tumors comparable in size and vessel density to those in “Dox” mice, yet less infiltrated with macrophages (Dox: 70±22 cells/LPF, late Dox: 51±17 cells/LPF, p=0.009); tumor spread was significantly reduced in "late Dox" mice compared to "Dox" mice (7.52e+7 vs 1.22e+8 P/s/mm2, p=0.01). "Dox" mice also had high levels of asTF in circulation (~1 ng/mL), which did not differ significantly from those in “late Dox” mice. The PDAC cell migration assay revealed a ~4 fold increase in the migration of Pt45P1/asTFi cells treated with Dox compared to untreated cells (p We here report that asTF can promote PDAC progression during early and late stages of the disease. asTF-β1 integrin interaction renders PDAC cells more motile, while Rb1 fully inhibits asTF-potentiated PDAC cell migration. Host-derived TF has no impact on the size of primary PDAC tumor, yet it contributes to tumor vascularization and, consequently, metastatic spread. In the presence of Rb1 in nude and/or SCID mice, Pt45P1 cells grow significantly smaller tumors with fewer monocytes and blood vessels that release less asTF in the circulation. Thus, antibody-based targeting of asTF may comprise a novel strategy to stem PDAC progression. Disclosures No relevant conflicts of interest to declare.

SapC-DOPS nanovesicles are a stable protein-lipid mixture assembled from saposin C (SapC) and dioleylphosphatidylserine (DOPS). It shows selective tumor-targeting activity in multiple animal tumor models and specific induction of apoptosis in various cancer cell lines. The preferential targeting is due to higher levels of exposed phosphatidylserine on the outer cell membrane of cancer cells. Importantly, induction of apoptosis by SapC-DOPS was shown to occur through intracellular ceramide elevation. Ceramide is known to be a tumor suppressor molecule capable of acting synergistically with Bax and regulating mitochondrial-mediated apoptosis. Effectors that mediate SapC-DOPS-induced apoptosis downstream of ceramide are currently unknown. Therefore, we hypothesized that mitochondria could play a major role during SapC-DOPS induced apoptotic cancer cell death. Cell death following SapC-DOPS treatment was assessed by several indices such as: G6PD release assay to estimate necrosis, DAPI staining and cell cycle analysis to estimate apoptosis and DNA fragmentation, JC-1 assay to estimate mitochondrial membrane potential (ΔΨM) and caspase-3 activation by Western blotting. SapC-DOPS treatment induced significant apoptosis as denoted by the appearance of a subG1 peak, loss of ΔΨM, mitochondrial release of Smac, Cyto c and AIF, as well as mitochondrial Bax translocation and oligomerization. ShRNA-mediated Smac knockdown and Bax inhibition with V5 peptide led to retention of ΔΨM and abrogation of apoptosis with decreased caspase-3 activation. Our results indicate that Smac and Bax play major roles in SapC-DOPS-induced mitochondria-mediated apoptotic pathway in human neuroblastoma cells

Despite significant advances in our understanding of the biology of CNS tumors, the translation of such knowledge to novel and effective therapeutic strategies has been slow. Consequently, both primary (e.g., glioblastoma multiforme) and secondary (metastatic) brain tumors remain among the most intractable and fatal of all cancers. We have shown that nanovesicles consisting of saposin C (SapC) and dioleylphosphatidylserine (DOPS) effectively target and kill brain tumor cells both in vitro and in vivo. These actions are a consequence of the affinity of SapC-DOPS for phosphatidylserine (PS), an acidic phospholipid abundantly present in the outer membrane of a variety of tumor cells and tumor-associated vasculature. Here, we characterize SapC-DOPS bioavailability in a human glioblastoma orthotopic mouse model and reveal a time-dependent, tumor-specific extravascular accumulation of fluorescently labeled SapC-DOPS. Glioblastoma targeting by SapC-DOPS is abrogated after in vivo exposure to lactadherin, a protein that binds PS with high affinity. We also demonstrate that SapC-DOPS selectively targets brain metastases-forming cancer cells both in vitro, in co-cultures with human astrocytes, and in mouse models bearing brain metastases derived from human breast or lung cancer. We finally that SapC-DOPS cytotoxic activity against metastatic breast cancer cells in vitro, and prolongs the survival of mice with breast cancer brain metastases. Taken together, these results suggest that surface-exposed PS can be potentially useful as a novel lipid marker for diagnosis, monitoring, and therapy of central nervous system cancer, and highlights the potential of SapC-DOPS in the diagnosis and treatment of primary and metastatic brain tumors.

NOV (Nephroblastoma OVerexpressed) C-ter (NOV C-ter) is the carboxy-terminal sequence (170aa) of NOV/CCN3 protein. NOV/CCN3 (357aa), a member of CCN family, is secreted by quiescent endothelial cells and is involved in the regulation of various cellular functions including angiogenesis, proliferation, differentiation, survival, adhesion and migration. Although NOV C-ter does not have a direct cytotoxic effect when tested on a large panel of human cell lines including the NCI 60 cell lines (NCI Drug Screening Program), our data demonstrates significant anti-angiogenic and anti-tumor effects in a variety of experimental models. NOV C-ter inhibits in vitro endothelial cell tube formation in a dose-dependent fashion, using endothelial cells derived from porcine aorta, human microvasculature, or human umbilical vein (HUVECs). In addition, NOV C-ter impairs HUVEC proliferation, migration, invasion and adhesion. Interestingly, inhibition of HUVEC proliferation occurs via interference with growth-promoting signals involving Apelin-13 and adenomedullin. Furthermore, NOV C-ter specifically reduced phosphorylation of PI3K/Akt in endothelial cells, with no significant effect on the p44/42 (ERK1/2) pathway. In vivo, NOV C-ter had significant anti-tumor effect as a single agent in a murine glioblastoma xenograft model using human X-12 culture-adapted cells and in a nude mouse xenograft model using human non-small-cell lung cancer cells (A549). In these two systems, NOV C-ter provided superior survival benefit compared to treatment with bevacizumab or vehicle (PBS). Similarly, NOV C-ter significantly increased overall survival versus vehicle (PBS), an effect which was comparable to temozolomide treatment, in a murine syngeneic orthotopic glioblastoma model using the GL-261 cell line. To directly study the effect of NOV C-ter on angiogenesis, we treated zebrafish embryos prior to the onset of angiogenesis, and found a considerable reduction in vessel formation compared to bevacizumab-treated or untreated zebrafish. NOV C-ter also reduced tumor neovascularization in human breast cancer (T47D cell line) implanted in zebrafish. In summary, NOV C-ter demonstrates anti-angiogenic and tumoricidal effects via a novel mechanism of action. Mechanistic studies of NOV C-ter are underway to evaluate its precise effects on key signaling pathways in endothelial cells. Preclinical pharmacology and toxicology studies are also underway with a plan to advance to early-phase clinical trials. Citation Format: Junfeng Luo, Yong Teng, Minghui Li, Theodore S. Johnson, Franck Cuttitta, Zhengtao Chu, Xiaoyang Qi, John K. Cowell, Olivier Rixe. NOV C-ter: A novel preclinical anti-angiogenic agent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1023. doi:10.1158/1538-7445.AM2014-1023

Introduction: Acidic phosphatidylserine-enriched membrane microdomains (PS-EMMs) are exposed on the membrane surface of cancer cells and tumor blood vessels. We have explored a novel PS-EMMs targeting mechanism by saposin C (SapC)-coupled dioleoylphosphatidylserine (DOPS) nanovesicles that are found to specifically target and destroy cancer and tumor-associated endothelial cells with little or no systemic toxicity (Qi et al. Clin. Cancer Res. 2009; Kaimal et al. Mol. Imaging Biol. 2011). Methods: We have developed a novel biotheragnositic agent, SapC-DOPS, which we assembled into cancer-selective nanovesicles. Surface exposed PS levels were analyzed by a flow cytometric method using fluorescently labeled annexin-V. Deparafinized tissue slides were stained using far-red fluorophore (CellVue Maroon, CVM) labeled SapC-DOPS nanovesicles, SapC-DOPS-CVM was used to determine cancer-selective targeting via PS-mediated membrane fusion in human cancer cells, patient-derived cancer tissue specimens, and preclinical animal tumor models, including brain, pancreatic, lung, and pediatric cancers. The cytotoxicity of SapC-DOPS was measured using the MTT assay. Live imaging for visualization of SapC-DOPS PS-targeting and survival curves were demonstrated in transgenic and xenograft mouse tumor models. Results: We demonstrated the specific targeting of SapC-DOPS in paraffin embedded patient-derived tumor tissue sections. Based on MTT assay and annexin-V labeled flow cytometry, we revealed that SapC-DOPS cytotoxic effects correlated to the level of surface exposed PS in a variety of human cancer cell lines. Kinetic studies indicated that SapC-DOPS fused with tumor cell membrane within a few minutes, followed by internalization into the cells. In vivo cancer cell targeting of SapC-DOPS was blocked by pretreatment with PS-specific binding proteins (Lact-C2 and beta-2 glycoprotin-1). Increased PS expression and externalization on cancer cell membrane surfaces after chemotherapy or extracellular stresses enhanced the SapC-DOPS response. SapC-DOPS showed cancer-selective targeting in numerous spontaneously formed and xenografted tumors in living mice using either a double-tracking optical or a contrast-enhanced magnetic resonance imaging system. Furthermore, we observed that tumor-bearing animals treated with intravenous SapC-DOPS had significant tumor reduction and increased survival. Tumor-free survivals were confirmed by tumor-luminescence imaging and at necropsy. Conclusion: We have demonstrated a novel biomarker for numerous human cancers that can be effectively targeted by using cancer-selective SapC-DOPS nanovesicles. The direct confirmation of tumor-targeting by SapC-DOPS provides powerful new evidence in support of our goal to develop PS-EMMs targeted nanovesicles as novel therapeutic agents for cancer. 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 1840. doi:1538-7445.AM2012-1840