bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract IA1: Metastasis and diversity in breast cancer
Authors: Polyak Kornelia
Published: August 2014

Genetic and phenotypic heterogeneity of cancer cells within tumors underlies metastatic progression and therapeutic resistance, thus, it is a major clinical problem. We have characterized intratumor genetic and phenotypic heterogeneity by immunoFISH (iFISH) in primary tumor, lymph node, and distant metastases from the same patient and breast tumor biopsies before and after treatment. We found that distant metastases were highly divergent in some patients and that intratumor diversity associated with tumor subtype and response to treatment. We have also developed preclinical models to assess the functional relevance of intratumor heterogeneity in breast cancer and found cooperative clonal interactions that may promote metastatic progression and contribute to therapeutic resistance. In addition, our analyses of associations between clonal frequency and drivers of tumor growth have revealed some unexpected findings. Our results highlight the importance of exploring and understanding tumors at the single cell level to be able to predict and overcome disease progression and therapeutic resistance.

Citation Format: Kornelia Polyak. Metastasis and diversity in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA1.
Abstract PR1: The role of the microenvironment protein cathelicidin LL-37 in pancreatic ductal adenocarcinoma
Authors: Sainz Bruno , Frias Javier , Cioffi Michele , Alcala Sonia , Heeschen Christopher
Published: August 2014

The tumor-associated microenvironment does not only provide structural support for tumor development, but more importantly the microenvironment provides cues to cancer initiating cells [i.e. cancer stem cells (CSC)] that regulate their self-renewal capacity and metastatic potential. Recent evidence also suggests that inflammatory molecules at the tumor-stroma interface may also play critical roles in the development and progression of numerous tumors, such as pancreatic cancer, and their effects may also be paracrine-mediated. Along these lines, we have discovered that the immuno-modulatory cationic antimicrobial protein 18 (hCAP-18 or LL-37) is highly expressed in microenvironment resident cells (cancer-associated fibroblasts, infiltrating immune cells, and pancreatic stellate cells) of patients with pancreatic cancer. It is also secreted by primary human macrophages co-cultured with pancreatic CSCs. Intriguingly, LL-37 was not detectable in tissue from patients with chronic pancreatitis or pancreatic intraepithelial neoplasia (PanIN) suggesting a bilateral crosstalk between LL-37 producing cells and fully transformed cancer (stem) cells. As we have recently shown a strong role for the tumor stroma and microenvironment-secreted factors in pancreatic CSC self-renewal and invasiveness, LL-37 may represent a new and uncharacterized PDAC microenvironment factor that may play a critical role in pancreatic cancer progression and metastasis. Indeed, treatment of pancreatic CSCs with LL-37 increased CSC pluripotency-associated gene expression, self-renewal, and invasion in vitro as well as tumorigenicity and metastasis in vivo. LL-37 exerted its biological functions through interaction with the G protein—coupled receptor, formyl peptide receptor—like 1, P2X(7) purinergic receptor. In addition, receptor independent activity was mediated by stabilization of the CXCR4 receptor in the cell membrane. Taken together, our data expands our understanding of the tumor microenvironment and the tumor host interactions that perpetuate pancreatic ductal adenocarcinoma. Even more importantly, these studies identify a new tumor-specific factor for identifying malignant lesions in the pancreas and predicting patient outcome. Targeting of LL-37 could improve our therapeutic armamentarium against this deadly disease.

This abstract is also presented as Poster A18.

Citation Format: Bruno Sainz, Jr., Javier Frias, Michele Cioffi, Sonia Alcala, Christopher Heeschen. The role of the microenvironment protein cathelicidin LL-37 in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr PR1.
Abstract A18: Mouse models of autochthonous cancer to study local immune subversion.
Authors: Bransi Ali , Yagita Hideo , Knuth Alexander , Broek Maries van den
Published: August 2014

The tumor environment delivers suppressive signals to the immune system, which impair the effector function of tumor-infiltrating lymphocytes. The identity of these signals, their targets and their mode of action, however, are largely unresolved.

We use TRAMP mice, a well-known autochthonous murine model for prostate cancer, to study the interaction of the adaptive immune system and cancer cells in order to investigate which cell types and molecules are crucial in deviating immune surveillance. Tumorigenesis in TRAMP mice is driven by SV40 large T (SV40LT), which allows monitoring of specific CD8 T cell responses to the tumor.

We analyzed quantitative and qualitative aspects of the tumor-specific T cell response in the spleen, the draining and non-draining lymph nodes and in the tumor of TRAMP mice. Interestingly, this mouse model shows CD8 T cell tolerance towards epitope IV but not epitope I of SV40LT when infected with recombinant vaccinia viruses expressing these epitopes. However, when transferring TCR transgenic CD8 T cells specific for epitope I, we observed rapid local and peripheral immune subversion of adoptively transferred cells in tumor-bearing animals. Homing and proliferation were not affected, but effector functions of these cells were greatly impaired. Furthermore, this immunosuppression was more severe locally at the tumor site than in the periphery. When using a combination of blocking antibodies for co-inhibitory molecules (PD-1, CTLA-4 and TIM-3), we could restore to some extent the function of transferred cells in the periphery, but not at the tumor site.

In parallel, we are in the process of generating an inducible CreERT/LoxP-based model to express the immunologically well-characterized oncogene SV40LT with the luciferase in a tissue specific fashion. This model will allow sensitive detection of arising tumors and precise dissection of tumor-specific immunity over time. By comparing fundamentally different target tissues, we may find mechanisms that are active in every malignancy and some that are organ-specific.

Citation Format: Ali Bransi, Hideo Yagita, Alexander Knuth, Maries van den Broek. Mouse models of autochthonous cancer to study local immune subversion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A18.
Abstract A24: Enhancing chemotherapy efficacy and antitumor immune activity by combined antisense downregulation of TS and IDO.
Authors: Vareki Saman Maleki , Koropatnick James
Published: August 2014

Chemotherapy plays a major role in treatment of many common cancers. However, it is limited by intrinsic and/or acquired tumor cell resistance to anticancer drugs, often mediated by increased expression of genes mediating that resistance. An approach to increase chemotherapy effectiveness is to reduce expression of such genes using small molecules or RNAi to silence specific targets that cancer cells rely on for escape from death due to exogenous treatment or immune cell attack. We have previously shown that siRNA downregulation of thymidylate synthase (TS), the enzyme responsible for the de novo synthesis of thymidylate and a common target for some anti-cancer drugs (5FUdR, pemetrexed, and others) enhances the activity of these drugs in vitro. Using lower concentrations of anticancer drugs has the potential to decrease deleterious side effects, including therapy-induced toxicity to T cells capable of recognizing and killing tumor cells. In addition, certain tumor proteins including indoleamine 2,3-dioxygenase [IDO] can regulate immunosurveillance by inhibiting antitumor T and NK cell activity. IDO can also mediate tumor cell resistance to some anticancer drugs, such as imatinib. To assess the capacity of combined siRNA knockdown of TS and IDO in human tumor cells to increase sensitivity to anticancer drugs, we report that: a) TS siRNA downregulates TS by 90% in HCT-15 colon carcinoma cells, over 90% percent in A549 lung carcinoma cells, and by 80% in H441 lung carcinoma cells, b) shRNA expression vectors stably expressing IDO siRNA in multiple clonally-derived populations of A549 reduces IFNγ-induced IDO protein expression by over 95%. Transiently-transfected TS siRNA is being used, alone and in combination with stable shRNA-mediated reduction in IDO, to assess the utility of targeting both mRNAs to enhance sensitivity to TS-targeting drugs (5-FU, 5-FUdR, pemetrexed), DNA-damaging anticancer drugs (cisplatin, melphalan) and both, both in vitro and against human A549-derived tumour xenografts in immunocompromised mice without T cells but competent with respect to NK cell activity. Combined antisense targeting of both TS and IDO in human tumor cells has the potential to increase the effectiveness of anti-TS and other drugs, both by increasing tumor cell drug sensitivity and by combined enhancement of sensitivity to drug treatment and reduced inhibition of NK cell activity.

Citation Format: Saman Maleki Vareki, James Koropatnick. Enhancing chemotherapy efficacy and antitumor immune activity by combined antisense downregulation of TS and IDO. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A24.
Abstract A3: Enhanced effector responses in CAR-redirected CD8+ T cells deficient in diacylglycerol kinases.
Authors: Riese Matthew , Wang Liang-Chuan , Ranganathan Anjana , Koretzky Gary , Albelda Steven
Published: August 2014

The use of chimeric antigen receptor (CAR)-redirected CD8+ T cells in the treatment of malignancy is a novel strategy that has shown tremendous promise; however, it remains likely this strategy could be enhanced when combined with additional approaches. We hypothesized that deletion of signaling mediators that negatively regulate TCR signal transduction might enhance CAR signaling and function, since CARs utilize the same machinery that transduces TCR signals. We tested CAR activity and function in T cells that lacked one or both lymphocyte-specific isoforms of diacylglycerol kinase (dgk), proteins that metabolize the second messenger DAG and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin demonstrated greatly enhanced cytokine production and cytotoxicity when co-cultured with a murine mesothelioma line that stably expresses mesothelin. Additionally, we found that dgk-deficient CAR transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells, and identify dgks as a potential target for improving the clinical potential of CARs.

Citation Format: Matthew Riese, Liang-Chuan Wang, Anjana Ranganathan, Gary Koretzky, Steven Albelda. Enhanced effector responses in CAR-redirected CD8+ T cells deficient in diacylglycerol kinases. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A3.
Abstract A38: T cell costimulation by TNFRSF4, TNFRSF18, and TNFRSF25 in the context of vaccination.
Authors: Schreiber Taylor H. , Gonzalez Louis , Wolf Dietlinde , Bodero Maria , Podack Eckhard R.
Published: August 2014

TNFRSF4 (OX40, CD134), TNFRSF18 (GITR) and TNFRSF25 are costimulatory receptors that influence CD4+ and CD8+ T cell responses to cognate-antigen. Independently, these receptors have been described to stimulate overlapping functions including enhanced proliferation and activation for both regulatory (CD4+FoxP3+, Treg) and conventional (CD4+FoxP3- or CD8+FoxP3-) T cells. To determine the relative functionality of TNFRSF4, TNFRSF18 and TNFRSF25 in T cell immunity, the activity of TNFRSF4, TNFRSF18 and TNFRS25 agonistic antibodies was compared in the context of both traditional protein/adjuvant (ovalbumin/alum) and CD8+ specific heat shock protein based (gp96-Ig) vaccine approaches. These studies demonstrate that both TNFRSF4, TNFRSF18 and TNFRSF25 independently and additively costimulate vaccine induced CD8+ T cell proliferation following both primary and secondary antigen challenge. In contrast, the activity of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4+ T cell immunity. TNFRSF4 and TNFRSF18 agonists were potent costimulators of ova/alum induced CD4+ conventional T cell proliferation but only weakly costimulated Treg proliferation and IgG2a production, while TNFRSF25 agonists were strong costimulators of Treg proliferation, production of IgG1, IgG2a and IgG2b and weak costimulators of CD4+ Tconv proliferation. Interestingly, antigen-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically effected by the presence of TNFRSF4, TNFRSF18 or TNFRSF25 costimulation. These studies highlight the overlapping but non-redundant activities of these three TNF receptors in T cell immunity, which may guide the application of receptor agonistic agents as vaccine adjuvants for infectious disease and tumor immunity.

Citation Format: Taylor H. Schreiber, Louis Gonzalez, Dietlinde Wolf, Maria Bodero, Eckhard R. Podack. T cell costimulation by TNFRSF4, TNFRSF18, and TNFRSF25 in the context of vaccination. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A38.
Abstract A60: NK cell activation and cytotoxicity can be enhanced with chemotherapy in a murine model of glioblastoma.
Authors: Pellegatta Serena , Cantini Gabriele , Pessina Sara , Finocchiaro Gaetano
Published: August 2014

Chemotherapy is currently regarded as a potential ally for cancer immunotherapy. Several anticancer agents, including classical chemotherapeutic compounds, are able to enforce tumor specific immune responses either by inducing the immunogenic death of tumor cells or by modulating key cells for immune suppression or activation.

To determine whether Temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma and malignant gliomas, could induce tumor immunogenicity, we treated murine GL261 cells with 50 and 150 microM TMZ in vitro for 2, 8 and 20 hours. Flow cytometry showed that the NKG2D ligand, involved in NKG2D-mediated NK cell recognition of tumor cells, was highly expressed by TMZ-treated GL261, whereas GL261 treated with vehicle showed only a weak expression (P < 0.01). B7-H3, an inhibitory molecule for NK cells, significantly decreased in TMZ-treated GL261 cells (P < 0.01). TGF-beta1 and TGF-beta2 concentrations in the supernatant from GL261 treated with TMZ significantly decreased, as shown by ELISA (from 23.2 ± 2.5 and 478.4 ± 19.3 in vehicle to 13.2 ± 2.2 and 283.8 ± 25.9 in TMZ-treated cells, respectively, P < 0.0001).

We therefore studied the effects of TMZ on anti-tumor NK cell response by treating mice 7 days after intracranial implantation of GL261 gliomas with intraperitoneal injections of TMZ (5mg/kg) or vehicle (control mice) for 5 days. Five mice were sacrificed 20 hours after treatment on days 1-5: brain, spleen and blood were harvested and analyzed by flow cytometry.

Trafficking of NKp46+ NK1.1+ CD3- NK cells in blood but not in spleen and their homing ability into the brain significantly increased in TMZ-treated compared to control mice after the second administration of TMZ (7.5 ± 1.4 vs 2.3 ± 1.2, P = 0.001; 10.9 ± 0.4 vs. 3.9 ± 0.6, P = 0.005, respectively). Notably TMZ led to an enrichment of CD11bhigh CD27high NK cells, the most potent effector cells.

To verify NK anti-glioma activity, NK1.1 positive cells were isolated from blood and brain of treated and control mice using magnetic separation and incubated with GL261 cells. NK cell cytotoxicity from TMZ-treated mice was significantly higher than that NK cells from control mice.

These results support the contention that chemotherapy may induce enhancement of NK cell response and open new opportunities to design novel combined therapies reversing the immune suppressive role of glioblastoma and unmasking the therapeutic potential of NK responses.

Citation Format: Serena Pellegatta, Gabriele Cantini, Sara Pessina, Gaetano Finocchiaro. NK cell activation and cytotoxicity can be enhanced with chemotherapy in a murine model of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A60.
Abstract A61: MDSC phenotypes but not Tregs predict patient survival in unresectable stage IV melanoma independently of the type of treatment applied.
Authors: Zelba Henning , Weide Benjamin , Martens Alexander , Pawelec Graham
Published: August 2014

Malignant melanoma is the most aggressive and treatment-resistant form of skin cancer with a steadily increasing incidence. Experimental immunotherapies may result in dramatic tumour regressions but only in a minority of patients. Defining biomarkers allowing identification of those who will survive long-term is crucial to understanding why this is so, and to patient selection. Here, we assessed myeloid-derived suppressor cells (MDSC) and regulatory T cells (Tregs) for their association with survival in late-stage melanoma. We investigated their prognostic value in peripheral blood by polychromatic flow cytometry in a prospective study of 78 patients with unresectable stage IV melanoma treated in a variety of different ways (different peptide vaccinations, ipilimumab, chemotherapy, etc). Thus far, this had only been analyzed in small cohorts and with equivocal results. We investigated four different populations of MDSCs (CD14+CD11b+HLA-DRlo, CD14+CD15+CD11b+, CD14+IL4Ra+ and CD14-CD15+IL4Ra+) that have been associated with poorer outcome in cancers other than melanoma. Tregs were identified as CD4+CD25+FoxP3+ and stratified into CD4+CD45RA+FoxP3lo (resting) and CD4+CD45RA-FoxP3hi (activated) Tregs. We found that the percentages of Tregs in patients were significantly greater than in healthy controls, but differences between patients in their frequencies were not associated with overall survival. In contrast, percentages of the four MDSC populations were not generally different in melanoma patients and controls. However, higher percentages of one population of MSDCs, CD14+CD11b+HLA-DRlo cells, did correlate with shorter survival in Kaplan-Meier analysis and were even superior to the M category in predicting patient survival. These results suggest the importance and potential usefulness of quantifying distinct MDSC populations in the peripheral blood of patients with melanoma for predicting the likelihood of responsiveness to therapy and long-term survival, independently of the nature of their treatment. This is consistent with the notion that anti-tumour immunity plays a crucial role in cancer survivorship, no matter which treatment is applied.

Citation Format: Henning Zelba, Benjamin Weide, Alexander Martens, Graham Pawelec. MDSC phenotypes but not Tregs predict patient survival in unresectable stage IV melanoma independently of the type of treatment applied. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A61.
Abstract A63: Melanoma-educated CD14+ monocytes become myeloid-derived suppressor cell-like and are potent inhibitors of autologous T cells through Cox-2 production and STAT-3 signaling.
Authors: Mao Yumeng , Poschke Isabel , Coaña Yago Pico de , Wennerberg Erik , Lundqvist Andreas , Kiessling Rolf
Published: August 2014

Tumors can suppress the host immune system through a variety of cellular mechanisms, such as regulatory T cells, tumor-associated macrophages and most recently discovered, myeloid-derived suppressor cells (MDSCs). We have previously demonstrated that CD14+HLA-DRlo/- cells accumulate in the peripheral blood of advanced melanoma patients and can suppress autologous T cells ex vivo in a STAT-3-dependent manner. However, the role of MDSCs in the melanoma tumor microenvironment remains to be elucidated. In the current study, early-passage human melanoma cell lines induce significant phenotypic changes on CD14+ monocytes resembling MDSCs in advanced stage melanoma patients. Moreover, such melanoma-educated monocytes strongly impair both autologous T cell proliferation and interferon-γ production. Of note, the induction of the suppressive functions in healthy donor-derived monocytes appears to require tumor contact and/or close proximity to the tumor cells, and is largely dependent on cyclooxygenase-2 (Cox-2) production and activation of STAT-3 signaling pathway. Intriguingly, we have also demonstrated that these two mechanisms are directly involved in the T cell suppression induced by these MDSC-like cells. Last but not least, CD14+ cells isolated ex vivo from advanced stage melanoma patient PBMCs (n=5) exhibited inhibition of autologous T cell functions in a dose dependent manner. Blockade of STAT-3 signaling (p=0.0358, n=4) and superoxide production (p=0.0195, n=4) significantly alleviated the T cell suppression. In conclusion, our data demonstrated the importance of STAT-3 and Cox-2 activity in inducing CD14+ monocytes to exhibit MDSC-like properties, which contributes to our knowledge of how immune suppression is initiated in the human melanoma tumor microenvironment.

Citation Format: Yumeng Mao, Isabel Poschke, Yago Pico de Coaña, Erik Wennerberg, Andreas Lundqvist, Rolf Kiessling. Melanoma-educated CD14+ monocytes become myeloid-derived suppressor cell-like and are potent inhibitors of autologous T cells through Cox-2 production and STAT-3 signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A63.
Abstract A66: IL-33 promotes CD8 T cell effector function and has a potent antitumor activity in vivo.
Authors: Gao Xin , Li Gang , Teng Bo , Yang Qianting , Zhu Yibei , Zhang Xueguang , Finn Olivera J. , Lu Binfeng
Published: August 2014

Interleukin-33 is a member of the IL-1 family of cytokines and is released by stressed cells, necrotic cells or activated innate immune cells such as macrophages during trauma or infection. Thus, IL-33 is thought to serve as an endogenous “danger signal” to trigger inflammation and promote cell mediated immune responses. Prior studies have supported the role of IL-33 in promoting type 2 immune responses but little was known for its role in type 1 immune responses such as the anti-tumor immune response. We have found that that ST2, a receptor for IL-33, is highly expressed in activated CD8 T cells. We have further revealed that, T-bet, a critical transcriptional factor for Th1 differentiation and effector functions, is required for ST2 expression by CD8 T cells, suggesting that ST2 is an integral part of the Th1 gene network in CD8 T cells. Furthermore, we have demonstrated that IL-33 synergized with TCR or IL-12 in increasing both human and mouse CD8 T cell functions such as IFN-γ production. Thus IL-33 can greatly enhance type 1 cell-mediated immune responses in both human and mice. The in vivo relevance of this pathway is examined in a preclinical model of tumor immunity. Our results support a potent function of IL-33 in inhibiting tumor growth and provide a novel therapeutic approach for tumor immunity.

Citation Format: Xin Gao, Gang Li, Bo Teng, Qianting Yang, Yibei Zhu, Xueguang Zhang, Olivera J. Finn, Binfeng Lu. IL-33 promotes CD8 T cell effector function and has a potent antitumor activity in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A66.
Abstract A82: Neutrophils recruit T-regulatory cells into the tumor via secretion of CCL17 – a new mechanism of tumor immune surveillance.
Authors: Mishalian Inbal , Bayuh rachel , Zolotarov Lida , Levy Liran , Fridlender Zvi Gregorio
Published: August 2014

Introduction: Regulatory T cells (T-regs) play an important role in immunological self-tolerance and are functionally immune-suppressive subsets of T cells. It has been repeatedly shown that T-regs are able to migrate in response to CCL22 and CCL17, and that depletion or inhibition of T-regs can enhance anti-tumor immunity. However, there is still limited data on the mechanisms of accumulation of T-regs within the tumor microenvironment. We have recently described the characteristics of “native” pro-tumorigemic N2 tumor-associated Neutrophils (TAN), and found that TAN significantly up-regulated many chemokines compared to bone-marrow (BM) naïve neutrophils. One of the highly up-regulated genes in TAN is CCL17.

Aims: We aimed to study the role of TAN in the secretion of CCL17 and the recruitment of T-regs into the tumor microenvironment. We hypothesized that the secretion of CCL17 by N2 TAN has an important role in the recruitment of T-regs, thus inhibiting the T-cytotoxic cell activity in the tumor.

Material and Methods: CCL17 production by N1 and N2 TAN and by BM naive Neutrophils was evaluated using ELISA, RT-PCR and IHC staining. We conducted in vivo and in vitro migration assays, and used depletion of TAN to reveal the role of CCL17 secreted from TAN in chemoattraction of T-regs.

Results: N2-TAN were more capable of producing CCL17 ex-vivo than N1-TAN or BM neutrophils. The level of CCL17 secreted from TAN increased during tumor development. In vitro migration assay using CD4+CD25+ cells purified from spleens of naïve mice revealed that migration of T-cells towards TAN were 5.27±1.76 times higher compared to BM neutrophils (p<0.05). Furthermore, migration was inhibited by neutralizing anti-mouse CCL17. In vivo migration assay using the air pouch model strengthen these results, demonstrating that more CD4+CD25+ cells migrated into the pouch when injecting TAN compared to BM neutrophils, again inhibited using anti-CCL17 mAb, suggesting that TAN-induced T-regs chemoattraction is CCL17 dependent. Systemic neutrophil depletion after tumor establishment reduced T-reg migration into the tumor.

Conclusions: Our results show that TAN from established tumors produced CCL17, increasing T-regs migration into the tumor. These results suggest a new indirect mechanism by which TAN may inhibit the anti-tumor immune activity, promoting tumor growth.

Citation Format: Inbal Mishalian, rachel Bayuh, Lida Zolotarov, Liran Levy, Zvi Gregorio Fridlender. Neutrophils recruit T-regulatory cells into the tumor via secretion of CCL17 – a new mechanism of tumor immune surveillance. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A82.
Abstract A88: Identification of factors modulating sensitivity to T cell infiltration in the breast cancer setting via next-generation sequencing.
Authors: Amos Sally , Leeuw Ron de , Kuklev Nikita , Kakal Juzer , Babinsky Sindy , Milne Katy , Kost Sara , Freeman Doug , Holt Rob , Watson Peter , Nelson Brad
Published: August 2014

Clinical and pre-clinical trials have established that killer (CD8+) T cells can recognize and destroy tumors. The challenge of our time is to address barriers to T cell infiltration into solid tumors and thereby make cancer immunotherapy a powerful, broadly applicable treatment modality. Bilateral breast cancer represents a unique opportunity to identify those proteins that modulate immune cell entry into solid tumors. Bilateral synchronous breast cancer is when tumors arise independently in separate breasts of the same patient at the same time. Similar to the powerful use of “twin studies” in epidemiology, bilateral tumors provide an opportunity to explore two independent tumorigenic events against a common genetic (and immunological) background. We hypothesise that the sensitivity of breast cancers to T cell infiltration can be predicted on the basis of their molecular profiles. To assess this question, a cohort of bilateral and unilateral breast cancer tissues has been assembled in collaboration with CTRNet-affiliated biobanks. Our objective was to identify 10 tumor pairs with differential T cell infiltration. Here we present the novel protocol for multi-colour brightfield immunohistochemistry (IHC) which was used to quantitate multiple lymphocyte subsets in these precious specimens at the same time. A workflow for automated image collection is presented, which is geared to the analysis of whole-tumour sections. Our results demonstrate that bilateral breast tumors often show differential lymphocyte infiltration despite being exposed to the same level of systemic immunity. Paired tumors will be subjected to whole transcriptome sequencing and lymphocyte receptor spectratyping to identify proteins that distinguish infiltrated from non-infiltrated tumors. Thus, patients with bilateral breast cancer have provided a powerful, translationally-relevant opportunity to study local barriers to T cell infiltration in solid tumors.

Citation Format: Sally Amos, Ron de Leeuw, Nikita Kuklev, Juzer Kakal, Sindy Babinsky, Katy Milne, Sara Kost, Doug Freeman, Rob Holt, Peter Watson, Brad Nelson. Identification of factors modulating sensitivity to T cell infiltration in the breast cancer setting via next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A88.
Abstract B1: Radiotherapy promotes tumor-specific effector CD8+ T cells via DC activation.
Authors: Gupta Anurag K. , Probst Hans Christian , Vuong Van , Landshammer Alexandro , Muth Sabine , Yagita Hideo , Schwendener Reto , Pruschy Martin , Knuth Alexander , Broek Maries van den
Published: August 2014

Radiotherapy is an important treatment for cancer. The main mode of action is thought to be the irreversible damage to tumor cell DNA, but there is evidence that irradiation mobilizes tumor-specific immunity and recent studies showed that the efficacy of high-dose radiotherapy depends on the presence of CD8+ T cells. We show here that the efficacy of radiotherapy given as a single, high dose (10 Gy) crucially depends on dendritic cells and CD8+ T cells, whereas CD4+ T cells or macrophages are dispensable. We show that local high-dose irradiation results in activation of tumor-associated dendritic cells that in turn support tumor-specific effector CD8+ T cells, thus identifying the mechanism that underlies radiotherapy-induced mobilization of tumor-specific immunity. We propose that – in the absence of irradiation – the activation status of dendritic cells rather than the amount of tumor-derived antigen is the bottleneck, which precludes efficient anti-tumor immunity.

Citation Format: Anurag K. Gupta, Hans Christian Probst, Van Vuong, Alexandro Landshammer, Sabine Muth, Hideo Yagita, Reto Schwendener, Martin Pruschy, Alexander Knuth, Maries van den Broek. Radiotherapy promotes tumor-specific effector CD8+ T cells via DC activation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B1.
Abstract B14: Minimum effective concentration of paclitaxel improves the effectivity of dendritic cells transfected with tumor RNA.
Authors: Camargo Marcela Rodrigues , Rodrigues Cecília Pessoa , Gorgulho Carolina Mendonça , Zambuzi Fabiana Albani , Galvão Victória Elizabeth , Frederico Juliana Cristina Longo , Penitenti Marcimara , Kaneno Ramon
Published: August 2014

The central role of dendritic cells (DCs) in antigen presentation and induction of antitumor immunity supported the development of antitumor vaccines based on these cells. In previous studies, we have observed that treatment with ultra-low doses of paclitaxel (PAC) modulated human DCs to express the phenotype of mature and activated cells, and stimulated their antigen presentation activity. At the same time, tumor cells treated with low concentrations of drugs became more immunogenic and more susceptible to the lytic activity of cytotoxic T lymphocytes in vitro. Therefore, we aimed to verify if the transfection of DCs with drug-treated tumor cells RNA enhances the effectiveness of DC-based vaccines and if the in vitro effects are reproducible in vivo. Murine colon tumor cells (MC-38) were subcutaneously inoculated in C57Bl/6 mice. At the 10th and 17th days after inoculation, they received a DC vaccine transfected with RNA of MC-38 cells pre-treated with minimum effective concentration of PAC. These DCs expressed 12% higher levels of CD40 and were able to delay the tumor growth when compared with non-treated animals. Although the differences were not significant, our results suggest that the treatment of tumor cells with low concentration of PAC can enhance the immunogenicity of tumor cells, which is transfered for DCs by RNA transfection, and delay tumor growth. Financial support: FAPESP 2009/18331-8; FAPESP 2010/06013-9.

Citation Format: Marcela Rodrigues Camargo, Cecília Pessoa Rodrigues, Carolina Mendonça Gorgulho, Fabiana Albani Zambuzi, Victória Elizabeth Galvão, Juliana Cristina Longo Frederico, Marcimara Penitenti, Ramon Kaneno. Minimum effective concentration of paclitaxel improves the effectivity of dendritic cells transfected with tumor RNA. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B14.
Abstract B20: Immunotherapy as treatment for dogs with glioma.
Authors: Goulart Michelle , Pluhar Elizabeth , Andersen Brian , Seiler Charles , Hunt Matthew , O'Sullivan Gerry , Zhang Zoe , Ohlfest John
Published: August 2014

Gliomas, the most invasive tumors of the brain, are a disparate group of oncological diseases for which there is currently no cure. Despite the use of combination therapy (surgery, radiation and chemotherapy) as the standard of care for patients, limited progress has been made in the control of the disease. Similar to that in humans, dogs develop spontaneous brain tumors that often carry a dismal prognosis regardless of therapeutic intervention. There is an obvious need for new therapies to treat dogs with brain tumors. Cancer immunotherapy, with the premise that tumor-reactive lymphocytes are able to infiltrate the brain to eradicate microscopic disease, is emerging as a promising strategy to treat glioma. To date, very little is known regarding canine glioma treatment outcome and there has been no reported randomized clinical trials examining the use of immunotherapy for treatment of glioma in dogs. A clinical trial using immunotherapy with autologous glioma cell lysate vaccines, combined with the TLR9 agonist CpG oligodeoxynucleotide to treat dogs with glioma, is underway in our laboratory. The objective of the treatment is to induce specific anti-tumor immunity in the brain and to prevent tumor recurrence. Our preliminary results have shown that vaccinated dogs exhibited extended survival when compared with historical controls. Based on western blot and flow cytometric analysis, this therapeutic approach induced tumor-reactive antibody responses with elevation of IgG antibody levels after vaccination. Thus, the examination of immune responses in therapeutic trials conducted in dogs with gliomas should greatly help predict outcomes of similar treatments in humans, and additionally provide an opportunity to accelerate the development of more efficient immunotherapeutic cancer vaccines for treatment of intracranial malignances.

Citation Format: Michelle Goulart, Elizabeth Pluhar, Brian Andersen, Charles Seiler, Matthew Hunt, Gerry O'Sullivan, Zoe Zhang, John Ohlfest. Immunotherapy as treatment for dogs with glioma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B20.
Abstract B25: Porous silicon microparticles exhibit immunomodulatory effects leading to suppression of tumor growth.
Authors: Meraz Ismail M. , Williams Laura , Yang Marie , Lavelle Edward C. , Serda Rita E.
Published: August 2014

Nanoparticles, such as polymeric delivery platforms, can exhibit intrinsic immunostimulant properties, dependent on size, charge, surface modification, and composition. To function as effective adjuvants, a balance between immunostimulatory properties and biocompatibility is essential. We have demonstrated that porous silicon (pSi) microparticles are effective delivery vehicles, with uptake by target cell populations. A peritonitis mouse model was used to access early innate immune responses 24 hours following administration of pSi microparticles. C57BL/6 mice were injected intraperitoneally with microparticles of various shapes and sizes and cytokine production and cell infiltration were assessed. pSi microparticles were found to have proinflammatory effects. The microparticles induced significant leukocyte infiltration into the site of injection and elevated IL-1β levels in lavage fluid.

As cancer progresses, immune responses become more tolerant and cancer cells become more refractory to chemotherapies. Select chemotherapeutics, including cyclophosphamide, doxorubicin, and paclitaxel, have immunodulatory effects. Based on the immunopotentiating effects of pSi microparticles, and the ability of monophosphoryl lipid A (MPL) adsorbed pSi microparticles to activate antigen presenting cells, we sought to potentiate the doxorubicin-mediated immune response through co-delivery of MPL-pSi microparticles. When tumors in mice bearing intramammary 4T1-luc breast tumors reached a volume of 100 mm3, mice were injected with doxorubicin loaded liposomes (5 mg/kg) and MPL-pSi microparticles (5x108 microparticles; 10 µg MPL equivalent) by means of tail vein injection. Tumor growth was monitored by calipher measurements and luciferase expression using the IVIS Imaging System 200. While MPL-pSi microparticle-treated mice exhibited reduced tumor growth, mice receiving MPL-pSi microparticles and doxorubicin-loaded liposomes exhibited arrest of tumor growth. Thus pSi microparticles are an attractive immunopotentiating platform, with applications for both drug and antigen delivery.

Citation Format: Ismail M. Meraz, Laura Williams, Marie Yang, Edward C. Lavelle, Rita E. Serda. Porous silicon microparticles exhibit immunomodulatory effects leading to suppression of tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B25.
Abstract B57: A natural plant-derived compound, Withaferin A, exploits the stress pathway to induce antitumor immunity.
Authors: Smith Annalise R. , Lopez Darlah , Andreansky Samita
Published: August 2014

Recent research involving chemotherapeutic drugs such as anthracyclines have aided in redefining the definition of apoptosis from a form of cell death that is immunologically silent to the one that can activate an anti-cancer immune response. This form of apoptosis has been characterized as immunogenic cell death (ICD) and possess certain hallmarks such as the cell surface expression of heat shock proteins (HSPs) including HSP70 and HSP90 as well as ER-stress mediated pre-apoptotic expression of the ER chaperone calreticulin (CRT). One of the mechanisms by which ICD occurs is linked to the induction of an unfolded protein response (UPR) due to prolonged chemotherapeutic stress on cancer cells. Our research focuses on the development of Withaferin A (WA), a steroidal lactone derived from the Indian ayurvedic medicinal plant Withania somnifera as a chemoimmunotherapeutic agent. The benefit of using natural plant derived products is their low toxicity, multimodal action and potential ability to enhance the immune system. We initially screened the effect of Withaferin A by microarray analysis on breast cancer cells. WA induced significant alteration of 1160 genes (p-value 0.005). A pathway analysis determined that WA induces a robust heat shock response through up-regulation of several genes related to this pathway including response to unfolded proteins. The gene expression data was confirmed by the induction of intracellular HSP70, HSP40 as well as the UPR marker Bip78 by western blot. Furthermore we are able to demonstrate that WA treatment results in the translocation of known immunogenic HSPs HSP70, CRT and gp96 to the cell surface of several cancer cell lines. Additionally, WA treatment results in the induction of apoptosis and these WA mediated apoptotic cells can confer immunological protection against re-challenge with live tumor cells in vivo. Our long-term goal is to elucidate the anti-tumor immunomodulatory mechanisms of WA.

Funded by: American Cancer Society (S.A.)

Citation Format: Annalise R. Smith, Darlah Lopez, Samita Andreansky. A natural plant-derived compound, Withaferin A, exploits the stress pathway to induce antitumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B57.
Abstract IA21: Using viruses to advance T cell therapy for malignancy.
Authors: Yanagisawa Miyuki , Ngo Minhtran , Rooney Cliona M. , Leen Ann Marie , Vera Juan , Heslop Helen E. , Brenner Malcolm K.
Published: August 2014

Virus-specific T cells effectively prevent and treat viral diseases after stem cell transplantation and have produced complete remissions (CRs) in patients with EBV-positive malignancy outside the transplant setting. The success of EBV-specific T cells is thought to be due to the strong viral antigens expressed by these tumors and the vaccine effect of endogenous EBV.

We tested if this endogenous viral vaccine effect could be exploited for the treatment of non-viral malignancy by engrafting chimeric antigen receptors specific for the disialoganglioside,GD2 (GD2.CAR) on EBV-specific T cells and comparing their in vivo expansion with that of GD2.CAR-engrafted CD3-activated T cells (ATCs) in patients with relapsed neuroblastoma. Transgenic EBV-CTLs were detected at higher levels than GD2.CAR-ATCs and 6 of 8 patients without disease at infusion remain tumor-free, while 6 of 11 with disease had tumor responses including 3 CRs. However the expected increase in frequency of EBV-specific T cells in patient blood was not observed, perhaps because EBV was well-controlled in these patients.1,2

To improve this vaccine effect, we looked to VZV, for which a potent, but safe commercially-available vaccine exists. We identified five VZV antigens that we predicted would be presented to the immune system early after infection, generated overlapping peptide libraries (pepmixes) for each and used them to stimulate T- cells from both naturally-infected and vaccinated individuals. T cells expanded in vitro and were antigen-specific as measured on day 9 using γ-IFN ELIspot analysis. T cells specific for all five antigens increased in frequency in response to VZV vaccination. VZV-specific T cells transduced with a GD2.CAR retained their ability to respond to VZV antigens and acquired the ability to eliminate GD2+ tumor cells.

Ultimately, we plan to combine GD2.CAR-engrafted, VZV-specific T cells with VZV vaccination for the treatment of patients with relapsed GD2+ tumors.

References:

1 Pule et al. Nat Med. 2008 Nov;14(11):1264-70

2 Louis et al. Blood. 2011 Dec 1;118(23):6050-6

Citation Format: Miyuki Yanagisawa, Minhtran Ngo, Cliona M. Rooney, Ann Marie Leen, Juan Vera, Helen E. Heslop, Malcolm K. Brenner. Using viruses to advance T cell therapy for malignancy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA21.
The Androgen-Regulated Protease TMPRSS2 Activates a Proteolytic Cascade Involving Components of the Tumor Microenvironment and Promotes Prostate Cancer Metastasis
Authors: Lucas Jared M. , Heinlein Cynthia , Kim Tom , Hernandez Susana A. , Malik Muzdah S. , True Lawrence D. , Morrissey Colm , Corey Eva , Montgomery Bruce , Mostaghel Elahe , Clegg Nigel , Coleman Ilsa , Brown Christopher M. , Schneider Eric L. , Craik Charles , Simon Julian A. , Bedalov Antonio , Nelson Peter S.
Published: August 2014

TMPRSS2 is an androgen-regulated cell-surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastases. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries, we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-MET receptor tyrosine kinase signaling, and initiated a proinvasive epithelial-to-mesenchymal transition phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment.

Significance: The vast majority of prostate cancer deaths are due to metastasis. Loss of TMPRSS2 activity dramatically attenuated the metastatic phenotype through mechanisms involving the HGF–c-MET axis. Therapeutic approaches directed toward inhibiting TMPRSS2 may reduce the incidence or progression of metastasis in patients with prostate cancer. Cancer Discov; 4(11); 1310–25. ©2014 AACR.

See related commentary by Rubin, p. 1262

This article is highlighted in the In This Issue feature, p. 1243
Recurrence of a Delayed-Onset Posttraumatic Renal Pseudoaneurysm after Initially Successful Transarterial Embolization
Authors: Meissnitzer Thomas , Hruby Stephan , Janetschek Günther , Meissnitzer Matthias Wolfgang
Published: August 2014

Hemodynamically stable patients with renal injury can be managed conservatively. Anatomy typically leads to physiologic confinement of urinomas or renal hematomas to the interfascial planes of the retroperitoneum. In the presented case there was unusual reperfusion 14 days after a successful embolization of a renal pseudoaneurysm, at this time 28 days after the initiating trauma. This article discusses the evolution, treatment options and possible underlying causes for the reperfusion of this late-onset renal pseudoaneurysm.