bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4763: Administration of a vaccine composed of dendritic cells pulsed with premalignant oral lesion lysate: immunologic effects in mice bearing carcinogen-induced premalignant oral lesions
Authors: Clark Anna-Maria A. , Young M R.
Published: February 2011

Most studies with dendritic cell vaccines have focused on eliminating or preventing the progression of already established malignancies. This is complicated by the immune suppression that is often exhibited in the presence of solid carcinomas. The goal of this study is to determine if an immune response can be elicited by administering a dendritic cell vaccine during the premalignant stages of oral squamous cell carcinoma (OSCC), prior to the development of systemic immune suppression. Mice treated with the carcinogen 4-nitroquinoline-1-oxide (4NQO) in drinking water develop premalignant oral lesions which subsequently progress to OSCC. As previous studies in our lab demonstrated that premalignant lesions and OSCC share overexpression of common tumor antigens, bone-marrow derived dendritic cells were pulsed with premalignant lesion lysate and administered to 4NQO-treated C57/BL6 mice exhibiting premalignant lesions. Immunohistochemical analysis demonstrated a similar increase in the number of CD8+ and CD4+ cells in the tongues of both the 4NQO-treated mice immunized with premalignant lesion-pulsed dendritic cells (DCpm) and the 4NQO-treated mice immunized with keratinocyte-pulsed dendritic cells (DCker) compared to 4NQO-treated mice injected with saline. However, the tongues of 4NQO-treated DCpm mice exhibited a dramatic decrease in the number of Foxp3+ cells compared to both 4NQO-treated control groups. In addition, splenocytes from 4NQO-treated DCpm mice released greater amounts of both IFN-γ and IL-2 in response to challenge with premalignant lysate compared to splenocytes from 4NQO-treated DCker mice in response to challenge with keratinocyte lysate. These studies indicate that stimulated dendritic cells, regardless of pulsing with premalignant lesions or keratinocytes, may be able to elicit an upregulation of T cells in the tongue. However, DCpm immunization specifically appears to result in a decrease in immune inhibitory regulatory T cells in the tongue and an increase in splenocyte secretion of immune stimulatory cytokines in response to antigenic challenge.

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 4763.
Abstract 4764: Vaccination of tumor-bearing mice with irradiated tumor cells and depot-formulated IL-2, IL-12, IL-4, GM-CSF and IFN-a eradicates the tumor cells and induces lasting immunity to the tumor
Authors: Falkenberg Frank W. , Pendzialek Dirk
Published: February 2011

In order to overcome the complex tumor-host interactions equally complex biological countermeasures have to be taken. By applying several cytokines in the vaccine the cytokine network is activated more efficiently then by using a single cytokine. To render the approach mote feasible, the cytokines were applied in depot Form. This allows formulation following pharmaceutical rules, and, at the same time, leads to steady release and keeps the inflammatory process active over a long period of time. Applying this concept, vaccines were designed that contained, besides irradiated tumor cells, a bouquet of cytokines in depot formulation. Due to their different biological activities IL-2, IL-4, IL-12, GM-CSF and IFNα were chosen.

The sequential steps of vaccine action:

The release of a multiplicity of cytokines accelerates the influx of inflammatory cells of the innate immune system.

Under the influence of IL-4. GM-CSF and IL-12 released from the depot, Dendritic cells are recruited, maturated and activated under physiological conditions in situ, at the inoculation site, circumventing tedious ex vivo procedures.

Tumor cell fragments generated by cells of the innate immune system are taken up by the locally generated CD86+ mature Dendritic Cells and transported to the lymph nodes where they are presented to T cells. In addition, cytokine-loaded depot particles are carried to the lymph nodes where they become involved in activation processes that are not understood yet.

Due to its angiogenic activity, depot-released GM-CSF induces the sprouting of capillaries into the inoculum, thus opening the way for recirculation of activated CD4+ and CD8+ T cells into the inoculum.

Depot-released IL-2 and IFNα further activate cells of the NK/NK-T cell lineage.

Applying Multi-Cytokine-Depot Tumor Vaccines, we have been able to cure mice from tumors induced by injection of a lethal dose of vital tumor cells.

Although the set-up of the vaccine is complex, its preparation is easy and relies on formulation following pharmaceutical principles rather than on trial and error.

For each cytokine as well as for the tumor cells a specific Dose-Response-relations has been determined. We are currently preparing for the application of this concept in clinical trials.

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 4764.
Abstract 4767: Neo-peptides from gene fusions as cancer vaccine antigens
Authors: Lee Hojoon , Rodenberry John-Charles , Day Kristen , Cano-Buendia Jose , Craciunescu Felicia , Chapuis Jean-Charles , Stafford Phillip , Sykes Kathryn , Carrigan Patricia E. , Johnston Stephen A.
Published: February 2011

Background: Of all possible sources of antigens for cancer vaccines, frameshift (FS) peptides are attractive in that they may represent neo-antigens. FS peptides may be induced by insertions/deletions or by chimeric transcripts that result from mis-splicing, chromosomal translocations or trans-splicing of mRNA. Aim: In order to discover FS neo-antigens, we have taken a bioinformatical approach at identifying tumor specific, neo-peptides that are generated when chimeric transcripts are translated. Specifically we have focused our attention on identifying chimeric transcripts that create a frameshift when the exons of two different genes are joined. As a result of this gene fusion, approximately 50% of the time a frameshift occurs thus creating a neo-peptide. Method: In order to quickly identify these types of transcripts that are specific to cancer and not present in normal tissue, we have developed an algorithm that can be used to screen publically available databases and high-throughput sequencing data. As proof of principle we have applied this algorithm to the NCBI Expressed Sequence Tag database that contains cDNA sequencing information for normal and cancer tissues. Results: Based on this approach, we have identified and validated 35 chimeric transcripts that are present in breast cancer cell lines and in primary breast tumors. In addition, 9 chimeric transcripts are also present in seven different pancreatic cancer cell lines. Of the 35 chimeric transcripts, 11 chimeric transcripts produce frameshift peptides that are at least nine amino acids or longer and are predicted by SYFPEITHI and Rankpep to be immunogenic and capable of being presented in the most frequent human class I MHC alleles. Moreover, our analyses also identified chimeric transcripts that have already been confirmed by 454 sequencing for breast and prostate cancer. Discussion: Defining the chimeric transcriptome in both healthy and cancer patients could be critical for the development of new therapeutic agents. Of more interest to us, is their potential contribution to a prophylactic cancer vaccine. (This work is supported by a DoD Innovator Award and a Keck Foundation grant to SAJ)

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 4767.
Abstract 4768: Investigation of frameshift peptides for cancer vaccines
Authors: Shen Luhui , Cano-Buendia Jose , Lee Hojoon , Bui Chu , Sykes Kathryn , Carrigan Patricia E. , Johnston Stephen A.
Published: February 2011

Background: Our overall goal is to develop a general, preventative cancer vaccine. We are systematically applying bioinformatics, proteomics and immunological methods to achieve this vision. One of our foci is the identification of framshifted (FS) neo-peptides in tumor cells as vaccine components. FS peptides are expressed in alternate reading frames which are activated by various gene mutations and abnormal splicings in tumor cells. Aim/Method: Our hypothesis is that FS peptides are recognized as foreign antigens to the immune system thus making them potential cancer vaccine candidates. We have identified a list of FS peptides by bioinformatic analysis using the publically available human tumor and normal EST databases. To set up a systematical approach of prophylactic cancer vaccine development, we evaluated one potential FS peptide on the list as cancer vaccine candidate which is produced by a splice variant in SMC1A (Structural Maintenance of Chromosomes 1A). Results: The SMC1A FS peptide is a 17 amino acid peptide which is encoded by an abnormal alternative splicing event between exon 1 and exon 4. It was detected in mouse tumor cell lines including melanoma (n=2) and a breast tumor cell lines. It was also detected in human tumor cDNAs assayed thus far including breast (n=34), pancreas (n=4), and lung cancer (n=1) cell lines and primary breast (n=7) and pancreatic tumors (n=4). The expression levels of the SMC1 FS variant increases in most primary tumors with ranges from 2 to 70 fold. Specifically we see an increased expression in 6/7 breast and 2/4 pancreatic tumors screened. Single genetic immunization with plasmids expressing SMC1 FS, mouse GM-CSF, and CpG2216 significantly delayed tumor growth in C57BL/6 mice transplanted with the B16F10 melanoma cell line. Both antibody and IFN-γ released splenocytes to SMC1 FS were detected in immunized mice. Discussion: These findings suggest that the SMC1 FS peptide can be a potential cancer vaccine candidate to prevent various tumors. We are applying this same basic approach to accumulate sufficient antigens to constitute a broadly protective, prophylactic cancer vaccine. (This research is supported by an Innovation Award from the DoD and The Keck Foundation to SAJ.)

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 4768.
Abstract 4771: Molecular, epigenetic, and phenotypic repolarization of human T lymphocytes using 5-aza-2′-deoxycytidine increases CD8 responsiveness and induces Th1 polarity
Authors: Dubovsky Jason A. , Powers John J. , Gao Yang , Pinilla-Ibarz Javier A. , Sotomayor Eduardo M.
Published: February 2011

The improper polarization of helper and cytotoxic T lymphocytes is a trademark immunosuppressive strategy employed by a litany of human malignancies. Mounting data implicates chromatin regulation, namely promoter methylation, in the plasticity of naïve human T cells. Very recent in-vitro evidence indicates that this plasticity may be phenotypically replicated by using methylation inhibitors which are approved for clinical use in certain types of cancer, however the longevity and therapeutic potential of repolarized cells remains to be elucidated. These results beg the question: can the ineffective polarization of T lymphocytes in the context of cancer be effectively modulated using methylation inhibitors in a sustainable therapeutic fashion? To answer this question our laboratory has focused on repolarizing helper and cytotoxic T lymphocytes using 5-aza-2′-deoxycytidine (5A2) tracking changes in well characterized molecular and epigenetic signaling pathways involved in effective polarization. Moreover, we sought to investigate the consequences of methylation inhibitor treatment on lymphocyte survival, activation intensity, and naïve cell polarization. Our studies relied upon intracellular staining, methylation specific PCR, and phosflow anlaysis. The data indicates that 5A2 treatment can repolarize Th2 cells to effectively secrete interferon gamma, signal via STAT4, and achieve demethylation of critical Th1 specific promoters. We also show that this repolarization can be maintained. Similar increases in cytotoxic signaling capability were obtained with CD8+ T cells after treatment with 5A2. Moreover, we demonstrate that 5A2 can force Th1 polarization of naïve T cells despite a strong IL-4 stimuli and a lack of IL-12. Finally, we have identified a cytotoxic threshold for 5A2 treatment which is above the concentration necessary to achieve effective and sustained molecular and epigenetic repolarization. All in all our data seeks to define a modality in which improper or ineffective T cell polarization can be altered ex post facto and continued immunotherapeutic intervention may benefit from the naïve polarization of anti-cancer responses.

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 4771.
Abstract 4776: Properties of allogeneic WT1126–134-specific CD8+ T cells generated across an HLA A2 barrier
Authors: Melenhorst Jan J. , Falkenburg Willem J.J. , Gostick Emma , Quigley Máire F. , Hensel Nancy F. , Douek Daniel C. , Price David A. , Barrett Austin J.
Published: February 2011

T cells recognizing self-antigens overexpressed in malignant cells such as the Wilms tumor 1 protein (WT1) may contribute to anti-tumor reactivity after both allogeneic and autologous stem cell transplantation. HLA A*0201-restricted WT1-reactive CD8+ T cells recognizing peptide-loaded HLA-A2+ target cells have been isolated from both HLA-A2 negative and positive donors. HLA-A2-restricted WT1-specific CD8+ T cells generated from HLA-A2 positive donors are likely to be specific for the WT1 peptide, but may show only low reactivity against targets presenting endogenous WT1 antigen due to thymic deletion of high avidity T cells. In contrast, HLA-A2-restricted WT1-specific T cells generated from HLA-A2 negative donors may be highly reactive against these targets, but with increased risk for cross-reactivity. To explore the possibility of generating highly WT1-reactive CTL across an HLA barrier, EBV-transformed lymphoblastic B cell lines (EBV-LCL) were transduced with a retroviral HLA-A2 construct, loaded with WT1126–134, and used to stimulate autologous CD8-selected T cells. Several T cell clones and lines were generated and sorted with wildtype WT1 tetramer. All T cell clones and lines selected for functional analysis stained brightly with WT1/A2 tetramers, but not with irrelevant HLA-A2 control tetramers (PR1, PRAME300–309, CMVpp65495–503), indicating specificity for the WT1 peptide presented in HLA-A2. Four lines screened against a panel of HLA-A2+ and HLA-A2- EBV-LCL recognized HLA -A2+ and one HLA-A2-, HLA-B57+ LCL. Autologous PHA blasts were not recognized, indicating specificity for EBV-derived antigens presented in the context of both HLA-A2 and -B57. Of five clones and one line tested only two showed true WT1126–134 specificity, whereas the other clones and line recognized any HLA-A2+ target cell without exogenously loaded WT1 peptide. Sequence analysis of the T cell receptor (TCR)-α and β genes showed that the WT1-specific CD8 T cell clones differed from the pan-HLA-A2-recognizing clones at only two positions in the TCRα third complementarity determining region. In conclusion, high avidity anti-WT1126–134 CD8 T cell responses could be generated in the HLA-A2 mismatch setting; however, the response was dominated by a WT1-independent, promiscuous HLA-A2 reactivity that could be hazardous after allogeneic transplantation.

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 4776.
Abstract 4784: CD8+ tumor-infiltrating lymphocytes predict favorable prognosis in malignant pleural mesothelioma
Authors: Yamada Noriyuki , Oizumi Satoshi , Kikuchi Eiki , Shinagawa Naofumi , Konishi Jun , Ishimine Atsushi , Aoe Keisuke , Gemba Kenichi , Kishimoto Takumi , Torigoe Toshihiko , Nishimura Masaharu
Published: February 2011

Purpose: Defect in human leukocyte antigen (HLA) class I expression may provide tumor cell with a mechanism to escape immune recognition, while T-cell infiltration is associated with good prognosis in many cancers. However, little has been known about the role of HLA class I expression and tumor-infiltrating lymphocytes (TILs) in malignant pleural mesothelioma (MPM). In this study, we investigated the immune profile and conducted outcome analyses in MPM patients.

Experimental Design: HLA class I expression and TILs (CD4+, CD8+, and NK cells) were detected by immunohistochemistry in a series of 44 MPM cases. To detect HLA class I expression, specimens were stained with the anti-pan HLA class I monoclonal antibody EMR8-5.

Results: The expression of HLA class I was positive in 33 patients (75%) and heterogenous in 11 patients (25%). There was no case that showed negative HLA class I expression. The density of CD4+ and CD8+ TILs were strongly correlated (R = 0.76, p<0.001). High density of CD8+ TILs were significantly better prognostic factor for survival of patients with extrapleural pneumonectomy (p<0.05). Multivariate analysis revealed high density of CD8+ TILs to be an independent prognostic factor. Conclusion: The presence of intratumoral CD8+ T cells correlated with improved clinical outcome, raising the possibility that CD8+ T cells might play a pivotal role in anti-tumor immune response against MPMs. Thus stimulation of CD8+ lymphocytes might offer new opportunities for efficacious immunotherapy in MPM.

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 4784.
Abstract 4785: Identification of HSP105 as a novel non-Hodgkin lymphoma (NHL) restricted antigen
Authors: Pupa Serenella M. , Zappasodi Roberta , Bongarzone Italia , Cabras Antonello , Ghedini Gaia C. , Castagnoli Lorenzo , Micciché Francesca , Gianni Massimo A. , Nicola Massimo Di
Published: February 2011

We recently reported that vaccination using dendritic cells (DCs) loaded with killed autologous tumor cells in relapsed indolent NHL patients induced objective clinical responses that correlate with multifaceted immunological responses. In particular, since responders (R) showed tumor-restricted humoral immunity in autologous setting, we exploited antibody (Ab) repertoires from vaccinated patients to evaluate whether the observed antibody responses was directed to indolent NHL-shared antigens (ags).

Immunohistochemistry and flow-cytometric analyses of biotinylated Immunoglobulins (Igs) from vaccinated patients’ serum samples preadsorbed on normal isolated B cells in the attempt to decrease Abs cross-reacting with normal auto-ags showed that only post-vaccine serum Ig of responders specifically reacted with higher intensity on allogeneic NHL tumors (either long term cell cultures or primary tumors from the same series) compared to matched biotinylated Igs from control samples (pre-vaccine serum from responders, pre- and post-vaccine serum from non-responders and healthy donors). In addition, only Abs contained in post-vaccine serum from R significantly inhibited NHL DOHH2 cells growth strongly suggesting the presence of therapeutic Abs in their serum samples. These findings indicate that Igs from R recognize tumor cell-surface associated molecules different from patient-specific idiotypes and support the potential expression of novel NHL-restricted ags. As a step toward identifying NHL-restricted ags as potential novel therapeutic targets, we applied a properly modified serological proteome analysis (SERPA) using as target DOHH2 cells and as probes pre- and post-vaccine Ab repertoires from our series. Proteins, fractionated using an OFFGEL IPG strip (pH 3-10), were separated on SDS-PAGE and then immunoblotted with pre- and post-vaccine Igs from R and controls. Differential hybridization analyses have revealed some potential candidate tumor-associated antigens which include heat shock protein 105 (HSP105), whose immunological role has been already proved in solid tumors. Preliminary data indicate that HSP105 is also expressed on cell membrane from different tumor B cell lines and experiments are in progress to evaluate its potential implication in tumor growth control. Partially supported by AIRC.

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 4785.
Abstract 4786: MiR-17-92 expression in differentiated T cells - Implications for cancer immunotherapy
Authors: Kohanbash Gary , Sasaki Kotaro , Hoji Aki , Fujita Mitsugu , Okada Hideho
Published: February 2011

As type-1 T cells have been shown to mediate effective anti-tumor immune response, we sought to identify microRNA (miRs) differentially regulated between type-1 and type-2 T cells, and determine how the expression of such miRs are regulated. We performed miR microarray analyses on in vitro differentiated murine T helper type-1 (Th1) and T helper type-2 (Th2) cells to identify differentially expressed miRs. We used RT-PCR to confirm the differential expression levels. WST-1, ELISA, and flow cytometry were used to evaluate the survival, function and phenotype of cells, respectively. Lastly, we employed mice transgenic for the identified miRs to determine the biological impact of the miR expression in T cells. Our miR microarray analyses revealed that the miR-17-92 cluster is one of the most significantly over-expressed miR in murine Th1 cells compared with Th2 cells. RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Disruption of the IL-4 signaling through either IL-4 neutralizing antibody or knockout of signal transducer and activator of transcription (STAT)6 reversed the miR-17-92 cluster suppression in Th2 cells. Furthermore, T cells from tumor bearing mice and glioma patients had decreased levels of miR-17-92 compared with cells from non-tumor bearing counterparts. CD4+ T cells derived from miR-17-92 transgenic mice demonstrated superior type-1 phenotype with increased IFN-γ production and very late antigen (VLA)-4 expression when compared with counterparts derived from wild type mice. Finally Jurkat T cells ectopically expressing increased levels of miR-17-92 cluster members demonstrated increased IL-2 production and resistance to activation-induced cell death (AICD). Collectively, our data suggest that the type-2-skewing tumor microenvironment can induce the down-regulation of miR-17-92 expression in T cells, thereby diminishing the persistence of tumor-specific T cells and tumor destruction. Genetic engineering of T cells to express miR-17-92 may prove to be a promising approach for cancer immunotherapy.

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 4786.
Abstract 4793: MUC1 immunogenicity in a triple transgenic mouse model for epithelial ovarian cancer
Authors: Budiu Raluca A. , Mantia-Smaldone Gina M. , Brozick Joan , Edwards Robert P. , Vlad Anda M.
Published: February 2011

Background: Ovarian cancer continues to be associated with a very high mortality rate. Improvements on the development of new therapeutic approaches, including antigen-specific immune therapies, depend in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. MUC1 mucin is a tumor associated antigen and a potential target for cancer immunotherapy. Human and murine mucin 1 share little homology in the extracellular region and transgenic mouse models with de novo lesions expressing the human antigen are needed.

Objective: To identify MUC1 antigen-specific immune regulation in mice with de novo malignant ovarian lesions.

Methods: In order to generate a human MUC1 antigen expressing ovarian cancer mouse model, we crossed the MUC1 transgenic mice which express human MUC1 under the endogenous promoter with the previously described KrasG12D/+PtenloxP/loxP mice that develop orthotopic ovarian tumors.

Results: The newly generated MUC1+/−KrasG12D/+ PtenloxP/loxP triple transgenic mice constitute a valuable Cre-loxP conditional in vivo model for MUC1 expressing ovarian tumors. Upon injection of Cre-encoding adenovirus under the ovarian bursa, the triple transgenic mice develop aggressive ovarian tumors with clear cell histology. The lesions express high levels of MUC1 and are estrogen receptor and cytokeratin 7-positive, markers classically used to diagnose malignant epithelial proliferation. The tumors are accompanied by ascites and numerous peritoneal implants, mimicking late stage disease clinically seen in women. We identified increased accumulation of Treg cells in the para-aortic (draining) lymph nodes and presence of Th17 cells in ascites, mimicking closely the immune phenotype of the human disease. In addition, using quantitative PCR T cell gene profiler arrays, we identified 5 significantly up- and 16 down-regulated genes in the spleen T cells of tumor-bearing mice. One of the upregulated genes was osteopontin (Spp1), recently validated as a marker in detecting human recurrent ovarian cancer. Among the down-regulated genes we identified, Cd4, Cd40ligand (Cd40lg), Interferon-g (Ifnγ), IL-23 receptor (IL-23ra) all suggestive of a shift in immune effectors, some of which may promote the chronic inflammatory background.

Conclusions: Our results reveal important immune regulatory mechanisms in mice with de novo MUC1-expressing ovarian tumors and provide unique opportunities for future in vivo testing of MUC1 vaccines as ovarian cancer therapy and potentially immune prevention.

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 4793.
Abstract 4795: Kupfer-type immunological synapse characteristics do not predict anti-brain tumor cytolytic T-cell function in vivo
Authors: Lowenstein Pedro R. , Yang Jieping , Sanderson Nicholas , Wawrowsky Kolja , Castro Maria G.
Published: February 2011

To analyze the in vivo structure of antigen-specific immunological synapses during an effective immune response, we established brain tumors expressing the surrogate tumor antigen ovalbumin, and labeled antigen-specific anti-glioma T cells using specific tetramers. Using these techniques we determined that a significant amount of antigen-specific T cells were localized to the brain tumor and surrounding brain tissue, and a large percentage could be induced to express IFNγ when exposed to the specific ovalbumin-derived peptide epitope SIINFEKL. Detailed morphological analysis of T cells immunoreactive for tetramers in direct physical contact with tumor cells expressing ovalbumin, indicated that the interface between T cells and target tumor cells displayed various morphologies, including Kupfer-type immunological synapses. Quantitative analysis of adjacent confocal optical sections was performed to determine if the higher frequency of antigen-specific anti-glioma T cells present in animals that developed an effective anti tumor immune response, could be correlated with a specific immunological synaptic morphology. Detailed in vivo quantitative analysis failed to detect an increased proportion of immunological synapses displaying the characteristic Kupfer-type morphology, in animals mounting a strong and effective anti-tumor immune response, compared to those experiencing a clinically ineffective one. We conclude that an effective cytolytic immune response is not dependent on an increased frequency of Kupfer-type immunological synapses

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 4795.
Abstract 4797: Dendritic cells loaded with autologous tumor lysate by electroporation effectively induce lysate specific IFN-γ producing T cells in glioblastoma patients
Authors: Takahara Masashi , Tomiyama Mai , Nieda Mie , Goto Shigenori , Peshwa Madhusudan V. , Kanemura Yonehiro , Maekawa Ryuji , Moriuchi Shusuke
Published: February 2011

Electroporation (EP) can facilitate antigen uptake by dendritic cells (DCs) with consequently enhanced antigen processing and presentation. Earlier we reported that DCs electroporated in the presence of tumor lysate are fivefold to 20-fold more effective in proliferation of tumor-lysate-specific T lymphocytes compared to controls, DCs incubated with the same lysate. In this study we examined the ability of EP-loaded DCs to induce tumor-lysate-specific IFN-γ producing T cells. DCs were prepared from peripheral CD14-positive monocytes by culturing and maturating in culture medium containing GM-CSF, IL-4, PGE2 and TNF-α. We electroloaded DCs from four glioblastoma patients who had been surgically treated. in the presence of autologous tumor lysates (final concentration of 2 mg/mL). In some experiments, we added zoledronate in the culture medium during DC maturating process in order to examine its enhanced effect on the induction of tumor-lysate-specific IFN-γ producing T cells. At 1.5 hrs after EP, we assessed such DCs’ ability to stimulate and proliferate T cells in comparison with DCs co-cultured with the same tumor lysate as control. We incubated autologous peripheral blood lymphocytes (1×106) with lysate-loaded DCs (1×105) for 14 days with IL-2 replenished every two to three days. Surface markers for T cell subsets were analyzed by flow cytometry on days 7 and 14 and IFN-γ secreting T cells by ELISPOT on day 14. EP-loaded DCs induced more IFN-γ producing T cells than control DCs (p<0.001). In two patients (Patient #2 & #3) that expressed higher levels of HLA class I molecules in resected tumor tissue, especially, IFN-γ producing T cells were tumor specific because such T cells significantly higher induced compared with DCs without lysate (p<0.001). Addition of zoledronate, further increased the induction of tumor lysate specific IFN-γ producing cells, concurrent to correlative expansion of the gamma-delta T cells in culture. We hypothesize that the gamma-delta T cells may play important roles in enhancement of tumor lysate specific IFN-γ producing cells. These results suggest that DCs electro-loaded with autologous tumor lysate in combination with zoledronate treatment induce more effective immune responses and may lead to development of more effective immunotherapies for treatment of glioblastoma 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 4797.
Abstract 4798: Cytotoxic T cells kill tumor cells in a contact and antigen-dependent manner, but the interaction affects neighboring, non-target cells via interferon gamma secretion
Authors: Sanderson Nicholas SR , Puntel Mariana , Baker Greg , Castro Maria G. , Lowenstein Pedro R.
Published: February 2011

The interaction between cytolytic T cells and antigen presenting targets involves stable intercellular contacts known as immunological synapses. It has been postulated that one function of immunological synapses might be to restrict the effector functions of the T cell to the target cell, while protecting bystanders (non-target cells). We wished to test this hypothesis in the context of an anti-tumor immune response. To model the three-way interaction between T cells, target cells and bystanders, we used OT-I ovalbumin-specific T cells, GL26 murine glioma cells expressing ovalbumin, and wild type mouse astrocytes. We confirmed the formation of immunological synapses between T cells and GL26 cells, and assessed whether two effector functions of T cells, i.e., cytolysis and cytokine action, were limited to the post-synaptic target cells. We found that cytolysis was strictly restricted to the target tumor cells, but that the effect of interferon gamma, as measured by Stat1 phosphorylation, was more pronounced on the bystander astrocytes. We conclude that a likely model of cytotoxic T cell action in tumor immunity involves direct lysis of tumor cells, and interferon gamma action on the tumor stroma. Our data has implications for the development of improved anti-tumor imunotherapies which will target both the tumor cells and the tumor microenvironment.

This work is supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) NIH/NINDS Grant 1R21-NS054143, 1UO1 NS052465 and 1RO1-NS057711 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193, RO1 NS061107 and 1R21 NS047298 to P.R.L.

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 4798.
Abstract 480: Establishment of human metastatic hepatocellular carcinoma HCCLM3 nude rat model for drug evaluation
Authors: Zhang Jing , Wu Haiyan , Zhang Ruihong , Zhang Yanmei , Shi Mei-shu , Duan Jeff , Zhang Kevin
Published: February 2011

Currently most of antitumor pharmacology studies are performed in mice using either human xenograft or murine tumors. However, the utility of rat tumor models has not been fully explored. Some of the obvious advantages of using rats remain to be validated, for example, the easy combination of pharmacokinetic study with an efficacy study, the easy performance of animal procedures, the more options for local dosing or interventions, etc. HCCLM3 is one of the human hepatocellular carcinoma(HCC) lines that are originally derived from Chinese HCC primary tumor and well established in mouse model system over last 20 years. To realize the potential utilities of rats for tumor models, especially for the need of evaluating various local treatments for the HCC, we examined the growth and metastasis of HCCLM3 tumor following subcutaneous and orthotopic xenograft in immunodeficient rats (RNU nude rats, Charles River). Following either subcutaneous or orthotopic inoculation, HCCLM3 was able to successfully grow up with 100% take-up rate. The subcutaneous tumor grew more quickly in nude rats than in BALB/c nude mice and the tumor weight reached about 10% of body weight 28 days after inoculation. The treatment with Sutent (28mg/kg, p.o. 5 times per week) significantly inhibited the subcutaneous tumor growth by 93% (P<0.05). In comparison, the orthotopic tumor grew rather slower but showed much stronger lung metastasis potential which was not observed in nude mice in 28 days. HCCLM3 in nude rats also demonstrated the growth pattern of the single nodule growth, different from the intrahepatic dissemination and multiple nodule development in the nude mice. Our established HCCLM3 subcutaneous and orthotopic xenograft rat models provided additional useful tools for evaluation of therapeutic profile of drug candidates and different interventional strategies for the treatment of human hepatocellular carcinoma.

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 480.
Abstract 481: METCAM promotes in vitro motility, invasiveness and colony formation, and in vivo tumorigenesis of human breast cancer cells
Authors: Wu Guang-Jer
Published: February 2011

METCAM (alternative names are MUC18, CD146, S-endo 1, MelCAM, and MCAM) is an integral membrane CAM in the Ig-like gene super-family because it has an immunoglobulin-like extra-cellular domain and a cytoplasmic domain, which contains five consensus sequences potentially to be phosphorylated by PKA, PKC, and CK2. METCAM, as predicted from the protein structure, is capable of performing typical functions of CAMs, such as cell-cell interaction, cell-extracellular matrix interaction, and cross-talk with many signaling pathways. Thus altered expression of METCAM should change cell motility and cellular invasiveness, alters angiogenesis, and affects cellular survival and growth. Consistent with this notion, aberrant expression of METCAM positively associates with the malignant progression of melanoma and prostate cancer. However, the role of METCAM in the progression of human breast cancer cells has been controversial. Two groups suggest that METCAM plays a tumor suppressor role in the development of breast cancer. Two other groups suggest the opposite role. To resolve this controversy, we reinvestigate the role of METCAM in the progression of breast cancer cells. We transfected the METCAM cDNA gene into two breast cancer cell lines, MCF-7 and SKBR3, and isolated G418-resistant clones, which expressed high levels of the protein, for testing the effect of enforced expression of the protein on their in vitro and in vivo cellular behaviors. We have found that METCAM is not expressed or very weakly expressed in five luminal breast cancer cell lines, MCF-7, T47D, BT-10, BT474, and SK-BR-3, but moderately expressed in two basal cell-like breast cancer cell lines, MDA-MB231 and 468. Enforced expression of METCAM in two breast cancer cell lines, MCF-7 and SKBR3, increased their in vitro motility and invasiveness and in vitro anchorage-independent colony growth, and promoted disorganized growth in 3D basement membrane culture. Enforce expression of METCAM also increased the in vitro motility and invasiveness of MDA-MB231 and MDA-MB-468 cells. Furthermore, the growth of MDA-MB231 and MDA-MB-468 in the 3D embedded basement membrane culture is inhibited by the anti-huMETCAM antibodies. Furthermore, enforced expression of METCAM dramatically promoted the tumor formation of SK-BR-3 cells in nude mice, but slightly promoted the tumor formation of MCF7 cells in SCID mice via orthotopic injection into mammary fat pads. Taken together, we concluded that over-expression of METCAM in breast cancer cell lines appeared to promote the tumorigenesis of breast cancer cells, suggesting its positive association with the progression of breast cancer cells, similar to melanoma and prostate cancer.

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 481.
Abstract 4810: Persistent migration of cancer cells in a 3-D mechanically confined microenvironment
Authors: Irimia Daniel
Published: February 2011

When cancer cells spread away from the primary tumour, they often follow the trajectories of lymphatic vessels, collagen fibres, white matter tracts, or other heterogeneous structures in tissues. To better understand this form of guided cell migration we designed a series of microfluidic devices that mechanically constrain migrating cancer cells inside microstructures with cross-section comparable to cell size and replicate critical 3D interactions that cancer cells would normally have inside tissues. We observed unexpectedly fast and persistent movement in one direction for several hours of cancer cells of different types. We observed the persistent motility in the absence of external gradients, suggesting the presence of intrinsic mechanisms driving cancer cell motility that are induced in conditions of mechanical confinement. To probe into the mechanisms responsible for this behaviour, we exposed cancer cells inside capillaries to drugs targeting the microtubules and measured significant reduction in the average migration speed in response. Surprisingly, a small numbers of cells appeared not to be affected by the treatment and displayed fast and persistent migration, comparable to untreated cells. The new matrix-free, 3D-confined motility assay is compatible with high-content, high-throughput analysis of cellular motility at single cell level and could provide useful insights into the biology of cancer cell migratory phenotype.

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 4810.
Abstract 4813: Modeling interactions between endothelial cells and metastatic breast cancer cells in a three-dimensional culture
Authors: Connor Yamicia , Harfouche Rania , Liu Cassie , Oh Michael , Sengupta Shiladitya
Published: February 2011

Standard two-dimensional model systems are limited in their ability to accurately capture the molecular mediators underlying breast cancer, and cannot be used to study interactions between multiple cell types. Previous studies have shown that three-dimensional systems provide a more physiologically relevant platform to study cancer progression in a structurally appropriate context 1. However, these systems do not capture the interactions between tumor cells and the angiogenic component, an essential step in tumor progression2. Here we describe, a novel three-dimensional co-culture system combining angiogenic endothelial cells and breast epithelial cells in a three-dimensional tumor matrix. Endothelial cells underwent angiogenic tubulogenesis on a matrigel matrix. Addition of metastatic green fluorescent protein+ve MDA-MB-231 resulted in a unique phenotype characterized by the alignment of the tumor cells on the endothelial tubes. We demonstrate FAK and B1 integrin signaling are important mediators of the observed phenotypes. Inhibiting FAK-B1 signaling disrupted the architecture, reducing the ability of the MDA-MB-231 cells to migrate and adhere to cultured vessels. Furthermore, treatment with small molecule inhibitors of phosphatidylinositol 3 kinase, LY294002 1.6uM, and mitogen activated protein kinase (MAPK), PD98059 20uM, also perturbed this architecture, indicating that these signaling pathways play a key role in controlling the observed phenotype. Furthermore, the inhibition of the PI3K and MAPK pathways, destroyed the tumor vasculature and inhibited progression and invasion of the MDA-MB-231 by 86% (p<0.001) while enhancing the cells’ susceptibility to the cytotoxic agent doxorubicin. Our study shows that a three-dimensional co-culture system provides a powerful in vitro model system to study the mechanisms underlying tumor metastasis and is a powerful tool to screen combination therapies. Supported by DoD BCRP.

1 Lee GY, et al. Nat Methods. 2007 Apr;4(4):359-65.

2Sengupta S, et al. FASEB J. 2004 Oct;18(13):1565-7.

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 4813.
Abstract 4818: Validation and clinical utility of a human hepatocellular carcinoma metastasis signature as prognostic classifier
Authors: Roessler Stephanie K. , Jia Hu-Liang , Budhu Anuradha S. , Qin Lun-Xiu , Ye Qing-Hai , Thorgeirsson Snorri , Tang Zhao-You , Wang Xin W.
Published: February 2011

Hepatocellular carcinoma (HCC) is the most frequent malignant tumor in the liver and the third leading cause of cancer death worldwide. The high mortality of HCC is mainly caused by intra-hepatic metastasis or the high rate of recurrence after surgery. Despite potentially curative surgical resection, eligibility is scarce and patients suffer from a high recurrence rate. Recently, we developed a tumor-based 153-gene signature that is predictive of HCC portal venous metastasis and survival in 40 HCC patients (Ye et al, Nat Med, 9: 416-23, 2003). This signature was based on cDNA microarray profiling of 20 well defined HCC cases, in which ten developed tumor thrombi in the major branch of portal vein and ten had no metastasis at one year follow-up. In this study, we sought to validate this signature for its utility in predicting HCC survival using two independent cohorts. The first cohort consisted of 247 predominantly HBV+ HCC cases analyzed by the Affymetrix U133A 2.0 platform. The second cohort included 139 cases with mixed etiology analyzed by the Human Array-Ready Oligo Set (version 2) array platform. We employed a survival risk prediction algorithm with 10-fold cross validation and 1000 random permutation to test whether the 153-gene signature is associated with survival. We found that this gene signature was able to predict prognosis in both independent HCC cohorts. Importantly, the risk prediction was independent of the microarray platform and etiology. In addition, survival prediction was successful in patients with small (<5 cm in diameter) and/or solitary tumor and the signature performed well on patients particularly with early recurrence (<2 years). Thus, the 153-gene signature was validated in three independent cohorts to be related to HCC patient survival. Furthermore, the risk prediction performed independently of clinical characteristics and microarray platform. We recommend that this signture may serve as a molecular diagnostic test useful to assess the risk that an HCC patient will recur within 2 years after resection, particularly for those with tumors <5cm and in a solitary presentation.

Supported by NIH intramural research funds

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 4818.