bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3479: Targeting FoxM1 selectively depletes hepatic cancer stem cells
Authors: Kopanja Dragana , Raychaudhuri Pradip
Published: August 2012

Over-expression of the Forkhead box M1 (FoxM1) transcription factor coincides with highly aggressive, poorly differentiated hepatocellular carcinoma (HCC). Over-expression of FoxM1 in HCC also correlates with poorer overall survival after hepatectomy. Since HCC progression involves hepatic cancer stem cells (HCSCs), we considered the roles of FoxM1 in HCSCs. We found that FoxM1 expression correlates with the expression of the stemness genes in human HCC cell lines. Interestingly, depletion of FoxM1 in human HCC lines causes a selective reduction in the population of the CD90+ HCSCs due to selective apoptosis. Moreover, FoxM1 silencing decreases sphere formation potential of the same cell lines, suggesting that FoxM1 is critical for survival of the HCSCs. Consistent with the loss of HCSCs, depletion of FoxM1 causes decrease in tumorigenic potential of the HCC cells in vitro as well as inhibition of tumor formation in mouse xenografts. Furthermore, we found that FoxM1 directly regulates expression of CD44, previously shown to be important for the tumorigenic potential of CD90+ HCC cells. These studies have exciting implications in further considerations of FoxM1 as a target of HCC therapy.

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 3479. doi:1538-7445.AM2012-3479
Abstract 3481: Dietary acetate supplementation as a means of inducing glioma stem cell growth arrest
Authors: Long Patrick M. , Tsen Andrew , Moffett John R. , Namboodiri Aryan M. A. , Viapiano Mariano S. , Jaworski Diane M.
Published: August 2012

Glioma, the most common primary brain tumor of the adult central nervous system, is associated with a poor prognosis due, in part, to the presence of chemoradiotherapy-resistant glioma stem-like cells responsible for inevitable post-surgical recurrence. N-acetyl-L-aspartate (NAA), one of the most concentrated metabolic sources of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors. NAA-derived acetate is converted to acetyl coenzyme A via acetyl-CoA synthetase (AceCS) for use in lipogenesis, protein/histone acetylation, and the TCA cycle. We propose that glyceryltriacetate (GTA), a FDA approved food additive with “generally regarded as safe” status, may be an effective means of reducing glioma growth via restoration of acetate levels. The effect of GTA on the growth of both established (Hs683, HOG) and stem-like (grade II OG33, grade III OG35) oligodendroglioma cell lines was assessed. In vitro, GTA induced growth arrest in all cells examined (i.e., increased proportion of cells in G0/G1 and reduced S phase cells by flow cytometry of propidium iodide labeled cells 24 hours after treatment and unbiased trypan blue exclusion based cytometry up to 5 days post-treatment). Growth arrest was not associated with apoptosis (lack of cleaved poly ADP-ribose polymerase immunolabeling), but differentiation (increased CNPase expression). ASPA expression was greater in stem-like cells when grown in stem cell media than differentiation media and was decreased in GTA-treated OG35 cells. Interestingly, GTA did not decrease ASPA expression in OG33 cells, but induced a novel 26 kDa ASPA isoform. ASPA and AceCS1 were co-localized within the nucleus. Nuclear, but not cytosolic, ASPA expression was decreased upon GTA addition in stem cell media, but not differentiation media. Finally, the effect of GTA on orthotopically grafted luciferase expressing OG33 and OG35 cells was assessed. Bioluminescence and tumor volume were reduced in GTA treated mice. These data suggest that the nuclear ASPA/AceCS1 co-localization provides acetate for histone acetylation to maintain cells in a progenitor/stem-like state and that decreased ASPA promotes gliomagenesis. Inasmuch as infants with Canavan Disease, a leukodystrophy due to ASPA mutation, treated with high dose GTA showed no significant side effects, GTA may prove an effective therapy to prevent recurrence by inducing growth arrest/differentiation of glioma stem-like cells.

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 3481. doi:1538-7445.AM2012-3481
Abstract 3483: Inhibition of Monocarboxylate Transporter 4 (MCT4) targets stem-like cells in glioblastoma
Authors: Lim Kah Suan , Lim Kah Jing , Orr Brent A. , Price Antoinette C. , Eberhart Charles G. , Bar Eli E.
Published: August 2012

Glioblastomas (GBM) are the most common adult malignant brain tumors and contain a hypoxic/necrotic core surrounded by proliferative cells. To unmask genes important in hypoxia, we exposed 2 GBM neurosphere lines, HSR-GBM1 and JHH-GBM10, to 1% and 21% oxygen levels for 24 hours and compared gene expression using Agilent oligonucleotide microarrays. We identified SLC16A3 (Monocarboxylate transporter-4, MCT4) as one of the most upregulated genes in response to hypoxia. To investigate the clinical importance of MCT4 in GBM, we examined the Kaplan-Meier survival curves of glioma patients using public databases. We found that patients with upregulation of MCT4 (≥2.0X) have a significantly shorter survival (p<0.0001) than patients with intermediate expression. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM (p<0.0001). We next examined MCT4 protein levels using immunohistochemical analysis of tissue microarrays, confirming that MCT4 protein levels were increased in high-grade as compared to lower grade astrocytomas (p<0.0001). These data clearly demonstrated that MCT4 is a clinically relevant target. We next tested the requirement of MCT4 in vitro. We found that when neurospheres were transduced with viruses encoding short hairpin RNA (shRNA) against MCT4, cell growth was inhibited by 30-69% in hypoxia. Interestingly, similar results were observed in normoxia, suggesting that MCT4 may be critical to tumor growth and survival independent of oxygen levels. We next explored the role that MCT4 may play in the maintenance of the stem-like population in our neurosphere lines. We found that MCT4 is overexpressed in sorted CD133-positive cells compared to CD133-negative cells in both HSR-GBM1 and JHH-GBM10. Furthermore, flow cytometric analysis of shMCT4-expressing neurospheres showed a decrease in the percentage of CD133-positive cells as compared to controls. This suggested to us that MCT4 silencing might inhibit stem-like cell proliferation/survival, offering a potential explanation for growth inhibition following MCT4 knockdown in normoxia. To test this hypothesis, we examined the effect of MCT4 inhibition functionally using a clonogenic assay. Cells were exposed to normoxia or hypoxia for 48 hours followed by recovery in normoxia. We found that MCT4 silencing resulted in significant reduction in sphere number and size in both normoxic and hypoxic cells, consistent with a loss of clonogenicity. In agreement with what we found in vitro, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo (p=0.009).

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 3483. doi:1538-7445.AM2012-3483
Abstract 3485: Inhibition of Notch signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells
Authors: Wei Ping , Qiu Ming , Jiang Ivy , Carroll Christopher , Han Guangzhou , Rymer Isha , Kraynov Eugenia , Wu Aidong , Thibault Stephane , Stone Donna , Gao Yijie , Sofia Susan , Gallo Jorge , White Janet , Li Gary , Yang Jennifer , Los Gerrit
Published: August 2012

Triple negative breast cancer (TNBC) is characterized by lack of estrogen receptor (ER), progesterone receptor (PR) or Her2/neu. It is known to be particularly aggressive and refractory to current chemotherapies. Tumor-initiating cells (TICs) have been postulated as responsible for treatment failure for this type of cancer. Recent evidence suggests that Notch signaling may play a role in regulation of TIC self-renewal and differentiation hence present a promising target for development of novel therapies for TNBC. We previously discovered a mouse monoclonal Ab (mu-hN1) that produces robust efficacy in both T-cell acute lymphoblastic leukemia and TNBC by specifically blocking human Notch1 signaling in tumor cells. We subsequently produced a humanized Notch1 mAb (hu-hN1) that retained similar characteristics and activities compared to the mouse prototype mu-hN1. To gain insights into the mechanisms of Notch1 blockade on TNBC tumor grow inhibition (TGI), we evaluated the effect of hu-hN1 on apoptosis, proliferation and TIC self-renewal in Notch-driven Sum149 and a patient derived xenograft (PDX) TNBC models. We showed that hu-hN1 inhibited Notch signaling by reduction of NICD (Notch intracellular domain) and target genes Hes-1 and cMyc in Sum149 tumors. Inhibition of Notch signaling led to significant TGI either as a single agent or in combination with Docetaxel. TGI of hu-hN1 appeared to be in part due to induction of apoptosis in the tumor cells. More interestingly, treatment with this Ab caused reduction in mammosphere formation and CD44+/CD133+/ESA+ cell population. It also resulted in decrease in tumor incidence and delay in tumor recurrence, suggesting an anti-TIC activity of the agent. Further investigation in a PDX TNBC model confirmed the robust tumor efficacy and anti-TIC effect of hu-hN1. Taken together, our findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional treatments by directly targeting the TIC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival.

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 3485. doi:1538-7445.AM2012-3485
Abstract 3486: Novel cell models of ovarian cancer for target discovery and validation obtained through stem cell culturing technology
Authors: Young Peter F. , Pan Zhuangyu , Roberts Penny , Hooley Jeff , Smith Doug , Chen Francine , Loo Deryk , Bonvini Ezio , Mather Jennie , Moore Paul A.
Published: August 2012

Introduction: Ovarian cancer is the leading cause of death from gynecological malignancies with greater than 50% of patients succumbing to the disease within 5 years of initial diagnosis. Contributors to the overall poor survival rate include late diagnosis, development of drug resistance and persistence of cancer stem cell (CSC) populations. With increasing evidence that CSC drive tumor initiation and metastases, development of model systems to better characterize such populations are needed. We recently reported use of stem cell culturing conditions to generate cancer stem like cells (CSLC) from colon and lung cancer that exhibit CSC properties (1-2). Here we apply a similar approach to ovarian cancer. Methodology: Freshly resected ovarian cancer tissue was obtained, processed and cultured using serum-free defined culture media evolved from conditions previously employed to culture tissue-specific progenitor stem cells. Expanded cell lines were characterized for their tumor initiating properties in NSG mice upon implantation under the sub-renal capsule. Xenografts were analyzed by H&E and immunostaining with ovarian cancer associated markers. Cell surface protein profile was examined by flow cytometry using mAbs against putative CSC markers and with those generated through de novo whole cell immunization of the putative ovarian cancer stem cell lines. Results: Ovarian CSLC were successfully cultured from independent serous ovarian tumor specimens. These lines can be indefinitely passaged in vitro while retaining ability to form tumors that faithfully recapitulate original patient tumor morphology and metastasize. The CSLC express EpCAM consistent with their epithelial origin and both CD44 and CD133. Analyses of mAbs generated from whole cell immunization identified B7-H3 as an additional target expressed on the ovarian CSLC. IHC analyses demonstrated B7-H3 expression also on primary and metastatic ovarian CSLC xenografts consistent with expression observed on patient derived specimens. Conclusion: Ovarian cancer cell lines have been generated using conditions previously established for tissue stem cells. These lines, which can be maintained indefinitely in vitro while retaining cancer stem cell properties, provide opportunity to identify and characterize novel ovarian cancer targeting strategies such as those designed against B7-H3. References: 1: Roberts (2011) AACR Abstract #5211; 2: Young (2011) AACR Abstract #502.

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 3486. doi:1538-7445.AM2012-3486
Abstract 3488: Targeting ligands and nanotherapeutics against acute myeloid leukemia stem cell
Authors: Zhang Hongyong , Luo Juntao , Li Yuanpei , Henderson Paul T , Wang Yanchun , Wachsmann-Hogiu Sebastian , Lam Kit S. , Pan Chongxian
Published: August 2012

Background: Acute myeloid leukemia (AML) stem cells (LSC) can survive the conventional-dose chemotherapy, generate more differentiated leukemic cells and cause recurrence. In order to cure AML, LSC must be eradicated. This project was to develop targeting therapeutics to overcome chemoresistance and eradicate LSC. Materials and Methods: The phage-display library was panned to identify ligands targeting C-type lectin-like molecule-1 (CLL1) expressed on LSC, but not on normal hematopoietic stem cells. A CLL1-targeting ligand was used to decorate the surface of micelle nanocarrier developed by our group to deliver chemotherapeutic drugs to LSC. Target-specific drug delivery was determined using A549 adherent cells expressing CLL1 and CD34+CD38- leukemia cells freshly isolated from clinical specimens. Toxicology study was performed in Balb/c mice. Results: After three rounds of panning against A549 cells expressing CLL1, 35 out of 36 clones contained the same amino acid motif LR(S/T). These ligands did not bind to any peripheral blood cells and normal hematopoietic stem cells. One ligand named CLL1-L1 was used to decorate the surface of nanomicelles loaded with chemotherapeutic drug daunorubicin (DNR). These targeting nanomicelles were approximately 15 nm in diameter. The loading capacity was around 5 mg of daunorubicin per 20 mg of nanomicelle, a clinically useful degree of drug loading. High-resolution tomography showed that targeting nanomicelles delivered the drug load directly to the intracellular space including nucleus much more efficiently than the non-targeting nanomicelles (p<0.05). These targeting nanomicelles could deliver the drug load to CD34+CD38- leukemic cells (containing LSC) isolated from clinical specimens, but did not bind to normal blood or normal hematopoietic stem cells. Chemotheapeutic drug formulated in these nanomicelles is about three times less toxic than the parental free drug, suggesting this nanoformulation may allow administration of high-dose chemotherapy without the associated severe toxicity. Conclusion: This is the first report on the development of targeting ligands and nanotherapeutics against AML stem cells. Nanomicelles decorated with CLL1-targeting peptides can potentially deliver high-dose chemotherapeutic drugs directly into LSC and eradicate this cell population, in addition to treating leukemic cells throughout the body and decreasing toxicity.

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 3488. doi:1538-7445.AM2012-3488
Abstract 349: Leptin-induced epithelial-mesenchymal transition in breast cancer cells requires β-catenin activation via Akt/GSK3-dependent and MTA1/Wnt1-dependent pathways
Authors: Yan Dan , Avtanski Dimiter B. , Saxena Neeraj K. , Sharma Dipali
Published: August 2012

Perturbations in the adipocytokines-profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide first molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1) and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of GSK3β-LKB1-Axin complex which leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors show increased expression of Wnt1, pGSK3β and Vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized crosstalk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells.

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 349. doi:1538-7445.AM2012-349
Abstract 3495: The novel phospholipid ether analog CLR1404 decreases glioblastoma stem cell proliferation, suppresses GBM growth, and improves survival
Authors: Clark Paul A. , Chen Hong-En , Mohamed Mohamed , Kandela Irawati K. , Longino Marc A. , Pinchuk Anatoly , Weichert Jamey P. , Kuo John S.
Published: August 2012

Most patients treated for glioblastoma multiforme (GBM), the most common primary brain malignancy, have less than 2 years survival due to rapid recurrence. The GBM stem cell (GSC) sub-population exhibits therapeutic resistance and is hypothesized to drive tumor recurrence. Therefore, GSC-specific targeting is likely critical for improving outcome. Phospholipid ether analog, CLR1404 and its radioiodinated isosteres, exhibit cancer cell-specific uptake and prolonged retention in 57/61 cancer cell lines and xenografts (including GBM) due to affinity for cancer cell lipid rafts. In this study, we investigated the therapeutic potential of CLR1404 against GBM and its GSC subpopulation. Multiple sphere-forming GSC lines were isolated from patient specimens with IRB approval, and rigorously validated for self-renewal, multi-lineage potential, and high efficiency orthotopic tumor initiation in NOD-SCID mice. Proliferation assays were performed by addition of CLR1404 in serum-free medium for 24 hours. To test effects on stem cell properties, GSCs were treated with CLR1404, dissociated to single cells, plated at 500 cells in a 96-well plate, and allowed to form spheres (≥2-4 weeks). CLR1404 inhibition of the AKT oncogenic signaling pathway in GSCs was assayed with immunoblot analysis. In vivo, GSCs were pre-treated for 24 hours with CLR1404 prior to orthotopic injection of 200,000 live cells into immunodeficient mice. Subsequent survival curves were then constructed. CLR1404 anti-proliferative effects were seen on all 7 different GSC and GBM lines tested with IC50 values ranging between 5-10 µM using MTS assay; control normal differentiated neural cells exhibited an IC50 of approximately 40 µM. CLR1404 treatment also decreased sphere-forming ability of multiple GSC lines with IC50 values between 5-10 µM. CLR1404 inhibition of AKT activation was observed using immunoblot analysis. CLR1404 pre-treatment of GSCs significantly increased survival time in an orthotopic mouse model (Control: 59±6.1 days; CLR1404: 94±4.4 days), suggesting CLR1404 treatment of the GSC tumor initiating cells improves outcome. This data of CLR1404's therapeutic potential against GBM and its GSCs, combined with previously demonstrated tumor cell targeting specificity of CLR1404 and its radioactive isosteres, provides strong evidence for the potential of novel CLR1404-based therapies to improve GBM outcomes.

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 3495. doi:1538-7445.AM2012-3495
Abstract 3507: Type 17-1-CD8+ T cells (Tc17-1) as potent effector cells for immunotherapy of brain tumors
Authors: Ohkuri Takayuki , Kosaka Akemi , Ikeura Maki , Litterman Adam , Ohlfest John , Okada Hideho
Published: August 2012

Malignant gliomas, such as glioblastoma multiforme (GBM), are the most common and dismal primary brain tumors, and there is a strong need for developing novel and effective therapeutic modalities. Although the central nervous system (CNS) and CNS tumors are often considered immunologically privileged, as demonstrated by studies on CNS autoimmunity, activated autoreactive T-cells, especially T helper (Th) 17 cells, infiltrate through the blood brain barrier (BBB) and mediate antigen-specific responses in the CNS. Especially, Th17 express high levels of CCR6, which has a crucial role in Th17 infiltration into the CNS. In addition to Th17 cells, a new putative subtype of IL-17-producing CD4+ T cells with CD4+IL-17+IFN-γ+ (Th17-1 cells) double-positive phenotype has also been identified. Based on our previous studies demonstrating a critical role of Th1-chemokine CXCL10 and its receptor CXCR3 for effective T-cell trafficking in CNS tumors, we hypothesized that Th17-1 cells and CD8+ T cells producing IL-17 and IFN-γ (hereby Type-17-1 CD8+ T-cells) will demonstrate superior CNS tumor infiltration compared with Th1 cells due to the activation of two CNS-relevant chemokine pathways, CXCL10-CXCR3 and CCL20-CCR6. Mouse Tc17, Tc17-1 and Tc1 cells generated from Pmel-1 mouse-derived naïve CD8+ T cells expressed high CCR6 and low CXCR3, high CCR6 and high CXCR3, and little CCR6 and high CXCR3, respectively. Although Tc17 displayed lower levels of antigen-specific cytotoxic activity compared with Tc17-1 and Tc1 in a 4 hr cytotoxic assay, Tc17 killed tumor cells more effectively than the other populations in long-term cultures (4 to 6 days). While Tc1 cells underwent apoptosis due to their exhausted state, Tc17-1 maintained strong cytotoxicity and proliferative activities even after 4 day co-culture with tumor cells. Moreover, the supernatant derived from activated Tc17 and Tc17-1 induced CCL20 and CXCL10 in the GL261 glioma cells while Tc1-derived supernatant induced CXCL10 but not CCL20. These results suggest a possibility that Tc17-1 may elicit persistent anti-CNS tumor responses and induce both CCL20 and CXCL10 in the CNS tumor microenvironment, thereby attracting additional effector cells to the site. In vivo studies in mice bearing CNS gliomas are warranted to determine whether adoptively transferred Tc17-1 infiltrate to the CNS tumor site and mediate superior anti-tumor response compared with Tc1.

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 3507. doi:1538-7445.AM2012-3507
Abstract 3509: Intracranial administration of activated NK-cells and mAb9.2.27 against NG2/CSPG4 activates microglial cytotoxicity and prolongs survival in glioblastoma xenograft model
Authors: Poli Aurelie , Wang Jian , Domingues Olivia , Yan Tao , Thorsen Frits , Brekke-Rygg Cecilie , Hentges Francois , Pedersen Paal Henning , Zimmer Jacques , Enger Per Øyvind , Chekenya Martha
Published: August 2012

Glioblastoma multiforme (GBM) is the most malignant brain tumour in adults with dismal prognosis despite multimodal treatment. We have previously shown that the NG2 proteoglycan mediates an aggressive disease course in GBMs and therefore aimed to target it using the monoclonal antibody 9.2.27 and adoptively transferred natural killer (NK) cells in athymic rats. We demonstrated that intracranial administration of activated NK cells and mAb9.2.27 eradicated established GBM and led to significantly increased survival, mediated by diminished tumour proliferation and increased cell death. The mechanisms involved sustained activation of pro-inflammatory ED1+CD8+ microglia and subsequent development of a Th1 immune response in the combination therapy compared to monotherapy controls. The Th1 response was characterized by increased tumour infiltration by CD4+ and CD8+ T cells, as well as augmented IFN-γ and TNF-α release into the cerebrospinal fluid. Furthermore, we showed that activated NK cells released IFN-γ and TNF-α, they shifted activated microglia from M2- to highly cytotoxic M1-like phenotype and preferentially killed M2-microglia compared to M1-microglia in vitro. These findings implicate NK cells in the pro-inflammatory environment that facilitated the tumour cytotoxicity by microglia in vivo. Our results suggest that the administration of activated NK cells and mAb9.2.27 is a promising approach to treat GBM.

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 3509. doi:1538-7445.AM2012-3509
Abstract 3517: Use of a novel synthetic biomaterial to induce mild whole body hyperthermia for the treatment of cancer in a preclinical model
Authors: Kodama Mitsuyama , De Velasco Marco A. , Yamamoto Yutaka , Shimizu Nobutaka , Nozawa Masahiro , Yoshimura Kazuhiro , Yoshikawa Kazuhiro , Uemura Hirotsugu
Published: August 2012

Hyperthermia is currently being investigated as an alternative form or cancer therapy. Numerous basic and pre-clinical published reports have shown the antitumor effects of hyperthermia in various cancer models. High temperatures, low duration (heat shock) will have a direct killing effect on exposed cells. The limitation to this approach is the restriction of tumor location and associated side effects. Low temperature, high duration whole body hyperthermia (WBH) is generally considered to be the least toxic but requires long exposures that are generally uncomfortable or inconvenient to the patients. In the present study, we show the efficacy of a synthetic far-infrared emitting biomaterial to induce and evaluate its therapeutic potential when used alone or in combination with chemotherapy. We used BioRubber (BR, Yamamoto Bio-Technology Inc., Anaheim, CA), a state of the art biomaterial that emits far infrared radiation in the 4-25 μm spectral ranges. To determine the efficacy to induce mild whole body hyperthermia, athymic nude mice were house in BR lined cages. Core body temperatures of mice housed in BR-lined cages were significantly higher than mice housed in unlined cages and were maintained up to 2 weeks during continuous exposure (37.70oC vs. 36.75oC, p=0.038, respectively.). To determine the antitumor effects of WBH, KU7/GFP-bearing nude mice were housed in cages lined with BR for 28 days. Non-significant tumor growth inhibition was observed in tumors of mice housed in BR. Histological and immunohistochemical analysis revealed increased granulocyte activity in tumors of BR-exposed mice. Additionally, increased levels of C-GSF, GM-CSF, TNFα and IL-12p70 corresponded to exposure to WBH. To determine the long term safety and efficacy of mild WBH and chemotherapy in deep tumors, we carried out a survival study using immunodeficient mice orthotopically inoculated with KU7/GFP cells and treated doxorubicin (Dox) alone or in combination with BR. Although no treatment efficacy was observed in mice treated with BR with regards to tumor size (tumor size according to the expression of GFP was 350.5, 309.7, 357.4, 257.3 for control, WBH, Dox and WBH/Dox groups, respectively), survival was improved for mice exposed to BR. Survival rates were 50% (5/10), 70% (7/10), 10% (1/10) and 50% (5/10), for control, WBH, Dox and WBH/Dox groups, respectively. It is unclear if mild WBH has a direct antitumor effect; however, it appears that BR-induced mild WBH may be capable of inducing immuno-stimulatory that may improve survival in a preclinical model.

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 3517. doi:1538-7445.AM2012-3517
Abstract 3519: Appropriate crosslinking is required for co-stimulatory activity of human CD27 antibody in a transgenic mouse model
Authors: He Li-Zhen , Prostak Naseem , Crocker Andrea , Weidlick Jeffery , Widger Jenifer , Sisson Crystal , Vitale Laura , Glennie Martin J. , Keler Tibor
Published: August 2012

CD27 is a member of TNFR superfamily. It constitutively expresses on the majority of T cell and a subset of NK cells, playing key roles in T cell activation and survival and in NK cell proliferation and cytotoxicity upon interaction with ligand CD70. Some antibodies to mouse CD27 have been reported that display agonistic and anti-tumor activities while other mAbs had less anti-tumor activity and were depleting. We hypothesized that differences in these antibodies may be due to Fc receptor engagement, as has recently been shown for the adjuvant and anti-tumor activities of agonistic mouse CD40 mAbs, which is also member of TNFR superfamily. We have developed and previously described a human anti-human CD27 antibody (1F5) and a human CD27 transgenic mouse model (hCD27-Tg) to explore the therapeutic potential of targeting CD27. In this study, we examined the effect of modifying the constant regions of the 1F5 mAb on its ability to enhance antigen specific T cell responses. With the original 1F5 hIgG1 as template, a panel of 1F5 variants was made including 1F5 mIgG1, 1F5 mIgG2a, 1F5 mIgG1D265A and 1F5 hIgG1N297S using molecular cloning techniques. All of the variants retained equal binding to hCD27 as shown by ELISA and flow cytometry studies. In addition, Biacore analysis confirmed the expected pattern of binding to human and mouse FcαRs. Co-injection of 1F5 or its variants with ovalbumin enhanced antigen-specific CD8 T cell response to different extents, as detected by SIINFEKL-specific IFNα-ELISPOT and ICS. The 1F5 mIgG1 induced the highest number of IFNα-producing CD8+ cells, whereas 1F5 mIgG2a was very weak at enhancing the CD8 T cell response. The hIgG1 version of 1F5 was intermediate in activity. Introduction of the D265A mutation that disrupts FcαRs binding into the mIgG1 eliminated the co-stimulatory function of 1F5. Similarly, the 1F5 hIgG1N297S also showed reduced activity compared to the original 1F5 hIgG1. The isotype-specific effects on our anti-hCD27 mAb are surprisingly consistent with the findings described for the agonistic anti-mCD40 mAbs, and imply that engagement of the inhibitory Fcα receptors (FcαRIIb) is driving the co-stimulatory activity in this model. Interestingly, the 1F5 hIgG1 triggered a significant T cell response, despite the lack of FcαRIIb binding by Biacore analysis. The effect of these variants on anti-tumor activity in hCD27 transgenic mice is currently being investigated. The 1F5 hIgG1 mAb (CDX-1127) is currently undergoing clinical evaluation in a phase 1 trial of patients with advanced cancers.

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 3519. doi:1538-7445.AM2012-3519
Abstract 3521: RECRUIT-TandAb AFM13: Overcoming limitations of monoclonal antibodies in Hodgkin lymphoma
Authors: Rajkovic Erich , Knackmuss Stefan , Reusch Uwe , Rothe Achim , Topp Max , Younes Anas , Ravic Miroslav , Hucke Christian , Zhukvosky Eugene , Little Melvyn
Published: August 2012

Introduction: AFM13 is a RECRUIT-TandAb (CD30xCD16A) for treating Hodgkin Lymphoma by recruiting NK-cells and macrophages to the specific CD30 surface antigen on Reed-Sternberg cells. This redirected immune response leads to potent tumor cell killing. RECRUIT TandAbs are tetravalent and bispecific, and address key deficiencies of monoclonal antibodies such as (i) V/F polymorphism and (ii) non-selective binding to immune effector cells vs. granulocytes via CD16B receptor. Furthermore, RECRUIT-TandAbs have significant advantages over other antibody fragment technologies such as (i) bivalent binding to the targets and, (ii) longer half-life. Methods: AFM13 binds with high affinity to both CD30 and CD16A and is able to rapidly induce the lysis of CD30+ cells at picomolar concentrations in the presence of PBMCs. Intensive in vitro characterization demonstrated that AFM13 is specific for the CD16A receptor, which becomes activated only in the presence of tumor cells: there is no systemic activation of NK-cells in the absence of target cells. A robust GMP production process in mammalian cells and a lyophilized formulation with excellent stability have been established. Toxicology testing in Cynomolgus monkeys did not reveal any toxicity. Currently, AFM13 is being investigated in a single arm phase I dose escalation trial for patients with relapsed and/or refractory Hodgkin Lymphoma (HL). Patients receive a single cycle of 4 weekly doses, which are escalated in cohorts of 3 patients at the dose levels of 0.01, 0.04, 0.15, 0.5, 1.5, 4.5 and 7.0 mg/kg. Study objectives are the assessment of safety and tolerability, PK, immunogenicity, antitumor activity, as well as the determination of the maximum tolerated dose (MTD) or the optimal biological dose (OBD). The safety of the MTD or OBD will be confirmed in further patients receiving 2 cycles of AFM13. Results and Conclusion: So far, AM13 was shown to be safe and well tolerated by highly pre-treated Hodgkin Lymphoma patients in weekly doses of up to 1.5mg/kg. Adverse events were generally mild, with the most frequent drug-related event being fever. A first objective response was achieved on the dose level 1.5mg/kg. Five cases of stable disease / minor response were observed on lower dose levels. Further patients are being treated in the ongoing dose escalation study. Preclinical and interim clinical data will be presented, showing that AFM13 may become a safe and effective targeted therapy for CD30 positive malignancies.

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 3521. doi:1538-7445.AM2012-3521
Abstract 3522: Preclinical development of an anti-CD30/anti-CD16A bispecific tetravalent TandAb antibody for the treatment of Hodgkin lymphoma
Authors: Knackmuss Stefan H. , Reusch Uwe , Burkhardt Carmen , Fucek Ivica , Gall Fabrice Le , Pauels Hans-Gerd , Zhukovsky Eugene A. , Little Melvyn
Published: August 2012

NK cells are professional cytotoxic immune effector cells and, unlike cytotoxic T cells, exist in a constitutively activated state not requiring pre- or co-stimulation. Thus, NK cells are attractive effectors to employ for cancer immunotherapy with bispecific antibodies. To improve the recruitment and activation of NK cells for lysis of tumor cells, we isolated a human antibody that was highly specific for the A isoform of FcαRIII (CD16A) and displayed substantially lower dissociation constant than native or Fc-optimized mAbs. The variable domains of this antibody were used to construct a tetravalent bispecific antibody, termed TandAb, consisting solely of antibody variable domains, with two valencies for CD16A (on effector cells) and two for CD30 (on target cells). CD30 represents a validated tumor target which is expressed on Hodgkin's Reed-Sternberg lymphoma cells, and on some activated immune cells. The binding properties of the TandAb and its efficacy for inducing tumor cell lysis were compared to those of a native anti-CD30 mAb, a mAb with enhanced Fc receptor binding properties, and an anti-CD30 bispecific diabody, all of which comprised the same anti-CD30 variable domains as the TandAb. Due to its bivalency for CD16A, the TandAb was retained longer on NK cells than the IgGs or the bispecific diabody, and relatively high concentrations of IgG were necessary to displace it from NK cells. The TandAb bound equally well to both CD16A alleles (158F, 158V) indicating a potentially broader patient response than that achieved with current therapeutic mAbs. The TandAb did not induce any systemic activation of NK cells even though it's binding was bivalent to CD16A. In a cytotoxicity assay, the TandAb displayed higher potency (EC50) and higher efficacy (maximal lysis) than the IgGs or the bispecific diabody. Cynomolgus monkeys were identified as the only relevant species for toxicity testing. Pharmacokinetics studies revealed half-lives for the TandAb up to 22.9 h after repeated dosing. No MTD was reached in a single dose escalation study to a maximum dose of 100 mg/kg. A pivotal 28 days toxicity study in which animals were dosed repeatedly up to 10 mg/kg every other day revealed an excellent safety profile. Several immunological parameters were assessed indicating no signs of immunotoxicity or systemic cytokine release. In conclusion, TandAbs may provide a more effective treatment for tumors that are refractory to treatment with IgGs and is currently being evaluated in the Phase I clinical trial.

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 3522. doi:1538-7445.AM2012-3522
Abstract 3529: A direct mass spectrometry approach for HPV T cell epitope identification
Authors: Blatnik* Renata , Hoppe* Stephanie , Wühl Martin , Grabowska Agnieszka K. , Riemer Angelika B.
Published: August 2012

To rationally design therapeutic cancer vaccines, it is important to know which T cell epitopes are present on cancer cells. Malignant transformation affects the cellular antigen processing machinery, thus not every epitope derived from intracellular proteins is necessarily presented by MHC molecules on the cancer cell surface. Up to now, T cell epitopes have mostly been defined by indirect methods, such as screening peptide libraries for MHC binders, and subsequent lymphocyte reactivity and cytotoxicity assays. However, all these assays with externally added peptides do not answer the question whether a candidate epitope is naturally processed and presented on the malignant cell. This can now be elucidated by using highly sensitive mass spectrometry approaches. For this study, we chose high-risk HPV-driven cancers as a model system. The induction and maintenance of the malignant phenotype in these tumors are dependent on two viral oncoproteins, E6 and E7, which are therefore present in all stages of HPV-driven cancers. We here aimed to assess the presence of T cell epitopes restricted by the major MHC class I supertypes, namely HLA-A1, A2, A3, A11, A24, B7, and B15, on HPV16-transformed cells. Prospective epitopes from the HPV16 E6 and E7 proteins for the above MHC supertypes were identified by high-throughput in silico epitope predictions, employing several web-based prediction algorithms. These peptides were tested for MHC binding in cellular binding assays on B-LCL of the respective HLA type. Finally, MHC-peptide complexes were immunoprecipitated from HPV16-transformed cells, and the presence of candidate epitopes was analyzed by nano-HPLC-MS2 and MS3 mass spectrometry. Binding of the majority of predicted peptides to the respective HLA allele was verified in the cellular assays. Several novel HPV16 MHC binders were identified. We optimized the immunoprecipitation protocol for epitope yield. Mass spectrometry analysis was successfully established and validated for HLA-A2, and is currently performed for the other supertypes. In conclusion, we show that ascertaining the actual cellular presentation of HPV T cell epitopes is feasible. As the included HLA supertypes have a cumulative population prevalence of >95%, the resulting set of peptides may be used for the formulation of a widely applicable therapeutic HPV vaccine, or for immunomonitoring purposes in other HPV immunotherapy studies. * These authors contributed equally to this study.

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 3529. doi:1538-7445.AM2012-3529
Abstract 3531: Papillary thyroid carcinomas with and without lymph node metastasis show differences in tumor-infiltrating leukocytes
Authors: Angell Trevor E. , Correa Adrian J. , Kang Kate , Bass Rikki S. , Church Connor H. , Epstein Alan L.
Published: August 2012

Papillary thyroid carcinoma is the most common endocrine malignancy with approximately 48,000 new cases expected in 2011. Additionally, the incidence has doubled over the last three decades. While 20-year survival rates are extremely high with usual therapy, 15% will have recurrence and 5-10% will die from their disease. If refractory to standard treatment, no sufficient adjuvant therapies are available. Cancer immunotherapy uses a patient's own immune system to recognize and eliminate malignant cells. The potential advantages of this approach over conventional treatments include the systemic trafficking of immune cells to treat primary and metastatic disease, inherent antigen-specificity of adaptive immunity to minimize collateral damage, and most importantly, the induction of immunological memory to prevent recurrent disease. The degree and type of immune response are poorly understood in thyroid cancer. In this study, papillary thyroid cancer samples, with normal tissue matched to the same specimen, from de-identified patients were obtained retrospectively from the Norris Cancer Center Tumor Bank and stained using immunohistochemistry with antibodies specific for CD8 and CD16. Sections were evaluated for tumor-infiltrating leukocyte location, intra-tumor versus invading margin, and percentage of CD8 or CD16 positive lymphocytes, respectively. The results were analyzed for correlation between immune infiltrate and clinical parameters. Nineteen cases of papillary thyroid cancer were studied. The mean patient age was 40 years (range 22-55). Seven of nineteen patients had lymph node metastasis. An organized lymphocytic infiltration was seen in seven samples and was significantly correlated with the presence with CD8+ and CD16+ cells, at the invading margin and tumor parenchyma, respectively. Age <40 years was significantly associated with this pattern of CD8+ and CD16+ cells compared to those older than 40. Lymph node metastasis was significantly association with an increased level of intra-tumoral CD8+ cell infiltration and there was a non-significant trend towards a positive association between nodal metastasis and intra-tumoral CD16+ cell infiltration. The present study demonstrates an increase in cell-mediated immune infiltration in samples that have nodal metastases versus those that do not, suggesting that immune therapy may be utilized in the future treatment of aggressive thyroid carcinoma to improve disease response.

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 3531. doi:1538-7445.AM2012-3531
Abstract 3535: Lymphoma-associated macrophages benefit for tumor progress via overexpressing legumain to degrade extracellular matrix in diffuse large B-cell lymphoma
Authors: Li Honghao , Xiong Min , Zhang Yan , Tan Xiaoyue , Xiang Rong
Published: August 2012

Crosstalk between macrophages in the tumor microenvironment and malignant cells has been implicated during the pathogenesis of many kinds of tumors. However, role of lymphoma-associated macrophages in the progress of lymphoma as well as its underlying mechanism is far from completely clear yet. Here we evaluated the effect of lymphoma-associated macrophages on the characteristics of diffuse large B-cell lymphoma (DLBCL). Immunohistological staining demonstrated that level of CD163 positive macrophages, so called M2 type macrophage, was negatively related with the prognosis of DLBCL. In addition, M2 macrophage overexpressed asparaginyl endopeptidase legumain, as shown by double-immunofluoresence staining. Then we set up human DLBCL xenograft model. Mice were sacrificed at the different stages of tumor progress (average volume are 0.8 or 1.4 cm3. respectively. n=4). Real-time PCR results showed that mRNA expression of CD206 (M2 macrophage marker in mouse model) and legumain significantly increased with the progress of tumor. However, contents of matrix significantly decreased as indicated by sirus red and Masson's staining. Overlapping existed in immunofluorescence staining of legumain and fibronectin/ collagen I. In vitro, we cultured monocytes U937 with supernatant from primary DLBCL cells. Real-time PCR and western blot demonstrated that mRNA and protein expression of M2 markers as well as legumain significantly increased in U937 after incubation of 48 hours. Further, we cultured the legumain overexpressed stable cell line in the wells coated with fibronectin or collagen I. Western blot showed that compared to control, legumain overexpression promoted the degradation of fibronectin and collagen I. Mice in human DLBCL xenograft model were randomly received clodronate liposome; PBS liposome or legumain inhibitor via i.v. injection (n=5). The results revealed that tumor growth was significantly delayed in the clodronate liposome group compared with PBS liposome control. Similarly, injection of legumain inhibitor mimicked the effect of clodronate liposome on the tumor progress. Our data suggests that lymphoma-associated macrophages polarized by lymophoma cells promote tumor progress and this effect might be mediated by degrading the matrix via overexpressed legumain.

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 3535. doi:1538-7445.AM2012-3535