bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5545: Functional antagonism between vorinostat and temozolomide when combined with ionizing radiation (IR) in glioblastoma
Authors: Lawrence Yaacov R. , Liu Yi , Andersen Barbara , Xia Xu , Dicker Adam P. , Wachsberger Phyllis
Published: February 2011

Background: Glioblastoma is the most common primary adult brain tumor. Surgery followed by radiation therapy in combination with temozolomide (Tmz) produces a median survival of 14.6 months. Tmz is a DNA akylating agent that leads to the mispairing of guanine residues with thymine. An intact mismatch-repair mechanism (MMR) converts the mispaired thymine into a lethal double-strand DNA break. Vorinostat (SAHA), an HDAC inhibitor, has been shown to act as a radiosensitizer, possibly through inhibition of DNA repair. SAHA has successfully been combined with a number of cytotoxic agents. We hypothesized that SAHA would further potentiate the radiosensitizing properties of Tmz in glioblastoma.

Materials/Methods: Experiments were performed with 2 glioblastoma cell lines (U87, LN18). DNA double strand breaks were quantified by both neutral comet assay and gamma-H2AX immunohistochemistry. Assays were performed immediately, and 24 hours after IR to assess DNA damage and repair respectively. For the neutral comet assay the dose of radiation was 30 Gy and the ‘olive moment’ was scored. The magnitude of the olive moment correlates with increasing DNA damage. For gamma-H2AX assay, the dose of radiation was 2 Gy and the percentage of cells with more than 3 foci was scored. Quantitative PCR and clonogenic cell survival were performed using standard techniques.

Results: In the absence of drug, radiation induced DNA-double strand breaks were almost completely repaired within 24-hours. In U87 both Tmz + IR, and SAHA + IR increased the olive moment compared to IR alone (24 hour post IR time-point). When Tmz and SAHA were combined together with IR, the olive moment decreased to 6.5 as compared to Tmz + IR alone 8.7 (p<0.05). Similarly, in the absence of IR, SAHA was found to decrease Tmz induced DNA damage. Comparable results were obtained with gamma-H2AX assay and with the LN18 cell line. Clonogenic radiation survival assay demonstrated Tmz + SAHA to be no more effective than Tmz alone in potentiating the cytotoxic effect of IR. On qPCR, SAHA was found to down-regulate MLH1, MLH3, MSH2 and MSH 3, key genes involved in MMR.

Conclusions: Both Tmz and SAHA individually potentiated the effect of IR, inhibiting the repair of DNA double strand breaks. However, when cells were exposed to both drugs together less DNA damage was seen than with Tmz alone. Based upon qPCR data we hypothesize that SAHA down regulates the MMR pathway. Our work demonstrates that the addition of an HDAC inhibitor to cytotoxic agents may produce unexpected antagonism. Research sponsored by Merck.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5545.
Abstract 5547: EF24 attenuates radiation-regulated eNOS→NO-dependent NFκB-mediated matrix metalloproteinases in human neuroblastoma
Authors: Madhusoodhanan Rakhesh , Veeraraghavan Jamunarani , Natarajan Mohan , Herman Terence S. , Awasthi Vibhudutta , Aravindan Natarajan
Published: February 2011

We recently reported that radiation (IR) induced NFκB in neuroblastoma cells and treatment with curcumin impedes the activation of NFκB and enhance IR-induced cell death. However, full potential of curcumin is yet to be realized because of its poor bioavailability. This prompted us to investigate the efficacy of a more potent synthetic analogue, EF24 in inhibiting IR-induced NFκB dependent cell survival and tumor progression and delineated the functional mechanism involved. Human SH-SY5Y and IMR-32 cells, either exposed to 2Gy or treated with 100nM EF24 were exposed to IR and examined after 1h, 3, 6, 24, 48 or 72h. IR profoundly inhibited both eNOS phosphorylation and transcriptional activation. Consistently, IR reduced the bioavailability of NO. EMSA analysis showed a robust and persistent NFκB activation at least up to 72h in IR exposed cells. eNOS inhibition (L-NAME) and NO induction (GSNO) studies demonstrated the influence of IR-regulated eNOS and NO in NFκB activation. Furthermore, IR significantly induced transcriptional levels of MMP-1, 2, 3, 7, 9, 11, 13 and 14 in neuroblastoma cells. NFκB knock-out (using RelA siRNA) or forced expression of NF-kB (p50, p65 over expression) exhibits that IR-induced NFκB mediates MMPs activation. Conversely, EF24 treatment significantly reverted IR-negated eNOS activity and enhanced NO availability in the cells. Moreover, EF24 profoundly inhibited IR-induced NFκB and this induced inhibition was sustained at least up to 72h. eNOS inhibition studies with EF24 clearly portraits that EF24 regulates induced NFκB in an eNOS-NO dependent manner. More importantly, EF24 completely suppressed IR-induced MMPs transcript levels in these cells. NFκB over expression studies validates that EF24 regulates IR-induced MMPs by muting IR-activated NFκB. In addition, MTT analysis revealed that EF24 significantly conferred IR-induced inhibition of cell survival in both SH-SY5Y and IMR-32 cells by targeting IR-induced NFκB. Taken together these results suggests that IR-regulated eNOS and reduction in NO availability activates NFκB and NFκB dependent transcriptional activation of MMPs. More importantly, reverting IR-regulated eNOS→NO with EF24 may significantly inhibit NFκB dependent survival advantage and, may serve as a potential deliverable in mitigating neuroblastoma relapse and progression after radiotherapy.

Funding Support: ACS- IRG-05-066-01; Presbyterian Health Foundation

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5547.
Abstract 5560: Biomarker Discovery and Pathway Mapping using Differential Phosphoprofiling of PI3K-Pathway Inhibitors: PRAS40 Correlates with AKT Activation, but not PTEN Expression in Lung and Breast Cancer
Authors: Paweletz Cloud P. , Andersen Jannik , Sathyanarayanan Sriram , Bacco Alessandra Di , Chi An , Zhang Theresa , Albert Chen , Feldman Igor , Kraus Manfred , Lynch Bethany , Rush John , Blume-Jensen Peter , Hendrickson Ronald C.
Published: February 2011

Protein phosphorylation plays a key role in cell signaling and pathophysiological alterations in protein kinases and phosphatases contribute to human diseases. We used stable isotope labeling by amino acids (SILAC) in cell culture combined with phospho-antibody based enrichment for quantitative mass-spectrometry-based identification of differentially phosphorylated proteins in response to small-molecule PI3K-pathway inhibition. We quantified over 500 non-redundant serine/threonine phosphopeptides (NSTPs) containing either the AKT-, MAPK-substrate, or PDK1-docking motif. Of these NSTPs, 71 phosphoproteins were modulated by inhibitors targeting either AKT, PDK1 or PI3K/mTOR and a common set of 11 were modulated by all three drugs. Bioinformatics analysis of the regulated phosphoproteins identified core components of the canonical PI3K pathway and showed enrichment in adaptor and scaffolding molecules involved in cell polarity (PAK4), cytoskeletal reorganization (Filamin, Stathmin, Dynamin) vesicle transport (LARP1, VPS13D, SLC20A1), protein translation (S6RP and PRAS40), and transcription (EIF4BP-1). These results have guided the rational selection of antibodies for biomarker and patient stratification assays and a newly developed phosphor-specific antibody against PRAS40T246 shows specificity for oncogenic PI3K-pathway activation in PTEN-deficient mouse prostate tumor tissue by immunohistochemistry. In addition, the phospho-PRAS40T246 biomarker was evaluated across a panel of 67 breast and 96 lung cancer cell lines and in triple negative human breast tumor tissue. In these datasets, phopspho-PRAS40T246 positively correlates with phospho-AKTS473, but not PTEN protein expression. As such, we have positioned PRAS40T246 as a clinically relevant biomarker for the identification of PI3K-pathway activated tumors to enable individualized cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5560.
Abstract 5567: Pancreatic disease-specific carriers of the CA 19-9 antigen identified by antibody microarrays and mass spectrometry
Authors: Yue Tingting , Hurley Mary C. , Brand Randel E. , Andrews Philip C. , Haab Brian B.
Published: February 2011

Distinguishing patients with malignant pancreatic tumors from those with benign conditions is critical for doctors to make proper treatment decisions. However, this process is usually costly and invasive due to the lack of a simple detection method such as a blood test. The current standard serological biomarker of pancreatic cancer, the CA 19-9 antigen, is not accurate enough for early diagnosis due to its occasional elevation in benign pancreatic diseases. The CA 19-9 antigen is a carbohydrate epitope found on multiple protein carriers. We sought to determine whether the protein carriers of the CA 19-9 antigen are different between benign and malignant pancreatic disease, and whether these differences could be harnessed to develop improved biomarkers. Two approaches were used to investigate this question. In the first, we used antibody microarrays to capture multiple different mucins from the sera of early-stage pancreatic cancer patients (n = 33), late-stage pancreatic cancer patients (n = 17), pancreatitis patients (n = 38), and healthy control subjects (n = 20), and we probed the level of the CA 19-9 antigen on the captured proteins using a monoclonal antibody. Pancreatic cancer progression was associated with a shift in CA 19-9 carriers from MUC16 to MUC1 and MUC5AC. Certain monoclonal antibodies captured glycoforms of MUC1 that predominantly carried CA 19-9 in the cancer patients. Because of that relationship, the detection of CA 19-9 on specific protein glycoforms improved the discrimination of cancer from control subjects over total CA 19-9 (AUC= 0.93 vs. AUC=0.86. In the second approach, we immunoprecipitated CA19-9 carrier proteins from pooled sera of late-stage cancer, early-stage cancer, pancreatitis, and control subjects, followed by deglycosylation and mass spectrometry (MALDI/TOF/TOF). Several proteins were identified in the sera of the cancer patients but not in the sera of pancreatitis or control subjects, indicating the possible identification of state-specific carriers of CA 19-9. Ongoing studies are aimed at verifying these identifications and testing performance as biomarkers of pancreatic cancer. These findings may lead to more accurate methods of discriminating benign from malignant pancreatic disease, which would improve the ability to render proper care to patients at the earliest possible stages.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5567.
Abstract 5581: Antigen-specific CTLA-4-regulated T-cell responses elicited in patients with prostate cancer treated by DNA immunization
Authors: McNeel Douglas G. , Jankowska-Gan Ewa , Burlingham William J.
Published: February 2011

Background: We and others have been interested in plasmid DNA vaccines as a means of eliciting tumor antigen-specific CD4+ and CD8+ T-cell responses. We have investigated plasmid DNA vaccines encoding prostatic acid phosphatase (PAP), a well-characterized prostate tumor antigen, in rodent models and found these to be effective in eliciting antigen-specific CD8+ T cells with evidence of anti-tumor efficacy. Multiple immunizations at high plasmid doses, however, elicited antigen-specific IL-10 secretion. We have recently completed a phase I clinical trial evaluating a DNA vaccine encoding PAP in patients with minimal residual prostate cancer. In that trial, while in vitro PAP-specific T-cell responses were elicited in multiple patients following six biweekly immunizations, others did not have evidence of an immune response, and some responses were not detectable until months later. The current study was conducted to determine whether immunization might have elicited an antigen-specific regulatory T-cell response in some individuals.

Methods: Peripheral blood mononuclear cells obtained from 21 patients prior to immunization, and two weeks after six biweekly immunizations, were evaluated for PAP-specific immune responses in a SCID mouse trans-vivo delayed-type hypersensitivity (DTH) assay. Specifically, DTH responses to PAP in PBMC were evaluated in the presence or absence of antibodies to regulatory molecules (IL-10, TGFß, CTLA-4, PD-1) to “uncover” antigen-specific regulatory mechanisms.

Results: 8/21 individuals had a PAP-specific T-cell response following immunization that was not detectable pre-immunization and which was “uncovered” by CTLA-4 blockade. This response was found to persist in at least one individual for several months. IL-10- and TGFß-regulated responses were not detected. Baseline regulatory PAP-specific T cells could be detected that suppressed concurrent tetanus-specific DTH responses. 6/8 individuals with a detectable CTLA-4-regulated response experienced a favorable increase in PSA doubling time.

Conclusions: Patients with prostate cancer can have PAP-specific regulatory T cells. Plasmid DNA immunization can elicit effector T cells specific for PAP that in many individuals is regulated by CTLA-4. Further studies are underway to characterize the regulatory T cell population(s), however these findings suggest that the efficacy of DNA vaccines might be further augmented by combination with anti-CTLA-4 blockade. In addition, these studies suggest that these trans-vivo DTH methods, in use for evaluating mechanisms of antigen-specific tolerance, could be more generally applied to the immune monitoring of anti-tumor vaccine clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5581.
Abstract 5587: Serum HPV 6, 11, 16 and 18 neutralizing antibody detection by multiplex liquid array system (luminex)
Authors: Bae Heejin , Bae Sumi , Lee Keun Ho , Han Sei Jun , Ahn Woong Shick
Published: February 2011

Recently two prophylactic HPV vaccines including types 6, 11, 16 and 18 are clinically available. Detection of HPV DNA in cervix and neutralizing antibody in serum are important before and after vaccination. A luminex technology facilitates the simultaneous evaluation of multiple immune mediators with advantages of high throughput, small sample volume, lower cost and short time than ELISA. We are already developed HPV DNA in cervix detection system using multiplex liquid array (Cancer science, 2007) and now established detection system using luminex.

For production the antigen, HPV 6, 11, 16 and 18 L1 genes were isolated from their genomic DNA by PCR, cloned into pGEM vector with simian virus 40 (SV 40) large T-antigen and expressed in E. coli. Purified GST fusion recombinant proteins are coupled to microbeads, respectively. The characteristics of the assay were optimized using monoclonal antibody (MAb) specificity, validated with several distinct serum panels and used to determine the serum neutralizing antibody.

Using this system, we tested 40 females who vaccinated quadrivalent prophylactic HPV vaccine 6 month ago (20) and sexually unexposed group in teenager (20). The value of neutralizing antibody was average 1800 (±200) MFI in vaccinated group. This high-throughput multiplex liquid array system is highly sensitive.

We can use population based validation and combination with HPV DNA test using luminex. By using multiplex luminex beads coupled HPV antigen, we can check whether neutralizing antibodies formation or not, and measure the titers of antibodies to each subtype of HPV. We found one possibility to check up the HPV DNA and serum neutralizing antibody before and after HPV prophylactic vaccination.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5587.
Abstract 5592: Lovastatin and low dose celecoxib decrease proliferation and inflammation in a cellular model of lung pre-malignancy
Authors: Grant Jeanette L. , Walser Tonya C. , Yanagawa Jane , Lee Gina , Minna John D. , Prasad Sri , Hazra Saswati , Dubinett Steven M.
Published: February 2011

Statin drugs, such as lovastatin and pravastatin, block the cellular synthesis of cholesterol by inhibiting the function of HMG-CoA reductase. In a prospective study, patients undergoing statin treatment for prevention of atherosclerosis were at a lower risk of developing certain cancers, including lung and breast, suggesting a possible use for lovastatin in the chemoprevention of lung cancer. Here, we show that lovastatin dose-dependently decreases proliferation in both histologically normal and Kras-mutated human bronchial epithelial cells (HBECs) through the inhibition of cholesterol production. However, increased expression of PGE2, which we and others have shown to contribute to lung cancer progression, was noted with lovastatin treatment alone. Lovastatin up-regulates the production of PGE2 by up-regulating COX-2 expression and simultaneously down-regulating metabolism of PGE2 via down-regulation of 15-PGDH expression. Treatment with the cholesterol precursor, mevalonic acid, inhibited up-regulation of PGE2 and blocked dose-dependent anti-proliferative activity of lovastatin, indicating sterol-dependence. Treatment with both lovastatin and low dose (0.1uM) celecoxib, a COX-2 inhibitor, decreased PGE2 protein production in vector control and Kras-mutated HBECs. These findings suggest that the combination of lovastatin and low dose celecoxib overcomes up-regulation of PGE2 by lovastatin alone and that the combination represents a potentially effective lung cancer chemopreventative regimen worthy of further investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5592.
Abstract 5594: Immunotherapy of metastastic renal cell carcinoma reveals a critical role for nitric oxide expression in the control of lung metastases but not primary tumors
Authors: Weiss Jonathan M. , Ridnour Lisa , Hussein Perwez , He Peijun , Back Timothy C. , Scarzello Tony , Maciag Anna E. , Keefer Larry , Murphy William J. , Harris Curtis C. , Wink David A. , Wiltrout Robert H.
Published: February 2011

Using an orthotopic mouse model of renal cell carcinoma, we showed previously that IL-2/αCD40 combination immunotherapy resulted in synergistic anti-tumor responses. In contrast, the use of either IL-2 or αCD40 as single agents mediated only partial, transient anti-tumor effects. We now show that treatment of tumor-bearing mice with IL-2/αCD40, but not either IL-2 or αCD40 alone, induces significant nitric oxide synthase (NOS2) expression within the tumor microenvironment through an interferon gamma-dependent pathway. In saline-control treated mice (low NO levels), NO inhibition reduced lung metastases by itself. However, in the context of immunotherapy (high NO levels), NOS2 inhibition completely abrogated the ability of IL-2/αCD40 treatment to reduce lung metastases but had no effect on the ability of IL-2/αCD40 to reduce primary kidney tumor burden. We further show that IL-2/αCD40 induces the NO-dependent decrease in matrix metalloproteinase (MMP) expression and activity, concomitant with an increase in the expression of tissue-inhibitor of metalloproteinase (TIMP)-1 within the tumor microenvironment. Finally, the treatment of tumor-bearing mice with the NO donor, JS-K significantly reduced lung metastases but had no effect on primary tumor size. These data distinguish the primary anti-tumor effects of IL-2/αCD40 immunotherapy, which are independent of NO expression, from the important ability of IL-2/αCD40 to inhibit lung metastases through the NO-dependent regulation of MMP and TIMP functions. Furthermore, the reduced MMP9 activity may be indicative of M1 polarized macrophages within the tumor microenvironment after IL-2/αCD40 treatment. Our data demonstrate the mechanistic basis for the IL-2/αCD40-mediated control of lung metastases and indicate that the context-dependent application of NO-inducing agents may hold considerable promise for the treatment of metastastic disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5594.
Abstract 560: Mesenchymal stem cells promote mammosphere formation in normal and malignant breast cells
Authors: Klopp Ann H. , Lacerda Lara , Gupta Anshul , Debeb Bisrat , Marini Frank , Andreeff Michael , Woodward Wendy A
Published: February 2011

Purpose: Normal and malignant breast tissue contains a rare population of cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC) in the case of tumors. These cells can be enriched by growth as “mammospheres” in three-dimensional cultures. We tested the hypothesis that mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.

Materials and Methods: Normal human mammary epithelial cells (HMEC), and breast cancer cells (MCF-7, SUM149 and IBC3) were grown as mammospheres, plated at low density in serum-free growth factor supplemented suspension cultures. MSC were added as an increasing percentage of total cells plated (0, 2, 5 and 10%). MSC-conditioned media was generated by growing MSC as spheroids for 5 days.

Results: MSC integrated into mammospheres, resulting in a dose-dependent increase in the number of mammospheres formed. Normal mammary epithelial cells formed 1.6 to 2.4 to 2.7-fold (p< 0.001) more mammospheres in the presence of 2, 5 and 10% MSC. Similarly, the number of MCF-7 derived mammospheres increased 5.9 to 8.5 to14.8-fold (p = ≤ 0.001) in the presence of 2, 5 and 10% MSC. A similar dose-dependent increase in sphere formation was seen for SUM149 cells and inflammatory breast cancer cells in short-term culture derived from pleural effusions. This effect was found to be mediated by secreted factors as MSC-conditioned media also increased sphere formation. SUM149 cells formed 6.4 to 14.2 to 13.7-fold more mammospheres when incubated for 5 days in 5, 25 and 50% MSC conditioned-media (p <0.0001). HMEC derived mammospheres had higher levels of expression of N-cadherin and RhoC and lower levels of expression of E-cadherin and Notch when formed in the presence of MSC conditioned media.

Conclusions: Mesenchymal stem cells increase mammosphere formation of normal breast epithelial cells, established cancer cell lines (MCF-7 and SUM149) and short term primary breast cancer cells (IBC3). This effect is mediated by MSC secreted factors and results in cadherin switching, which may promote breast cancer progression and resistance to therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 560.
Abstract 5602: MSC-mediated intratumoral cytokine delivery augments anti-tumor immunotherapy of glioma
Authors: Gunnarsson Salina , Svensson Andreas , Siesjö Peter , Darabi Anna , Bengzon Johan
Published: February 2011

Glioblastoma multiforme is the most common and aggressive form of primary brain tumor. In spite of surgical resection, combined radiation therapy and chemotherapy, the mean survival is less than 2 years following diagnosis. Using the established N32 animal model of glioma, shown to have many similarities with the human tumor, we are able to study and develop gene therapy strategies based on multipotent mesenchymal stromal cells (MSCs) as an efficient vector system.

MSCs derived from adult rat bone marrow have been shown to selectively target tumors when inoculated intracranially and these cells can be used as cellular vector systems for gene therapy of the tumor. Our group has previously shown that the cells, when transplanted into an established tumor, migrate within the tumor as well as along infiltrating tumor arms and to distant micro-satellites. We have transformed these rat MSCs to produce the cytokine interleukin 7 (IL-7), a growth factor involved in the development of immune cells. When IL-7 secreting MSCs are injected intratumorally following peripheral immunizations with IFNγ-secreting autologous irradiated tumor cells, a synergistic effect on anti tumor immunity is noticed. Tumor size is significantly smaller in treated animals compared to control as recently reported by our group. These studies have now been extended with survival experiments which show that there is an increased survival in the group receiving MSC-IL7 + IFNγ-immunization compared to IFNγ-immunization alone, 45% and 21% (animals alive) respectively at day 120 post tumor inoculation. In addition, MSCs transduced to express IFNγ in combination with peripheral IFNγ-immunization result in a 40% survival in the same study.

The use of MSCs from adult bone marrow has several advantages, it can be retrieved relatively easy and the patient self can donate it. Combining immunization with tumor cells together with MSC-delivered cytokines intratumorally might be further developed into a clinically useful treatment strategy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5602.
Abstract 5605: Targeting cancer stem cells in squamous cell carcinoma of the head and neck with combinatorial adoptive immunotherapy
Authors: Visús Carmen , Wang YangYang , Szczepanski Miroslaw J. , Ferris Robert L. , Whiteside Theresa L. , Ferrone Soldano , Wang Xinhui , DeLeo Albert B.
Published: February 2011

According to current views, in order for cancer immunotherapy to be successful it must target cancer stem cells (CSC) and counteract the multiple mechanisms involved in immune escape. Guided by these concepts, we have developed a combinatorial adoptive immunotherapeutic strategy which utilizes a monoclonal antibody (mAb) and cytotoxic T lymphocytes (CTL) in combination with continuous administration of low dose cyclophosphamide (metronomic chemotherapy) to target CSC and differentiated tumor cells, as well as activated pericytes in the tumor microenvironment. The targets selected are aldehyde dehydrogenase 1 family member A1 (ALDH1A1) and chondroitin sulfate proteoglycan 4 (CSPG4). We now report that ALDHbright cells in squamous cell carcinoma of the head and neck (SCCHN) cell lines, which express high levels of ALDH1A1, are tumorigenic at low inoculum in immunodeficient mice, and particularly sensitive to recognition by ALDH1A1-specific T cells. The ALDHbright cells express CSPG4, a cell surface antigen functional in adhesion and migration. CSPG4 is also expressed on tumor associated activated pericytes which stabilize tumor blood vessels and provide vascular endothelial cells with survival signals. Administration of CSPG4-specific mAb and metronomic chemotherapy combined with intralesional adoptive transfer of ALDH1A1 peptide-specific CTL to mice bearing human SCCHN PCI-13-derived xenografts significantly reduced tumor growth and number of tumor cells with CSC phenotype. The antitumor effects of the immunotherapy we have developed were significantly higher than those of its individual components. These results argue in favor of the possibility that this combinatorial strategy may enhance the clinical efficacy of adoptive immunotherapy in SCCHN patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5605.
Abstract 5613: Beta-mannosylceramide: a novel NKT cell agonist which synergizes with alpha-galactocylceramide to induce protection against tumors
Authors: O'Konek Jessica J. , Illarionov Petr , Khursigara Deborah Stewart , Ambrosino Elena , Castillo Bernard F. , Raju Ravinder , Khalili Maryam , Kim Hee-Yong , Howell Amy R. , Besra Gurdyal S. , Porcelli Steven A. , Berzofsky Jay A. , Terabe Masaki
Published: February 2011

Type I natural killer T (NKT) cells are a unique subset of T cells which express phenotypic and functional characteristics of both NK cells and T cells. NKT cells recognize glycolipid antigens presented by the non-classical MHC-like molecule CD1d. Upon stimulation with the prototypical type I NKT cell antigen α-galactosylceramide (α-GalCer), type I NKT cells promote tumor immunity through IFN-γ-dependent mechanims. In this study the ability of glycosylceramides with alterations of the sugar moiety to induce anti-tumor immune responses in an in vivo model of CT26 colon carcinoma metastasis to lungs of BALB/c mice was investigated. β-mannosylceramide (β-ManCer) induced strong tumor protection, while α-mannosylceramide and α-fucosylceramide did not. This is the first evidence of a glycosylceramide with a β-linked sugar moiety capable of inducing significant NKT cell-dependent anti-tumor immunity. We also confirmed that β-ManCer could protect at least as well as α-GalCer in a model of B16F10 melanoma lung metastasis in C57BL/6 mice. Interestingly, β-ManCer induced little cytokine production and retained protective activity in IFN-γ KO mice, unlike α-GalCer analogs which where completely dependent upon IFN-γ. The protection did not require CD8+ T cells, and was largely independent of NK cells. Blockade of nitric oxide synthase or TNF-α significantly reversed the β-ManCer-induced protection but had no effect on tumor protection for the α-GalCer analogs. Thus, contrary to α-GalCer, β-ManCer protects through a novel TNF-α and NOS-dependent, IFN-γ-independent mechanism. Consistent with the conclusion that β-ManCer and α-GalCer induce tumor immunity by distinct mechanisms, simultaneous administration of sub-therapeutic doses of these glycolipids resulted in synergistic reduction in the number of lung metastases. The distinct mechanism of action and ability to synergize with α-GalCer may suggest that β-ManCer represents a new class of NKT agonists with novel clinical applications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5613.
Abstract 5614: Interleukin-10 inhibits the induction of macrophage cytotoxicity against bladder cancer cells by Mycobacterium bovis bacillus Calmette-Guérin (BCG)
Authors: Luo Yi , Han Ruifa , Evanoff David P. , Chen Xiaohong
Published: February 2011

Intravesical BCG therapy has been used for the treatment of superficial bladder cancer for three decades. However, the mechanisms underlying BCG therapy currently remain elusive. We have focused on BCG induction of macrophage cytotoxicity against bladder cancer cells. Previously, we observed that macrophages are cytotoxic to bladder cancer cells upon stimulation with BCG in vitro. However, we found that macrophages from C57BL/6 mice are less potent than those from C3H/HeN mice for the killing of both bladder cancer MBT-2 cells and MB49 cells. This study was to determine whether macrophage-derived interleukin (IL)-10 is responsible for this discrepancy between these two commonly used murine bladder cancer models. Thioglycollate-elicited peritoneal exudate cells were harvested from mice 3 days after intraperitoneal injection of 2 ml 3% thioglycollate. Cells were incubated for 3 hours and non-adherent cells were removed after incubation. The remaining cells were 96% F4/80-positive cells as determined by flow cytometry. The prepared macrophages were then used for the analysis of BCG induction of cytotoxicity, cytokines, and nitric oxide (NO) in vitro. Studies demonstrated that BCG-stimulated C57BL/6 macrophages produced a substantially high level of IL-10 compared to BCG-stimulated C3H/HeN macrophages. This high IL-10 production correlated with reduced production of tumor necrosis factor (TNF)-α, IL-6 and NO in BCG-stimulated C57BL/6 macrophages. Neutralizing endogenous IL-10 during BCG stimulation increased C57BL/6 macrophage cytotoxicity against MB49 cells by 3.2-fold, along with increased production of TNF-α by 6.4-fold and NO by 3.6-fold, respectively. Macrophages from genetically modified C57BL/6 mice lacking IL-10 (IL-10−/−) also exhibited increased killing of MB49 cells and production of TNF-α and NO upon BCG stimulation. In addition, supplementation of exogenous recombinant IL-10 reduced BCG-induced C3H/HeN macrophage cytotoxicity against both MBT-2 cells and MB49 cells in a dose-dependent manner. Taken together, our results reveal the inhibitory role of IL-10 in BCG-induced macrophage cytotoxicity, suggesting that blockage of IL-10 may potentially enhance the effect of BCG in the treatment of bladder cancer patients, particularly for BCG non-responders who are often associated with high IL-10 production in response to BCG treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5614.
Abstract 5619: Natural killer cell function in multiple myeloma is impaired by dexamethasone and can not be rescued by immunomodulatory drugs
Authors: Hsu Andy , Quach Hang , Tai Tsin , Prince H M. , Smyth Mark J. , Trapani Joseph A. , Neeson Paul J. , Ritchie David S.
Published: February 2011

The immunomodulatory (IMiD) drug Lenalidomide (Len) is known to stimulate natural killer (NK) cells and enhance anti-tumor responses. Len plus Dexamethasone (Dex) therapy (Len-Dex) is an effective treatment for multiple myeloma (MM). As part of a prospective clinical trial of monthly cycles of Len-Dex therapy we assessed NK cell number and function in 25 MM patients. Whilst NK numbers increased from a mean of 2×105/ml (baseline) to a mean of 3.9×105/ml (cycle 6) (p=0.05), the mean NK cell-mediated cytotoxicity in chromium assays against K562 significantly decreased from 48.9% to 27.6% respectively (p=0.0028). Further analysis showed that when NK cell cytotoxicity was assessed from MM patient peripheral blood mononuclear cells (PBMC) during therapy, no enhancement of cytotoxicity could be observed when cultured in vitro with Len. We hypothesized that Dex potently suppressed the Len-mediated enhancement of NK function. In vitro, Len increased normal donor (n=6) NK cytotoxicity from 38.5% (untreated) to 53.3%. Dex severely impaired NK cytotoxicity to <10%, this failed to increase (<13%) in the presence of Len. Only the addition of high dose IL-2 (500U/ml) rescued cytotoxicity of Dex-treated NK cells (28.6%). The suppressive effect of Dex on NK cytotoxicity was not permanent as NK function returned to normal levels within 3 days of drug removal. However, these dex-‘washout’ PBMCs could no longer respond to Len with increased NK function, suggesting a permanent Dex-induced NK impairment. In cell sorting and co-culture experiments we found that Len enhancement of NK cytotoxicity was only mediated by CD4+ T cells (NK alone 76.6% vs NK+CD4 85%) and the effect of CD4+ T cells was IL-2 dependent. Furthermore IL-2 production by CD4+ T cells in response to anti-CD3/CD28 was also suppressed by Dex. Following Len-Dex therapy in patients we observed less IL-2 and IFN-γ production from T cells in response to anti-CD3/CD28 at cycle 6 compared to baseline.

Therefore, Len-mediates enhanced NK function through CD4+ T cell production of IL-2. The NK impairment observed in MM patients receiving Len-Dex is due to CD4+ T cell suppression. Our findings indicate that in order to harness both the anti-tumor and immunomodulatory capacity of Len the dose and timing of Dex therapy should be carefully reconsidered.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5619.
Abstract 5625: In vitro and in vivo pharmacology of MEDI-565 (MT111), a novel CEA/CD3-bispecific single-chain BiTE antibody in development for the treatment of gastrointestinal adenocarcinomas
Authors: Fuhrmann Stacy R. , Amann Maria , Mulgrew Kathy , Lorenczewski Grit , Baffa Raffaele , Wolf Andreas , Oberst Michael , Rattel Benno , Lutterbuese Petra , Baeuerle Patrick , Jallal Bahija , Trail Pamela , Richman Laura , Coats Steven , Hammond Scott A.
Published: February 2011

MEDI-565 (MT111) is a novel bispecific single-chain antibody of the BiTE (Bispecific T cell engager) class that transiently links carcinoembryonic antigen (CEA) on cancer cells with human CD3 on T cells. MEDI-565 specifically binds to human CEA with an affinity of 8.5 nM but not to any other member of the CEACAM family. In targeted expression studies on cryopreserved and formalin-fixed paraffin-embedded sections, MEDI-565 demonstrated membrane-localized binding specifically to epithelial cells in human cancerous tissues. The highest prevalence of MEDI-565 binding (∼90%) was in adenocarcinomas of gastrointestinal origin.

In in vitro killing assays, MEDI-565 recruited T cells via the CD3 antigen (affinity of 310 nM) in a process that required concomitant binding to CEA positive tumor cells for T cell activation. As a consequence, T cells expanded, increased cell surface expression of the activation markers CD69 and CD25, and released perforin and granzymes. The release of cytotoxic granule content led to a Ca2+-dependent activation of pro-caspases and subsequent apoptosis of CEA-expressing tumor cells. Efficient target cell lysis by MEDI-565 was predominantly mediated by CD8+ T cells and occurred within a wide range of effector-to-target ratios (80:1 to 5:1) for T cells derived from various human donors. BiTE mediated killing was not affected by the presence of soluble CEA (≤5 µg/mL).

The in vivo activity of MEDI-565 was investigated in subcutaneous xenograft models using immunocompromised SCID mice inoculated with mixtures of human T cells and human cancer cell lines. The antibody was eliminated from the serum with a half-life of a few hours following a single intravenous (IV) or subcutaneous (SC) injection. Despite a relatively short serum half-life, daily IV or SC bolus treatments over 5 days provided sufficient levels of exposure to inhibit the growth of CEA-positive tumors in a dose-dependent manner. Inhibition of growth was observed for tumors of different tissue origins and was dependent on the presence of human T cells and CEA expression by tumor cells. There were no MEDI-565-related in-life observations following treatment. These studies demonstrated that MEDI-565 has potent and selective anti-cancer activity in vitro and in vivo and provides evidence that MEDI-565 may be an effective monotherapy to treat CEA-expressing malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5625.
Abstract 5633: Two-compartment pharmacokinetics model of radioimmunotherapy with 131I-3F8 directly administered into the cerebrospinal fluid
Authors: He Ping , Kramer Kim , Smith-Jones Peter , Zanzonico Pat , Humm John , Larson Steven M. , Cheung Nai-Kong V.
Published: February 2011

Introduction

Injection of 131I-3F8 directly into the cerebrospinal fluid (CSF) through an Ommaya reservoir is a safe form of radioimmunotherapy (RIT) treatment for leptomeningeal metastasis (JCO, 25:5465, 2007). Recently, we described a one-compartment pharmacokinetics model that showed good fitting to CSF sampling data from 6 patients (JNM 50:1324, 2009). We now describe an improved two-compartment model to take into account the ventricular reservoir before 131I-3F8 distribution into the subarachnoid space and to explore limiting factors that may significantly affect the efficacy of the therapy.

Methods

The distribution and binding of 131I-3F8 in the cerebral spinal fluid were modeled using a system of rate equations derived from the principle of mass conservation. The model was fitted to CSF sampling data from an expanded cohort of patients (39 injections in 25 patients). Three sets of criteria were used to examine the effect of each parameter on the efficacy of the treatment. These criteria were (1) the area under the radioactivity concentration curve of the bound antibody (AUC[CIAR]) (2) the unbound antibody AUC[CIA], and (3) their therapeutic ratio (AUC[CIAR]/AUC[CIA]). In addition to parameters tested in the previous model, immunoreactivity, tumor distribution, and nonspecific binding were also tested.

Results

The two-compartment model showed improved fitting to patient data when compared to the one compartment model (R=0.92±0.11 versus 0.77±0.21, p=0.005). Predictions from the one compartment model were confirmed. Furthermore, we made these new observations: (1) Increasing immunoreactivity of 131I-3F8 from 10% to 90% increased both (AUC[CIAR]) and therapeutic ratio ([AUC[CIAR]/AUC[CIA]) by 7.4 fold, (2) The presence of ≤ 5% tumor cells in the ventricles or ≤ 10% of nonspecific 131I-3F8 binding to normal tissues had no appreciable effect on the therapeutic ratio. When extrapolated to a clinical setting, the model predicted that if 131I-3F8 could be split into 4 doses of 1.4 mg each and given at ≥ 24 hours apart, an antibody affinity of KD of 4 × 10−9 and immunoreactivity of 50% were adequate in order to deliver ≥10,000 Rad to tumor cells while keeping normal CSF exposure to <1,000 Rad.

Conclusion

The two compartment model of intra-ommaya radioimmunotherapy showed better fitting to patient data than the one-compartment model. This model predicted that immunoreactivity and affinity were crucial in improving therapeutic efficacy. Nevertheless, an optimal scheduling of antibody injections should permit highly favorable therapeutic ratio even with 4 nM affinities and suboptimal immunoreactivity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5633.
Abstract 564: A >50KDa soluble factor from the stroma-derived conditioned media contributes to resistance towards BCR-ABL inhibitors in CML via the JAK-STAT3 pathway
Authors: Nair Rajesh R. , Tolentino Joel H. , Argilagos Raul F. , Pinilla-Ibarz Javier , Hazlehurst Lori A.
Published: February 2011

Soluble factors and extracellular matrices from the bone marrow stroma, provides the essential microenvironment that contributes to the persistence of minimal residual disease found in patients with Chronic Myeloid Leukemia (CML), under treatment with tyrosine kinase inhibitors. We previously showed that in CML cells, bone marrow stroma-derived conditioned medium (CM) is sufficient to cause resistance to BCR-ABL inhibitors in a STAT3-dependent manner. In this study, we show that development of resistance in CM correlated with increased STAT3 phosphorylation and increased STAT3 DNA binding in CML cells. The activation of Stat3 in the context of CM was persistent even in the presence of BCR-ABL inhibitors. Moreover, the increase in DNA binding resulted in increased transcriptional activity of STAT3 and increased expression of MCL-1, Bcl-xl and Cyclin D1 in cells exposed to CM. We also found that CM-mediated increase in STAT3 activation requires JAK activity, since use of a pan JAK inhibitor sensitized the cells to BCR-ABL inhibitor in CM. Surprisingly, neutralizing the activity of IL-6, G-CSF and VEGF in the CM did not reverse the protection afforded by CM against BCL-ABL inhibitors. Furthermore, utilizing size exclusion filter columns, we were able to show that CM retentates that were higher than 50 kDa, was sufficient to duplicate the protective effects of CM against BCR-ABL inhibitor. Finally, we could show that CD34+ cells from BCR-ABL patients when exposed to CM showed an increase in STAT3 phosphorylation and this correlated with resistance to Nilotinib-mediated cell death. In conclusion, a greater than 50 KDa factor in CM leads to JAK-STAT3 activation, which is necessary and sufficient to confer resistance to CML cells against BCR-ABL inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 564.
Abstract 5642: Investigation of effective combination of anti-cancer drugs and radiation therapy against non-small cell lung cancer using NCI-H520 cell line
Authors: Oh In-Jae , Ban Hee-Jung , Kwon Yong-Soo , Kim Kyu-Sik , Lim Sung-Chul , Kim Young-Chul , Song Sang-Yun , Na Kook-Joo , Yoon Mee-Sun , Ahn Sung-Ja , Choi Song , Kim Yun-Hyeon , Choi Yoo-Duk , Choi Chan
Published: February 2011

Background: Radiotherapy (RT) is used for to treat unresectable locally advanced non-small cell lung cancer (NSCLC). For better disease control, RT has recently been combined with chemotherapy (CTx) in a variety of ways. This study examined the most effective combination methods of CTX and RT against NSCLC.

Material and Methods: Human lung cancer cell line (NCI-H520) and various chemotherapeutic agents (paclitaxel, docetaxel, gemcitabine, and cisplatin) were used in this study. The radiation doses used were 0, 2, 4 and 8 Gy. After processing of various combinations according to the radiation dose and concentrations of chemotherapeutic agents, the level of cell survival was quantified by a MTT assay. The combination index was calculated using the Chou and Talalay method on Calcusyn software and used to examine the synergism between CTx and RT.

Results: Among the various combinations of chemotherapeutic agents and radiation doses, concurrent chemoradiation therapy (CCRT) led to the highest rate of apoptosis and synergism. CTx followed by RT was the most effective combination when taxane was administrated. When high-dose chemotherapeutic agents were added to CCRT, induction CTx resulted in higher rate of apoptosis rate and synergism than consolidation CTx.

Conclusions: CCRT is the most effective combination when RT is combined with CTx in the NCI-H520 cell line. Induction CTx is more effective than consolidation CTx if a high-dose chemotherapeutic agent is added to CCRT.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5642.