Sophie Baumgart

MicroRNAs with Prognostic Potential for Metastasis in Clear Cell Renal Cell Carcinoma: A Comparison of Primary Tumors and Distant Metastases

AbstractBackground MicroRNAs (miRNAs) are regulators of gene expression in tumor development and progression. However, their influence on metastasis of clear cell renal cell carcinoma (ccRCC) is less understood. To determine the role of miRNAs in metastatic progression, miRNA expression in primary ccRCC was compared to distant metastases. MethodsTotal RNA of 53 primary ccRCCs, 35 distant metastases from lung, bone, brain, and abdomen, as well as 17 normal kidney tissues was isolated from fresh frozen tissue and formalin-fixed paraffin-embedded (FFPE) samples. The miRNA microarrays were performed based on fresh frozen tissue. Results were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on fresh frozen tissue and FFPE samples. Real-time cell analyses and transwell invasion assays were carried out after transient transfection of microRNA-30c (miR-30c) in cell line 786-O.ResultsThere were 14 miRNAs differently expressed in metastatic primary ccRCC and distant metastases compared to non-metastatic primary tumors. A strong correlation of miRNAs to progression-free- and cancer-specific 5-year-survival was determined. Specific miRNAs were differently expressed in distant metastases compared to primary ccRCC. A miRNA signature distinguished lung metastases from other metastatic sites. Overexpression of miR-30c increased adherence and decreased migration and invasion in the ccRCC cell line.ConclusionsMiRNAs are deregulated in metastatic primary ccRCC and could be promising prognostic markers for an early prediction of metastasis. Alterations in miRNA expression characterize distant metastases of different metastatic sites. Furthermore, our study suggests a functional role of miR-30c in metastasis. The miRNAs could be a helpful tool for individual follow-up prediction and personalized therapy selection.

Exosomes of invasive urothelial carcinoma cells are characterized by a specific miRNA expression signature

Muscle-invasive bladder cancer (MIBC) represents a highly aggressive tumor type compared to non-muscle-invasive tumors. MIBC is characterized by specific molecular alterations, which may also modulate extracellular tumorigenic effects. Tumor-associated exosomes, especially exosomal miRNAs, are important regulators in the interaction between tumor cells and tumor microenvironment by affecting tumor-promoting processes in target cells. It is important to analyze whether their exosomal patterns also reflect the specific molecular characteristics of MIBC. The aim of this study was to compare the miRNA expression in secreted exosomes from urinary bladder cancer cells (UBC) with different degrees of invasiveness. By electron microscopy, nanotracking analysis and western blot we proofed a high quality of isolated exosomes. Microarray analysis identified an invasion-associated signature of 15 miRNAs, which is significantly altered in exosomes of invasive UBC compared to non-invasive counterparts. Therefrom, 9 miRNAs are consistent differently expressed in both, invasive cells and their secreted exosomes. The remaining 6 exosome-specific miRNAs are only deregulated in exosomes but not in their parental cells. MiRNA alterations were verified by qPCR in cell culture and urinary exosomes. In conclusion, we showed that exosomes from invasive UBC cells are characterized by a specific miRNA signature. Further analyses have to clarify the functional relevance of exosomal miRNAs secreted by invasive bladder cancer cells for modification of the tumor microenvironment and their putative role as molecular markers in liquid biopsies.Muscle-invasive bladder cancer (MIBC) represents a highly aggressive tumor type compared to non-muscle-invasive tumors. MIBC is characterized by specific molecular alterations, which may also modulate extracellular tumorigenic effects. Tumor-associated exosomes, especially exosomal miRNAs, are important regulators in the interaction between tumor cells and tumor microenvironment by affecting tumor-promoting processes in target cells. It is important to analyze whether their exosomal patterns also reflect the specific molecular characteristics of MIBC. The aim of this study was to compare the miRNA expression in secreted exosomes from urinary bladder cancer cells (UBC) with different degrees of invasiveness. By electron microscopy, nanotracking analysis and western blot we proofed a high quality of isolated exosomes. Microarray analysis identified an invasion-associated signature of 15 miRNAs, which is significantly altered in exosomes of invasive UBC compared to non-invasive counterparts. Therefrom, 9 miRNAs are consistent differently expressed in both, invasive cells and their secreted exosomes. The remaining 6 exosome-specific miRNAs are only deregulated in exosomes but not in their parental cells. MiRNA alterations were verified by qPCR in cell culture and urinary exosomes. In conclusion, we showed that exosomes from invasive UBC cells are characterized by a specific miRNA signature. Further analyses have to clarify the functional relevance of exosomal miRNAs secreted by invasive bladder cancer cells for modification of the tumor microenvironment and their putative role as molecular markers in liquid biopsies.

Abstract 5182: Characterization of miRNA expression pattern from in-vitro obtained exosomes of different urinary bladder cancer cell lines

Introduction & objectives: Interaction of tumor cells and the tumor microenvironment (TME) plays an important role in tumor development and progression. It was shown that microRNAs (miRNAs), packed in exosomes, can affect cell-cell communication at the site of origin as well as the TME. The aim of the project is the identification of a specific miRNA expression pattern from in-vitro obtained tumor-derived exosomes of different UBC cell lines in correlation to their malignant potential. Furthermore, we want to analyze the effect of these exosomal miRNAs on tumor-associated fibroblasts (TAFs). Materials & methods: Exosomes were isolated from invasive (T-24,253J-BV, J82) and non-invasive (RT-112, 5637) UBC cell lines. The number and size of vesicles were measured by NTA. The vesicles were examined for exosomal and contamination markers by Western blotting. TotalRNA was isolated from the exosomes upon treatment with RNase. MiRNA expression pattern was analyzed from exosomes secreted by invasive and non-invasive UBC cells using miRNA microarray. The validation of significantly differently expressed miRNAs was performed by using qPCR. Exosome-mediated miRNA transfer between cancer cells and TAFs was verified by 1) transfection of donor UBC cells with the C. elegans-specific miRNA, cel-miR-39, 2) Exosome isolation and RNAse treatment, 3) Transfer to recipient TAFs, and 4) miRNA-specific qRT-PCR analysis using totalRNA from the recipient TAFs. Results: The isolated exosomes from UBC cells exhibited a high amount of exosomal markers (CD63, CD81, syntenin). 16 miRNAs were identified, which distinguishes invasive UBC cells from non-invasive cells. Exosomes secreted by invasive UBC cells are characterized by a specific miRNA signature of 25 miRNAs compared to exosomes from non-invasive UBC cells. The validation of differently expressed miRNAs is ongoing. After successful transfection of RT-112 and T-24 with cel-miR-39, cel-miR-39 was detected in RT-112 and T-24 exosomes as well as in recipient TAFs cultivated in the presence of these exosomes. Conclusion: Exosomes secreted by UBC cells exhibit a specific miRNA signature depending on the invasive potential of the originating cells. We could proof an exosome-mediated transfer of miRNAs between tumor cells and TAF. These results emphasize the role of exosomal miRNAs for the interaction between tumor cells and the tumor microenvironment. Further studies have to show the functional relevance of selected exosomal miRNAs. Citation Format: Sophie Baumgart, Joana Heinzelmann, Michael Stoeckle, Marie Stampe Ostenfeld, Kerstin Junker. Characterization of miRNA expression pattern from in-vitro obtained exosomes of different urinary bladder cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5182. doi:10.1158/1538-7445.AM2015-5182

MP54-15 UROTHELIAL BLADDER CANCER CELLS AFFECT TUMOR-PROMOTING PROCESSES IN NORMAL BLADDER FIBROBLASTS AND SUPPORT TUMORIGENESIS BY SECRETION OF TUMOR-ASSOCIATED EXOSOMES

trials, has not been utilized in treatment of bladder cancer. The purpose of this study was to measure the cell death effect of HAL/Blue light on patient-derived bladder cancer organoids. METHODS: Fresh bladder cancer specimens were collected as part of an IRB approved protocol (AAAN8850) and grown as 3D bladder cancer organoids per lab protocol. Organoids were treated with saline 100uM, HAL 10uM, HAL 100uM, camptothecin 10uM, or camptothecin 100uM, then incubated for 4 hours. One group of HAL treated organoids at each drug concentration was exposed to blue light while the groups remained in the incubator. The blue light source was calibrated to deliver a peak wavelength of 410nm at an energy of 1J/cm2. Cell viability was assessed using the Promega CellTiter-Glo luminescent cell viability assay at 18 hours and caspase-3 activity was assessed on histologic specimens at 6, 12, and 18 hours. RESULTS: Bladder cancer organoid cell viability was significantly reduced by 95% and 97% for HALþBlue Light treated organoids at drug concentrations of 10uM and 100uM, respectively. HAL treatment alone reduced cell viability by 19% and 21% for concentrations of 10uM and 100uM, respectively. P-value < 0.0001 for all comparison groups (figure). Histologic staining for caspase-3 was sparse and did not show significant difference between saline controls, HAL alone, or HALþBlue Light treated organoids but was increased in the camptothecin treated group. CONCLUSIONS: The combination of HAL and Blue Light drastically reduces cell survival in patient-derived bladder cancer organoids. These results suggest that beyond the diagnostic utility of Blue Light Cystoscopy with HAL there is a potential for therapeutic benefit. Further investigation into the apoptotic pathway is warranted based on IHC staining results.

Abstract 3162: Blood-based exosomal miRNAs as biomarkers for diagnosis and prognosis of clear cell renal cell cancer

Introduction & objectives: In previous studies we identified specific miRNA alterations in tumor tissues of clear cell renal cell cancer (ccRCC) with diagnostic and prognostic value relating to the presence of metastasis. For few years it has been known, that miRNAs are actively packed in exosomes which can be released into body fluids. Therefore, we hypothesize that in a simple blood based test we are able to use specific miRNA signatures as minimal invasive biomarkers for confirmation of the diagnosis and evaluation of the metastatic risk in ccRCC. Materials & methods: Initially, exosomes were isolated from 1 ml serum of 10 ccRCC patients (metastatic and non metastatic tumors) and 10 healthy volunteers using appropriated exosome isolation kit protocol (Thermo Fisher Scientific). Quality of exosomes was proven using exosomal markers (CD63, CD81, Alix, Synthenin) and Nano Particle Tracking Analysis. Exosomal totalRNA was isolated using miRNeasy Mini Kit (Qiagen). For miRNA analyses total RNA was reverse transcribed using TaqMan Reverse Transcription Kit (Thermo Fisher Scientific), cDNA was preamplified using TaqMan® PreAmp Master Mix (Thermo Fisher Scientific) and real time PCR was performed using Gene Expression master mix (Thermo Fisher Scientific). Expression differences were calculated using REST Software and SPSS. Results: We have shown that 1 ml serum is sufficient to analyze miRNA expression in exosomes. Exosomes isolated from serum exhibit high amount of exosomal markers and possess the typical exosomal size (30-120 nm). CcRCC serum samples are characterized by downregulation of several miRNAs (including miR-10b and miR451). Furthermore, specific miRNAs (including miR-30c) differentiate patients with or without metastases as well as healthy volunteers. Conclusion: These initial data confirm our hypothesis that the tissue based miRNA signature could be used as biomarkers for detection of aggressive ccRCC analyzing exosomes from liquid biopsy. This minimal invasive blood based test is easy to handle in clinical practice and could be a powerful tool for early detection and monitoring of metastatic disease. To validate these results the expansion of the sample set is ongoing. Citation Format: Joana Heinzelmann, Sophie Baumgart, Sebastian Hoelters, Martin Janssen, Michael Stockle, Kerstin Junker. Blood-based exosomal miRNAs as biomarkers for diagnosis and prognosis of clear cell renal cell cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3162.

Abstract 1493: Specific miRNA signatures characterize different metastatic sites in clear cell renal cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: MiRNAs are regulators of gene expression in tumorigenesis and progression. To identify miRNAs associated with metastasis miRNA expression in distant metastases was compared to primary clear cell renal cell carcinoma (ccRCC). Material and methods: Total RNA of 27 primary ccRCC samples and 25 distant metastases (lung, bone and brain) was isolated from formalin-fixed paraffin-embedded (FFPE) samples Microarray analyses were performed for a global miRNA expression profiling. Results were validated by qPCR. For miRNA target identification a ccRCC cell line (786-O) was transfected with miRNAs differently expressed in metastatic primary tumors and metastases. Results: We identified 9 miRNAs (including miR-30c and miR-126) with a similar expression in metastatic primary ccRCC and distant metastases from different metastatic sites compared to non-metastatic primary ccRCC. 11 miRNAs (including miR-10b and miR-204) were differently expressed in distant metastases compared to primary ccRCC. Furthermore, each metastatic site is characterized by a specific miRNA signature. Results were verified on selected miRNAs using qPCR. In vitro studies identified potential miRNA targets and associated metastasis related pathways. Discussion: These data suggest that miRNAs play an important role in metastatic processes of ccRCC. Furthermore, our results regarding different metastatic sites suggest two important statements. Specific miRNAs characterize distant metastases in general. On the other hand, miRNA expression is associated with specific conditions at different metastatic sites. Thus, the data presented in this study give the base for a better understanding of the involvement of miRNAs as regulators of metastasis which opens new possibilities for new targeted therapy options. Citation Format: Joana Heinzelmann, Franzsika Stolzenbach, Robert Schneeweiss, Ulrike Wickmann, Sophie Baumgart, Mieczyslaw Gajda, Michael Stockle, Kerstin Junker. Specific miRNA signatures characterize different metastatic sites in clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1493. doi:10.1158/1538-7445.AM2014-1493