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A protocol for the isolation and long-term propagation of adult rat Sertoli cells (SCs) using conditional reprogramming (CR) was developed and the formation of tight junctions as an in vitro model for the blood testis barrier (BTB) was studied. Three pure primary SC lines were isolated successfully and maintained for several months without significant changes in expression levels of SC-typical markers such as SRY-box transcription factor 9 (SOX9), transferrin, clusterin, androgen receptor (AR), and GATA binding protein 1 (GATA1). In addition to AR expression, the tight junction proteins, zonula occludens-1 (ZO-1) and the junctional adhesion molecule-3 (JAM-3), were upregulated and the SC barrier integrity was enhanced by testosterone. Peritubular/myoid cells did not increase the tightness of the SC. The cytokines, interleukin-6 (IL-6), bone morphogenetic protein-2 (BMP2), and transforming growth factor beta-3 (TGF-β3), negatively affected the tightness of the SC barrier. We have established a protocol for the isolation and long-term propagation of highly pure primary adult rat SCs, which are able to respond to androgen treatments, to form tight junctions and to maintain the mRNA expression of SC-specific genes. By applying this new method, adult SCs can now be analyzed in more detail and might serve as an in vitro model for the study of many SC functions.

Fabian Eberle, 1–4 Stefan Lautenschläger, 4 Rita Engenhart-Cabillic, 1, 3, 4 Alexandra D Jensen, 5 Barbara Carl, 6 Marco Stein, 7 Jürgen Debus, 1–3, 8–11 Henrik Hauswald 1–4, 8, 10, 11 1Department of Radiation Oncology, Heidelberg University Hospital, Heidelberg, Germany; 2Heidelberg Institute for Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), Heidelberg, Germany; 3Marburg Ion-Beam Therapy Center (MIT), Department of Radiation Oncology, Heidelberg University Hospital, Marburg, Germany; 4Department of Radiation Oncology, Marburg University Hospital, Marburg, Germany; 5Department of Radiation Oncology, Gießen University Hospital, Gießen, Germany; 6Department of Neurosurgery, Marburg University Hospital, Marburg, Germany; 7Department of Neurosurgery, Gießen University Hospital, Gießen, Germany; 8Heidelberg Ion-Beam Therapy Center (HIT), Department of Radiation Oncology, Heidelberg University Hospital, Heidelberg, Germany; 9German Cancer Consortium (DKTK), Heidelberg, Germany; 10National Center for Tumor Diseases (NCT), Heidelberg, Germany; 11Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center (DKFZ), Heidelberg, GermanyCorrespondence: Henrik HauswaldDepartment of Radiation Oncology, Heidelberg University Hospital, INF 400, Heidelberg 69120, GermanyTel +49-6221-56-8201Email henrik.hauswald@med.uni-heidelberg.deBackground: Patients with recurrent glioma after prior radiotherapy have a poor prognosis. Carbon ion beam radiotherapy offers highly conformal dose distributions and more complex biological radiation effects eventually resulting in optimized normal tissue sparing and improved outcome. The aim of this study was to analyze toxicity, local control and overall survival after reirradiation of recurrent high-grade glioma with carbon ion radiotherapy.Methods: Between 10/2015 and 12/2018, 30 patients (median age: 59 years) with recurrent high-grade glioma were reirradiated with carbon ion beams and retrospectively analyzed. Diagnosis of recurrent glioma was based on magnetic resonance imaging. Thirteen patients had repeated resection prior to reirradiation and 24 patients underwent additional chemotherapy. The median initial radiation dose was 60 Gy and the median time interval between the initial and repeated radiotherapy was 10 months. The reirradiation dose was 45 Gy (relative biological effectiveness) applied in 15 fractions. All patients received regular follow-up imaging after reirradiation. Kaplan-Meier estimation, log rank test and Cox regression analysis were used for statistical assessment.Results: Applying common toxicity criteria, there were no grade 5 or 4 adverse events, while 8 patients showed grade 3 adverse events. The median follow-up after reirradiation was 11 months and the median overall survival after diagnosis of recurrent high-grade glioma was 13 months. The 6-, 12- and 24-month overall survival rates after diagnosis of recurrent high-grade glioma were 76%, 50% and 19%, respectively. Upon multivariate Cox regression analysis, a Ki67 score of the initial tumor histology of less than 20% was prognostic. Repeated resection or chemotherapy for the recurrent disease did not result in significantly prolonged survival.Conclusion: Carbon ion reirradiation in recurrent high-grade glioma is safe and feasible. No radiation-associated grade 4 toxicities were documented and treatment was tolerated well.Keywords: glioma, glioblastoma, reirradiation, carbon ion beam therapy, C12, particle beam therapy, radiotherapy