bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3100: Characterisation of novel chalcone derivatives, CTR compounds as tubulin polymerisation inhibitors
Authors: Lindamulage Indeewari K. , Vu Hai-Yen , Trivedi Dr. Piyush , Lee Dr. Hoyun
Published: August 2015

Agents targeting colchicine-binding site are recognized as valuable lead compounds in the development of new anticancer drugs. Although colchicine can effectively inhibit cell proliferation, its use as an anticancer agent has not been approved by FDA due to its inherent toxicity. To develop colchicine-binding site targeting agents with low or no toxicity, we have created and examined several chalcone derivatives, from which we have identified CTR-17 and CTR-20 as promising leads. We have examined their anti-proliferative activities using three human breast cancer cell lines (MDA-MB-468, MDA-MB-231 and MCF-7) and two matching non-cancer breast cell lines (184B5 and MCF10A). In addition, we also examined their efficacy using one leukemic cell line (K-562) and one cervical cell line (HeLa). Data from this study showed that CTR-17 and CTR-20 preferentially kill cancer cells 10-100 times over non-cancer cells. Data obtained from flow cytometry, confocal microscopy and Western blotting showed that CTR-17 and CTR-20 induced prolonged mitotic arrest prior to killing cancer cells by apoptosis. We also found that both CTR-17 and CTR-20 inhibit tubulin polymerisation and bind to purified tubulin fibers with a dissociation constant of 3.4±1.7 μM and 5.9±1.9 μM, respectively. CTR-17 competitively inhibits the binding of colchicine to tubulin with an inhibitory concentration of 8.67 μM, suggesting that CTR-17 binds to tubulin at a site close to the colchicine binding site. Molecular docking studies confirmed this binding which occurs via three hydrogen bonds and Van der Waals interactions. In our xenograft model of the human breast cancer cells, the treatment of mice with CTR-17 or CTR-20 (30 mg/kg) twice per week for 30 days effectively inhibits tumor growth. The combination of CTR compounds (15 mg/kg/week) and paclitaxel (5 mg/kg/week) increases paclitaxel's anti-tumor activity by 40-60%. Most importantly, both CTR-17 and CTR-20, when used alone or in combination with paclitaxel, showed no notable toxicity to vital organs (spleen, liver, kidney and lung). Therefore, the novel CTR-17 and CTR-20 chalcone derivatives possess substantial potential as safe and effective anticancer drugs.

This project was supported in part from the funds by the Northern Ontario Heritage Funds Corporation, Northern Cancer Foundation, and The City of Greater Sudbury Development Corporation. IKL is a recipient of the Ontario Trillium Scholarship.

Citation Format: Indeewari K. Lindamulage, Hai-Yen Vu, Dr. Piyush Trivedi, Dr. Hoyun Lee. Characterisation of novel chalcone derivatives, CTR compounds as tubulin polymerisation inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3100. doi:10.1158/1538-7445.AM2015-3100
Abstract 3108: MicroRNA-200B inhibits arsenic-transformed cell migration by targeting protein kinase Cα signaling pathway
Authors: Wang Zhishan , Humphries Brock , Yang Chengfeng
Published: August 2015

Arsenic is one of the most common environmental pollutants and long term exposure to arsenic is associated with increased risk of multiple types of cancer including lung cancer. Despite extensive studies, the mechanism of arsenic carcinogenicity has not been elucidated. MicroRNAs (miRNAs) are a large class of small non-coding RNAs and have been shown to play important roles in many fundamental processes of cancer. MiRNA-200b (miR-200b) is a member of miR-200 family, which contains 5 members. The miR-200 family has been shown to inhibit tumor cell migration; however, the underlying mechanisms are not clearly defined. We recently found that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells, which are highly migratory and invasive. To further study the mechanism by which miR-200 inhibits tumor cell migration, we investigated the effects of stably expressing miR-200b on arsenic-transformed cell migration. We found that stably expressing miR-200b drastically inhibits arsenic-transformed cell migration. Wnt5b and protein kinase Cα (PKCα) are highly expressed in arsenic-transformed cells. Stably expressing miR-200b significantly down-regulated protein levels of both Wnt5b and PKCα. Combining bioinformatics screening and 3′UTR luciferase reporter assays, we identified PKCα but not Wnt5b as a new direct target of miR-200b. We also found that siRNA knocking down Wnt5b greatly reduces PKCα activation while inactivating PKCα drastically decreases Wnt5b level, revealing a positive feedback loop between Wnt5b and PKCα. Knocking down Wnt5b or PKCα phenocopy the effect of stably expressing miR-200b on cell migration. Moreover, enforced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration. These findings not only reveal a novel mechanism showing that miR-200b inhibits tumor cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop, also suggest that deregulation of miR-200b, Wnt5b and PKCα plays an important role in arsenic carcinogenesis.

Citation Format: Zhishan Wang, Brock Humphries, Chengfeng Yang. MicroRNA-200B inhibits arsenic-transformed cell migration by targeting protein kinase Cα signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3108. doi:10.1158/1538-7445.AM2015-3108
Abstract 3113: Primate-specific miR-663 suppresses glioblastoma progression and predicts patient prognosis
Authors: Shi Yu , Ping Yi-fang , Bian Xiu-wu
Published: August 2015

Purpose: miRNAs (microRNAs) have recently been recognized as important regulators of cancer biological behavior. However, the expression patterns and prognostic significance of miRNAs in GBM remain elusive due to the lack of large pools of clinical specimens for screening. This study was performed to identify miR-663 as a potential regulator of glioblastoma (GBM) malignant behaviors from miRNA profiles. The prognostic significance of miR-663 in GBM, its effect in tumor progression and the underlying mechanism were determined.

Methods: Specimens from 256 cases of glioma patients, including 239 patients with follow-up information, were used to analyze the association between miR-663 and patients’ prognosis by Kaplan-Meier and multivariate Cox regression analyses. The effects of miR-663 on GBM cell proliferation and invasion were examined both in vitro and in vivo. Bioinformatics prediction and signal network analysis were applied to identify the putative targets of miR-663, which were further verified by luciferase reporter assay, rescue experiments as well as the immunohistochemistry (IHC) and Western blotting examination of downstream effectors. Quantitative reverse transcriptase-PCR (qRT-PCR) and IHC were applied to investigate the clinical association between miR-663 and its target in human GBM specimens.

Results: miR-663 was identified from miRNA profiles and inversely correlated with glioma grades but positively correlated with patients’ survival. Furthermore, two distinct subgroups of GBM patients with different prognoses were identified based on miR-663 expression in our specimens and that from TCGA database. Overexpression of miR-663 significantly suppressed the proliferation and invasion of GBM cells in vitro and in vivo. Mechanistically, we discovered PIK3CD as a direct target of miR-663 and found that phosphorylated AKT and three key downstream effectors of PIK3CD, were down-regulated by miR-663 overexpression. Moreover, PIK3CD was inversely correlated with miR-663 in GBM specimens and predicted poor prognosis of GBM patients.

Conclusion: miR-663 is a novel prognostic biomarker and a potential therapeutic candidate for GBM.

Citation Format: Yu Shi, Yi-fang Ping, Xiu-wu Bian. Primate-specific miR-663 suppresses glioblastoma progression and predicts patient prognosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3113. doi:10.1158/1538-7445.AM2015-3113
Abstract 3125: miR-9-3p plays a tumor-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells
Authors: Higashi Takaaki , Hayashi Hiromitsu , Takeyama Hideaki , Kaida Takayoshi , Arima Kota , Taki Katsunobu , Okabe Hirohisa , Nitta Hidetoshi , Hashimoto Daisuke , Chikamoto Akira , Beppu Toru , Baba Hideo
Published: August 2015

Background: Hepatocellular carcinoma is the sixth most prevalent cancer and the third most frequent cause of cancer-related death. Despite recent advance in cancer treatment including the development of molecular target drugs, the prognosis in patients with hepatocellular carcinoma (HCC) still remains poor.

The inactivation of the Hippo-pathway lead to TAZ/YAP overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). While there are several reports of miR targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC.

Methods: miR expression was analyzed using the Human miFinder 384HC miScript miR PCR Array and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumor-suppressor miR targeting TAZ using miRBase. We examined the functional role of miR-9-3p targeting TAZ using miR-9-3p mimic and inhibitor in HCC cell lines (HLF and HuH1 with high and low TAZ expressions, respectively).

Results: In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions by real-time PCR. By western blotting, TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. TAZ nuclear localization was indeed inhibited with a treatment with miR-9-3p mimics in HLF cells, and was inversely accelerated with a treatment of miR-9-3p inhibitors in HuH1 cells.

Treatment of miR-9-3p mimic inhibited cell proliferative ability by downregulating the phosphorylation of Erk1/2 and Akt signals in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared to control in HuH1. On the other hand, cell invasiveness was not affected by miR-9-3p.

Conclusions: miR-9-3p was identified as the tumor-suppressor miR targeting TAZ expression in HCC cells. miR-9-3p played a crucial role in cell proliferation, but not in invasion, via Akt and Erk1/2 signals in HCC cells.

Citation Format: Takaaki Higashi, Hiromitsu Hayashi, Hideaki Takeyama, Takayoshi Kaida, Kota Arima, Katsunobu Taki, Hirohisa Okabe, Hidetoshi Nitta, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. miR-9-3p plays a tumor-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3125. doi:10.1158/1538-7445.AM2015-3125
Abstract 3128: MicroRNA-mediated reprogramming of myeloid cells by targeting TGFβ signaling and its associated molecular network
Authors: Vodnala Suman K.
Published: August 2015

Tumor derived soluble factors result in accumulation of Gr-1+CD11b+ immature myeloid cells at tumor site and premetastatic organ. These cells are known to promote tumor progression and metastasis. Gr-1+CD11b+ immature myeloid cells and tumor-associated macrophages display a type 2 phenotype (M2) and produce immune suppressive cytokines including TGFβ. We have previously shown that immature myeloid cells are vicious producers of TGFβ ligand and express high levels of TGFβR2 receptor for eliciting immune suppressive function. Specific deletion of TGFβR2 in myeloid cells resulted in increased antitumor immunity and decreased metastasis in the lungs of cancer mouse models. microRNAs (miRs) are abundant class of small non-coding RNAs that have recently emerged as powerful epigenetic regulators of gene expression in pathophysiological conditions. One of the critical properties of miR is to target a large number of genes frequently within the context of a network, making them very efficient in regulating distinct biological cell processes. In the current project, we have identified three miRs that target myeloid TGFβR2 associated molecular network including p38 and cAMP pathways, which are critical in type 2 cytokine production. We have successfully transduced bone marrow stem cells with lentivirus expressing these miRs driven by a myeloid specific promoter CD11b. Transplantation of these modified bone marrow into lethally irradiated mice decreased breast cancer metastasis. The underlying mechanisms involve the reduction in type 2 cytokines such as IL-4, IL-10 and IL-13 and activation of IFN producing CD8 cytotoxic lymphocytes. Interestingly, the miRNAs are also differentially regulated by TGFβ signaling in TGFβR2+/+ and TGFβR2−/− myeloid cells, suggesting a feedback loop between TGFβ and miRNA. These results demonstrate that the candidate miRNA can reprogram tumor-associated myeloid cells by altering the cytokine milieu and enhance host antitumor immunity. Our work points out that endogenous miRNA targeting myeloid TGFβ signaling and its molecular network provides an important therapeutic option.

Note: This abstract was not presented at the meeting.

Citation Format: Suman K. Vodnala. MicroRNA-mediated reprogramming of myeloid cells by targeting TGFβ signaling and its associated molecular network. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3128. doi:10.1158/1538-7445.AM2015-3128
Abstract 3133: Deciphering the novel role and underlying mechanism of let-7i repression in stem-like properties of head and neck cancer cells
Authors: Liao Tsai-Tsen , Yang Muh-Hwa
Published: August 2015

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the major male cancers worldwide, and the main therapeutic strategies include surgery, radiotherapy and chemotherapy. Cancer stem cells are a small population of tumor cells that are responsible for tumorigenesis, tumor recurrence and treatment resistance. let-7 family microRNAs have been shown to function as a tumor suppressor. Here we aim to investigate the role of let-7i in regulating stem cell properties in HNSCC. Methods: Luciferase reporter assay were used to identify the targets of let-7i; Western blot to analysis the correlation between ARID3B and Oct4; ChIP assay confirm the binding of ARID3B to Oct4 proximal promoter; Quantitative RT-PCR to analyzed the level of ARID3B, Oct4, and let-7i in the 52 pairs of samples containing primary tumor tissues and adjacent normal epithelium from head and neck cancer patients with different stage receiving surgery.

Results: ARID3B, a member of the AT-rich interaction domain family proteins which plays an essential role during embryogenesis, is a bona fide target of let-7i. ARID3B promotes stem-like properties of HNSCC cells and upregulates a panel of stemness genes, and Oct4 is one of the most prominent ones to be upregulated. ARID3B direct binds to the AT-rich region of Oct4 promoter to activate its transcription. Downregulation of let-7i in HNSCC samples correlates with a corresponding upregulation of Oct4 and ARID3B.

Conclusions: We propose a mechanism of let-7i repression-mediated stem-like properties through releasing the suppression of a novel let-7i target ARID3B, which leads to transcriptional activation of stemness genes. Let-7i coordinates the function of ARID3B through repressing targets related to ARID3B activity, such as ARID3A and KPNA4. Furthermore, we elucidate of the regulatory mechanism of the stemness factors by ARID3B through epigenetic regulation. The results provide useful information in elucidating the mechanism of HNSCC progression and also provide potential targets in developing HNSCC-specific treatment.

Citation Format: Tsai-Tsen Liao, Muh-Hwa Yang. Deciphering the novel role and underlying mechanism of let-7i repression in stem-like properties of head and neck cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3133. doi:10.1158/1538-7445.AM2015-3133
Abstract 3142: Inhibition of the transcription factor Ikaros augments the tumoricidal capacity of CD8+ T cells expressing chimeric antigen receptors
Authors: O'Brien Shaun , Wang Liang-Chuan , Newick Kheng , Thomas Rajan M. , Wells Andrew , Albelda Steven M.
Published: August 2015

Recent success of adoptive transfer of T cells expressing chimeric antigen receptors (CARs) in patients with blood-borne malignancies has resulted in growing enthusiasm to apply the same approach to treat solid tumors. CAR-expressing T cells, however, will likely face many challenges that will limit their efficacy in solid tumors. One possible obstacles will be the intrinsic negative feedback systems that prevent continuing T cell responses. The transcription factor Ikaros which has been shown to suppress endogenous T effector functions through epigenetic remodeling of cytokine gene loci may be one such negative regulator. Here we show that anti-mesothelin CAR (mesoCAR) T cells with reduced levels of Ikaros consistently mediate better in vitro cell killing and in vivo tumor regression than their wild-type counterparts. This increased anti-tumor activity was accompanied by increased persistence of mesoCAR T cells, as well as increased IFN-g. Enhanced cytolytic activity was also evidenced in mesoCAR T cells with reduced Ikaros, which could be partially explained by increased CD107a upregulation and Granzyme B production. Reduction in the Ikaros level in mesoCAR T cells did not affect T cell receptor and CAR signaling, but was able to render them more resistant to soluble inhibitory factors like TGF-B and adenosine. In summary, inhibition of the transcriptional repressor Ikaros in CAR T cells could be an attractive strategy to overcome immunosuppressive tumor microenvironments and enhance their tumoricidal capacity. Further clinical development is warranted.

Citation Format: Shaun O'Brien, Liang-Chuan Wang, Kheng Newick, Rajan M. Thomas, Andrew Wells, Steven M. Albelda. Inhibition of the transcription factor Ikaros augments the tumoricidal capacity of CD8+ T cells expressing chimeric antigen receptors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2015-3142
Abstract 3144: Development of immunotherapy for anaplastic large cell lymphoma (ALCL) using CSF1R retargeted human T lymphocytes
Authors: Achkova Daniela , Spicer James , Maher John
Published: August 2015

Anaplastic large cell lymphoma (ALCL), a type of T/null-cell lymphoma, represents about 20-30% of paediatric lymphomas and has an aggressive clinical course with frequent relapse. Recently, high-level expression of CSF1R on malignant cells in ALCL has been linked to shorter progression free survival, providing a rationale to test adoptive C28ζ-mediated T-cell therapy. This approach encompasses the genetic modification of T-cells to express a chimeric antigen receptor (CAR) named C28ζ, a fusion receptor coupling the recognition of CSF1R on the tumour cell surface to the delivery of tailored T-cell activation signal provided by a fused CD28+CD3ζ endodomain. To optimize this approach for human translation, the C28ζ CAR was co-expressed with the chimeric cytokine receptor 4αβ (combination termed “C4”), which allows for selective expansion and enrichment of CAR-transduced T-cells using IL-4. PBMCs of healthy donors were isolated and activated using anti-CD3/CD28 beads. Following retroviral T-cell transduction and 14 days expansion of C4+ T-cells in the presence of IL-4, co-cultures with a panel of ALCL cell lines (2 ALK positive and 2 ALK negative ALCL cell lines) were set up to test C4 efficacy in vitro. Successful re-targeting of C4+ T-cells was confirmed by monitoring target cell destruction. Conditioned media was collected and analysed for IFN-γ and IL-2 production. Cytotoxicity, cytokine release and T-cell proliferation data demonstrated Ag-specific activation of the C4+ cells, which contrasted with T-cells expressing a control CAR with truncated endodomain. This data provides for the first time the basis for successful application of CAR-based immunotherapy against CSF1R-expressing malignancies.

Citation Format: Daniela Achkova, James Spicer, John Maher. Development of immunotherapy for anaplastic large cell lymphoma (ALCL) using CSF1R retargeted human T lymphocytes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3144. doi:10.1158/1538-7445.AM2015-3144
Abstract 3147: Generation of exhaustion-resistant memory CD8 T-cells by simultaneous downmodulation of mTORC1 and PD-1 in activated CD8 T-cells
Authors: Berezhnoy Alexey , Levay Agata , Rajagopalan Anugraha , Puplampu-Dove Yvonne , Gilboa Eli
Published: August 2015

CD8 memory T cells play a crucial role in controlling chronic infections and certain malignancies. It is possible to expand the population redirecting early-activated T-cells toward memory differentiation by altering intracellular signaling of mTORC1 pathway. Indeed, treatment with mTORC1 inhibitor rapamycin increased formation of memory cells as published previously; however, animals treated with the drug exhibited signs of immune suppression and rapamycin-generated CD8 memory T-cells failed to protect animals against melanoma challenge. We introduced highly efficient RNAi-based method to downmodulate mTORC1 signaling specifically in target cells. siRNA against raptor, a critical component of the signaling complex, was delivered in vivo into activated T-cells by conjugating to the 4-1BB-binding RNA aptamer resulting in expansion of CD8+ memory T-cells formation.

Memory T-cells are generally resistant to immune suppression, yet in vivo, the population suffers from the gradual attrition mediated by PD-1 signalling. We developed the conjugate with two highly effective siRNAs against raptor and PD-1 linked to the same 4-1BB aptamer molecule. In vitro, both siRNAs efficiently dissociated from the conjugate inside target cells and simultaneously downregulated its respective mRNAs. In vivo, using OT-I adoptive transfer model, we demonstrated that treatment with 4-1BB-raptor-PD1 conjugates increased formation of memory CD8+ T-cells (as measured by absolute number of OT-1 cells 30 days after the treatment) in a rate similar to the 4-1BB-raptor conjugate. There was, however, qualitative difference between two populations, as CD8+ memory T-cells expanded with 4-1BB-raptor-PD1 conjugates expressed significantly less PD-1 on cell surface, and were, therefore, resistant to PD-1-mediated immune suppression. Thus, we introduced a new method to generate memory cells with enhanced functional potential and currently exploring its therapeutic usefulness in various models.

Citation Format: Alexey Berezhnoy, Agata Levay, Anugraha Rajagopalan, Yvonne Puplampu-Dove, Eli Gilboa. Generation of exhaustion-resistant memory CD8 T-cells by simultaneous downmodulation of mTORC1 and PD-1 in activated CD8 T-cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2015-3147
Abstract 3158: Ex vivo expansion and activation of human lymphocytes with a selective activator of effector cells
Authors: Lopes Jared E. , Fisher Jan L. , Losey Heather C. , Ernstoff Marc S. , Alvarez Juan C.
Published: August 2015

RDB 1450 is an engineered fusion protein of circularly permuted IL-2 and IL-2Rα that is selective for the intermediate-affinity IL-2 receptor. The intermediate-affinity receptor is comprised of the IL-2 receptor beta chain (CD122) and common gamma chain (CD132) expressed on lymphocytes with effector function, whereas the high-affinity receptor, which additionally includes the alpha chain (CD25), is expressed primarily on immunosuppressive CD4+CD25+ regulatory T cells (Tregs). Ex vivo proliferation of subsets of human lymphocytes in response to RDB 1450 or rhIL-2 were evaluated. RDB 1450 supports the selective activation and expansion of effector lymphocytes based on higher observed memory CD8 T/Treg and NK/Treg ratios. Both RDB 1450 and IL-2 drive expansion of NK cells, however, a greater proportion of CD25+ NK cells are observed in response to RDB 1450. Furthermore, unlike IL-2, RDB 1450 does not effectively expand highly suppressive ICOS+ Tregs.   The immunological profile resulting from the ex vivo treatment of human PBMCs with RDB 1450 supports its use as a novel immunotherapy for cancer.

Citation Format: Jared E. Lopes, Jan L. Fisher, Heather C. Losey, Marc S. Ernstoff, Juan C. Alvarez. Ex vivo expansion and activation of human lymphocytes with a selective activator of effector cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3158. doi:10.1158/1538-7445.AM2015-3158
Abstract 3159: In vitro characterization of third-generation chimeric antigen receptor T cells directed toward GD2-expressing and BRAF-inhibitor resistant melanoma target cells
Authors: Gargett Tessa , Brown Michael P.
Published: August 2015

Advances in the treatment of metastatic melanoma include the recent approval of the immunotherapy ipilimumab, an anti-CTLA-4 monoclonal antibody, and of the targeted therapies vemurafenib, dabrafenib and trametinib, which are mitogen-activated protein kinase (MAPK)-pathway inhibitors. Cancer immunotherapy also includes adoptive transfer of autologous tumor-specific chimeric antigen receptor (CAR) T cells. CAR T cells are responsible for significant tumor responses in leukemia and neuroblastoma patients. We have manufactured third-generation GD2-specific CAR T cells to be used in our open CARPETS Phase I clinical trial of autologous GD2-CAR T cells in patients with advanced GD2-expressing melanoma (ACTRN12613000198729). However, although these promising new targeted- and immuno-therapies can produce complete or partial tumor responses, tumor resistance via alteration or bypass of the targeted molecules inevitably occurs.Combination therapies have been proposed as a way to exploit the high-level but short-term responses associated with kinase-inhibitor therapies and the low-level but longer-term responses associated with immunotherapy, and could potentially overcome tumor resistance by targeting multiple tumor-associated molecules. To this end, we have investigated the effects of vemurafenib, dabrafenib and trametinib on the function of our GD2-CAR T cells and found that dabrafenib does not inhibit effector functions at concentrations equivalent to reported plasma concentrations in treated patients. We have also observed that although GD2-CAR T cells have reduced killing of BRAF inhibitor -resistant melanoma cell lines, their function is not inhibited by PD1 blockade in vitro.These effects depend on whether GD2-CAR T-cell activation occurs via the native TCR or the CAR and upon the nature of apoptosis resistance in the GD2-expressing melanoma target cells.

Citation Format: Tessa Gargett, Michael P. Brown. In vitro characterization of third-generation chimeric antigen receptor T cells directed toward GD2-expressing and BRAF-inhibitor resistant melanoma target cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3159. doi:10.1158/1538-7445.AM2015-3159
Abstract 316: Development of a novel 3D cancer model by cultivation of malignant effusions in a 3D cell culture system
Authors: Amann Arno , Zwierzina Marit , Huber Julia M. , Gamerith Gabriele , Koeck Stefan , Bitsche Mario , Lorenz Edith , Zwierzina Heinz
Published: August 2015

Introduction: Metastatic spread and the occurrence of tumour cell resistance to therapeutic interventions can be induced by different molecular processes. Among those, tumour stroma interactions play a crucial role. So far only few cell culture models exist to study these cell processes.

Therefore, we describe the development of a hanging drop technology for 3D culture of primary cells isolated from malignant effusion obtained from cancer patients.

Methods: A 96-well hanging drop microtiter plate (InSphero AG, Zürich, Switzerland) was applied for the 3D cultivation of cells. Primary cells were isolated either from malignant ascites or pleura effusion of cancer patients. Cells were seeded either into 2D cell culture plates or into hanging drops for an incubation period of ten days. Apart from conventional histology (H&E), protein expression was displayed immunohistochemically (IHC) and by flow cytometry. The applied antibodies displayed the expression of e-cadherin, vimentin, Ki67, CD31, CD45, Epcam, Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) and α-smooth muscle actin (α-SMA). In addition, viability of all samples was determined by an AnnexinV/Propidium Iodide staining using flow cytometry.

Results: Microtissue aggregation differed significantly depending on whether the malignant cell effusions displayed expression of Epcam. Effusions consisting of a high number of Epcam positive cells formed solid and viable microtissues. The viability of microtissues remained high (>80%) similar to cells cultivated in 2D cell culture plates. Tumour cells, defined as e-cadherin and Epcam positive by flow cytometry were still detected in microtissues after ten days of incubation in 3D.

Furthermore, endothelial cells, defined as CD31 and Lgr5 positive could also be discriminated in microtissues.

Conclusion: We demonstrate that our method is a promising tool for the generation of tumour microtissues consisting of primary isolated cells from malignant effusions. Therefore, our technology represents a promising method to investigate tumour stroma interactions in an in vitro system that reflects more closely the in vivo environment.

Citation Format: Arno Amann, Marit Zwierzina, Julia M. Huber, Gabriele Gamerith, Stefan Koeck, Mario Bitsche, Edith Lorenz, Heinz Zwierzina. Development of a novel 3D cancer model by cultivation of malignant effusions in a 3D cell culture system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 316. doi:10.1158/1538-7445.AM2015-316
Abstract 3166: The role of CD4 T cells in murine model of NASH-promoted HCC
Authors: Ma Chi , Felsher Dean , Greten Tim
Published: August 2015

Aim: Non-alcoholic steatohepatitis (NASH), a common condition in obese patients, is an important risk factor for hepatocellular carcinoma (HCC). However, only little information exists about the immunological mechanisms of how NASH promotes HCC. This study was designed to better understand the role of adaptive immunity during the process of NASH-promoted hepatocarcinogenesis.

Experimental procedure: Methionine choline deficient (MCD) diet is widely used to induce NASH. In this study we fed Lap-tTA-myc mice, which develop HCC, with MCD diet. Choline-deficient and amino acid-defined (CDAA) diet and high fat (HF) diet was also used to induce NASH in B6 mice. GK1.5 antibody was used to deplete CD4 T cells. Gas chromatography-mass spectrometry was performed to identify free fatty acids. Difference immune cell subsets were identified by flow cytometry. Cell death was measured by 7AAD/Annexin V staining.

Results: MCD diet accelerated tumor development in MYC-on mice. A robust selective decrease of hepatic CD4 but not CD8 T cells was observed in NASH mice, which was exacerbated by MYC transgene expression. The selective decrease of hepatic CD4 T cells was also observed in B6 mice treated with CDAA or HF diet suggesting it's a general phenomena in NASH. As expected higher level of cell death was detected in hepatic CD4 T cells after NASH. Phenotypic analysis demonstrated that these CD4 T cells had a CD44highCD62Llow effector memory phenotype and produced more IFNγ. Selective CD4 T cell depletion further accelerated HCC development suggesting an important role of these cells for tumor development. Next, we studied the cause of CD4 T cell death. In co-culture experiments we demonstrated that lipid-laden hepatocytes from mice on MCD diet induced selective CD4 but not CD8 T cells death through a contact-independent mechanism. FFAs were found from hepatocyte culture medium to be responsible for T cell cytotoxicity, and linoleic acid recapitulates the selective toxicity to CD4 T cells. Blocking ROS abolished linoleic acid-induced CD4 T cell death. In vivo study showed that N-acetyl cysteine reversed the NASH-induced hepatic CD4 T cell decrease and delayed NASH-promoted tumor development.

Conclusion: Our results suggest the critical role of CD4 T cells in the disease progression of NASH to HCC, and provide a new link between lipid metabolism dysfunction with impaired anti-tumor surveillance.

Citation Format: Chi Ma, Dean Felsher, Tim Greten. The role of CD4 T cells in murine model of NASH-promoted HCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3166. doi:10.1158/1538-7445.AM2015-3166
Abstract 3172: CCL9 induction in myeloid cells of the premetastatic niche enhances tumor cell survival and metastatic colonization
Authors: Yan Hannah HY , Pang Yanli , Hunter Kent , Khanna Chand , Yang Li
Published: August 2015

Tumor cell survival in the hostile distant organ is a rate-limiting step in cancer metastasis. Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, it is unclear whether these myeloid cells in the premetastatic site have any direct effect on tumor cell survival. Here we report that chemokine CCL9 was highly induced in Gr-1+CD11b+ immature myeloid cells and in premetastatic lung of mice bearing 4T1 mammary tumor and B16 melanoma. Knockdown of CCL9 in myeloid cells decreased tumor cell survival and metastasis. The expression level of CCL23, the human orthologue for CCL9, in peripheral blood mononuclear cells correlated with progression and survival of cancer patients. Our studies identify CCL9 as a key pro-survival factor for cancer cells in the premetastatic organ.

Interestingly, the expression of CCL9 in myeloid cells is regulated by TGF-β signaling as overexpression of CCL9 in myeloid cells lacking TGF-β signaling rescued the tumor metastasis defect observed in mice with myeloid-specific deletion of TGF-β signaling. These data together with our previous report of a fundamental role of myeloid TGF-β signaling in host compromised immune surveillance (Cancer Discovery 2013); demonstrate that myeloid-specific TGF-β signaling constitutes an essential component of the metastasis-promoting puzzle of TGF-β. This finding is different from TGF-β signaling in fibroblasts and a variety of epithelial cells, as well as T cells in which the TGF-β signaling functions as a tumor suppressor. This new understanding of TGF-β at the interface of tumor and host may provide insight into how TGF-β switches from a tumor suppressor to a tumor promoter, a long-standing challenge in cancer biology field. Our study demonstrates that CCL9 could serve as a good candidate for anti-metastasis treatment by targeting the rate-limiting step of cancer cell survival. Additionally, targeting CCL9 may avoid the adverse effects of TGF-β-targeted therapy.

Citation Format: Hannah HY Yan, Yanli Pang, Kent Hunter, Chand Khanna, Li Yang. CCL9 induction in myeloid cells of the premetastatic niche enhances tumor cell survival and metastatic colonization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3172. doi:10.1158/1538-7445.AM2015-3172
Abstract 3174: Retinoblastoma protein 1 implication in the monocytic - granulocytic transition
Authors: Mastio Jerome , Condamine Thomas , Gabrilovich Dmitry
Published: August 2015

Alterations in the myeloipoiesis during cancer lead to production of myeloid-derived suppressor cells (MDSC). MDSC are comprised of two subsets: monocytic MDSC (M-MDSC) and polymorphonuclear MDSC (PMN-MDSC). Both subsets contribute to the immunosuppressive tumor microenvironment. Therefore elimination of MDSC could potentially improve the clinical success of cancer immune therapy. Direct targeting of MDSC progenitors is an attractive possibility. Our laboratory has recently discovered that M-MDSC from the bone-marrow can give rise to PMN-MDSC and that this phenomenon is mediated by the loss of retinoblastoma protein 1 (Rb1) expression. However, a full phenotypic and molecular characterization of these MDSC progenitors has not been established. To identify the subpopulation able to give rise to PMN-MDSC within the M-MDSC population, we used Rb1-GFP reporter mice to track Rb1 expression in vivo. We found that Rb1 low M-MDSC express the stem cell marker c-Kit and low levels of CD115. These Rb1 low c-Kit + M-MDSC are substantially expanded in tumor-bearing mice. Interestingly, this population is also present in naive mice, albeit at very low frequency. Rb1 low c-Kit + monocytic cells from naïve and tumor bearing mice can give rise to granulocytic cells in vitro. These preliminary results indicate that these cells are indeed granulocytic progenitor and are expanded during tumor development. Although this monocyte-granulocyte conversion is associated with a decrease in Rb1 expression, it seems that a minimal Rb1 level is required to ensure this process, at least in naïve mice. Further characterization of the phenotype and molecular profiles of this new MDSC progenitor could lead to the development of new ways to target MDSC.

Citation Format: Jerome Mastio, Thomas Condamine, Dmitry Gabrilovich. Retinoblastoma protein 1 implication in the monocytic - granulocytic transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3174. doi:10.1158/1538-7445.AM2015-3174
Abstract 3175: CD38-expressing myeloid-derived suppressor cells promote tumor growth in a murine model of esophageal cancer
Authors: Karakasheva Tatiana A. , Waldron Todd J. , Eruslanov Evgeniy , Lee Ju-Seog , O'Brien Shaun , Basu Devraj , Singhal Sunil , Malavasi Fabio , Rustgi Anil K.
Published: August 2015

Myeloid derived suppressor cells (MDSCs) are an immunosuppressive population of immature myeloid cells found in advanced stage cancer patients and mouse tumor models. MDSCs suppress T cell-mediated tumor clearance by production of inducible nitric oxide synthase (iNOS) and arginase, as well as through other mechanisms. The purpose of this study was to identify the factor(s) contributing to the pro-tumorigenic role of MDSCs in esophageal squamous cell carcinoma (ESCC).

We have used an unbiased approach to search for changes in global gene expression profile of MDSCs derived from conditional p120-catenin knockout mice (L2-cre;p120ctnf/f, a model of oral-esophageal cancer; Stairs D.B., et al Cancer Cell 2011), compared to immature myeloid cells from age-matched wild-type mice. We have detected a dramatic increase in expression of the Cd38 gene in MDSCs, compared to control cells. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in several mouse tumor models, and CD38high MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38high MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38low MDSCs, likely as a result of increased iNOS production. We have identified novel tumor-derived factors, including IL-6, IGFBP-3 and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have demonstrated that monoclonal antibody-mediated targeting of CD38 results in suppressed tumor growth in vivo in a subcutaneous transplant model of ESCC.

In conclusion, we have shown that expression of CD38 plays an important role in MDSC biology by promoting an enhanced immunosuppressive capacity and a more immature phenotype in vivo. We have also detected an expansion of CD38-positive MDSCs in peripheral blood of advanced stage cancer patients and propose targeting CD38 in vivo as an approach to cancer therapy.

Supported by NCI P01CA098101.

Citation Format: Tatiana A. Karakasheva, Todd J. Waldron, Evgeniy Eruslanov, Ju-Seog Lee, Shaun O'Brien, Devraj Basu, Sunil Singhal, Fabio Malavasi, Anil K. Rustgi. CD38-expressing myeloid-derived suppressor cells promote tumor growth in a murine model of esophageal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3175. doi:10.1158/1538-7445.AM2015-3175
Abstract 3176: Linking suppressive activity and ER-Stress in Myeloid Derived Suppressor Cells
Authors: Condamine Thomas C. , Kumar Vinit , Gabrilovich Dmitry I.
Published: August 2015

In recent years, immunotherapies have shown great promise for the treatment of cancer patients. However, the presence of a very potent immunosuppressive microenvironment in the tumor site remains one of the major obstacles in fully harnessing the potential of immunotherapeutic strategies. Myeloid Derived Suppressor cells (MDSC) represent one of the main suppressive populations inside the tumor. These cells can be divided in two sub-populations: PMN-MDSC and Monocytic-MDSC. They suppress immune response using various well-known mechanisms and accumulate to a large extent in tumor-bearing hosts. Despite the increasing body of literature on MDSC, which mechanisms drives their suppressive capacity remains unclear. Our group recently reported that the ER-Stress response is activated in MDSC isolated from tumor bearing mice and cancer patients. In this study, we investigated the possibility that the ER-stress response could be responsible for the immune suppressive capacities of MDSC. To test this hypothesis, we induce ER-Stress using well-known chemical inducer into non suppressive myeloid cells (PMN and monocyte) and assess their ability to suppress T cells function. Our results show that, following ER-Stress induction, normal myeloid cells were able to suppress T cells response (in murine and human system). To better understand how the ER-Stress response can induce suppressive capacities, we performed microarray analysis following treatment with ER-Stress inducer. Interestingly, we found that arginase-1, one the major enzyme implicated in MDSC suppressive capacities, was induced by more than 5 fold. Altogether, our results suggest that activation of the ER-Stress response in MDSC could be responsible for the up-regulation of arginase-1 and the suppressive phenotype of MDSC. These results suggest that targeting the ER-Stress response in cancer patients could inhibit the suppressive activity of MDSC and improve the efficacy of current immunotherapies.

Citation Format: Thomas C. Condamine, Vinit Kumar, Dmitry I. Gabrilovich. Linking suppressive activity and ER-Stress in Myeloid Derived Suppressor Cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3176. doi:10.1158/1538-7445.AM2015-3176
Abstract 3185: Complement C5 promotes male bias of pancreatic neuroendocrine tumor metastasis
Authors: Contractor Tanupriya , Chan Chang , Kobayashi Shinta , Clausen Richard , Sun Yvonne , da Silva Edaise , Vosburgh Evan , Levine Arnold J. , Tang Laura , Harris Chris R.
Published: August 2015

Among patients with neuroendocrine tumors, males are more likely than females to develop liver metastatic disease. We show that this male bias toward liver metastasis is also observed in a mouse model for pancreatic neuroendocrine tumorigenesis (PanNETs). Interestingly, primary tumors from mice with metastatic disease display hepatomimicry, expressing genes normally expressed in liver. Both male and female primary tumors from mice with metastatic disease show hepatomimicry, although one of the liver genes, complement C5, is only induced in males. In mice knocked out for the complement C5 gene, the frequency of liver metastasis dropped significantly; moreover, males and females lacking a gene for complement C5 developed liver metastasis at the same frequency. Liver metastasis was also prevented by PMX53, a small molecule antagonist of C5aR1/CD88, which is the receptor for complement C5a. Thus complement C5 is epistatic to metastasis and promotes gender bias of liver metastatic disease.

Citation Format: Tanupriya Contractor, Chang Chan, Shinta Kobayashi, Richard Clausen, Yvonne Sun, Edaise da Silva, Evan Vosburgh, Arnold J. Levine, Laura Tang, Chris R. Harris. Complement C5 promotes male bias of pancreatic neuroendocrine tumor metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2015-3185