Sebastian Hölters

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.

Robust method for isolation of tumor infiltrating lymphocytes with a high vital cell yield from small samples of renal cell carcinomas by a new collagenase-free mechanical procedure

BACKGROUND Tumor-infiltrating lymphocytes (TIL) play an important role in the pathogenesis of renal cell carcinoma. Characterization of TIL requires efficient isolation procedures, especially in early stage disease when the tumor is of small in size. Conventional methods for isolating TIL are based on enzymatic tissue digestion, most frequently with collagenase. Collagenase isolation is limited by poor cell recovery, altered expression of cell-surface molecules, and impaired TIL-functionality. To overcome these limitations, we developed and optimized conditions for a robust collagenase-free mechanical procedure for improved isolation of TIL from renal cell carcinoma samples. METHODS TIL from tumor samples and T cells from peripheral blood were collected from 12 patients undergoing partial or radical nephrectomy. Samples were subjected to an enzymatic reference protocol and to a newly established mechanical isolation protocol. After viability staining, TIL-subpopulations were quantified and phenotyped by immunohistochemistry and flow-cytometric analysis, and were compared to characteristics of peripheral blood T cells. As a marker for TIL-functionality, T-cell cytokine induction was quantified after polyclonal stimulation. RESULTS We show that this new technique is rapid and allows identification of CD4 and CD8 T-cell subpopulations including CD4, CD8, and regulatory T cells expressing anergy markers such as programmed death-1 (PD-1) or B- and T-lymphocyte attenuator. When compared to the reference protocol involving collagenase digestion, the yield of TIL after mechanical isolation was higher and the expression of cell-surface markers was better preserved. Moreover, although antitumor activity was not assessed, mechanically isolated TIL are at least equally functional as T cells from peripheral blood, as polyclonal stimulation induced cytokines such as interferon-γ and tumor necrosis factor-α in both TIL and T cells from peripheral blood. CONCLUSION The mechanical procedure may be applied as a robust and rapid alternative to collagenase digestion for isolation of high amounts of phenotypically and functionally intact TIL from fresh tumor samples.

Abstract 901: Plasma proteins characterize second-line therapy response in progressive renal cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction & Objectives: For patients with metastatic renal cell carcinoma (mRCC) vascular endothelial growth factor (VEGF)-targeted therapy represents a promising first-line therapy after tumour nephrectomy. For patients who progress during first-line therapy, inhibitors of mammalian target of rapamycin (mTOR) represent an option for second-line therapy. Within the framework of the MARC-2 clinical trial, the aim of our study is the identification and validation of marker proteins associated with second-line therapy (Everolimus) response. Material & Methods: We included blood plasma samples from 19 mRCC patients receiving Everolimus. Therapy response was defined as stable disease and partial remission. We analysed the samples by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) using CM10 and Q10 arrays. For the identification of potential marker proteins, 2-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and antibody array screening are on-going. Results: We identified 20 mass peaks by SELDI-TOF MS with Q10 and 16 with CM10 chips for the discrimination between responders (defined as stable disease or partial remission after two month of therapy) and non-responders (defined as progressive disease after 2 month of therapy). With the Q10, but not with the CM10 chip, we obtained five peaks for the discrimination between both patient groups before the first drug application. These peaks could be useable to preselect patients for the second-line therapy with Everolimus. For therapy monitoring, we found seven mass peaks (four with Q10 and three with CM10 chips) after two weeks and 24 mass peaks (11 with Q10 and 13 with CM10 chips) after four weeks of therapy to differentiate between responders and non-responders. These mass peaks could be useful to estimate the therapy response for the patients. Conclusion/Outlook: Our preliminary results argue for the presence of blood plasma proteins which potentially predict therapy response to Everolimus before as well as during therapy. The confirmation and identification of the SELDI-TOF MS outcomes by 2-dimensional gel electrophoresis and MALDI-TOF MS is on-going as well as the antibody array screening for angiogenesis related proteins. Sponsored by: iOMEDICO AG Citation Format: Sebastian Holters, Lothar Bergmann, Viktor Grunwald, Ulrich Keilholz, Carsten Ohlmann, Michael Staehler, Diana Schmerler, Kerstin Junker. Plasma proteins characterize second-line therapy response in progressive 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 901. doi:10.1158/1538-7445.AM2014-901