Joana Heinzelmann

Specific protein and miRNA patterns characterise tumour-associated fibroblasts in bladder cancer

PurposeTumour development and progression are strongly affected by interaction of tumour cells and tumour stroma. Different tumour models demonstrate a supportive effect of tumour-associated fibroblasts (TAF) on the tumour genesis. Aims of the present study are the isolation of TAF from primary urinary bladder tumour specimens and the proteomic and epigenetic characterisation.MethodsTAF were isolated from cultured urinary bladder tumour specimens. Therefore, primary tumour material was treated with EDTA followed by two separated detachment steps. Non-tumour fibroblasts were isolated from foreskin and normal bladder tissues. Proteins and total RNA were isolated from cultured fibroblasts. Protein pattern analyses were carried out by SELDI–TOF–MS. The miRNA expression profile was analysed by miRNA microarray.ResultsBy optimising cell culture routines, we achieved to isolate and subsequently cultivate TAF from primary tumour material of the urinary bladder. SELDI–TOF–MS measurements reveal distinct differences in the proteomic patterns of TAF and non-tumour fibroblasts. Microarray analyses indicate specific expression of several miRNAs in TAF and non-tumour fibroblasts.ConclusionIn summary, we determined proteomic and epigenetic differences between non-tumour fibroblasts and TAF of urinary bladder carcinoma and identified specific protein expression patterns as well as miRNA profiles of TAF in comparison with non-tumour fibroblasts. These findings provide more insights into the complex tumour network and a good starting point for the identification of markers for the prediction of tumour development and progression based on specific TAF expression patterns.

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.

Extracellular Vesicles and Their Role in Urologic Malignancies

CONTEXT Research has increased significantly on small vesicles secreted by healthy and diseased cells. Recent discoveries have revealed their functional and biomarker roles in urologic diseases. Whether and how this knowledge of extracellular vesicles (EVs) affects translational research and clinical practices have become pertinent questions. OBJECTIVE To provide an overview of the currently available literature on the rising field of EVs, focusing on function and pathogenesis in urologic cancers and the usefulness of EVs as biomarkers. EVIDENCE ACQUISITION A systematic literature search was conducted using PubMed to identify original articles, review articles, and editorials regarding EVs in different types of urologic tumor diseases. Articles published between 2005 and 2015 were reviewed and selected with the consensus of all authors. EVIDENCE SYNTHESIS Besides soluble factors, different types of EVs are involved in the complex cross talk between different cell types. EVs regulate normal physiologic processes like spermatogenesis and renal function, as well as disease-specific processes including bladder, kidney, and prostate cancer. The content of EVs is derived from the cytoplasm of the donor cell. The proteins and RNAs within these EVs can be isolated from body fluids (eg, urine and blood) and represent potential diagnostic and prognostic biomarkers. EVs are also candidate therapeutic targets and potentially useful as therapeutic vehicles. CONCLUSIONS The current data suggest that EVs are important regulators of cell-cell communication. The growing knowledge about their roles in urologic malignancies provides the basis for novel therapeutic strategies. In addition, nucleic acid and the protein content of EVs holds promise for the discovery of urine- or serum-based biomarkers for kidney, bladder, and prostate cancer. PATIENT SUMMARY Normal and cancer cells secrete small vesicles that contain proteins and RNAs from the cell of origin. Changes in the diseased cells can be detected by examining the altered content of these vesicles when secreted in body fluids, for example, blood and urine. The recently discovered roles of extracellular vesicles (EVs) provide new options to detect malignancy in the urine and blood. The uptake of EVs may be blocked therapeutically and thereby potentially impede cancer progression.

Prognostic and predictive miRNA biomarkers in bladder, kidney and prostate cancer: Where do we stand in biomarker development?

PurposeDefining reliable biomarkers is still a challenge in patients with urological tumors. Because miRNAs regulate diverse important cellular processes, these noncoding RNAs are putative molecular candidates. This review intends to give a critical overview about the current state of miRNAs as biomarkers in urological cancers with respect to prognostic stratification as well as for individual treatment selection.MethodsA comprehensive review of the published literature was conducted focusing at the clinical relevance of miRNAs in tissues and body fluids of prostate, bladder and kidney cancer. Using electronic database, 91 articles, published between 2009 and 2015, were selected and discussed regarding the robustness of miRNAs as valid biomarkers.ResultsA number of miRNAs have been identified with prognostic and predictive relevance in different urologic tumor types. However, the inconsistency of the published results and the lack of multivariate testing in independent cohorts do not allow an introduction into clinical decision making at present.ConclusionmiRNA-based biomarkers are a promising tool for future personalized risk stratification and response prediction in urological cancers.

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.

Experimental imaging in orthotopic renal cell carcinoma xenograft models: comparative evaluation of high-resolution 3D ultrasonography, in-vivo micro-CT and 9.4T MRI

In this study, we aimed to comparatively evaluate high-resolution 3D ultrasonography (hrUS), in-vivo micro-CT (μCT) and 9.4T MRI for the monitoring of tumor growth in an orthotopic renal cell carcinoma (RCC) xenograft model since there is a lack of validated, non-invasive imaging tools for this purpose. 1 × 106 Caki-2 RCC cells were implanted under the renal capsule of 16 immunodeficient mice. Local and systemic tumor growth were monitored by regular hrUS, μCT and MRI examinations. Cells engrafted in all mice and gave rise to exponentially growing, solid tumors. All imaging techniques allowed to detect orthotopic tumors and to precisely calculate their volumes. While tumors appeared homogenously radiolucent in μCT, hrUS and MRI allowed for a better visualization of intratumoral structures and surrounding soft tissue. Examination time was the shortest for hrUS, followed by μCT and MRI. Tumor volumes determined by hrUS, μCT and MRI showed a very good correlation with each other and with caliper measurements at autopsy. 10 animals developed pulmonary metastases being well detectable by μCT and MRI. In conclusion, each technique has specific strengths and weaknesses, so the one(s) best suitable for a specific experiment may be chosen individually.

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 1961: Characterization of MicroRNA Expression during the Development of Resistance to a mTOR-Inhibitor in renal cell carcinoma

Introduction: Second line therapy of metastatic renal cell carcinoma (RCC) using the mTOR inhibitor RAD001 (everolimus) is hampered by the appearance of drug resistance. The cellular mechanisms leading to drug resistance are not yet clarified. Although the influence of miRNAs is proven for other systemic treatments, their impact on mTOR inhibitors is not investigated. Therefore, we analyzed miRNA alterations in correlation to development of resistance to RAD001 in RCC cell culture models. Material and Methods: The RCC cell line 786-O was treated with 1μM RAD001 for up to 20 passages after developing sunitinib resistance. TotalRNA was isolated at defined intervals. Resistance-development as well as drug efficacy during the treatment was monitored by proliferation assays. MiRNA Microarrays (Agilent Technologies) were performed using RAD001 sensitive and RAD001 resistant cells to identify differently expressed miRNAs. Results were validated by qRT PCR. Functional analyses are performed after transfection of RAD001 sensitive cells with miRNA candidates. Results: The Microarray analyses revealed a miRNA signature including 9 differently expressed miRNAs in RAD001 resistant cells compared to sensitive cells. QRT-PCR results showed that especially miR-302e was strongly up-regulated by the factor of four. At present, functional analyses of miR-302e are ongoing. Conclusion: To our knowledge this is the first study analyzing miRNA alterations during the development of resistance to mTOR inhibitors. We have shown that miR-302e expression is associated with RAD001 resistance in RCC cells. Since miR-302e is a known tumor suppressor modulating radio sensitivity and drug sensitivity in several tumors we are proving now if miR-302e can regulate the mTOR inhibitor resistance of RCC. Citation Format: Joana Heinzelmann, Franziska Bauer, Gerhard Unteregger, Darja Schendel, Anne Weiland, Michael Stockle, Kerstin Junker. Characterization of MicroRNA Expression during the Development of Resistance to a mTOR-Inhibitor in renal cell carcinoma. [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 1961.

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.