bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2790: Establishment and characterization of human skingraft model in immunodeficient mice.
Authors: Serin Guillaume , Morgand Loïc , Leblanc Marie , Bichat Francis
Published: August 2014

The humanization process of mice with various tissues named Chi-mice aimed to reproduce better the human situation and to be more predictive than conventional models. In order to evaluate new targeting therapies or adverse side effects involving skin components, pre-clinical studies need to be assayed with new molecules in adequate and validated mouse models bearing human skin.

The collection of skin samples was done under ethically approved master agreements and with the signed consent of each patient. The patient's clinical history, the serology results (HIV, HBV and HCV) and tissue banking were centralized in our internal approved biological resource center. To develop such models, we used skins of different origins including foreskin, breast and abdomen that were isolated by different methods (full recovery of about 1 cm depth sample or dermatomization of the 1-2 mm epidermal/dermal layer). These samples were xenografted on immune deficient NOD-SCID mice under different conditions including matrigel grafting. After one month implantation, skingrafts were collected and observed microscopically to confirm the preservation of human organization of skin by full histology. Also, the species nature of vessels was characterized by immunochemistry to evaluate the host penetration within human skingraft and both CD-31 positive mouse and human vessels were found in the skingraft. In the context of vascular leak syndrome (VLS) induction by IL2 treatment, we have used the wet/dry ratio of skingraft to measure the induced-edema and we have measured the blue evans uptake in those injured skingraft to appreciate the epidermal vasculature leaking. Anapathological comparisons were also performed to reveal the histological modifications observed during VLS.

These different characterizations performed during development of the skingraft model enabled us to obtain human skin xenografted on mice that retained the human characteristics of the primary material. The co-implantation of other human tissue (tumor) with skin in same mice which refined better the humanized model will be presented.

The use of fresh skin and various tissues in drug discovery and early preclinical development of new therapies aimed at corroborating results with clinical reality. Altogether, these processes from the clinical sample collection to the in vivo drug efficacy study through ex vivo assays should help the preclinical drug selection, development and clinical positioning as well as companion biomarker identification.

Citation Format: Guillaume Serin, Loïc Morgand, Marie Leblanc, Francis Bichat. Establishment and characterization of human skingraft model in immunodeficient mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2790. doi:10.1158/1538-7445.AM2013-2790
Abstract 2795: Establishment and molecular characterization of cell lines from Japanese patients with malignant pleural mesothelioma.
Authors: Tada Ryuhei , Yamamoto Hiromasa , Furukawa Masashi , Shien Kazuhiko , Hashida Shinsuke , Soh Junichi , Asano Hiroaki , Tsukuda Kazunori , Toyooka Shinichi , Miyoshi Shinichiro
Published: August 2014

Malignant pleural mesothelioma (MPM) is a poor prognostic malignancy that is distinctly resistant to the conventional therapies. Cell lines are useful materials for studying the biological characteristics of the tumors, thus the establishment of MPM cell lines is valuable for exploring the new therapeutic strategies of MPM. We established 4 MPM cell lines (YUMC8, YUMC44, YUMC63 and YUMC64) from Japanese patients, consisting of 2 epithelioid and 2 sarcomatoid types for histological subtype. We analyzed the status of methylation, mutation, copy number gains (CNGs), and protein expression in representative genes and the sensitivity to several drugs in these 4 cell lines. We detected the complete methylation of P16 in 3 cell lines, in which the protein expression of p16 was lost. Methylation of RASSF1A was observed in 3 of 4 cell lines. Copy number gains (CNGs) in EGFR, HER2 and MET were not detected in all the cell lines. Mutations in various genes including EGFR, KRAS, HER2, BRAF, and PIK3CA, which are frequently detected in non-small cell lung cancer, were not detected in all 4 cell lines. As for microRNA, miR-34b/c was completely methylated in 2 of 4 MPM established cell lines. About cell adhesion molecules, E-cadherin expression was detected in two epithelioid type MPM cell lines, whereas N-cadherin expression was detected in all four established cell lines by Western blot analysis. Vimentin was strongly expressed in two sarcomatoid type MPM cell lines. All established MPM cell lines showed no dramatic responses to the drugs tested including NVP-AUY922, 17-DMAG, Trichostatin A and Vorinostat. Although new molecular findings were not observed in the process of the current characterization of these new MPM cell lines, they seem to be useful for the future extensive analyses of biological behavior of MPM, so as to the development of new therapeutic strategies.

Citation Format: Ryuhei Tada, Hiromasa Yamamoto, Masashi Furukawa, Kazuhiko Shien, Shinsuke Hashida, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichi Toyooka, Shinichiro Miyoshi. Establishment and molecular characterization of cell lines from Japanese patients with malignant pleural mesothelioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2795. doi:10.1158/1538-7445.AM2013-2795
Abstract 2799: Regulation of tumor cell attachment, spreading and migration by the GPR4 receptor and related G protein pathways.
Authors: Justus Calvin R. , Yang Li V.
Published: August 2014

The tumor microenvironment has a characteristic low pH due to inadequate blood perfusion, hypoxia, altered cellular metabolism, and inflammation. The effects of acidic pH on cancer progression are complex. It has been shown that low pH increases the metastatic spread of cancer by assisting in the degradation of extracellular matrix. Other studies show that acidic microenvironments decrease metastatic potential in B16F10 melanoma cells upon activation of the GPR4 receptor and also inhibit Akt activity in breast cancer cells. In this study we have investigated the attachment, spreading, and migration of B16F10 cells that have been genetically modified to express the GPR4 receptor at a high level (named B16F10/GPR4 cells). The results showed that upon stimulation of GPR4 in B16F10/GPR4 cells with low pH, cell spreading, membrane ruffling, and migration were significantly decreased. The phosphorylation levels of paxillin Y118 and focal adhesion kinase (FAK) Y397 following one-hour cell attachment were also decreased, and the spatial localization of the phosphorylated paxillin and FAK was substantially altered. Further tests were performed to identify the G-protein signaling pathway responsible for this effect. By using a G13 dominant negative construct or inhibiting Rho activation with C3 transferase (CT04) in B16F10/GPR4 cells, the cell spreading and membrane ruffling abilities were almost completely restored back to the level of B16F10/vector control cells, whereas the inhibition of the Gs and Gq pathways had little effect. These results suggest that activation of the GPR4 receptor and subsequently the G13/Rho downstream pathway are responsible for decreased cell attachment, spreading and membrane ruffling, which may cause decreased metastatic potential of B16F10/GPR4 cells found previously.

Citation Format: Calvin R. Justus, Li V. Yang. Regulation of tumor cell attachment, spreading and migration by the GPR4 receptor and related G protein pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2799. doi:10.1158/1538-7445.AM2013-2799
Abstract 2802: Salmonella typhimurium A1-R inhibits brain metastasis in an orthotropic breast cancer nude-mouse model.
Authors: Zhang Yong , Zhang Nan , Hoffman Robert M. , Zhao Ming
Published: August 2014

We have previously developed tumor-targeting Salmonella typhimurium A1-R (A1-R), which has shown efficacy against multiple cancer types in nude mouse models. In this study, we demonstrated that S. typhimurium A1-R can inhibit and eradicate brain metastasis in an orthotopic metastatic BALB/c mouse model of breast cancer. To establish the model, 4T1 breast cancer cells expressing RFP (5.0 × 106/100 μL) in 100 μl serum-free solution were injected slowly into the right second mammary gland. Two weeks after cells implantation, the primary tumors were removed. Approximately 30% of post-surgery nude mice were found with brain metastasis using fluorescence imaging 4-5 weeks after injection of 4T1-RFP cells. When the mice were found with brain metastasis, S. typhimurium A1-R [5×107 CFU/100 μl] was injected into the tail vein once a week for 3 weeks. Inhibition of brain metastatic growth was observed noninvasively by the Maestro fluorescence imaging system (Perkin-Elmer Inc.) compared with untreated mice. Intravenous administration of S. typhimurium A1-R improved survival 70%. This study shows the important potential of bacterial therapy of brain metastasis.

Citation Format: Yong Zhang, Nan Zhang, Robert M. Hoffman, Ming Zhao. Salmonella typhimurium A1-R inhibits brain metastasis in an orthotropic breast cancer nude-mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2802. doi:10.1158/1538-7445.AM2013-2802
Abstract 2806: CD146, a suppressor of breast cancer, is a novel target of CD44-signaling.
Authors: Ouhtit Allal , Abdraboh Mohamed E. , Hollenbach Andrew D. , Gupta Ishita , Shanmuganathan Somya , Raj Madhwa HG
Published: August 2014

Cancer cell invasion and metastasis is the worst aspect of cancer, and disruption in the cell adhesion molecules (CAM) represents the initiating event in tumor cell invasion. While the hyaluronan receptor CD44 promotes breast cancer (BC) metastasis, CD146 is a melanoma CAM that appears to suppress BC (although it promotes other cancers). In addition, the molecular mechanism by which CD146 suppresses BC is still unclear. To shed light on this discrepancy, we initially used subtractive hybridization combined with northern blotting, and identified CD146 as a potential downstream target of CD44-signaling in the MDA-435 BC cell line. Here, we provide both structural and functional evidence indicating that CD146 is a downstream target for CD44, in a way that CD146 expression is regulated according to tumor microenvironment. Finally, this study demonstrates a new role for CD44 in regulating neovascularization and promoting cancer cell transmigration of blood vessels via CD146.

Citation Format: Allal Ouhtit, Mohamed E. Abdraboh, Andrew D. Hollenbach, Ishita Gupta, Somya Shanmuganathan, Madhwa HG Raj. CD146, a suppressor of breast cancer, is a novel target of CD44-signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2806. doi:10.1158/1538-7445.AM2013-2806
Abstract 2808: Morphine modulates the paracrine interaction between breast tumor cells and macrophages.
Authors: Afshar Imani Banafsheh , Baran JoAnne , Cabot Peter J. , Parat Marie-Odile
Published: August 2014

While opioids are highly effective perioperative analgesics, there have been concerns about their effects on the growth and spread of tumor cells when used in cancer surgery patients. Using a mouse syngeneic model of breast cancer, we studied the effect of morphine on breast tumor dissemination to lungs. Intraperitoneal injection of morphine (10mg/kg) to BALB/c mice (n=8) every 12h for 3 consecutive days, caused a reduction in the number of tumor foci in lungs as measured 18 days after intravenous tumor inoculation. Morphine treatment also caused a reduction in circulating extracellular matrix (ECM)-degrading matrix metalloproteinase-9 (MMP-9) measured by gelatin zymography. One of the major contributors to tumor cell invasiveness is the immune cells infiltrating the tumors, particularly macrophages. Therefore, we tested the effect of morphine on the production of gelatinases by murine breast cancer cells 4T1 and macrophages RAW264.7 alone or cultured together. The levels of MMP-9 as well as its endogenous inhibitor tissue inhibitor of metalloproteinase-1 (TIMP-1) were shown to be elevated in the conditioned media of the co-cultures. Interestingly, we found out that although morphine treatment did not have any significant effect on the level of gelatinases produced by individual cells, in the co-cultures it caused a significant decrease in MMP-9 while increasing TIMP-1.We observed the same effect when breast cancer cells were incubated with the conditioned media of morphine-treated macrophages, indicating that cell-cell interaction was not required. Our in vitro data thus suggest that the anti-tumor effect of morphine observed in our animal studies is at least in part mediated through modulating the paracrine communication between tumour cells and tumor associated macrophages in their microenvironment rather than an effect on tumor cells alone. Randomized prospective clinical studies are required to determine how opioids, as a part of the perioperative pain management, could affect breast tumor recurrence and metastasis in patients.

Citation Format: Banafsheh Afshar Imani, JoAnne Baran, Peter J. Cabot, Marie-Odile Parat. Morphine modulates the paracrine interaction between breast tumor cells and macrophages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2808. doi:10.1158/1538-7445.AM2013-2808
Abstract 2809: Deficiency of Notch-1 signaling in liver increases neuroblastoma hepatic metastases.
Authors: Banerjee Debarshi , Garcia Alejandro , Andrews John , Sbiroli Emily , Kandel Jessica J. , Yamashiro Darrel J.
Published: August 2014

Purpose: The Notch signaling pathway has been extensively studied for its role in tumor angiogenesis but its function in tumor metastasis is largely elusive. In the present study we investigated the role of a notch receptor, Notch-1, in the regulation of neuroblastoma liver metastatic process.

Methods: To investigate the function of Notch-1 in neuroblastoma liver metastasis we first generated immunodeficient (rag2gammaC deletion) Notch-1 heterozygous (+/-) mice. We crossed a Notch1 heterozygous (+/-) mouse with a rag2/gamma-c double knockout (-/-) mouse. The mouse with Notch1(+/-)rag2(+/-)gammaC(+/-) genotype was then backcrossed with a rag2gammaC double knockout (-/-) mouse to generate rag2(-/-)gammaC(-/-)Notch1(+/-) mouse or rag2(-/-)gammaC(-/-)Notch1(+/+)control mouse . These mice (n=5) were intracardically injected with 105 neuroblastoma NGP (MYCN amplified) cells expressing Firefly luciferase. The mice were subjected to IVIS- bioluminescence imaging, once in a week, to monitor the metastatic spread. All mice were sacrificed at week 8. At the time of sacrifice, mice were injected with Luciferin, sacrificed and liver was then dissected out, imaged and bioluminescence flux was measured (ex vivo imaging). The liver tissues were also sectioned (5um, paraffin) and H&E stained at multiple levels to further confirm the presence of metastases.

Result: Intracardically injected NGP cells formed increased liver metastases in rag2(-/-)gammaC(-/-)Notch1(+/-) mice compared to littermate control rag2(-/-)gammaC(-/-)Notch1(+/+) mice. 4 out of 5 (80%) mice in Notch-het group and only 1 out of 5 (20%) mice in control group developed hepatic metastases. Notch-het livers showed multiple metastatic lesions with diameter ranged from 1-5 mm. Ex vivo bioluminescence imaging showed total flux (photon/sec) of Notch1 (+/-) livers were significantly higher (p<0.01) compared to Notch1 (+/+) control livers. Immunohistochemical staining against blood vessel markers such as Collagen IV, endomucin (endothelial cells) and aSMA (pericyte) showed extensive network of angiogenesis in Notch1 (+/-) liver metastases.

Conclusion: Deficiency of Notch1 signaling in liver microenvironment presented an increase in metastases in Notch-het mice in NGP intracardiac injection metastatic model. Therefore our study indicates that Notch1 acts as tumor suppressor in neuroblastoma liver metastasis.

Citation Format: Debarshi Banerjee, Alejandro Garcia, John Andrews, Emily Sbiroli, Jessica J. Kandel, Darrel J. Yamashiro. Deficiency of Notch-1 signaling in liver increases neuroblastoma hepatic metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2809. doi:10.1158/1538-7445.AM2013-2809
Abstract 2814: Sonic hedgehog in gastric cancer metastasis; MAP Kinase/NFκB-dependent signaling pathway.
Authors: Kang Myoung Hee , Kim Bo Ram , Park Joo Sung , Jeon Jung Guk , An Hyun Sook , Jang You Jin , Kim Sun Il , Kim Jun Suk , Oh Sang Cheul
Published: August 2014

Up-regulation of Sonic hedgehog and their receptors by tumor is an important hallmark in cancer progression, as it contributes through autocrine and paracrine mechanisms to tumor development, invasion, and metastasis, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of Mitogen-activated protein kinase (MAPK) signaling, nuclear factor (NF)-κB, and matrix metalloproteinase (MMP) expression in Shh-mediated metastatic function in gastric cancer. We found that stimulation of Shh in gastric cancer cells enhanced the phosphorylation of JNK and p38. Accompanying activation of JNK and p38 kinase, Shh also enhanced phosphorylation/degradation of IκBα and the nuclear translocation/activation of NF-κB.

Interestingly, blockade of JNK and p38 signaling using SP600125 and SB202190, respectively, significantly inhibited Shh-induced motility and invasiveness in association with the activation of NF-κB. In addition, phosphorylation of JNK, p38 and of IκBα, IκBα degradation, and the nuclear translocation of NF-κB was enhanced by treatment with N-shh, but not cyclopamin or shh neutralization antibodies. Furthermore, Shh-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with MAPK, JNK, or NF-κB inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of shh and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the Shh signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the JNK or MAPK pathway followed by the induction of NF-κB and MMP-9 activity, indicating that shh has the potential to be a therapeutic molecular target to decrease metastasis. Overall, our studies suggest that Shh promote motility and invasion of gastric cancer cells by activating MAPK/ NF-κB signaling and that targeting for these signaling may provide therapeutic opportunities in preventing metastasis mediated by Shh.

Citation Format: Myoung Hee Kang, Bo Ram Kim, Joo Sung Park, Jung Guk Jeon, Hyun Sook An, You Jin Jang, Sun Il Kim, Jun Suk Kim, Sang Cheul Oh. Sonic hedgehog in gastric cancer metastasis; MAP Kinase/NFκB-dependent signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2814. doi:10.1158/1538-7445.AM2013-2814
Abstract 2817: The contribution of bone marrow cells to carcinogenesis in the chronic inflammatory lung conditions.
Authors: Takikawa Sachiko , Powell Charles
Published: August 2014

The role of inflammation in lung carcinogenesis is suggested by the independent association between chronic obstructive lung disease, a systemic inflammatory process, and lung cancer risk. To investigate that chronic inflammation in the lung potentiates tobacco smoke-induced lung carcinogenesis, we utilized two murine models of chronic lung inflammation: a model of sustained intranasal exposure to lipopolysaccharide (LPS) and a transgenic model of inducible interleukin-1β overexpression in the lung. In preliminary experiments, nicotine-derived nitrosamine (NNK) tobacco carcinogen induced tumor multiplicity is significantly increased in stress markers in the lung tumors with chronic inflammation.

To determine the role of bone marrow-derived stem cells (BMDCs) in mediating the oxidative stress response in this model of chronic lung inflammation, we performed lineage tracing experiments. BMDCs are capable of self-renewal and recruited to the end organs where they differentiate into various terminal cells. In inflammation-associated gastric cancer, BMDCs contribute to the tumor microenvironment both by direct proliferation of epithelial cells and by differentiation into α-SMA positive cancer-associated fibroblasts. We hypothesize that BMDCs are recruited to inflammatory sites and contribute to the process of lung carcinogenesis through differentiation. We performed bone marrow transplantation from male GFP tagged donors to female chronic lung inflammation models. We investigated the localization and quantification of BMDCs contributions to subsequent lung tumors by immunohistochemistry of GFP and FISH for the Y chromosome. Additionally, we examined the differentiation of BMDCs in the tumor and associated microenvironment.

This study has the potential to identify novel pathways of lung carcinogenesis in cigarette carcinogen-induced lung cancers arising in the setting of chronic inflammation.

Citation Format: Sachiko Takikawa, Charles Powell. The contribution of bone marrow cells to carcinogenesis in the chronic inflammatory lung conditions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2817. doi:10.1158/1538-7445.AM2013-2817
Abstract 2821: GRK6 deficiency promotes tumor aggressiveness and metastasis in a murine model of human lung cancer .
Authors: Sutton Natalie , Smith Nikia , Thomas Ariel J. , Raghuwanshi Sandeep K. , Richardson Ricardo M.
Published: August 2014

G protein coupled receptor kinases (GRKs) phosphorylate the activated form of G protein coupled receptors (GPCRs) followed by the association of β-arrestin (βarr) with the phosphorylated receptor, desensitization and down-regulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis and wound healing. In this study, we sought to investigate the role of GRK6 in CXCR2-mediated tumorigenesis using a murine model of human lung cancer. Mice deficient in GRK6 (GRK6−/−) exhibited a significant increase in Lewis lung cancer (LLC) growth and metastasis relative to control littermates (GRK6+/+). GRK6 deletion had no effect on the expression of pro-angiogenic chemokine or vascular endothelial growth factor (VEGF), but up-regulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs and microvessel density. Treatment of the mice with a CXCR2 inhibitor (SB225002) or a murine anti-CXCR2 antibody blocked the effect of GRK6 deletion. Surprisingly, a GRK6−/−/βarr2−/− double knockout animal model exhibited faster tumor growth relative to GRK6−/− or βarr2−/− animals. Exposure of GRK6−/−/βarr2−/− mice to the anti-CXCR2 antibody totally inhibited tumor growth. This suggest that the effects of βarr2 and GRK6 knockdown in CXCR2-mediated tumor development are additive. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment thereby promoting angiogenesis and metastasis. Since GRK6−/−/βarr2−/− mice showed greater tumor growth relative to GRK6−/− or βarr2−/− mice, the data also suggest that CXCR2 couples to a different mechanism to mediate tumor progression and metastasis.

Citation Format: Natalie Sutton, Nikia Smith, Ariel J. Thomas, Sandeep K. Raghuwanshi, Ricardo M. Richardson. GRK6 deficiency promotes tumor aggressiveness and metastasis in a murine model of human lung cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2821. doi:10.1158/1538-7445.AM2013-2821
Abstract 2827: A platform to study metastatic cancer.
Authors: Wong Andrew D. , Searson Peter C.
Published: August 2014

Metastasis is responsible for over 90% of cancer related deaths. While significant advances in visualizing metastasis have been made in vivo, the details of the biological and physical processes that govern invasion and intravasation remain poorly understood. The difficulty in studying metastasis stems from the complexity of the interface where invasion and intravasation take place, between the tumor's local tissue microenvironment and the vascular system. To elucidate the mechanistic events taking place during invasion and intravasation, we have developed a platform that positions tumor cells adjacent to an artificial vessel embedded in an extracellular matrix (ECM). Using live-cell, fluorescence microscopy, we study the complex interplay between highly metastatic cancer cells and a functional artificial microvessel lined with endothelial cells during tumor migration and intravasation. We hypothesize that an engineered platform that recapitulates the interactions between a tumor and a physiologically relevant artificial vessel within an extracellular matrix will allow the systematic study of the physical and biological properties that regulate invasion and intravasastion. Since there remain many gaps in our understanding of the biology and physics of invasion and intravasation, further insight into these poorly understood processes may provide new strategies to prevent the spread of cancer and reduce the high mortality rates associated with metastasis.

Citation Format: Andrew D. Wong, Peter C. Searson. A platform to study metastatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2827. doi:10.1158/1538-7445.AM2013-2827
Abstract 283: Cancer MET at the metastatic lesion preserves the malignant features acquired via EMT.
Authors: Kudo-Saito Chie , Kawakami Yutaka
Published: August 2014

Purpose: Cancer EMT is one of the mechanisms to acquire malignant features including invasiveness, drug resistance and stemness. However, the subsequent mechanism after EMT toward progression has not been fully understood yet. Based on the EMT-centered theory, it is simply thought that the reversal event of EMT, “MET," is caused in the tumor cells at the distant metastatic lesions. However, the molecular mechanism still remains unclear. In this study, we attempted to clarify the “post-EMT” mechanism in tumor cells.

Results: Firstly, we prepared tumor cells harvested from metastatic lesions of mice after tumor implantation provisionally as “post-EMT” tumor cells. Murine metastatic melanoma B16-F10 cells were transduced with a neo resistant gene, and were subcutaneously (s.c.) implanted in many C57BL/6 mice to obtain spontaneous tumor metastasis. Three weeks later, various tissues including bone marrow were harvested from the mice, and the single-cell suspensions were cultured in G418-supplemented medium for tumor selection. To characterize the post-EMT tumor cells, RT-PCR, immunostaining and cellular functional assay were conducted. As compared to the primary tumor cells harvested from s.c. tumor tissues, these tumor cells proliferated extremely slowly, and were resistant to chemotherapeutics, but rapidly grew in vivo after implantation. However, these tumor cells were very adhesive accompanied by increase of epithelial markers such as E-cadherin, but not mesenchymal markers such as Snail, indicating epithelial features. In addition, its chemoresistance and in vivo tumorgenesis activity of these tumor cells were superior to those of even Snail-transduced B16-F10 tumor cells, which were harvested from the metastatic lesions in a similar way. DNA microarray analysis revealed the significant upregulation of epithelial markers and drug resistant genes such as c-kit and GDF15 in the post-EMT tumor cells, as compared to those of the Snail transfectant. These imply that “MET” may not be just a reversion of EMT.

Conclusions: These results suggest that tumor cells revert into an epithelial state preserving malignant features acquired via EMT even after Snail lost at the metastatic lesions toward progression. We are now attempting to elucidate the molecular mechanism specific for the unique transition utilizing DNA microarray data.

Citation Format: Chie Kudo-Saito, Yutaka Kawakami. Cancer MET at the metastatic lesion preserves the malignant features acquired via EMT. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 283. doi:10.1158/1538-7445.AM2013-283
Abstract 2840: Dendritic cell-based immunotherapy targeting WT1 and MUC1 for advanced biliary tract cancer.
Authors: Kobayashi Masanori , Sakabe Tomoyo , Tanii Mitsugu , Abe Hirofumi , Takahashi Hedenori , Tsujitani Shun-ichi , Shimodaira Shigetaka , Yonemitsu Yoshikazu , Okamoto Masato , Nagaya Masaki
Published: August 2014

Objectives:Despite intensive chemotherapy for biliary tract cancer (BTC), the outcome is not enough. Dendritic cells are the powerful antigen-presenting cells that have attracted attention from the efficacy of DC-based immunotherapy against many types of cancers. The objective of this study was to clarify the safety and efficacy of dendritic cell therapy for BTC.Methods: This retrospective study selected patients who underwent DC-based vaccine for BTC between May of 2009 and February of 2012. The safety of this treatment, median survival time (MST), 1-year survival rate, objective response rate (ORR), and disease control rate (DCR) were evaluated. The univariate and multivariate analyses were performed to identify the efficacy of DC by a Cox proportional hazards analysis.Results:Twenty-seven patients were selected for the study. The patients, and all but one patient had previous therapy, 13 patients had undergone prior surgical resection, and 21 patients went on to receive chemotherapy. No serious treatment-related adverse events were observed during the study period. One-year survival rate from 1st vaccination was 22 % and from the initial diagnosis was 67%. The MST from 1st vaccination and from the initial diagnosis were 8.2 months and 18.5 months, respectively. DCR and ORR were 37% and 11%,respectively. None of 27 patients showed complete response (CR), but we have found out 3 cases of partial responses (PR) and 7 cases of stable disease (SD).Furthermore, LDH, CRP, DTH, Performance Status (PS), combination with lymphokine-activated killer, chemotherapy were found to be statistically significant independent risk factors in multivariate analysis.Conclusion: DC-based immunotherapy for BTC was safe and prolonged the MST of the patients with BTC compared to chemotherapy alone,comparing the result from UK ABC-02 trial. DC-based immunotherapy for BTC may another option for BTC patient who has no chance for curative surgery.

Citation Format: Masanori Kobayashi, Tomoyo Sakabe, Mitsugu Tanii, Hirofumi Abe, Hedenori Takahashi, Shun-ichi Tsujitani, Shigetaka Shimodaira, Yoshikazu Yonemitsu, Masato Okamoto, Masaki Nagaya. Dendritic cell-based immunotherapy targeting WT1 and MUC1 for advanced biliary tract cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2840. doi:10.1158/1538-7445.AM2013-2840
Abstract 2843: Recombinant Vaccinia virus expressing CD40L: a multipotent antitumor immunogenic reagent.
Authors: Panopoulos Evangelos , Trella Emanuele , Raafat Nermin , Mengus Chantal , Spagnoli Giulio , Zajac Paul
Published: August 2014

Recombinant vaccinia virus (rVV) is one of the most immunogenic viral vectors. Its immunogenicity can be further modulated by the insertion of transgenes or deletion of viral genes which are involved in immune responses or specific cellular pathways. We generated a rVV encoding human CD154 (CD40L) and confirmed an efficient licensing of antigen presenting cells (APC), upon infection with a non-replicating (psoralen UV-inactivated) CD40L-rVV, as demonstrated by increased expression of (i) surface ligand/receptors (MHC I/II, CD80), (ii) Th1 cytokines (IL-12, IL-15, TNFa) and most importantly (iii) an increased capacity to stimulate TAA specific CTL proliferation. The hereby presented results are further characterizing some aspects of the pleiotropic roles in immuno-oncology of CD40 triggering via CD40L expressing recombinant vaccinia virus.

One of the immuno-modulatory functions of CD40L-CD40 is to play a key role during CD8+ priming for the generation of memory responses. Using sorted naive CD8+ Tcells (CD45RA+CD62L+) primed with allogeneic CD14+ infected with rVV 40L we observed an increased central memory phenotype (CD45RO+ CD62L+) as compared to culture primed with wild type virus infected cells. Moreover, rVV-CD40L stimulation of CD14+ cells also led to the inhibition of co-cultured tumor cells’ proliferation, correlating with increased TNFα expression. Finally, apoptosis of CD40+ tumor cell lines was induced upon direct infection with this CD40L non-replicating recombinant virus.

These data are confirming the potent capacities of recombinant vaccinia viruses expressing CD40L to efficiently trigger multiple CD40 mediated anti-tumor pathways. Not only this vector enhances the generation of tumor specific CD8+ T cells effector and memory responses, but it also promotes anti-tumoral functions of innate immune cells and is even able to directly induce apoptosis in CD40+ tumor cells. These in-vitro observations underline the strong clinical potential of recombinant vaccinia virus expressing CD40L in cancer viro- and immunotherapies.

Citation Format: Evangelos Panopoulos, Emanuele Trella, Nermin Raafat, Chantal Mengus, Giulio Spagnoli, Paul Zajac. Recombinant Vaccinia virus expressing CD40L: a multipotent antitumor immunogenic reagent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2843. doi:10.1158/1538-7445.AM2013-2843
Abstract 2846: Safety and efficacy of a genetic vaccine targeting telomerase plus chemotherapy for the therapy of canine B-cell lymphoma .
Authors: Gavazza Alessandra , Lubas George , Fridman Arthur , Peruzzi Daniela , Impellizeri Joseph A. , Luberto Laura , Marra Emanuele , Roscilli Giuseppe , Ciliberto Gennaro , Aurisicchio Luigi
Published: August 2014

Background: We have recently shown that a genetic vaccine targeting dog telomerase (dTERT) and based on Ad/DNA-EGT technology can induce strong cell-mediated immune responses against this tumor antigen and increase overall survival of dogs affected by B-cell lymphosarcoma (LSA) in comparison with historical controls when combined with COP chemotherapy regimen.

Objectives: Here, we have conducted a double arm clinical trial with an extended number of LSA patients, measured the antigen-specific immune response and evaluated potential toxic effects of the immunotherapy along with a follow up of patients survival for three years and half.

Methods: The immune response was measured by ELISPOT. The expression of dTERT was quantified by quantitative PCR. Changes in hematological parameters, local/systemic toxicity or organic dysfunction and fever were monitored over time during the treatment.

Results: dTERT-specific cell mediated immune responses were induced in almost all treated animals No adverse effects were observed in any dog patient that underwent treatment. The overall survival time of vaccine/COP treated dogs was significantly increased over the COP-only treated cohort (>76.1 vs 29.3 weeks, respectively, p<0.0001). There was a significant association between dTERT expression levels and overall survival (OS) among vaccinated patients. Conclusions and Clinical relevance: Ad/DNA-EGT-based cancer vaccine against dTERT in combination with COP chemotherapy is safe and significantly prolongs the survival of LSA canine patients. These data confirm the therapeutic efficacy of dTERT vaccine and support the evaluation of this approach in human trials and for other cancer types.

Citation Format: Alessandra Gavazza, George Lubas, Arthur Fridman, Daniela Peruzzi, Joseph A. Impellizeri, Laura Luberto, Emanuele Marra, Giuseppe Roscilli, Gennaro Ciliberto, Luigi Aurisicchio. Safety and efficacy of a genetic vaccine targeting telomerase plus chemotherapy for the therapy of canine B-cell lymphoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2013-2846
Abstract 2847: Molecular determinants of sensitivity and resistance to tumor-directed antibody-dependent cellular cytotoxicity (ADCC).
Authors: Murray Joseph C. , Nasto Rochelle E. , Jablonski Sandra A. , Tang Yong , Weiner Louis M.
Published: August 2014

Monoclonal antibodies can induce antibody-dependent cellular cytotoxicity (ADCC) through immune effector cells, such as natural killer (NK) cells. Although clinical evidence suggests a role for ADCC in effective antibody therapy of cancer, molecular determinants of ADCC responsiveness—in oncogenic signaling networks, for example—have not been well characterized. Using an in vitro model system including an NK-like cell line (NK92-CD16V), cetuximab, and A431 cells, we screened 60 genes from an EGFR gene network by RNAi to assess for synthetic enhancement of ADCC. Primary and validation screens, and correlative characterization studies, identified four genes whose knockdown enhanced ADCC: GRB7, PRKCE, RET, and ABL1. Inhibition of Abl kinase activity enhanced ADCC, phenocopying the effects of ABL1 knockdown and providing pre-clinical evidence for combining Abl inhibitors and cetuximab. Next, we derived an ADCC-resistant cell population (A431/ADCC-R) by treating parental A431 cells with over 30 successive ADCC treatments. We describe the development, screening, and characterization of A431/ADCC-R. Our screening strategy has revealed molecular determinants of both tumor cell sensitivity and resistance to ADCC in vitro that may translate into increased monoclonal antibody efficacy in the clinic.

Citation Format: Joseph C. Murray, Rochelle E. Nasto, Sandra A. Jablonski, Yong Tang, Louis M. Weiner. Molecular determinants of sensitivity and resistance to tumor-directed antibody-dependent cellular cytotoxicity (ADCC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2847. doi:10.1158/1538-7445.AM2013-2847
Abstract 2849: Pharmacodynamic biomarkers of anti-PD-L1 activity in pre-clinical mouse tumor models.
Authors: Boe Marigold , Huseni Mahrukh , Jiang Brittany , Koeppen Hartmut , Maecker Heather , Irving Bryan , Hegde Priti , Kowanetz Marcin
Published: August 2014

Programmed cell death 1 ligand 1 (PD-L1, CD274, B7-H1) is an immune checkpoint molecule expressed in various tissues, including multiple peripheral blood mononuclear cells and placenta. PD-L1 binds its receptor PD-1 on activated T cells to negatively regulate their function in both physiological and pathological conditions. PD-L1 also binds B7.1 (CD80), further down modulating immune responses. Importantly, numerous cancers resist T-cell mediated cytotoxic activity by over-expressing PD-L1.

The goal of this study was to identify pharmacodynamic biomarkers of response to agents blocking PD-L1/PD-1 pathway in pre-clinical models. CT-26 and MC-38 murine mouse tumor models were selected for this analysis, based on their PD-L1 expression and in-vivo sensitivity to anti-PD-L1 treatment. Tumors have been collected at various time points following treatment with an antibody blocking PD-L1 binding to PD-1 and B7.1. Subsequently, FACS, IHC, qPCR and a multiplex qPCR-based expression assay measuring a panel of ∼90 immune-related biomarkers have been used to evaluate changes in major immune cell subtypes and their activation status. We show that the intra-tumoral immune environment undergoes a time-sensitive dynamic modulation in response to PD-L1 pathway blockade, including an early and potent Th1 driven CTL response, as measured by relevant markers. Additionally, our findings show that targeting PD-L1 in pre-clinical tumor models can reinvigorate the host immune responses against the tumor and lead to T-cell mediated tumor killing. Our data also support identification of potential pharmacodynamic biomarkers that can be used to monitor activity of agents targeting the PD-L1 pathway in the clinic.

Citation Format: Marigold Boe, Mahrukh Huseni, Brittany Jiang, Hartmut Koeppen, Heather Maecker, Bryan Irving, Priti Hegde, Marcin Kowanetz. Pharmacodynamic biomarkers of anti-PD-L1 activity in pre-clinical mouse tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2849. doi:10.1158/1538-7445.AM2013-2849
Abstract 2850: Predicting anti-tumor responses to phosphatidylserine targeting antibodies using tumor imaging.
Authors: Gong Jian , Archer Richard , Nguyen Van , Hughes Christopher C.W. , Hutchins Jeff , Freimark Bruce
Published: August 2014

Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed on tumor vascular endothelial cells and tumor cells in response to chemotherapy, irradiation and oxidative stresses in the tumor microenvironment. Binding of antibodies targeting PS on the tumor endothelial cells and tumors induces the recruitment of immune cells and engages the immune system to destroy tumor vasculature. The antibodies also enhance anti-tumor immunity by blocking the immunosuppressive action of PS. A chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in Phase II trials. Fully human antibody PGN635 binds PS through the interaction of beta-2-glycoprotein 1 (β2GP1) in the same manner as bavituximab. Using human PC-3 prostate tumor xenografts in SCID mice, we demonstrate that targeting of PS in tumors by PGN635 is enhanced by chemotherapy. Combination of PGN635 with docetaxel inhibited tumor growth compared to the control IgG plus docetaxel group (p<0.05). Near-infrared optical imaging of PS in tumors with PGN650, an F(ab’)2 antibody fragment of PGN635, showed tumor growth inhibition in mice treated with docetaxel correlates with PS expression levels in the tumors. Maximal uptake of the PS imaging was observed when chemotherapy was given 24 hours before the imaging probe.

Citation Format: Jian Gong, Richard Archer, Van Nguyen, Christopher C.W. Hughes, Jeff Hutchins, Bruce Freimark. Predicting anti-tumor responses to phosphatidylserine targeting antibodies using tumor imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2850. doi:10.1158/1538-7445.AM2013-2850
Abstract 2855: Anti-PSMA x anti-CD3 SCORPIONTM molecule inhibits tumor growth in vivo in mouse models of prostate cancer.
Authors: Miller Robert , Fang Hang , Luke Jessica , Aguilar Megan , Chenault Ruth , Kumer John , Wiens Jennifer , Algate Paul , Bienvenue David , McMahan Catherine , Nguyen Holly , Vessella Robert , Blankenship John
Published: August 2014

Background: Treatment of metastatic, castrate-resistant prostate cancer (CRPC) remains a highly unmet medical need. We have developed a bispecific SCORPIONTM (multi-specific protein therapeutic) molecule that redirects T cell cytotoxicity against cells expressing PSMA (Prostate Specific Membrane Antigen), a common prostate-cancer related antigen. The SCORPION molecule described here contains two pairs of binding domains, each targeting a unique antigen, linked to opposite ends of an immunoglobulin Fc domain to extend the half-life of the molecule in vivo. In these studies, we examine the ability of a SCORPION molecule targeting both CD3 and PSMA, to inhibit the growth of PSMA expressing tumors in vivo.

Materials and Methods: NOD/SCID mice were implanted with the PSMA(+) prostate tumor cell line C4-2B and purified human T cells in one of two models. In a first model of residual disease, C4-2B cells were coimplanted subcutaneously with human T cells, and treatment with drug was initiated immediately. This model was run twice with T cells from two different human donors. In a second model of established disease, C4-2B cells were implanted subcutaneously and allowed to develop for 20 days, after which human T cells were injected intra-peritoneally, with subsequent administration of drug. Tumor growth was assessed by tumor volume and serum PSA, and tumor volume and weight loss were used as endpoints.

Results: SCORPION molecules targeting PSMA and CD3 inhibited growth of PSMA(+) tumors in both model settings and significantly improved overall survival. Reduction in circulating serum PSA was also observed. Repeat studies using a second human T cell donor showed similar inhibition of tumor growth and overall survival. Inhibition of tumor growth was seen at doses in the low pmol range per mouse.

Conclusions: These studies show that anti-PSMA x anti-CD3 SCORPION therapeutics redirect T cell cytotoxicity in in vivo xenograft models and merit further investigation as potential therapeutics in CRPC.

Citation Format: Robert Miller, Hang Fang, Jessica Luke, Megan Aguilar, Ruth Chenault, John Kumer, Jennifer Wiens, Paul Algate, David Bienvenue, Catherine McMahan, Holly Nguyen, Robert Vessella, John Blankenship. Anti-PSMA x anti-CD3 SCORPIONTM molecule inhibits tumor growth in vivo in mouse models of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2855. doi:10.1158/1538-7445.AM2013-2855