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353 Natural Killer Cell Responses to Tumor Priming
S86
european journal of cancer 48, suppl. 5 (2012) S25–S288
351 A Twist1 Code of P53 Inactivation S. Piccinin1 , E. Tonin1 , S. Sessa1 , F. Pivetta1 , C. Rosano2 , S. Rossi3 , A.P. Dei Tos3 , C. Doglioni4 , R. Maestro1 . 1 CRO-National Cancer Institute, Experimental Oncology 1, Aviano, Italy, 2 IST National Cancer Institute, Nanotechnology Unit, Genova, Italy, 3 Treviso General Hospital, Pathology, Treviso, Italy, 4 San Raffaele Scientific Institute, Pathology, Milano, Italy Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we identified an unprecedented mechanism of destabilization of p53. We show that Twist1, by its C-terminus called Twist box, bounds the C-terminal regulatory domain of p53 and physically hinders p53 key phosphorylations facilitating MDM2:p53 complex formation and p53 degradation. This study suggests the existence of a Twist code of p53 inactivation in sarcomas and provides the proof of principle that targeting the Twist:p53 interaction may offer additional avenues for cancer treatment. 353 Natural Killer Cell Responses to Tumor Priming M. Sabry1 , M.W. Lowdell1 . 1 University College London, Academic Haematology, London, United Kingdom Background: Human Natural Killer (NK) cells are lymphocytes that play an important role in host protection against viral infections and tumours. Our group previously hypothesized a two-stage process for resting NK cell activation. A ‘priming’ signal can be delivered by an activating cytokine such as IL-2 to generate a lymphokine-activated NK cell (LAK). Similarly, conjugation to a tumour cell expressing an appropriate intensity and combination of ligands, can also result in NK cell priming and the generation of a tumour-activated NK cell (TaNK). The second ‘triggering’ signal is more specific to prevent autoreactivity, and initiates NK cell degranulation and/or cytokine release. The CTV-1 leukemic cell line is capable of priming NK cells to kill NK-resistant tumours. In this set of experiments we have investigated NK cell responses unique to tumour priming by measuring TaNK cell surface expression, cytotoxic capacity and secretome profiles compared to cytokine-primed NK cells. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from consenting normal healthy donors by density gradient separation and CD56+ purification was performed using magnetic microbeads. NK cells were incubated overnight with whole CTV-1 cells, IL-2, IL-7, IL-15, IL-12 or IL-21. Using 8 colour flowcytometric immunostaining NK cell receptor expression was analyzed. The NK cell secretome was examined using the cytometric bead array (CBA) assay and the CD107a assay was performed to determine NK cell degranulation. Results and Discussion: Tumour-priming of NK cells is equivalent to cytokine-priming as demonstrated by the upregulation of the activation markers CD69 and CD25, as well as the enhancement of NK cell cytotoxicity and cytokine production. However, NK cell responses to in vitro stimulation by CTV-1 appeared different to all the cytokines tested. Uniquely, TaNKs upregulated the inhibitory receptor NKG2A and downregulated the activation receptors CD16, NKG2D, DNAM-1, ICAM-1, NKp44 and NKp46. Moreover, NK cell subsets defined phenotypically by their CD56, CD2, CD57 and CD16 expression, responded differently to the various stimuli in terms of their activation marker expression, cytokine production and degranulation. Conclusion: Tumour-mediated priming of NK cells is different from cytokinemediated priming as evidenced by unique cell surface protein expression patterns, secretome profiles and killing capacities. 354 Cellular and Molecular Characteristics of Novel Colon Cancer Cell Line G. Bogdanovic1 , S. Usaj-Knezevic1 , M. Krajnovic2 , V. Kojic1 , Z. Nikin3 , T. Petrovic4 , K. Krtolica2 , M. Popsavin5 , A. Krtolica6 . 1 Oncology Institute of Vojvodina, Experimental Oncology Department, Sremska Kamenica, Serbia, 2 Institute for Nuclear Research Vinca, Laboratory for Radiobiology and Molecular Genetics, Belgrade, Serbia, 3 Oncology Institute of Vojvodina, Pathology Department, Sremska Kamenica, Serbia, 4 Oncology Institute of Vojvodina, Surgery Department, Sremska Kamenica, Serbia, 5 University of Novi Sad Faculty of Sciences, Chemistry Department, Novi Sad, Serbia, 6 StemLifeLine, San Carlos CA, USA Background: Cell lines represent important resource for research in a variety of fields and disciplines. Continuous cell lines used in experimental settings are a valuable tool for understanding the biology, pathogenesis, and treatment modalities for various malignancies. We report on characteristics of a novel human cancer cell line derived from primary colon tumor. Material and Method: Surgical tumor specimen was obtained from 35-year old Caucasian who has undergone complete resection of tumor at the Oncology Institute of Vojvodina. Tissue sample was minced and further dissociated with complex enzyme mixture. The cells were cultured in DMEM/F12 medium supplemented with FBS, L-glutamine, and antibiotics.
Sunday 8 − Tuesday 10 July 2012
Classic cytological analyses, immunocytochemical analyses, and molecular genetic procedures were used for cell line characterization. Proliferation capacity and cell chemosensitivity were analyzed by MTT assay. Results: The cultured cells formed morphologically homogenous population of epithelial-like cells. Immunocytochemical analyses showed mild to strong staining for CD133, CD166, ESA, CKAE1-AE3, Ki67, p53, that was similar to original tumor tissue. Compared to original tumor sample, the cultured cells did not express VEGF and EGFR. We examined methylation status of CpG islands in 5 promoter region of p15, p16, MGMT and DAPK genes. Our results obtained by MSP revealed lack of methylation and the presence of unmethylated pattern of all of the four analyzed genes, in both, colorectal carcinoma tissue sample and the cell line. Detection of mutations in K-ras (codons 12, 13, 60, and 61) and BRAF (V600E) genes is in progress. The cell line was found sensitive to common antitumor drugs and to several new tiazofurin analogues. Conclusion: A new colon cancer cell line retained many molecular characteristics of the original tumor tissue and can serve as a model for evaluation of both biology and treatment options for colon cancer. 355 Clonogenicity and in Vivo Growth Identify Two Types of Stem-like Glioblastoma Cells (SLGC) Differing in Tumorigenicity and Invasiveness Z. Gal1 , B. Campos1 , M.R. Farhadi1 , T. Schmoch1 , B. Lahrmann2 , P. Beckhove3 , N. Grabe2 , V. Eckstein4 , A. Unterberg1 , C. Herold-Mende1 . 1 University of Heidelberg, Department of Neurosurgery, Heidelberg, Germany, 2 Institute of Medical Biometry and Informatics, Hamamatsu Tissue Imaging and Analysis Center (TIGA), Heidelberg, Germany, 3 German Cancer Research Center, Division of Tumor Immunology, Heidelberg, Germany, 4 University of Heidelberg, Department of Medicine V, Heidelberg, Germany Introduction: Glioblastoma is the most common and aggressive primary brain malignancy. Stem-like tumor cells have been shown to more closely resemble the patient tumor. However, they vary markedly regarding growth and marker expression. Therefore the aim of the following study was to carefully characterize SLGC-lines based on their in vitro self renewal potential, in vivo tumorigenicity and expression of the surrogate marker AC133. Material and Method: Neural colony-forming cell assay (NCFCA) was performed in a collagen matrix on 8 SLGC lines to quantify their self renewal potential. Then 104 cells of each cell line were implanted orthotopically into non-obese diabetic immunodeficient (NOD SCID) mice and monitored for a period of 8 weeks (short-term). In addition, for 6/8 cell lines observation period was prolonged (long-term) and for 4/8 SLGCs the implanted cell number was increased to 106 cells/mouse. Cryosections of the brains were analyzed for tumorigenicity, invasiveness, neoangiogenesis and proliferative potential. Furthermore DiI-labeled SLGC tumorspheres were implanted into a cranial window chamber in nude mice. Tumor growth, cell migration and angiogenesis were monitored using a fluorescence intravital microscope (IVM) during a 17days observation period. Results and Discussion: NCFCA revealed clonogenicity ranging from 0.13% to 18%. Concordantly, in our short-term approach 4/5 highly clonogenic cell lines developed tumors with 100% penetrance resulting in a decreased symptom-free survival. Tumor penetrance was associated with a higher AC133 content. In our long-term approach and after implanting increased cell numbers even AC133low/negative cell lines developed tumors. However, the latter were less dense and more infiltrative. In contrast, superficial implantation in the window chamber model revealed an aberrant vascular network with microvascular proliferation for all SGLC lines but was not able to delineate growth differences observed after intracranial implantation. Conclusion: Our findings indicate a critical influence of the tumor-surrounding brain and the observation period length on tumorigenicity and invasiveness of SLGCs which needs to be taken into account when planning in vivo experiments with SLGCs. 356 Plexin B1 Trafficking in Prostate Cancer A. Legendre1 , M. Williamson1 , J.R. Masters1 . 1 Prostate Cancer Research Center, University College London, London, United Kingdom Background: Prostate cancer is a leading form of cancer in men with 324,000 cases diagnosed/year in the European Union. Plexin B1 is a cell-membrane receptor for semaphorins, its ligand is Semaphorin 4D. Plexin receptors are involved in axon guidance, angiogenesis, immunity as well as cancer biology. We have identified a high frequency of somatic missense mutations in the plexinB1 gene and overexpression of the protein in prostate cancer. The mutations found in prostate cancer are functionally significant, altering signalling pathways downstream of plexinB1 and affecting cell phenotype. These results suggest that plexinB1 has an important role in prostate cancer progression. Previous studies have documented the unexpected localisation of cell membrane receptors such as EGFR and ErbB2, in the nucleus and their role as transcription factors. Preliminary immunohistochemistry results in the
european journal of cancer 48, suppl. 5 (2012) S25–S288
351 A Twist1 Code of P53 Inactivation S. Piccinin1 , E. Tonin1 , S. Sessa1 , F. Pivetta1 , C. Rosano2 , S. Rossi3 , A.P. Dei Tos3 , C. Doglioni4 , R. Maestro1 . 1 CRO-National Cancer Institute, Experimental Oncology 1, Aviano, Italy, 2 IST National Cancer Institute, Nanotechnology Unit, Genova, Italy, 3 Treviso General Hospital, Pathology, Treviso, Italy, 4 San Raffaele Scientific Institute, Pathology, Milano, Italy Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we identified an unprecedented mechanism of destabilization of p53. We show that Twist1, by its C-terminus called Twist box, bounds the C-terminal regulatory domain of p53 and physically hinders p53 key phosphorylations facilitating MDM2:p53 complex formation and p53 degradation. This study suggests the existence of a Twist code of p53 inactivation in sarcomas and provides the proof of principle that targeting the Twist:p53 interaction may offer additional avenues for cancer treatment. 353 Natural Killer Cell Responses to Tumor Priming M. Sabry1 , M.W. Lowdell1 . 1 University College London, Academic Haematology, London, United Kingdom Background: Human Natural Killer (NK) cells are lymphocytes that play an important role in host protection against viral infections and tumours. Our group previously hypothesized a two-stage process for resting NK cell activation. A ‘priming’ signal can be delivered by an activating cytokine such as IL-2 to generate a lymphokine-activated NK cell (LAK). Similarly, conjugation to a tumour cell expressing an appropriate intensity and combination of ligands, can also result in NK cell priming and the generation of a tumour-activated NK cell (TaNK). The second ‘triggering’ signal is more specific to prevent autoreactivity, and initiates NK cell degranulation and/or cytokine release. The CTV-1 leukemic cell line is capable of priming NK cells to kill NK-resistant tumours. In this set of experiments we have investigated NK cell responses unique to tumour priming by measuring TaNK cell surface expression, cytotoxic capacity and secretome profiles compared to cytokine-primed NK cells. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from consenting normal healthy donors by density gradient separation and CD56+ purification was performed using magnetic microbeads. NK cells were incubated overnight with whole CTV-1 cells, IL-2, IL-7, IL-15, IL-12 or IL-21. Using 8 colour flowcytometric immunostaining NK cell receptor expression was analyzed. The NK cell secretome was examined using the cytometric bead array (CBA) assay and the CD107a assay was performed to determine NK cell degranulation. Results and Discussion: Tumour-priming of NK cells is equivalent to cytokine-priming as demonstrated by the upregulation of the activation markers CD69 and CD25, as well as the enhancement of NK cell cytotoxicity and cytokine production. However, NK cell responses to in vitro stimulation by CTV-1 appeared different to all the cytokines tested. Uniquely, TaNKs upregulated the inhibitory receptor NKG2A and downregulated the activation receptors CD16, NKG2D, DNAM-1, ICAM-1, NKp44 and NKp46. Moreover, NK cell subsets defined phenotypically by their CD56, CD2, CD57 and CD16 expression, responded differently to the various stimuli in terms of their activation marker expression, cytokine production and degranulation. Conclusion: Tumour-mediated priming of NK cells is different from cytokinemediated priming as evidenced by unique cell surface protein expression patterns, secretome profiles and killing capacities. 354 Cellular and Molecular Characteristics of Novel Colon Cancer Cell Line G. Bogdanovic1 , S. Usaj-Knezevic1 , M. Krajnovic2 , V. Kojic1 , Z. Nikin3 , T. Petrovic4 , K. Krtolica2 , M. Popsavin5 , A. Krtolica6 . 1 Oncology Institute of Vojvodina, Experimental Oncology Department, Sremska Kamenica, Serbia, 2 Institute for Nuclear Research Vinca, Laboratory for Radiobiology and Molecular Genetics, Belgrade, Serbia, 3 Oncology Institute of Vojvodina, Pathology Department, Sremska Kamenica, Serbia, 4 Oncology Institute of Vojvodina, Surgery Department, Sremska Kamenica, Serbia, 5 University of Novi Sad Faculty of Sciences, Chemistry Department, Novi Sad, Serbia, 6 StemLifeLine, San Carlos CA, USA Background: Cell lines represent important resource for research in a variety of fields and disciplines. Continuous cell lines used in experimental settings are a valuable tool for understanding the biology, pathogenesis, and treatment modalities for various malignancies. We report on characteristics of a novel human cancer cell line derived from primary colon tumor. Material and Method: Surgical tumor specimen was obtained from 35-year old Caucasian who has undergone complete resection of tumor at the Oncology Institute of Vojvodina. Tissue sample was minced and further dissociated with complex enzyme mixture. The cells were cultured in DMEM/F12 medium supplemented with FBS, L-glutamine, and antibiotics.
Sunday 8 − Tuesday 10 July 2012
Classic cytological analyses, immunocytochemical analyses, and molecular genetic procedures were used for cell line characterization. Proliferation capacity and cell chemosensitivity were analyzed by MTT assay. Results: The cultured cells formed morphologically homogenous population of epithelial-like cells. Immunocytochemical analyses showed mild to strong staining for CD133, CD166, ESA, CKAE1-AE3, Ki67, p53, that was similar to original tumor tissue. Compared to original tumor sample, the cultured cells did not express VEGF and EGFR. We examined methylation status of CpG islands in 5 promoter region of p15, p16, MGMT and DAPK genes. Our results obtained by MSP revealed lack of methylation and the presence of unmethylated pattern of all of the four analyzed genes, in both, colorectal carcinoma tissue sample and the cell line. Detection of mutations in K-ras (codons 12, 13, 60, and 61) and BRAF (V600E) genes is in progress. The cell line was found sensitive to common antitumor drugs and to several new tiazofurin analogues. Conclusion: A new colon cancer cell line retained many molecular characteristics of the original tumor tissue and can serve as a model for evaluation of both biology and treatment options for colon cancer. 355 Clonogenicity and in Vivo Growth Identify Two Types of Stem-like Glioblastoma Cells (SLGC) Differing in Tumorigenicity and Invasiveness Z. Gal1 , B. Campos1 , M.R. Farhadi1 , T. Schmoch1 , B. Lahrmann2 , P. Beckhove3 , N. Grabe2 , V. Eckstein4 , A. Unterberg1 , C. Herold-Mende1 . 1 University of Heidelberg, Department of Neurosurgery, Heidelberg, Germany, 2 Institute of Medical Biometry and Informatics, Hamamatsu Tissue Imaging and Analysis Center (TIGA), Heidelberg, Germany, 3 German Cancer Research Center, Division of Tumor Immunology, Heidelberg, Germany, 4 University of Heidelberg, Department of Medicine V, Heidelberg, Germany Introduction: Glioblastoma is the most common and aggressive primary brain malignancy. Stem-like tumor cells have been shown to more closely resemble the patient tumor. However, they vary markedly regarding growth and marker expression. Therefore the aim of the following study was to carefully characterize SLGC-lines based on their in vitro self renewal potential, in vivo tumorigenicity and expression of the surrogate marker AC133. Material and Method: Neural colony-forming cell assay (NCFCA) was performed in a collagen matrix on 8 SLGC lines to quantify their self renewal potential. Then 104 cells of each cell line were implanted orthotopically into non-obese diabetic immunodeficient (NOD SCID) mice and monitored for a period of 8 weeks (short-term). In addition, for 6/8 cell lines observation period was prolonged (long-term) and for 4/8 SLGCs the implanted cell number was increased to 106 cells/mouse. Cryosections of the brains were analyzed for tumorigenicity, invasiveness, neoangiogenesis and proliferative potential. Furthermore DiI-labeled SLGC tumorspheres were implanted into a cranial window chamber in nude mice. Tumor growth, cell migration and angiogenesis were monitored using a fluorescence intravital microscope (IVM) during a 17days observation period. Results and Discussion: NCFCA revealed clonogenicity ranging from 0.13% to 18%. Concordantly, in our short-term approach 4/5 highly clonogenic cell lines developed tumors with 100% penetrance resulting in a decreased symptom-free survival. Tumor penetrance was associated with a higher AC133 content. In our long-term approach and after implanting increased cell numbers even AC133low/negative cell lines developed tumors. However, the latter were less dense and more infiltrative. In contrast, superficial implantation in the window chamber model revealed an aberrant vascular network with microvascular proliferation for all SGLC lines but was not able to delineate growth differences observed after intracranial implantation. Conclusion: Our findings indicate a critical influence of the tumor-surrounding brain and the observation period length on tumorigenicity and invasiveness of SLGCs which needs to be taken into account when planning in vivo experiments with SLGCs. 356 Plexin B1 Trafficking in Prostate Cancer A. Legendre1 , M. Williamson1 , J.R. Masters1 . 1 Prostate Cancer Research Center, University College London, London, United Kingdom Background: Prostate cancer is a leading form of cancer in men with 324,000 cases diagnosed/year in the European Union. Plexin B1 is a cell-membrane receptor for semaphorins, its ligand is Semaphorin 4D. Plexin receptors are involved in axon guidance, angiogenesis, immunity as well as cancer biology. We have identified a high frequency of somatic missense mutations in the plexinB1 gene and overexpression of the protein in prostate cancer. The mutations found in prostate cancer are functionally significant, altering signalling pathways downstream of plexinB1 and affecting cell phenotype. These results suggest that plexinB1 has an important role in prostate cancer progression. Previous studies have documented the unexpected localisation of cell membrane receptors such as EGFR and ErbB2, in the nucleus and their role as transcription factors. Preliminary immunohistochemistry results in the