bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 314: A rapid 3D tumor spheroid analysis method using the Celigo imaging cytometry
Authors: Chan Leo Li-Ying , Cribbes Scott , Vinci Maria , Kessel Sarah , Patterson Lisa , Eccles Sue
Published: August 2015

The current 2D methods for cancer drug discovery have had some difficulty in identifying potential drug candidates that can be used for clinical testing. To overcome this challenge, there has been an increase in research of 3D tissue culture that facilitated the development of new in-vitro tumor model assays. Traditional 2D and 3D analysis method relied heavily on visual observation using microscopy. However, the method is time-consuming and has high variations. Automated plate-based imaging cytometer can be employed to rapidly analyze and characterize 3D tumor spheroids, which can be used to generate both quantitative and qualitative results. In this work, we demonstrate a novel 3D tumor spheroid analysis method using the Celigo imaging cytometer for spheroid counting, size analysis, tumor migration and invasion, tumor viability, and dose response of drug induced/inhibited tumor growth. The plate-based imaging cytometer utilizes bright-field and three fluorescence channels (Blue, Green, and Red) for multi-channel analysis. By utilizing the F theta lens technology, uniform bright-field image is captured for more accurate counting of the entire well. In addition, Celigo analysis software is used to report numerous parameters allowing detailed spheroid characterization. In addition to direct spheroid counting in the well, the use of specific fluorescent dyes and probes allow the user to define viable and hypoxic areas within spheroids and monitor migration and invasion on or into supporting cells and/or tissues. The results showed that Celigo imaging cytometer can accurately count and measure spheroid sizes in response to drug induction. Furthermore, tumor migration and invasion were clearly observed and quantified in the captured images. By utilizing the 3D spheroid imaging cytometry method, researchers can quickly characterize and quantify tumor spheroids, which can improve the efficiency of identification of potential cancer drug candidates.

Citation Format: Leo Li-Ying Chan, Scott Cribbes, Maria Vinci, Sarah Kessel, Lisa Patterson, Sue Eccles. A rapid 3D tumor spheroid analysis method using the Celigo imaging cytometry. [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 314. doi:10.1158/1538-7445.AM2015-314
Abstract 3153: Critical roles of chemo-resistant effector and regulatory T cells in cancer immunotherapy during hemostatic proliferation
Authors: Sato Ko , Watanabe Satoshi , Saida Yu , Tanaka Tomohiro , Baba Junko , Ohtsubo Aya , Shoji Satoshi , Ishikawa Daisuke , Kondo Rie , Okajima Masaaki , Miura Satoru , Tanaka Junta , Kagamu Hiroshi , Yoshizawa Hirohisa , Narita Ichiei
Published: August 2015

Antitumor immunity has been well established to be augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naïve and effector T cells proliferate extensively and show enhanced antitumor effects during homeostatic proliferation when they were adoptively transferred into tumor-bearing hosts that were lymphodepleted with cytotoxic agents or by whole body irradiation. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8+ T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents, such as fludarabine, cisplatin, etoposide, paclitaxel or gemcitabine; however, the percentage of CD4+CD25+Foxp3+ Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemo-resistant Tregs following CPA treatment and transfer of naïve CD4+ T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8+ T cells were responsible for this augmentation. Using Rag2−/− mice or depletion of recipient CD8+ T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4+ T cells enhanced the proliferation of CD8+ T cells and the priming of tumor-specific CD8+ T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies.

Citation Format: Ko Sato, Satoshi Watanabe, Yu Saida, Tomohiro Tanaka, Junko Baba, Aya Ohtsubo, Satoshi Shoji, Daisuke Ishikawa, Rie Kondo, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Critical roles of chemo-resistant effector and regulatory T cells in cancer immunotherapy during hemostatic proliferation. [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 3153. doi:10.1158/1538-7445.AM2015-3153
Abstract 3154: Identification of CD4+ T cell epitopes specific for the breast cancer associated antigen NY-BR-1
Authors: Gardyan Adriane , Osen Wolfram , Agawal Maria , Zörnig Inka , Ruggiero Eliana , Schmidt Manfred , Schneeweiss Andreas , Jäger Dirk , Eichmüller Stefan B.
Published: August 2015

Questions: Whether CD4+ T cell epitopes specific for the breast cancer associated antigen NY-BR-1 can be identified using HLA-transgenic (HLAtg) mice.

Introduction: Adoptive transfer of TCR-transduced autologous tumor antigen-specific T cells provides an innovative strategy for cancer immunotherapy. The differentiation antigen NY-BR-1 is over-expressed in approx. 60% of all invasive mammary carcinomas, thus representing a potential target for cancer immunotherapy. We thus screened NY-BR-1 for the presence of CD4+ T cell epitopes.

Methods: Splenocytes from DNA immunized HLA-DRB1*0301 and HLA-DRB1*0401 tg mice were screened ex vivo by IFNγ ELISpot assay against a NY-BR-1-derived peptide library. In silico predicted candidate epitopes present among recognized library peptides were used to establish CD4+ T cell lines as a read tool to prove HLA-DR-restriction and natural processing of these epitopes by human cells. NY-BR-1-specific CD4+ T cell reactivity among PBMC of breast cancer patients was tested after long term in vitro restimulation with synthetic 15mers by intracellular cytokine staining.

Results: Three HLA-DRB1*0301-restricted and four HLA-DRB1*0401-restricted NY-BR-1-specific CD4+ T cell epitopes were identified in HLAtg mice. HLA-DR-restriction was confirmed upon specific recognition of peptide-loaded T2/DR3 and T2/DR4 target cells by epitope-specific CD4+ T cell lines established from HLAtg mice. These T cell lines specifically recognized human dendritic cells loaded with cell lysates from Ad5-NY-BR-1 infected melanoma cells, showing natural processing of the epitopes. Moreover, upon deep sequencing of TCRs from these murine CD4+ T cell lines two NY-BR-1-specific TCRs were identified which could be considered for the generation of autologous TCR-transduced T cells lines. Finally, CD4+ T cells reactive against the NY-BR-1 specific epitopes were also detected among PBMCs of HLA-DR matched breast cancer patients.

Conclusion: NY-BR-1-specific, HLA-DRB1*0301 and HLA-DRB1*0401 restricted CD4+ T cell epitopes could be identified using HLAtg mice and CD4+ T cells reactive against these epitopes are present among PBMCs of breast cancer patients. Finally, murine HLA-restricted CD4+ T cell lines might serve as source of NY-BR-1-specific TCRs for adoptive immunotherapy approaches.

Citation Format: Adriane Gardyan, Wolfram Osen, Maria Agawal, Inka Zörnig, Eliana Ruggiero, Manfred Schmidt, Andreas Schneeweiss, Dirk Jäger, Stefan B. Eichmüller. Identification of CD4+ T cell epitopes specific for the breast cancer associated antigen NY-BR-1. [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 3154. doi:10.1158/1538-7445.AM2015-3154
Abstract 3156: Construction and validation of an activating and inhibitory chimeric antigen receptor (CAR) system
Authors: Chicaybam Leonardo , Bonamino Martin H.
Published: August 2015

Introduction: Chimeric antigen receptors (CARs) consists a promising type of adoptive immunotherapy. CARs consist of an antigen recognition unit (scFv), a transmembrane (TM) region and an intracellular activation domain. However, on target/off tumor responses due to target recognition in normal cells limits the widespread application of this therapy. In B-cell precursor acute lymphoblastic leukemia, lymphocytes expressing anti-CD19 CARs (19BBz) are being used in the clinical setting. However, the expression of CD19 as a pan-B marker can lead to undesired side effects such as depletion of mature B cells. As mature B cells express both CD19 and CD20 antigens, we propose the creation of an inhibitory anti-CD20 CAR, that when used in combination with 19BBz would be able to discriminate between leukemic blasts and normal B cells. Methods and results: Three inhibitory CARs were constructed containing signaling domains of CTLA-4, PD-1 or BTLA. Jurkat T cells expressing the plasmid pGL4.30 - expressing luciferase controlled by a NFAT responsive promoter - were generated. K562 cell line was used as target cells and modified to express CD19 (K5-19), CD20 (K5-20) or CD19 and CD20 (K5-19/20). In coculture experiments, Jurkat cells expressing 19BBz showed induction of luciferase activity when cultured with K5-19 or K5-19/20. However, Jurkat expressing both activation and inhibitory CAR showed inhibition of luciferase activity when incubated with K5-19/20 while maintained high activity when cultured with K5-19 cells. Furthermore, all three inhibitory CARs were able to inhibit the expression of the activation marker CD69 induced by 19BBz. In primary human T lymphocytes, the 20PD1 CAR did not inhibit proliferation or cytotoxicity induced by 19BBz. Surprisingly, T cells expressing 19BBz and 20PD1 showed preferential lysis of NALM6 CD19+ CD20+ cells, suggesting that the addition of PD1 domain may increase T-cell function in specific contexts. This effect was partially reversed by using a mutant PD1 domain where the tyrosines were replaced by phenylalanine and was completely reversed by using an anti-CD20 CAR truncated at the TM region, suggesting that this phenomenon is dependent of signaling. Conclusions: Additional experiments are needed to evaluate the activity of 20PD1 in primary human T cells. Experiments with inhibitory CARs 20LAG3 and 20PD1LAG3 are ongoing and can serve as an alternative to CAR 20PD1 in the construction of a conditional response system.

Citation Format: Leonardo Chicaybam, Martin H. Bonamino. Construction and validation of an activating and inhibitory chimeric antigen receptor (CAR) 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 3156. doi:10.1158/1538-7445.AM2015-3156
Abstract 3168: A role for tumor-derived CCL2 in directing leukocyte infiltration and stromal heterogeneity in pancreatic ductal adenocarcinoma
Authors: Tooker Graham M. , Gladney Whitney L. , Beatty Gregory
Published: August 2015

Tumor heterogeneity within solid tumors is a recognized barrier to the efficacy of standard therapies. In pancreatic ductal adenocarcinoma (PDAC) the microenvironment is characterized by varying degrees of cytokine/chemokine production, stromal extracellular matrix, and immune cell infiltrates (Hezel et al. 2006). To investigate stromal phenotypical differences in PDAC, we have used the KrasG12D-; Trp53R172H; Pdx-1 Cre (KPC) genetically engineered mouse model which recapitulates tumor heterogeneity seen in human PDAC (Hingorani et al. 2005). Gross histological analysis of KPC tumors allowed us to stratify tumors into 2 broad groups: stromal and non-stromal. We found that stromal tumors were comprised of a significant macrophage infiltrate, whereas non-stromal tumors were generally void of macrophages. We modeled this stromal heterogeneity using tumor cell lines derived from KPC tumors. We found that, when implanted under the skin of wild-type mice, some cell lines produced stromal tumors rich in macrophages, while others formed non-stromal tumors with few macrophages within the microenvironment. Supernatant cultured from these KPC tumor cell lines in vitro revealed elevated CCL2 production, specifically in cell lines from stromal tumors. As CCL2 is a known chemo-attractant for monocytes, we hypothesized that tumor-derived CCL2 contributes to monocyte recruitment and stromal heterogeneity within the tumor microenvironment. To test this hypothesis, we have used an in vitro trans-well migration assay. With this assay, we found that more bone marrow derived cells migrate toward supernatant from stromal tumors relative to non-stromal tumors. This finding was lost in the presence of a CCL2 inhibitor. Ongoing studies are examining a role for tumor-derived CCL2 in regulating tumor-associated stroma in PDAC. Our findings suggest the importance of tumor-immune cell interactions in defining stromal heterogeneity and may offer insight into mechanisms that regulate the stromal compartment in human PDAC.

Citation Format: Graham M. Tooker, Whitney L. Gladney, Gregory Beatty. A role for tumor-derived CCL2 in directing leukocyte infiltration and stromal heterogeneity in pancreatic ductal adenocarcinoma. [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 3168. doi:10.1158/1538-7445.AM2015-3168
Abstract 3186: Role of Interleukin 1 signaling in tumor elicited inflammation and colon cancer
Authors: Dmitrieva Oxana , Possoco David , Francescone Ralph A. , Hou Vivianty , Vendramini-Costa Debora B. , Grivennikov Sergey
Published: August 2015

Colorectal cancer (CRC) is the 2nd most common cause of cancer deaths in the United States and other developed countries, despite important advances in detection, surgery and chemotherapy. It becomes increasingly clear that not only cell autonomous events in cancer cells are required for tumor growth and progression. Growing CRC tumors possess the ability to recruit inflammatory cells and upregulate inflammatory cytokines - a phenomenon recently introduced as “Tumor elicited inflammation”(TEI). The mechanisms of TEI induction and tumor promotion are not yet well defined. IL23/IL17 pathway is one of the established regulators of TEI. While in mice the ablation of IL-23 signaling results in a uniform loss of IL-17 response and in a decrease in tumor growth; in humans IL23/IL-23R gene expression signature is not always consistent with elevated IL-17 signature, implicating that there should be other regulators of IL-17 and TEI induction in tumors. IL-1 pathway is important for the induction of T helper IL-17 producing cells (Th17) and for the production of IL-17 by innate lymphoid cells (ILC), both in mice and in humans. IL-1 pathway is also implicated in host defense, immunity and tumorigenesis, with some of its functional characteristics similar to IL-23 pathway. In addition, IL-1, much alike IL-23, can be also induced by various microbial derived and host derived ‘danger signals’ readily available within the tumor microenvironment. Our own data suggests that components of IL-1 pathway are upregulated in CRC tumors. Therefore, we hypothesized, that IL-1 pathway might be an important regulator of TEI and CRC tumorigenesis. Using genetic tools and animal models, we examined if IL-1 has the potential role in different stage of CRC and whether it serves as an important regulator of TEI and CRC tumor cells proliferation and survival.

Citation Format: Oxana Dmitrieva, David Possoco, Ralph A. Francescone, Vivianty Hou, Debora B. Vendramini-Costa, Sergey Grivennikov. Role of Interleukin 1 signaling in tumor elicited inflammation and colon 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 3186. doi:10.1158/1538-7445.AM2015-3186
Abstract 3202: Single cell analysis of ascites macrophages in ovarian cancer
Authors: He Huanhuan Mahsa , Yu Feiqiao Brian , Quake Stephen R. , Dorigo Oliver
Published: August 2015

Macrophages compose a major part of the tumor microenvironment (TME) in several types of cancer, including ovarian cancer [1]. Studies have shown that a high percentage of tumor associated macrophages (TAMs) correlates with a poor prognosis in ovarian cancer [2], due to the crucial role of TAMs in promoting tumor growth and angiogenesis [3,4]. Yet not much investigation has been executed to reveal the function of macrophages in ovarian cancer ascites, a hallmark of ovarian cancer.

Previous gene expression analysis in TAMs often used bulk samples [5,6]. However, due to the complexity of the peritoneal environment, this approach had the effect of “dampening” rare and potentially informative signals from a heterogeneous population such as ascites macrophages. Therefore, we resorted to single-cell expression analysis to dissect the heterogeneity of ascites macrophages.

Recent breakthroughs in single cell RNA-seq techniques have made it more cost effective to process a large number of samples at single cell resolutions. Here we used Fluidigm's Single-Cell Auto Prep System, a state-of-art technology for automate single cell cDNA synthesis [7]. We sequenced single ascites macrophages from ID8 murine ovarian cancer model and single alternatively activated bone marrow derived macrophages (BMDM-M2). When comparing the single cell expression profiles of ascites macrophages to BMDM using principle component analysis (PCA), we observed that the ascites macrophages scattered broadly and could be further divided into two groups, whereas BMDM were tightly clustered into one group. Furthermore, gene oncology (GO) analysis of highly expressed genes in each group revealed that ascites macrophages had a distinct gene expression profile from BMDM, highlighting the metabolic pathways, especially catabolic process. We are currently working on validating these findings in human patients with ovarian carcinoma. In summary, this work sheds light on the understanding of the role of ascites macrophages in ovarian cancer.

1. Qian BZ, Pollard JW. Cell. 2010;141:39-51.

2. He YF, Zhang MY, Wu X, et al. PLoS One. 2013;8:e79769.

3. Mantovani A, Germano G, Marchesi F, et al. Eur J Immunol 2011;41:2522-25.

4. Murray PJ, Wynn TA. Nat Rev Immunol, 2011;11:723-37.

5. The Cancer Genome Atlas Research Network. Nature. 2014;511:543-50.

6. The Cancer Genome Atlas Research Network. Nature. 2014;513:202-9.

7. Wu AR, Neff NF, Kalisky T, et al. Nat Methods. 2014;11:41-6.

Citation Format: Huanhuan Mahsa He, Feiqiao Brian Yu, Stephen R. Quake, Oliver Dorigo. Single cell analysis of ascites macrophages in ovarian 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 3202. doi:10.1158/1538-7445.AM2015-3202
Abstract 3253: Metastasis suppression by non-KP54 kisspeptins
Authors: Hampton Kelsey R. , Pounds Keke M. , Tremblath Andrew P. , Welch Danny R.
Published: August 2015

The KISS1 metastasis suppressor blocks metastasis by inhibiting colonization at secondary sites. KISS1 activity requires secretion and extracellular processing by furin into kisspeptins. Kisspeptin-54 (KP54; G68 to F121) binds a G-protein coupled receptor, KISS1R, and the mechanism of metastasis suppression has been assumed to be through the receptor. However, KISS1 is cleaved into kisspeptins outside of the cell, and KISS1R expression is not required for metastasis suppression. Therefore, an alternate hypothesis is that a non-KISS1R binding kisspeptin(s) is responsible for metastasis suppression. To test this hypothesis, we engineered all ten of the possible kisspeptins predicted from known dibasic cleavage sites (M1-Q145; M1-R56; M1-R67; M1-R124; R56-R67; G68-F121; E55-F121; E55-Q145; G68-Q145; E125-Q145) downstream of the KISS1 secretion signal peptide (designated KMP1 to KMP10, respectively) and expressed each individually in B16-F10 melanoma cells. Expression was confirmed by immunoblot and/or RT-QPCR analysis. Following intravenous injection, experimental lung metastases were quantified. KP54 expression suppressed metastasis. Some KMP lacking the KISS1R ligand suppressed metastasis as well as KP54 and full-length KISS1 (p<0.05). Specifically, the N-terminal kisspeptin (M1 - R56) dramatically suppressed metastasis. This suggests that the mechanism of action for metastasis suppression by KISS1 does not necessarily require activation of KISS1R by the peptide ligand but also leaves the possibility for additional receptors. Alternatively, KISS1/KMP interactions with other intracellular proteins may be required for metastasis suppressor activity. Support: CA134981, Natl Fndn Cancer Res, Komen SAC11037, KS Bioscience Auth, P30-CA168524, Steiner Family Fund.

Citation Format: Kelsey R. Hampton, Keke M. Pounds, Andrew P. Tremblath, Danny R. Welch. Metastasis suppression by non-KP54 kisspeptins. [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 3253. doi:10.1158/1538-7445.AM2015-3253
Abstract 3255: The role of Hdj2 protein in metastatic progression of experimental intracranial glioma
Authors: Meshalkina Darya A. , Shevtsov Maxim A. , Margulis Boris A. , Guzhova Irina V.
Published: August 2015

Glioblastoma is one of the most malignant cancer types with median survival of 15 months regardless of the application of combined radio- and chemotherapy. Expression of Hdj2 negatively correlates with the increase of tumor aggressiveness. For assessment of Hdj2 role in tumor progression, invasiveness and metastasis formation in gliomas we developed three C6-based cell lines with Hdj2 and related proteins knock-down: C6 shHsp70, C6 shHdj1 and C6 shHdj2 itself. Following intracranial injection of the modified tumor cells the animals’ survival was estimated. As compared to control C6 (25.4±3.9 days) and C6 shHdj1 (25.5±3.8) we observed a nearly 1.5-fold decrease in survival in C6 shHdj2 (16.8±3.5 days) (P<0.05). On the contrary, in C6 shHsp70 group the survival increased up to 42.5±12.0 days (the increase is completely explainable by the slower growth rate of the culture). Subsequent MR imaging and histological analysis of the tumors demonstrated elevated invasiveness and metastatic activity in C6 shHdj2 group in comparison to C6 shHsp70, C6 shHdj1 and control C6. High migration activity and the ability of detached C6 shHdj2 cells to settle on the substrate was proved in wound-healing assay, colony forming assay and calcium-dependent cell aggregation assay. Immunofluorescence analyses showed loss of membrane-expressed N-cadherin and loss of intercellular contacts mediated by N-cadherin in C6 shHdj2 cells in comparison to other considered cell lines. Actin staining with rhodamine-falloidin revealed highly abundant leading edges in C6 shHdj2 culture.

Decrease of Hdj2 expression in gliomas has proved to be of high importance in tumor progression and formation of metastasis.

Citation Format: Darya A. Meshalkina, Maxim A. Shevtsov, Boris A. Margulis, Irina V. Guzhova. The role of Hdj2 protein in metastatic progression of experimental intracranial glioma. [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 3255. doi:10.1158/1538-7445.AM2015-3255
Abstract 3257: NME1 mediates a switch in expression of beta integrin subunits that correlates with prolonged patient survival
Authors: Leonard M. Kathryn , Novak Marian , Yang Xiuwei , Belkin Alexey , Kaetzel David
Published: August 2015

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the molecular mechanisms underlying this activity are not completely understood. Herein we report a novel mechanism through which NME1 modulates focal adhesion dynamics via regulation of integrin β1. Stable expression of NME1 significantly altered focal adhesion turnover at the cell periphery. Interestingly, over-expression of NME1 resulted in a switch in from predominantly fast recycling α4β1 integrins to slower recycling αvβ3 integrins. Contrary to its regulation of other cell surface receptors, the inhibition of integrin β1 by NME1 was found to occur at the transcriptional level rather than through dynamin-mediated endocytosis. This effect of NME1 required both its 3-5′ exonuclease and nucleoside diphosphate kinase (NDPK) activities, which are also required for its metastasis suppressor activities in vivo. Furthermore, an inverse correlation was observed between NME1 and integrin β1 mRNA in a large cohort of primary melanoma biopsies. The inverse correlation of NME1 and integrin β1 RNA was also a strong predictor of prolonged distant disease-free and overall survival in patients with the basal-like subtype of breast carcinoma. Together, these data strongly suggest NME1 suppresses metastasis of human melanoma and some types of breast cancers by inhibiting integrin β1 expression, which reduces recycling of focal adhesions and suppresses cell motility.

Citation Format: M. Kathryn Leonard, Marian Novak, Xiuwei Yang, Alexey Belkin, David Kaetzel. NME1 mediates a switch in expression of beta integrin subunits that correlates with prolonged patient survival. [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 3257. doi:10.1158/1538-7445.AM2015-3257
Abstract 3258: Breast Metastasis Suppressor 1 (BRMS1) phosphorylation appears necessary for metastasis suppressor activity
Authors: Contreras Lellys M. , Sarcevic Boris , Pounds Keke M. , Welch Danny R.
Published: August 2015

Breast cancer is the most common cancer among women worldwide. BRMS1 is a metastasis suppressor that suppresses metastasis without significantly affecting the growth of a primary tumor. BRMS1 contains two coiled-coil motifs important for protein-protein interactions and two putative nuclear localization sequences (NLS - aa198-205 (NLS1) and aa239-245 (NLS2)) and one nuclear export signal which appear to mediate nuclear-cytoplasmic shuttling. Although NLS2 is not necessary for active transport into the nucleus, it is required for metastasis suppression (PMC3563580). A serine immediately upstream of NLS2 (S237) can be phosphorylated by cdk2 (PMC3037622). Therefore, we hypothesized that NLS2 and adjacent amino acids are critical mediators of metastasis suppression. To test this hypothesis, NLS2 (aa231-248) epitope-tagged with HA and GST was transduced into metastatic MDA-MB-231 and -435 breast carcinoma cells expressing green fluorescent protein and stable colonies were selected. BRMS1S237A-expressing MDA-MB-231 cells were no longer metastasis suppressed, suggesting a critical role for phosphorylation of BRMS1 in its metastasis suppressor activity. Studies are underway with full-length BRMS1S237D (phosphomimetic) as well as expression with truncated wild-type NLS2 and mutant (NLS2wt/S237A/S237D) to test whether the BRMS1 phosphorylation or the NLS2 domain itself is critical for metastasis suppressor activity. Support: CA87728, CA134981, P30-CA168524, Natl Fndn Cancer Res, Komen SAC11037, KS Bioscience Auth, NHMRC

Citation Format: Lellys M. Contreras, Boris Sarcevic, Keke M. Pounds, Danny R. Welch. Breast Metastasis Suppressor 1 (BRMS1) phosphorylation appears necessary for metastasis suppressor activity. [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 3258. doi:10.1158/1538-7445.AM2015-3258
Abstract 3265: Thrombopoietin and megakaryocytes in breast cancer metastasis to bone
Authors: Jackson Walter , Mastro Andrea M. , Sosnoski Donna M.
Published: August 2015

Bone is one of the preferred sites for breast cancer cells to metastases. Once patients are diagnosed with skeletal metastases, the relative five-year survival rate falls from 90% to less than16%. Thrombocytosis is a sign of poor prognosis for survival, and thromboembolisms are one of the leading causes of death in all cancers patients. These conditions are due to high numbers and/or high platelet activity. Megakaryocytes (MKs) are responsible for platelet production, but also play a role in bone metabolism and skeletal homeostasis.

We had determined in two mouse models of experimental metastasis (MDA-MB-231 in athymic mice, and 4T1.2 in Balb/c mice), that bone metastasis was associated with increased numbers of MKs. Under normal conditions MKs represent less than 1% of the total bone marrow cell population, but after analysis of the femurs of the mice with metastasis, we found at least a two-fold increase in the numbers of MKs present in the bone marrow in the MDA-MB-231mice. The Balb/c mice injected in the mammary glands with mouse 4T1.2 cells showed a four-fold increase in MKs in the spleen (extrameduallary hematopoiesis). Therefore we hypothesized that increased megakaryocytosis was linked to metastasis, and that decreasing the number of MKs would decrease metastasis. We transferred a thrombopoietin (TPO) expression deletion mutation to a Balb/c background and inoculated the mice with 4T1.2 cells. These TPO -/- mice showed approximately a 90% decrease in bone marrow MKs compared to the wild type mice. The numbers of blood platelets was also decreased by 90%. We tested our hypothesis by inoculation of 4T1.2LUC cells into the mammary gland, and then followed the course of the cancer and metastasis by luciferase imaging. Contrary to our hypothesis the TPO -/- mice developed metastasis to the bone more rapidly than the heterozygotes or wild type mice. Literature suggests that platelets interact with tumor cells to contribute to hematogenous metastasis and angiogenesis. In the TPO-/- mice with very few platelets, metastasis was more aggressive and developed more rapidly than in the wild type or the heterozygote mice. The next step will be to separate the role of the megakaryocytes from the platelets.

This work was sponsored by the U.S. Army Medical Research and Materiel W81XWH-10-1-0253

Citation Format: Walter Jackson, Andrea M. Mastro, Donna M. Sosnoski. Thrombopoietin and megakaryocytes in breast cancer metastasis to bone. [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 3265. doi:10.1158/1538-7445.AM2015-3265
Abstract 3289: Skeletal consequences of bone resorption inhibitors (zoledronic acid and RANKL blocking antibody) injections during growth: mouse strain disparities and synergic effect
Authors: Lezot Frederic P. , Navet Benjamin , Choi YongWon , Yagita Hideo , Berdal Ariane , Mueller Christopher G. , Redini Francoise , Heymann Dominique
Published: August 2015

Abstract

Background: High doses of bone resorption inhibitors are currently evaluated for the treatment of malignant primary bone tumors. The impact of such an intensive treatment on growing skeleton is a critical question in the context of pediatric oncology.

Methods: Protocols adapted from pediatric treatments were developed for mice (a total of 4 or 5 injections of Zoledronic acid (50 μg/kg) or IKK-22.5 antibody (50 μg/injection) every two or three days). Their impact on tibias, skull bones and teeth growth was analyzed by micro-CT and histology up to 3 months after the last injection. Potential disparities between mouse strains were evaluated using both CD1 and C57BL/6J mice, as was the possible synergic effect of the two inhibitors.

Results: In both mouse strains, bone resorption inhibitors administrations induced a transient delay of tibia and skull bone growth. Indeed, three months after the last injection all bone parameters were back to normal. Regarding tooth eruption and root elongation, both were severely impaired in C57BL/6J mouse whatever the inhibitor considered while in CD1 mouse highly more important doses are necessary to observe similar effects. Combination of both inhibitors, at half-doses in CD1 mouse, induced severe delay of tooth eruption demonstrating the existence of synergic effect between the two inhibitors.

Conclusions: In mouse, high doses of bone resorption inhibitors disturbed teeth eruption and elongation, and delayed tibias and skull bone formation. Difference of sensitivity to these inhibitors was observed between mouse strains which understanding will require further studies. A synergic effect was also observed for these inhibitors. In human, similar treatments may impact the permanent teeth eruption. These observations are crucial for the follow-up of pediatric patients treated with high doses of bone resorption inhibitors in ongoing European and American protocols.

Citation Format: Frederic P. Lezot, Benjamin Navet, YongWon Choi, Hideo Yagita, Ariane Berdal, Christopher G. Mueller, Francoise Redini, Dominique Heymann. Skeletal consequences of bone resorption inhibitors (zoledronic acid and RANKL blocking antibody) injections during growth: mouse strain disparities and synergic effect. [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 3289. doi:10.1158/1538-7445.AM2015-3289
Abstract 3308: Enhanced expression of galectin-3 isassociated with the alternative activation of macrophages and development oflung fibrosis following radiation
Authors: Zhao Weiling , Zhang Jing-Hua , Lopes Ingrid J.
Published: August 2015

Radiotherapy is a major treatment approach to non-operable lung cancer patients. Unfortunately, radiation-induced lung injury occurs in 5-20% of lung cancer patients following radiotherapy. Radiation-induced lung fibrosis contributes to an increased risk of death or a decreased quality of life.

The mechanism(s) for the development of radiation-induced lung fibrosis is not fully understood, and no interventions to prevent/ameliorate it have proven successful. Macrophages are the first line of defense for airways and alveolar surfaces and play a key role in the initiation and orchestration of inflammatory responses. Two distinct macrophage subsets have been identified; classically activated macrophages (M1) and alternatively activated macrophages (M2).

Increased numbers of activated M2 macrophages have been seen in human fibrotic conditions and rodent models of fibrosis. However, the mechanism(s) driving the persistent activation of M2 macrophages and how this leads to the development of fibrosis remains undefined. Recent studies indicate that galectin-3 (gal-3) is potentially a key regulator of M2 macrophage activation; inhibition of gal-3 expression prevents M2 macrophage activation. In this paper, we show elevated numbers of Ym-1 positive alveolar macrophages in the irradiated lung. Increased expression of gal-3 cells was found in the fibrotic lung, which was associated with fibrotic lesions. M2 macrophages were the majority of cells that expressed gal-3.

In our in vitro studies, we found that knocking-down gal-3 using Mission lentiviral transduction particles prevented radiation-induced increases in the expression of YM-1 in Raw 264.7 macrophage. Mouse recombinant gal-3 stimulated mouse lung fibroblast cell proliferation and expression of α-SMA. These data suggest that gal-3 appears to regulate alternative activation of M2 macrophage and play an important role in the development of radiation-induced lung injury.

Citation Format: Weiling Zhao, Jing-Hua Zhang, Ingrid J. Lopes. Enhanced expression of galectin-3 isassociated with the alternative activation of macrophages and development oflung fibrosis following radiation. [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 3308. doi:10.1158/1538-7445.AM2015-3308
Abstract 3309: Co-inhibition of ALK and EGFR and/or c-MET on cell growth and response to radiation in ALK-positive NSCLC cells
Authors: Li Chunrong , Huang Shyhmin , Ma Fang , Armstrong Eric A. , Francis David , Werner Lauryn , Harari Paul M.
Published: August 2015

ALK (anaplastic lymphoma kinase), EGFR (epidermal growth factor receptor) and c-MET are molecular drivers for a subset of non-small cell lung cancers (NSCLC). Approximately 4-7% of NSCLCs carry the EML4-ALK chromosomal rearrangement. We identified overexpression of EGFR and c-MET in EML4-ALK-positive NSCLC cell lines H3122 and H2228. ALK, EGFR, and c-MET may function cooperatively to modulate tumor growth, stress response, and survival in EML4-ALK-positive cells and co-inhibition may favorably impact tumor control. We therefore investigated the effects of co-inhibition of ALK and EGFR or c-MET on cell survival and response to radiation in EML4-ALK-positive NSCLC cells.

Compared with ALK-negative cell lines (H226, A549 and H520), the ALK-positive cell lines (H3122, H2228) were highly sensitive to the ALK inhibitors crizotinb, ceritinib and alectinib, although with differing sensitivity to radiation. Crizotinib, ceritinib and alectinib sensitized H3122 and H2228 to radiation, as evaluated by clonogenic assay. However, knockdown of ALK by siRNA in H3122 and H2228, or overexpression of EML4-ALK in NIH-3T3 cells, had only moderate effects on radiosensitivity. Simultaneous knockdown of ALK and MET, or ALK and EGFR, increased radiosensitivity as assessed by clonogenic survival in H3122 and H2228 cells, while also increasing inhibitory effects on cell growth and proliferation. These results were confirmed in cells treated with the combination of ceritinib and su11274 (c-MET inhibitor) or with ceritinib and erlotinib (EGFR inhibitor). We further assessed the impact of drug alone or in combination with radiation on cell cycle, cell survival and DNA damage repair pathways. To further evaluate ALK function in the cellular response to radiation in NSCLC, we are developing EML4-ALK stable expression cell lines. Evaluation of drug-radiation interactions in xenograft model systems are also under consideration.

These data indicate that co-inhibition of ALK and EGFR or c-MET can enhance growth inhibitory effects in ALK-positive cells and augment radiosensitivity in-vitro, suggesting the potential value for ALK inhibitors combined with EGFR/c-MET-targeting for therapeutic benefit in ALK-positive NSCLC patients with or without radiation.

Citation Format: Chunrong Li, Shyhmin Huang, Fang Ma, Eric A. Armstrong, David Francis, Lauryn Werner, Paul M. Harari. Co-inhibition of ALK and EGFR and/or c-MET on cell growth and response to radiation in ALK-positive NSCLC 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 3309. doi:10.1158/1538-7445.AM2015-3309
Abstract 3331: Gene expression profiling after radiation in human breast cancer specimens and breast cancer cell lines
Authors: Siamakpour-Reihani Sharareh , Chen Wei , Lee Chen-Ting , Zhou Yingchun , Owzar Kouros , Chi Jen-Tsan , Horton Janet K.
Published: August 2015

Background: Breast radiotherapy is currently “one size fits all” regardless of breast cancer subtype (eg. luminal, basal). Emerging clinical data suggests that these distinct subtypes of breast cancer have unique patterns of response to radiation. There is a clinical need to identify radiation response biomarkers in order to provide individualized and optimal radiotherapy. Here we report on gene expression profiles associated with radiation response from: i) a rare clinico-genomic data in women treated with preoperative breast radiation and, ii) data from a pilot study of 16 biologically diverse breast tumor cell lines. Methods: Microarray analysis was done using the Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) and the Affymetrix HU133A2.0 arrays. RNA was harvested from paired pre- and post-radiation formalin-fixed paraffin-embedded breast cancer tissue in 26 patients treated with radiation prior to surgical resection. In addition, RNA was obtained from 16 biologically diverse breast cancer cell lines exposed to radiation treatment (5Gy and 0Gy). The genes with the highest coefficient of variation were selected and a paired sample t-test was used to evaluate the significance of these changes. We compared the top 100 significantly induced human genes with genes identified as differentially expressed in the 16 breast cancer cell lines. Findings of interest were validated using qPCR, IHC and wWestern blotting assays. Results: Fourteen genes had significant adjusted p-values for differential expression in the human data and were differentially expressed among the radiation responsive versus non-responsive cell lines. Differential expression of genes involved in: apoptosis, cell cycle and MAPK signaling pathways were observed in both the luminal human tumors and the largely luminal radiation responsive cell lines. Differential expression of FAS, a critical modulator of programmed cell death, was highly significant. Conclusion: The known function of FAS suggests a biologically plausible role in breast subtype specific radiation response. Induction of FAS is seen in human tissue, and preferentially in luminal breast cancer cell lines.

Citation Format: Sharareh Siamakpour-Reihani, Wei Chen, Chen-Ting Lee, Yingchun Zhou, Kouros Owzar, Jen-Tsan Chi, Janet K. Horton. Gene expression profiling after radiation in human breast cancer specimens and breast cancer cell lines. [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 3331. doi:10.1158/1538-7445.AM2015-3331
Abstract 3338: Pharmacological inhibition of MRK/ZAK kinase for the treatment of medulloblastoma
Authors: Ruggieri Rosamaria , Markowitz Daniel , Powell Caitlin , Tran Nhan , Vanan Magimairajanissai , He Mingzu , Al-Abed Yousef , Symons Marc
Published: August 2015

Medulloblastoma is a cerebellar tumor and the most common pediatric brain malignancy. Radiation therapy is part of the standard care for this tumor, but its effectiveness is accompanied by significant neurocognitive sequelae due to the deleterious effects of radiation on the developing brain.

We have previously shown that the protein kinase MRK/ZAK protects tumor cells from radiation-induced cell death by regulating cell cycle arrest after ionizing radiation. Here we show that siRNA-mediated MRK depletion sensitizes medulloblastoma primary cells to radiation. We have, therefore, designed and tested a small molecule inhibitor of MRK, M443, which binds to MRK in an irreversible fashion and inhibits its activity. We found that M443 strongly radio-sensitizes UW228 medulloblastoma cells as well as IMB226 patient-derived primary cells. M443 also inhibits radiation-induced activation of both p38 and Chk2, two proteins that act downstream of MRK and are involved in DNA damage-induced cell cycle arrest. We also tested the effect of M443 in an animal model of medulloblastoma that employs orthotopic implantation of IMB226 medulloblastoma cells in nude mice. Intra-tumoral delivery of M443 alone significantly extended animal survival by 5 days compared to vehicle treatment. Combination treatment of M443 with radiation at 2 × 3 Gy, that is not effective on its own (1 day of additional survival over control), achieved a synergistic increase in survival (15 days over the control survival time). Western blotting of tumor lysates demonstrated strong inhibition of MRK activity.

In conclusion, we have developed a new small molecule inhibitor of MRK/ZAK that radio-sensitizes medulloblastoma cells. We hypothesize that combining radio-therapy with M443 will allow us to lower the radiation dose while maintaining therapeutic efficacy, thereby minimizing radiation-induced side effects.

Citation Format: Rosamaria Ruggieri, Daniel Markowitz, Caitlin Powell, Nhan Tran, Magimairajanissai Vanan, Mingzu He, Yousef Al-Abed, Marc Symons. Pharmacological inhibition of MRK/ZAK kinase for the treatment of medulloblastoma. [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 3338. doi:10.1158/1538-7445.AM2015-3338
Abstract 3339: Silibinin improves radiotherapeutic efficacy in prostate cancer by reducing IR-induced toxicity and EMT
Authors: Nambiar Dhanya K. , Rajamani Paulraj , Jain Anil , Deep Gagan , Agarwal Rajesh , Singh Rana P.
Published: August 2015

Radiotherapy of prostate carcinoma is well established and frequently utilized in a significant proportion of cancer patients. However, two major concerns with radiotherapy is the effect on normal tissues and development of more invasive-radioresistant population. We have previously shown that the use of a plant flavonoid, silibinin can radiosensitize prostate cancer cells preferentially in vitro, by inhibiting DNA damage repair. We extended this study further, to validate the effects of silibinin in improving the radiotherapeutic efficacy in vivo. Our results showed that, in vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P<0.01) with higher apoptotic response (10-fold, P<0.01) and reduced repair of DNA damage as evidenced by reduced Chk2 activation and enhanced pH2A.X foci in tumor tissues. Interestingly, we also observed that silibinin could rescue the mice from IR-caused hematopoietic injury and normal tissue toxicity. Studies have shown that ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of radiation induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. In the current study, we demonstrate that silibinin can also radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it significantly reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. We have found that most of the pro-angiogenic, migratory and EMT promoting proteins were up regulated in response to IR in PCa cells. All of these invasive and EMT promoting actions of IR were markedly decreased by silibinin. Further, we found that potentiated effect was an end result of attenuation of IR activated mitogenic and pro-survival signaling, including Akt, Erk1/2 and STAT-3 by silibinin. Therefore, the study not only underlines the preferential radiosensitizing ability of silibinin in PCa, but also shows its efficacy in modulating IR-induced toxicity in normal cells and EMT in PCa cells.

Citation Format: Dhanya K. Nambiar, Paulraj Rajamani, Anil Jain, Gagan Deep, Rajesh Agarwal, Rana P. Singh. Silibinin improves radiotherapeutic efficacy in prostate cancer by reducing IR-induced toxicity and EMT. [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 3339. doi:10.1158/1538-7445.AM2015-3339