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MP-02.07 Calcium Depending Bone Metastases in Renal Cell Carcinoma
POSTER PRESENTATIONS
tracorporeal circulation, and, at 36°C, cardiac movement was DC-induced. Extracorporeal circulation was discontinued, and the wound closed. Results: Patients consisted of 7 males and 1 female with a mean age of 59.4 years. The mean surgical time was 773 min, mean bleeding volume 3673 ml, mean duration of extracorporeal circulation 258 min, and mean discontinuation of circulation 37.4 min. Surgical death was observed in 1, and cancer death in 6. One patient died of cerebral infarction without recurrence. The mean survival period was 22.4 months. Conclusions: The surgical field is bloodless using the hypothermic circulatory arrest, and the possibility of residual tumor lesions decreases. Because of marked invasiveness, patient selection is important.
MP-02.07 Calcium Depending Bone Metastases in Renal Cell Carcinoma Brenner W1, Schneider E1, Junker K2, Roos F1, Thüroff J1 1 Dept. of Urology, University Medical Center, Johannes Gutenberg University, Mainz, Germany; 2Dept. of Urology, University Hospital Friedrich Schiller University, Jena, Germany Introduction and Objective: Although a number of molecules have been implicated in the process of cancer metastasis, the organ-selective nature of cancer cells is still poorly understood. Renal cell carcinoma (RCC) often metastasize into bones, a calcium enriched tissue. We analyzed the influence of the calcium sensing receptor (CaSR) in RCC and an enhanced calcium concentration in processes of metastasis. Material and Methods: In 27 matched clear cell RCC specimens, 9 non-metastasized, 9 metastasized into the lung and 9 metastasized into bones, CaSR were quantified by Western blot analyses. To analyze the influence of calcium on RCC, RCC cells were isolated from patients developing bone, lung and no metastasis (each 3 cell lines). Cell proliferation after calcium treatment (2.5, 5 and 10 M) was quantified by BrdU incorporation and cell migration in a Boyden chemotaxis assay with calcium as chemotactical attractant (10 M). Expression and activity of intracellular signal transduction mediators were analyzed by a human phospho kinase array and Western blot analyses. Results: Compared to non-metastasizing RCC, we found an 85% enhanced CaSR
expression in tumor specimens of patients who developed bone metastasis within 5 years after surgery. In cells isolated from patients developing bone metastasis CaSR was expressed 3.5-fold higher compared to cells isolated from patients without metastases. Calcium treatment of RCC cells of bone metastasizing specimens, but not of non-metastasizing specimens, induced a concentration dependent enhancement of proliferation. In a Boyden chamber with calcium as chemo-attractant, RCC cells from patients with bone metastases showed a 7.5-fold higher migration rate compared to cells from patients without metastases. In cells with lung metastases, migration was 2.5-fold enhanced. Treatment of RCC cells with calcium induced an enhanced activity of Src and its downstream targets STAT 3 and c-Jun, leading to enhanced proliferation. Furthermore, in tumors metastasizing into bones PTEN was reduced expressed and subsequently its downstream target Shc was enhanced active, inducing cell migration. Conclusion: Our results indicate that bone metastasis of RCC is caused by enhanced expression of CaSR and that calcium has an enhancing effect on cell migration and proliferation of bone metastasizing RCC cells via Src and Shc signalling.
MP-02.08 Identification of Cancer-Stem-Cell-Like Cells in a Papillary Renal Cell Carcinoma-Derived Cell Line Börger V1, Rottke D5, Sorg U2, Heikaus S3, Opalka B6, Ackermann R4, Wernet P1, Sorg R1 1 Institute For Transplantation Diagnostics & Cell Therapeutics; 2 Institute For Medical Microbiology & Hospital Hygiene; 3Institute of Pathology, 4 Dept. of Urology, University Hospital Düsseldorf, Düsseldorf, Germany; 5Dept. of Urology, University of Hamburg, Hamburg, Germany; 6Dept. of Hematology, University of DuisburgEssen, Essen, Germany Introduction and Objectives: Cancer stem cells play an important role in the development und progression of tumors. They are defined as a subpopulation of cells within a tumor which shows self– renewal capacity, pseudo/differentiation activity and most importantly, they are able to initiate tumor growth when xenotransplanted into immunocompromized mice. In papillary renal cell carcinoma, cancer stem cells have not been identified so far.
UROLOGY 78 (Supplement 3A), September 2011
Materials and Methods: We have newly established a tumor cell line, MG-1, from a papillary renal cell carcinoma. This cell line as well as subclones obtained after cell sorting have been characterized by flow cytometry, RT-PCR expression analysis, in vitro differentiation assays and by assessing tumorigenicity in immunocompromized mouse models to identify cells with characteristics of cancer stem cells. Results: MG-1 showed an epithelial morphology. They expressed epithelial markers like EpCam and cytokeratins as well as markers on at least subpopulations which have been identified on cancer stem cells of other tumor entities, including CD44 and CD133. MG-1 could be differentiated along the osteogenic pathway. They formed tumor-spheres when single cells were plated in growth-factor supplemented serum-free medium. Moreover, MG-1 was tumorigenic in nude, NOD/ SCID and NOD/SCID IL2rc␥null mice when 1x106 cells were transplanted. Serial transplantations confirmed self-renewal activity. After CD133-based cell sorting, MG-1 subclones were generated. All tumorigenic clones were derived from CD133⫺ cells, indicating that the cancer stem cells reside within the CD133⫺ population. One CD133⫺ subclone, M-G1/F7, was highly tumorigenic: Tumor development was observed when as few as 1000 tumor cells were transplanted. MG-1/F7 showed a phenotype similar to the parental line but lacked EpCAM and CD133 expression. It revealed osteogenic differentiation potential but no sphere-forming activity. Interestingly, although expression of the stem cell marker Oct-4 was observed in about half of the clones, it was not associated with tumorigenicity, and MG-1/F7 cells were Oct-4 negative. Conclusion: The newly established papillary renal cell carcinoma cell line MG-1 and particularly its CD133⫺ subclones share typical features with cancer stem cells, including immunophenotype, tumorigenicity, self-renewal and differentiation potential.
MP-02.09 Long-Term Results of Percutaneous Radiofrequency Ablation (PRFA) of Small Renal Tumors in High-Risk Patients: A Single Institution Experience Brausi M, Gavioli M, Peracchia G, De Luca G, Verrini G, Viola M, Simonini G, Giliberto G, Ferrari F, Romano A Dept. of Urology, AUSL Modena, Carpi, Italy
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tracorporeal circulation, and, at 36°C, cardiac movement was DC-induced. Extracorporeal circulation was discontinued, and the wound closed. Results: Patients consisted of 7 males and 1 female with a mean age of 59.4 years. The mean surgical time was 773 min, mean bleeding volume 3673 ml, mean duration of extracorporeal circulation 258 min, and mean discontinuation of circulation 37.4 min. Surgical death was observed in 1, and cancer death in 6. One patient died of cerebral infarction without recurrence. The mean survival period was 22.4 months. Conclusions: The surgical field is bloodless using the hypothermic circulatory arrest, and the possibility of residual tumor lesions decreases. Because of marked invasiveness, patient selection is important.
MP-02.07 Calcium Depending Bone Metastases in Renal Cell Carcinoma Brenner W1, Schneider E1, Junker K2, Roos F1, Thüroff J1 1 Dept. of Urology, University Medical Center, Johannes Gutenberg University, Mainz, Germany; 2Dept. of Urology, University Hospital Friedrich Schiller University, Jena, Germany Introduction and Objective: Although a number of molecules have been implicated in the process of cancer metastasis, the organ-selective nature of cancer cells is still poorly understood. Renal cell carcinoma (RCC) often metastasize into bones, a calcium enriched tissue. We analyzed the influence of the calcium sensing receptor (CaSR) in RCC and an enhanced calcium concentration in processes of metastasis. Material and Methods: In 27 matched clear cell RCC specimens, 9 non-metastasized, 9 metastasized into the lung and 9 metastasized into bones, CaSR were quantified by Western blot analyses. To analyze the influence of calcium on RCC, RCC cells were isolated from patients developing bone, lung and no metastasis (each 3 cell lines). Cell proliferation after calcium treatment (2.5, 5 and 10 M) was quantified by BrdU incorporation and cell migration in a Boyden chemotaxis assay with calcium as chemotactical attractant (10 M). Expression and activity of intracellular signal transduction mediators were analyzed by a human phospho kinase array and Western blot analyses. Results: Compared to non-metastasizing RCC, we found an 85% enhanced CaSR
expression in tumor specimens of patients who developed bone metastasis within 5 years after surgery. In cells isolated from patients developing bone metastasis CaSR was expressed 3.5-fold higher compared to cells isolated from patients without metastases. Calcium treatment of RCC cells of bone metastasizing specimens, but not of non-metastasizing specimens, induced a concentration dependent enhancement of proliferation. In a Boyden chamber with calcium as chemo-attractant, RCC cells from patients with bone metastases showed a 7.5-fold higher migration rate compared to cells from patients without metastases. In cells with lung metastases, migration was 2.5-fold enhanced. Treatment of RCC cells with calcium induced an enhanced activity of Src and its downstream targets STAT 3 and c-Jun, leading to enhanced proliferation. Furthermore, in tumors metastasizing into bones PTEN was reduced expressed and subsequently its downstream target Shc was enhanced active, inducing cell migration. Conclusion: Our results indicate that bone metastasis of RCC is caused by enhanced expression of CaSR and that calcium has an enhancing effect on cell migration and proliferation of bone metastasizing RCC cells via Src and Shc signalling.
MP-02.08 Identification of Cancer-Stem-Cell-Like Cells in a Papillary Renal Cell Carcinoma-Derived Cell Line Börger V1, Rottke D5, Sorg U2, Heikaus S3, Opalka B6, Ackermann R4, Wernet P1, Sorg R1 1 Institute For Transplantation Diagnostics & Cell Therapeutics; 2 Institute For Medical Microbiology & Hospital Hygiene; 3Institute of Pathology, 4 Dept. of Urology, University Hospital Düsseldorf, Düsseldorf, Germany; 5Dept. of Urology, University of Hamburg, Hamburg, Germany; 6Dept. of Hematology, University of DuisburgEssen, Essen, Germany Introduction and Objectives: Cancer stem cells play an important role in the development und progression of tumors. They are defined as a subpopulation of cells within a tumor which shows self– renewal capacity, pseudo/differentiation activity and most importantly, they are able to initiate tumor growth when xenotransplanted into immunocompromized mice. In papillary renal cell carcinoma, cancer stem cells have not been identified so far.
UROLOGY 78 (Supplement 3A), September 2011
Materials and Methods: We have newly established a tumor cell line, MG-1, from a papillary renal cell carcinoma. This cell line as well as subclones obtained after cell sorting have been characterized by flow cytometry, RT-PCR expression analysis, in vitro differentiation assays and by assessing tumorigenicity in immunocompromized mouse models to identify cells with characteristics of cancer stem cells. Results: MG-1 showed an epithelial morphology. They expressed epithelial markers like EpCam and cytokeratins as well as markers on at least subpopulations which have been identified on cancer stem cells of other tumor entities, including CD44 and CD133. MG-1 could be differentiated along the osteogenic pathway. They formed tumor-spheres when single cells were plated in growth-factor supplemented serum-free medium. Moreover, MG-1 was tumorigenic in nude, NOD/ SCID and NOD/SCID IL2rc␥null mice when 1x106 cells were transplanted. Serial transplantations confirmed self-renewal activity. After CD133-based cell sorting, MG-1 subclones were generated. All tumorigenic clones were derived from CD133⫺ cells, indicating that the cancer stem cells reside within the CD133⫺ population. One CD133⫺ subclone, M-G1/F7, was highly tumorigenic: Tumor development was observed when as few as 1000 tumor cells were transplanted. MG-1/F7 showed a phenotype similar to the parental line but lacked EpCAM and CD133 expression. It revealed osteogenic differentiation potential but no sphere-forming activity. Interestingly, although expression of the stem cell marker Oct-4 was observed in about half of the clones, it was not associated with tumorigenicity, and MG-1/F7 cells were Oct-4 negative. Conclusion: The newly established papillary renal cell carcinoma cell line MG-1 and particularly its CD133⫺ subclones share typical features with cancer stem cells, including immunophenotype, tumorigenicity, self-renewal and differentiation potential.
MP-02.09 Long-Term Results of Percutaneous Radiofrequency Ablation (PRFA) of Small Renal Tumors in High-Risk Patients: A Single Institution Experience Brausi M, Gavioli M, Peracchia G, De Luca G, Verrini G, Viola M, Simonini G, Giliberto G, Ferrari F, Romano A Dept. of Urology, AUSL Modena, Carpi, Italy
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