bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3235: Validation of anti-human PD1 and PD-L1 antibodies in MiXeno mouse models
Authors: Zhang Juan , Qiao Meng , Zhang Lan , Shi Qian
Published: August 2015

As evidenced by strong clinical data from antibodies targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death protein-1 (PD-1), and its ligand PD-L1, cancer immunotherapy is now a major breakthrough in cancer treatment. Meanwhile, new immunotherapeutic agents with various targets and mechanisms, as well as new drug combinations are under active preclinical development. However, reliable humanized animal models are of increasing importance as more programs are reaching the in vivo stage, where direct evaluation of therapeutics with anti-human targets are necessary. We have previously reported our efforts in generating mouse MiXeno models that harbor human immune cells by engrafting immuno-deficient mice with human PBMC (the MiXenoTM model) to evaluate anti-PD-L1 antibody. In the current study, we carried out extensive validation of these models in vivo by generating multiple cell line-derived MiXeno models and testing them with anti-human PD-1 or PD-L1 antibodies in different animal strains (NOD/SCID vs. NPG). Our studies demonstrated that MiXeno models can successfully reconstitute human T cells in mice to support the anti-tumor activity of the immuno-therapeutic agents. MiXeno model is simple and fast, and may become valuable tools for in vivo evaluation of immunotherapeutic agents.

Citation Format: Juan Zhang, Meng Qiao, Lan Zhang, Qian Shi. Validation of anti-human PD1 and PD-L1 antibodies in MiXeno mouse models. [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 3235. doi:10.1158/1538-7445.AM2015-3235
Abstract 3254: Promotion of colon cancer invasion and metastasis via activation of Notch-Dab1-Abl-RhoGEF protein Trio
Authors: Taketo Mark
Published: August 2015

We have recently identified a metastasis suppressor gene for colorectal cancer (CRC); AES/Aes which encodes an endogenous inhibitor of Notch signaling. When Aes is knocked out in the adenomatous epithelium of intestinal polyposis mice, their tumors become malignant, showing marked submucosal invasion and intravasation. Here we show that one of the genes induced by Notch signaling in CRC is DAB1/Dab1. Genetic depletion of Dab1 suppresses cancer invasion and metastasis in the Notch signaling-activated mice. Dab1 is phosphorylated by Abl tyrosine kinase, which activates Abl reciprocally. Consistently, inhibition of Abl suppresses cancer invasion in mice. Furthermore, we show that one of the targets of Abl Tyr-kinase is Rac/Rho-GEF protein Trio, and that phosphorylation at its Tyr residue 2681 (pY2681) causes Rho activation in CRC cells. Its unphosphorylatable mutation Trio(Y2681F) reduces the RhoGEF activity, and inhibits invasion of CRC cells. Importantly, Trio(pY2681) correlates with significantly poorer prognosis of CRC patients after surgery. These results indicate that Trio(pY2681) is one of the downstream effects by Notch signaling activation in CRC, and can be a prognostic marker, helping determine the therapeutic modality of CRC patients.

Citation Format: Mark Taketo. Promotion of colon cancer invasion and metastasis via activation of Notch-Dab1-Abl-RhoGEF protein Trio. [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 3254. doi:10.1158/1538-7445.AM2015-3254
Abstract 3269: Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level
Authors: Celik Haydar , Sajwan Kamal P. , Pai Amrita V. , Marsh Ben J. , Saygideger Kont Yasemin , Rahim Said , Han Jenny , Minas Tsion , Toretsky Jeffrey A. , Uren Aykut
Published: August 2015

Ezrin is a member of the ezrin-radixin-moesin (ERM) family of actin-membrane linker proteins that play key roles in regulating cell shape, movement, adhesion and signal transduction pathways. The expression of ezrin is linked to the metastatic progression in several cancers including osteosarcoma (OS). We discovered a small molecule, NSC305787, that inhibits ezrin activity and metastatic phenotype both in vitro and in vivo. We hypothesized that the anti-metastatic effects of NSC308787 could be mediated through preventing specific protein-protein interactions involving ezrin. In this study, we used affinity pull-down coupled with mass spectrometry-based proteomic approach to unravel putative ezrin interactors that are competed away by NSC305787. We identified a number of candidate ezrin binding proteins that are associated with metastatic behavior and implicated in the regulation of stress granule dynamics and protein translation initiation. We selected DDX3, a DEAD-box RNA helicase, as a candidate for further analysis. We confirmed that ezrin directly binds to DDX3. Depletion of ezrin protein expression by RNA interference in several cancer cell lines resulted in substantial reduction in DDX3 protein levels without affecting its transcription, which suggested that ezrin is required for post-transcriptional maintenance of DDX3 in the cell. Paradoxically, recombinant ezrin specifically inhibited the RNA duplex unwinding activity and stimulated the ATPase activity of DDX3. Our data suggest that ezrin regulates the translation of mRNAs with 5′ secondary structure through DDX3, at least in part, through maintaining its intracellular protein level and/or modulating its unwinding and ATPase activities. Therefore, our findings suggest that a novel function of ezrin regarding the regulation of mRNA translation exists that is independent from its classical role as a cytoskeletal cross-linker protein at the plasma membrane.

Citation Format: Haydar Celik, Kamal P. Sajwan, Amrita V. Pai, Ben J. Marsh, Yasemin Saygideger Kont, Said Rahim, Jenny Han, Tsion Minas, Jeffrey A. Toretsky, Aykut Uren. Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level. [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 3269. doi:10.1158/1538-7445.AM2015-3269
Abstract 327: 3D dynamics of the response to cell cycle checkpoint targeting drugs in multicellular tumour spheroids
Authors: LOBJOIS Valérie , MONDESERT Odile , FRONGIA Céline , DESMAISON Annaick , GOMES Aurélie , CAZALES Martine , Ducommun Bernard
Published: August 2015

Major recent advances have recently been made in the understanding of the molecular pathways regulating the cell cycle machinery and their deregulations in cancer. These results open new avenues for the development of innovative antitumor pharmacological strategies targeting the cell cycle and its checkpoints.

MultiCellular Tumor Spheroid (MCTS) are now considered as invaluable models to study cancer cell biology and for the preclinical development of new antiproliferative drugs.

To fully exploit the features of MCTS, it is essential to preserve the 3D regionalization level of analysis when investigating the effect of a drug. We will report here a breakthrough in the use of MCTS with new technological developments that can be used to explore the regionalization and the live dynamics of the effects of anticancer drugs in 3D.

Spheroids expressing fluorescent cell cycle reporters (i.e. Fucci) and biosensors were engineered and used to monitor cell cycle arrest and DNA damage Response (DDR) pathway activation induced by chemotherapeutic agents. The kinetics and regionalized aspects of the response were investigated on genetically modified spheroids using innovative 3D imaging strategy (based on 3D light sheet microscopy).

We will present our latest results on the characterization of the effects of several anticancer drugs including DNA damaging agents alone or in association with checkpoint abrogating compounds.

This study opens new perspectives for original 3D assays dedicated to the identification of new original targets and compounds and for the investigation of the dynamics of the 3D response to novel antiproliferative agents.

Citation Format: Valérie LOBJOIS, Odile MONDESERT, Céline FRONGIA, Annaick DESMAISON, Aurélie GOMES, Martine CAZALES, Bernard Ducommun. 3D dynamics of the response to cell cycle checkpoint targeting drugs in multicellular tumour spheroids. [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 327. doi:10.1158/1538-7445.AM2015-327
Abstract 3279: Expression of chemerin and chemerin receptors in neuroblastoma: implications in tumorigenesis
Authors: Tuemmler Conny , Snapkov Igor , Moens Ugo L. , Kogner Per , Johnsen John Inge , Sveinbjørnsson Baldur
Published: August 2015

Chemerin is an adipokine and immunomodulating factor that promotes chemotaxis of immature dendritic cells, natural killer cells, macrophages and endothelial cells. Secreted as prochemerin with low activity, it can be C-terminally processed by different proteases expressed by a broad range of cell types and tissues. The resulting isoforms vary in receptor affinity and biological activity and are natural ligands for the G protein-coupled receptors (GPCRs) CMKLR1, GPR1 and CCLR2. To date, the activation of CMKLR1 (chemokine-like receptor 1) by chemerin and its role in metabolism and metabolic disorders as well as inflammation is best understood.

Neuroblastoma (NB) is a malignancy of the sympathetic nervous system and the most common extracranial solid pediatric tumor. Several chemoattractant GPCRs have been suggested to promote tumor progression, angiogenesis and metastasis in NB. Although for some cancers a potential function has been suggested, the role of chemerin and its receptors in the NB tumor microenvironment remains unknown.

In our study, the screening of microarray databases and analysis of neuroblastoma expression data showed a correlation between high CMKLR1, GPR1 and CCLR2 expression and a reduction in the overall survival probability. Expression of CMKLR1, GPR1, and chemerin was shown in nine neuroblastoma cell lines using RT-PCR, Western blot and immunocytochemistry. Furthermore, chemerin and CMKLR1 were also detected in neuroblastoma tumor tissue by immunohistochemistry. Stimulation of NB cell lines with active chemerin induced calcium mobilization and increased phosphorylation of MEK1/2 and ERK1/2 indicating an activation of the MAPK signaling pathway. Furthermore, chemerin stimulation led to increased NF-κB phosphorylation and translocation to the nucleus. The induction of NF-κB mediated signaling was observed by luciferase reporter assay. TNFα, IL-1β or serum stimulation increased chemerin protein expression and secretion in neuroblastoma cells.

To assess the functional significance of chemerin and its receptors in neuroblastoma tumorigenesis, cell clones overexpressing or silenced for CMKLR1/ Chemerin/ GPR1 are used in NB animal models.

Citation Format: Conny Tuemmler, Igor Snapkov, Ugo L. Moens, Per Kogner, John Inge Johnsen, Baldur Sveinbjørnsson. Expression of chemerin and chemerin receptors in neuroblastoma: implications in tumorigenesis. [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 3279. doi:10.1158/1538-7445.AM2015-3279
Abstract 3307: Chemopotentiation by low dose fractionated radiation therapy in colon cancer cells
Authors: Smith Teresa , Parekh Palak R. , Chang Elizabeth T. , Chuong Michael , Carrier France
Published: August 2015

Response rates for patients with primary and metastatic colorectal cancer treated with modern chemotherapy regimens such as 5-FU, leucovorin, oxaliplatin (FOLFOX) have routinely exceeded 50% and are frequently as high as 60-70%. However, as in most chemotherapy regimens, toxicity to normal cells is often a significant limitation. Tumors of the gastrointestinal (GI) track are also sensitive to radiation but the inability to combine radiation therapy (RT) with full-dose chemotherapy can be detrimental to patients, particularly those that may have disseminated micrometastatic disease. Therefore, new treatment strategies that incorporate chemotherapy that could increase tumor response without increasing toxicity are highly desirable. Laboratory and clinical data suggest that using low-dose fractionated radiation therapy (LDFRT) as a chemopotentiator may allow for full-dose chemotherapy with improved efficacy without adding to the toxicity of the systemic treatment. Here we show that twice daily radiation doses as low as 0.15 Gy for three consecutive days can significantly increase FOLFOX killing efficiency in human colon cancer RKO cells. In addition, our data indicate that the chemopotentiation by LDFRT may be mediated by the stress inducible hnRNP A18 protein. Down regulation of hnRNP A18 sensitizes RKO cells to LDFRT and FOLFOX independently and increases further the chemopotentiation by LDFRT. Moreover, overexpression of hnRNP A18 increases radio-resistance and chemo-resistance to FOLFOX. hnRNP A18 is a new regulator of protein translation in cancer cells that specifically targets transcripts devoted to confer growth advantages. This data suggest that hnRNP A18 could be targeted to increase further the systemic effect of chemopotentation by LDFRT. The identification of hnNRP A18 as a new cellular pathway responsive to low dose radiation and its contribution to chemopotentiation provide a novel opportunity for better measurement of the therapeutic response and could contribute to the rationale designs of new mechanism based clinical trials.

Citation Format: Teresa Smith, Palak R. Parekh, Elizabeth T. Chang, Michael Chuong, France Carrier. Chemopotentiation by low dose fractionated radiation therapy in colon 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 3307. doi:10.1158/1538-7445.AM2015-3307
Abstract 3343: Podophyllotoxin acetate (PA) enhancing γ-ionizing radiation-induced apoptosis via the ROS/p38/caspase pathway
Authors: Choi Jae Yeon , Hwang Sang-Gu , Um Hong-Duck , Park Jong Kuk
Published: August 2015

To develop a new radiosensitizer candidate capable of increasing the efficiency of radiotherapy and to investigate the underlying molecular mechanisms in vitro and in vivo. To develop a new radiosensitizer against non-small cell lung cancer cells, we screened a natural product library for growth-inhibitory compounds. Podophyllotoxin acetate (PA) was found to be cytotoxic toward NCI-H460 cells, and its IC50 value was determined. The radiosensitizer effects of PA were tested at its IC50 value in clonogenic and cell-counting assays. The intracellular mechanism underlying this effect was determined by immunoblotting and by measuring propidium iodide uptake and ROS generation. The radiosensitizer activity of PA in vivo was tested in nude mice by treating with PA and IR, and measuring tumor volume and assessing apoptosis. PA, tested at its experimentally determined IC50 value (12 nM), enhanced γ-ionizing radiation (IR)-induced death of NCI-H460 cells by increasing apoptosis, yielding a mean calculated dose-enhancement ratio of 3.8. Combination with PA and IR also increased the production of reactive oxygen species (ROS), which subsequently induced phosphorylation of p38, suppressed phosphorylation of ERK, and activated caspase-3, -8, and -9. Notably, inhibition of ROS production prevented p38 phosphorylation, and inhibition of ROS production or p38 activation blocked caspase activation and apoptosis. In a xenograft assay, combination with PA and IR delayed tumor growth by 11.4 days compared with controls, yielding an enhancement factor of 1.48. Collectively, these results indicate that PA functions as a radiosensitizer by enhancing apoptosis through activation of a ROS/p38/caspase pathway and suppression of ERK.

Citation Format: Jae Yeon Choi, Sang-Gu Hwang, Hong-Duck Um, Jong Kuk Park. Podophyllotoxin acetate (PA) enhancing γ-ionizing radiation-induced apoptosis via the ROS/p38/caspase 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 3343. doi:10.1158/1538-7445.AM2015-3343
Abstract 3347: Radiation-induced gastrointestinal (GI) syndrome as a function of age
Authors: Li Hongyan , Kucharavy Herman , Hajj Carla , Hua Guoqiang , Glass Ryan , Zeng Zhaoshi , Fuks Zvi , Paty Philip B. , Kolesnick Richard , Hubbard Karen , Haimovitz-Friedman Adriana
Published: August 2015

The incidence of cancer increases with age and is generally thought to be an age-related disease. In prior studies we model tumor response to radiation therapy (RT) by studying its effect on normal tissue damage. We showed that RT-induced gastrointestinal (GI) syndrome represents coupled damage to intestine's micovasculature and the parenchymal epithelial cells. In addition, we showed that 15Gy irradiation is a lethal dose 100 for C57Bl6 mice treated with whole body irradiation (WBI) as it induces apoptosis in endothelial cells (EC) and initiates the GI syndrome in the small intestine. Our objective is to determine whether the microvasculature and/or the cycling crypt base columnar cells (CBCs), the small intestine stem cells, in an aging population (29 months mice, approx. 69 years in humans) are more sensitive to radiation effects than in young adults (3 months mice, approx. 20 years old human) with regards to normal tissue toxicity.

Radiation-induced premature senescence and apoptosis were evaluated in EC and in CBCs in old mice versus young adult mice. Our results show enhanced endothelial apoptosis in crypts of older mice compared to young adult mice. There was a higher death rate in older versus young adult mice due to radiation-induced GI syndrome (90% vs 40% at 14Gy and 100% vs 60% at 15Gy). Crypt microcolony survival assays showed that 29 months-old mice had a lower number of regenerating crypts in all three doses tested (13Gy, 14Gy, 15Gy). In addition, there was an increase in CBC senescence in older mice compared to young adult mice. Therefore, microvasculature damage and an increase in CBCs apoptosis and senescence are the main events contributing to the enhanced rate of development of the RT-induced GI syndrome in the older mice. Ongoing studies are testing whether there is more senescence in the endothelial cells in older mice than in the mature adults, and if RT enhances senescence in these cells in older vs younger adult mice.

These studies will be extended to include animals with various cancer burdens to explore the role of age in the efficacy of radiation treatment.

Citation Format: Hongyan Li, Herman Kucharavy, Carla Hajj, Guoqiang Hua, Ryan Glass, Zhaoshi Zeng, Zvi Fuks, Philip B. Paty, Richard Kolesnick, Karen Hubbard, Adriana Haimovitz-Friedman. Radiation-induced gastrointestinal (GI) syndrome as a function of age. [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 3347. doi:10.1158/1538-7445.AM2015-3347
Abstract 3348: Tumor irradiation induced a phenotype change of monocytes to facilitate vascular regrowth
Authors: Deng Lei , Stafford Jason , Chernikova Sophia , Liu Shie-chau , Sun Jianguo , Brown J. Martin
Published: August 2015

We and others have previously shown that tumor irradiation mobilizes CD11b+ myeloid cells from bone marrow into the tumor thereby facilitating vascular regrowth and tumor recurrence. However, how these CD11b+ cells promote vasculature growth remains largely undefined. In this study, we found that CD11b+ cells in a 12Gy-irradiated U251 glioblastoma tumor model were largely Ly6g negative, suggesting that these cells are not in the granulocyte lineage, but rather are monocyte/macrophages (CD11b+Ly6g-). Interestingly, we found that the proportion of Ly6c+ monocytes in the irradiated tumor remained unchanged compared to that in the pre-irradiate tumor. However, the proportion of Ly6c- monocytes rose, driving the increase of total CD11b+ cells post-irradiation. Compared to Ly6c+ monocytes, Ly6c- cells in irradiated tumors were the major cells expressing VEGF. In vitro culture of murine bone marrow enriched Ly6c+ monocyte with colony stimulating factor-1 (CSF-1) downregulated Ly6c expression and increased in vitro angiogenic capability as demonstrated by the tube formation assay. Adding PLX3397, a CSF-1 receptor inhibitor to irradiation, which has been shown to delay tumor growth previously, decreased the Ly6c- monocytes more than the Ly6c+ monocytes. Given that we and others have previously shown that CSF-1 expression was increased in irradiated tumors, we conclude that tumor irradiation induced a phenotype change of monocytes from Ly6c+ to Ly6c- and that the latter, which are more pro-angiogenic, promote tumor vascular regrowth and recurrence post-irradiation.

Citation Format: Lei Deng, Jason Stafford, Sophia Chernikova, Shie-chau Liu, Jianguo Sun, J. Martin Brown. Tumor irradiation induced a phenotype change of monocytes to facilitate vascular regrowth. [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 3348. doi:10.1158/1538-7445.AM2015-3348
Abstract 3353: Prostate cancer microenvironment modulates macrophage phagocytosis
Authors: Weagel Evita G. , Anderson Nicholas B. , Meng Wei , Davis Josh , Robison Richard A. , O'Neill Kim L.
Published: August 2015

Macrophages play an important role in the body's immune system. There are two main phenotypes of macrophages, M1 and M2. M1 polarized macrophages participate in tumorcidal activity and pro-inflammatory responses. M2 macrophages perform anti-inflammatory responses and promote tissue repair, angiogenesis, and tumor growth. Macrophages within the vicinity of cancer cells may be influenced by the microenvironment and change their gene expression accordingly. Macrophages can express different cytokine profiles depending on their polarization state (M1 or M2). We investigated whether macrophages exposed to cancer cells demonstrate a M2 or M1 phenotype. We stimulated U937 cells with phorbol 12-myristate 13-acetate (PMA) for 24 hours to allow for adhesion and differentiation. We co-cultured PMA-stimulated U937 macrophages with PC3 and DU145 prostate cancer cell lines in a 1:1 ratio with a transpermeable membrane to allow for cell to cell contact and communication, and with PC3 and DU145 conditioned media for 48 hours. We isolated the RNA from the treated macrophages and performed qPCR to assess gene expression levels of the following cytokines: IL-10, a cytokine demonstrating a M2 phenotype was upregulated by 10 and 15 fold for PC3 and DU145 cells respectively and by 3 fold in both cell lines exposed to conditioned media. We also found a trend of downregulation with the cytokines IL-1, IL-12, and TNF - alpha, which also depict a M2 phenotype. We also assessed macrophage function by measuring engulfment by flow cytometry. Treated macrophages were allowed to phagocytose fluorescent microbeads for 1 hour. Flow cytometry analysis revealed that total bead engulfment decreased by up to 80% in macrophages treated with PC3 cells. Moreover, the population of macrophages that engulfed 3+ beads decreased significantly from both the cells and media treatments. Macrophages treated with DU145 cells and spent media also showed a similar trend to macrophages exposed to PC3 cells. The results show that macrophages exposed to PC3 and DU145 cancer cell lines polarized towards an M2 phenotype and could be important in the success of cancer within the microenvironment. Further research will reveal whether this process of a M2 polarization of macrophages is mediated by cytokines and cell vesicles or other factors.

Citation Format: Evita G. Weagel, Nicholas B. Anderson, Wei Meng, Josh Davis, Richard A. Robison, Kim L. O'Neill. Prostate cancer microenvironment modulates macrophage phagocytosis. [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 3353. doi:10.1158/1538-7445.AM2015-3353
Abstract 3361: CD73 expression on tumor-infiltrating breast cancer leukocytes
Authors: Buisseret Laurence , Garaud Soizic , Allard Bertrand , Cousineau Isabelle , Chouinard Guillaume , Sotiriou Christos , Willard-Gallo Karen , Stagg John
Published: August 2015

Recent studies have shown that CD73 could be a new promising target in breast cancer (BC). CD73 is a cell surface ecto-nucleotidase, expressed by both tumor cells and immune cells, that catalyses the hydrolysis of AMP into adenosine in the tumor microenvironment. Adenosine has immunosuppressive and tumor-promoting effects and is therefore implicated in the mechanism of tumor immune escape. High CD73 gene expression has recently been associated with a poor prognosis in triple negative BC.

While tumor expression of CD73 is well documented, expression of CD73 on tumor-infiltrating immune cells remains poorly characterized. We evaluated the expression of CD73 on immune cells present in fresh tissue homogenates (GentleMACS with no enzymatic digestion) from untreated primary breast tumors and compared to its expression to peripheral blood mononuclear cells (PBMC) from patients with breast cancer. In the peripheral blood, CD73 was found mostly expressed on CD4+ (mean percentage: 13.24±12.88%) and CD8+T cells (mean percentage: 40.78±19.42%) and a major subset of B cells (mean percentage: 51.93±21.83%). Analysis of fresh tumor tissues revealed that less than 25% of CD45+ tumor immune infiltrates express CD73 (mean percentage: 17.63± 5,1%) and was relatively similar to its expression on PBMC (mean percentage: 14.87± 9.5%). In the tumor immune infiltrates, B cells were the immune subset with the higher proportion of CD73+ cells (mean percentage: 53.27±14.63%) while the proportion of CD73+ was lower on tumor-infiltrating CD8+ T cells compared to CD8+ PBMC (p value: 0.022). Notably, we observed that CD73 expression was significantly higher on tumor-infiltrating NK cells and CD14+ myeloid cells compared to peripheral cells (p = 0.005 for NK cells, and p = 0.015 for CD14+ cells). Immunofluorescence analysis confirmed that CD73 is expressed by both tumor cells, CD45+ immune cells and stromal cells in breast cancer.

These results suggest that CD73 is differentially expressed on tumor-infiltrating leukocytes compared to PBMCs. Modulation of CD73 activity on tumor-infiltrating leukocytes may therefore contribute to the mechanism-of-action of anti-CD73 therapies.

Citation Format: Laurence Buisseret, Soizic Garaud, Bertrand Allard, Isabelle Cousineau, Guillaume Chouinard, Christos Sotiriou, Karen Willard-Gallo, John Stagg. CD73 expression on tumor-infiltrating breast cancer leukocytes. [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 3361. doi:10.1158/1538-7445.AM2015-3361
Abstract 3376: Ras activation in aberrant crypt foci is associated with decreased expression of cell cycle regulators and disruption of stromal homeostasis
Authors: Mo Allen , Horelik Nicole A. , Jackson Stephen , Devers Thomas J. , Rosenberg Daniel W.
Published: August 2015

Aberrant crypt foci (ACF) are the earliest detectable premalignant lesion within the colon. A majority of ACF harbor somatically acquired mutations to cancer-related genes. ACF may be an indicator for a colonic mucosa at-risk for cancer development, however only a small subset of ACF have the potential to progress to malignancy. We believe that the majority of ACF activate growth inhibitory programs that ultimately result in their growth arrest and regression. In order to begin to define the gene expression changes that occur within these early microscopic lesions and their underlying stroma, we have developed a novel method that combines laser capture micro-dissection with targeted RNA-seq analysis. In the following study, gene expression profiles of thirteen human ACF biopsy specimens harboring known oncogenic mutations (K-ras, B-raf, N-ras, and APC) were compared to matched normal mucosa. Two gene panels were used to assess the status of important cell regulatory pathways: an apoptosis panel comprised of 267 genes and a customized cell senescence panel consisting of 20 genes. In the apoptosis panel, the expression of 81 genes were differentially expressed in ACF compared to matched normal mucosa. Expression of HRK, a potent activator of apoptosis, was the most significantly down-regulated gene (6.75-fold, q <0.01) in every ACF when compared to normal mucosa. Inactivation of HRK function occurs in a variety of tumor types, including prostate cancer, astrocytomas and lymphomas. ACF with mutations occurring within the MAP-K pathway (K-ras, B-raf, N-ras) showed strong activation of downstream ras-associated genes and down-regulation of cell cycle regulators (CDK4 (p = 0.01), CCNB1 (p = 0.04), CCNA2 (p = 0.01), and TP53 (p = 0.04)). Interestingly, these expression panels were distinct from ACF harboring somatic mutations to APC. Analysis of microdissected stroma directly underlying the aberrant crypt structures revealed 25 differentially expressed genes, with dysregulation of several inflammatory mediators (IL2, IL2RA, IL6, TGFB2, BMP4) and extracellular matrix remodeling targets (TIMP1, TIMP2). Our findings suggest that ras-activated ACF may cause significant changes in cell cycle regulation, as well as alterations in stromal extracellular matrix maintenance and inflammatory response.

Citation Format: Allen Mo, Nicole A. Horelik, Stephen Jackson, Thomas J. Devers, Daniel W. Rosenberg. Ras activation in aberrant crypt foci is associated with decreased expression of cell cycle regulators and disruption of stromal homeostasis. [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 3376. doi:10.1158/1538-7445.AM2015-3376
Abstract 3377: Co-spheroid analysis reveals attenuating effect of HS27A stroma cells on DLD1 colon carcinoma susceptibility to MEK kinase inhibitor trametinib
Authors: Hoffmann Claudia , Feger Daniel , Siedentopf Oliver , Weber Holger , Umber Sarah , Ehlert Jan E.
Published: August 2015

In cancer treatment, stroma-derived microenvironmental cues are suspected to exert an adversary impact on anti-cancer drug efficacy. In order to take such influences into account in cellular drug screening campaigns, we have set up a high-throughput-compatible co-spheroid model, enabling the analysis of various combinations of cancer and stroma cell lines. This assay system allows for cell analysis in a three-dimensional (3D) close-to-physiological environment with simultaneous detection of cancer and stroma cell viability based on differential luciferase cell-labelling. Our data revealed that growth characteristics strongly depend on the specific cell types combined. Stroma cells in contrast to tumor cells usually ceased to proliferate or even showed dramatic loss of viability possibly due to metabolic hijacking. We report on the optimization of the cellular test systems to ensure significant viable stroma cell counts during the drug testing phase. In such an optimized co-spheroid model of Firefly-luciferase labelled DLD-1 colon cancer cells with Renilla-luciferase labelled HS27A stroma cells, we identified the MEK kinase inhibitor Trametinib as a compound showing a 10-30fold attenuated potency as compared to the DLD-1 mono-spheroid model. Based on these results we suggest integrating optimized co-spheroid studies into early drug development to focus on compounds that remain active under microenvironmental conditions.

Citation Format: Claudia Hoffmann, Daniel Feger, Oliver Siedentopf, Holger Weber, Sarah Umber, Jan E. Ehlert. Co-spheroid analysis reveals attenuating effect of HS27A stroma cells on DLD1 colon carcinoma susceptibility to MEK kinase inhibitor trametinib. [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 3377. doi:10.1158/1538-7445.AM2015-3377
Abstract 338: Immunohistochemical expression of CD44/ERM complex proteins in penile carcinoma
Authors: de Andrade Juliana B. , Soares Fernando A.
Published: August 2015

Background: The loco-regional involvement is considered the main complication and cause of death in the natural history of penile carcinoma. Understanding the molecular processes that may be involved in metastatic dissemination would improve the prognostic tools to better evaluate the patients, avoiding the morbidity of unnecessary surgery. Some studies have emphasized the influence of cellular adhesion in the development of tumorigenesis and metastasis as the proteins of complex CD44/ERM (Ezrin/Radixin/Moesin). Therefore, this study aimed to analyze the expression of proteins CD44 and its variant CD44v6, Ezrin and Moesin in squamous cell penile carcinoma, correlating their expression with the clinical-pathological criteria.

Material and Methods: Eighty four patients were selected, retrospectively, undergoing surgery for squamous cell penile carcinoma at AC Camargo Cancer Center. The expression evaluation for CD44, CD44v6 Ezrin and Moesin was performed by immunohistochemistry using Ventana® equipment, subsequently analyzed by digital microscopy in Aperio® system. The immunoreactivity of each protein was categorized as positive and negative using a cut-off criteria established by the average percentage obtained.

Results: CD44 expression was present in 19 (22.6%) positive cases, CD44v6 in 50 (59.5%), Ezrin in 30 (35.7%) and Moesin in 53 (63.1%). It was observed that the loss of CD44 expression was significantly correlated with recurrence (p = 0.002) and with the glans invasion (p = 0.035); the positive expression of CD44v6 was significantly correlated with the tumor size (p = 0.009) and with recurrence (p = 0.018); the loss of Ezrin expression was significantly correlated with macroscopic invasion (p = 0.028) and with foreskin of invasion (p = 0.015); the positive expression of Moesin was significantly correlated with macroscopic invasion (p = 0.010) and the loss of its expression was significantly correlated with recurrence (p = 0.035). Disease-free survival was significantly influenced by CD44, CD44v6 and Moesin.

Conclusion: The expression of CD44, CD44v6, Ezrin and Moesin seems to significantly influence the prognostic factors of penile carcinoma and could be an important molecular marker in the identification of recurrence risk.

Citation Format: Juliana B. de Andrade, Fernando A. Soares. Immunohistochemical expression of CD44/ERM complex proteins in penile carcinoma. [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 338. doi:10.1158/1538-7445.AM2015-338
Abstract 3383: Correlation of mutations detected in liquid and tissue biopsies
Authors: White Douglas H. , Horejsh Douglas , Accola Molly , Rehrauer William , Urh Marjeta
Published: August 2015

Circulating cell-free DNA (ccfDNA) in plasma can be used to detect biomarkers that show great promise for diagnosis and monitoring of cancer, giving rise to the possibility of liquid biopsies that obviate the need for invasive tissue collection. The low concentration and highly fragmented nature of ccfDNA, coupled with the low frequency of potential oncogenic biomarkers, presents challenges and will require a purification method that is efficient and highly reproducible.

Here we describe a method for purifying nucleic acids based on novel surface and binding chemistries. The combination of these two approaches allows for increased binding of fragmented DNA. The method can be partially automated to ensure highly reproducible results. Up to 4mls of plasma can be processed and eluted in as little as 15ul, giving DNA concentrations of 1-10ul. This greatly facilitates use in Next Generation Sequencing.

Using the automated method, ccfDNA was purified from the plasma of 7 patients who had previously undergone surgical resection for malignancy. DNA was also purified from the FFPE malignant tissue off of slides, following macrodissection, from the same patients. NGS was used to interrogate both sample types for potentially oncogenic variants. Several laboratory developed tests, all including COLD-PCR, were also employed to verify the presence or absence of variants. The two types of samples showed excellent correlation on mutations, suggesting that use of a less invasive liquid biopsy has the potential to enable actionable mutation detection without using more invasive solid tumor biopsy means.

Citation Format: Douglas H. White, Douglas Horejsh, Molly Accola, William Rehrauer, Marjeta Urh. Correlation of mutations detected in liquid and tissue biopsies. [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 3383. doi:10.1158/1538-7445.AM2015-3383
Abstract 3384: Cancer Core 125 Panel for quantitative expression and mutation profiling
Authors: Chenchik Alex , Makhanov Mikhail , Dolganov Gregory , Jeffrey Stefanie
Published: August 2015

Molecular profiling of heterogeneous circulating tumor cells (CTCs) and tumor biopsy samples at the single-cell level is critical for identifying different cancer cell subpopulations and understanding their value in predicting metastatic cancer progression and responses to treatment. Unfortunately, the isolation and comprehensive characterization of hundreds to thousands of individual cancer cells present formidable analytical and technical challenges.

To this end, we developed a high-throughput (HT) HiCellex technology to obtain the genome-wide expression and mutation profiles of hundreds to thousands of epithelial cancer cells at the single cell level. Importantly, HiCellex allows selective molecular profiling of a complete set of viable cells at the single-cell level in the presence of contaminating leukocytes without physical separation of individual cells. The genetic profiling technology uses unique cell-specific molecular barcodes to label mRNAs in individual viable cancer cells by a spliceosome-mediated trans-splicing mechanism. Barcoded trans-splicing constructs, necessary for the labeling of the entire population of pre-mRNAs, are delivered and expressed in a pool of target cancer cells with lentiviral vectors. Digital expression data are generated by HT sequencing of barcoded amplified cDNAs and easily cluster to each cell in silico using cell-specific barcodes.

This presentation will provide results of development and validation of genome-wide and multiplex RT-PCR profiling assay of the 500 most informative subtyping and metastatic signature genes (MGS) in model cancer cell lines, CTC and tumor samples. The developed HiCellex 500 MGS profiling assay is a cost-effective approach to effectively characterize, subtype and identify prognostic and diagnostic biomarkers in heterogeneous cancer cells at the single-cell level.

Citation Format: Alex Chenchik, Mikhail Makhanov, Gregory Dolganov, Stefanie Jeffrey. Cancer Core 125 Panel for quantitative expression and mutation profiling. [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 3384. doi:10.1158/1538-7445.AM2015-3384
Abstract 3386: The development of a highly specific monoclonal antibody against Ki67 useful for immunohistochemistry
Authors: Chen Caiwei , Yuan Kehu , Chu Boyang , Shu Youmin , Chen Jian , Stafford Joe , Fu Wei , He Wei-Wu , Chambers Ross , Ma Donghui
Published: August 2015

Ki-67 is a nuclear protein that is a useful marker of proliferation. Ki-67 antibodies are used to generate a Ki-67 labeling index, which is the percentage of cells with nuclear immunostaining in immunohistochemistry. The Ki-67 labeling index may be useful as a prognostic and predictive marker in cancer. The Ki-67 labeling index relies on the use of antibodies with a suitable level of sensitivity and specificity, and many Ki-67 antibodies have been developed over the last two decades. One of the most widely used Ki-67 antibodies is the mouse monoclonal MIB1. However, there have been many reports showing that in some cases MIB1 detects an unexpected membranous and cytoplasmic staining pattern in immunohistochemistry. Since Ki-67 is exclusively expressed in the nucleus this raises questions about the specificity of MIB1. We have created a nearly comprehensive microarray of about 17,000 human proteins to investigate the specificity of MIB1 and identify cross reacting proteins. The protein microarray was also used to screen new panels of mouse monoclonals against Ki-67. We have identified the antibody UMAB107 that in immunohistochemistry performs with similar sensitivity as MIB1, but unlike MIB1, UMAB107 is highly specific.

Citation Format: Caiwei Chen, Kehu Yuan, Boyang Chu, Youmin Shu, Jian Chen, Joe Stafford, Wei Fu, Wei-Wu He, Ross Chambers, Donghui Ma. The development of a highly specific monoclonal antibody against Ki67 useful for immunohistochemistry. [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 3386. doi:10.1158/1538-7445.AM2015-3386
Abstract 3397: ￼A novel clinical tool that provides quantitative and accurate measurement of Met protein
Authors: Cecchi Fabiola , Liao Wei-Li , Thyparambil Sheeno , Darfler Marlene , Krizman David , Hembrough Todd , Burrows Jon , Bottaro Don , Catenacci Daniel V.T.
Published: August 2015

BACKGROUND:

Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue.

METHODS:

After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH).

RESULTS:

Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/μg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/μg to 4669.5 amol/μg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of >1500 amol/μg was 100% sensitive (95% CI 0.69-1) and 100% specific (95% CI 0.92-1) for MET amplification.

CONCLUSIONS:

The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC.

Citation Format: Fabiola Cecchi, Wei-Li Liao, Sheeno Thyparambil, Marlene Darfler, David Krizman, Todd Hembrough, Jon Burrows, Don Bottaro, Daniel V.T. Catenacci. ￼A novel clinical tool that provides quantitative and accurate measurement of Met protein. [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 3397. doi:10.1158/1538-7445.AM2015-3397
Abstract 3415: PAICS is the prognostic marker in colorectal cancer patients with stage III
Authors: Kobayashi Yusuke , Kumamoto Kensuke , Matsumoto Yoshiko , Saito Motonobu , Shimura Tatsuo , Takenoshita Seiichi
Published: August 2015

PAICS (Phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase) is an important bifunctional enzyme in de novo purine biosynthesis. It could be an attractive target for anticancer drug design, since rapidly dividing cancer cells rely on the purine through de novo pathway for synthesis of adenine and guanine, whereas normal cells favor the salvage pathway.

Previous reports demonstrated that PAICS expression was up-regulated in ALL, lung cancer, and glioblastoma. In the present study, we first performed the comparison of PAICS mRNA expression between cancer tissue and normal colonic mucosa in 83 colorectal cancer (CRC) patients by realtime RT-PCR. We further investigated PAICS expression by immunohistochemical staining in 258 CRC patients and analyzed the association between PAICS expression and clinicopathological factors and prognosis. To understand the biological significance of PAICS expresssion, knockdown experiments of PAICS expression were performed by a siRNA method using the colon cancer cell line, HCT116. We analyzed the relationship between PAICS mRNA expression and genetic status and prognosis using several databases.

PAICS mRNA expression in CRC tissue was significantly enhanced when compared to that in normal tissue. In immunohistochemical examination, PAICS expression was observed in cytoplasm of cancer cells. PAICS expression was detected in 55% cases of colorectal cancer patients. There were significant association between PAICS expression and histology (p = 0.049). There was not significant correlation between PAICS expression and overall survival in CRC patients. Regarding CRC patients with stage III, patients with positive expression of PAICS had significantly good prognosis as compared to those with negative expression (p = 0.015). Knockdown of PAICS expression acquired the ability of cellular invasion using colon cancer cell lines, HCT116 and SW480. In all CRC databases we analyzed, stage III patients with high expression of PAICS had good prognosis as compared to those with low expression of PAICS.

We concluded that PAICS expression was up-regulated in approximately 55% CRC, and higher level of PAICS expression might serve as a good prognostic marker in advanced CRC patients.

Citation Format: Yusuke Kobayashi, Kensuke Kumamoto, Yoshiko Matsumoto, Motonobu Saito, Tatsuo Shimura, Seiichi Takenoshita. PAICS is the prognostic marker in colorectal cancer patients with stage III. [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 3415. doi:10.1158/1538-7445.AM2015-3415