Ein neuartiges Zellkulturmodell des high risk Prostatakarzinoms

Abstract

Das Prostatakarzinom steht an erster Stelle der malignen Erkrankungen des Mannes. Es tritt vorwiegend im hohen Lebensalter auf und ist durch eine hohe Morbidität und Mortalität gekennzeichnet. Vor dem Hintergrund einer alternden Gesellschaft wird die Relevanz dieser Erkrankung in Zukunft weiter zunehmen. Die verfügbaren diagnostischen und therapeutischen Möglichkeiten sind begrenzt, da die Biologie dieser Erkrankung noch unzureichend verstanden ist. Dies hängt insbesondere mit dem Fehlen geeigneter präklinischer Modelle in der Prostatakarzinomforschung zusammen. Die in vitro Kultivierung sowohl lokal begrenzter als auch metastasierter Tumorstadien hat sich über die letzten Jahrzehnte hinweg als ausgesprochen schwierig erwiesen. Viele Eigenschaften des Prostatakarzinoms stehen einer in vitro Kultivierung entgegen. Hierzu gehört das nur sehr limitierte Tumorgewebe, welches pro Patient für Forschungszwecke zur Verfügung gestellt werden kann, sowie das vergleichsweise langsame Zellwachstum. Zudem benötigen die Zellen sehr spezielle Umgebungsbedingungen, die bisher noch unzureichend erforscht wurden. Sowohl aus diagnostischen, therapeutischen wie auch gesundheitsökonomischen Gesichtspunkten wäre es von großer Bedeutung, humane Primärtumore in vitro zu kultivieren und die Progression zu fortgeschrittenen Tumorstadien nachzuvollziehen. Unter Berücksichtigung neuer Ansätze der in vitro Forschung aus anderen Tumorentitäten sowie der embryonalen Stammzellforschung konnte aus seriell transplantierten Mausxenografts der LuCaP Reihe ein neuartiges Zellkulturmodell entwickelt werden, bei dem es gelungen ist, das Zellmaterial über mehrere Passagen hinweg in Zellkultur zu halten. Die Innovationen, die dabei zum Erfolg führten, waren zum einen die permanente Beibehaltung von Zell-Zell Kontakten sowie die Zugabe sogenannter small molecules (Y27632), welche die Zell-Zell Interaktionen verstärken. In der vorliegenden Arbeit wurde ein an Xenograftmodellen metastasierter Tumorproben etabliertes Modell auf direkt nach OP gewonnenes Patientenmaterial übertragen. Das Zellmaterial wurde aus Prostataresektaten nach radikaler Prostatektomie sowie in einem Fall durch palliative TUR-P gewonnen und nach limitiertem Verdau als Sphäroidsuspensionskultur kultiviert. So konnten die entstandenen Sphäroide über mehrere Monate hinweg in Kultur gehalten werden. Die Morphologie der Sphäroide wurde lichtmikroskopisch und histologisch untersucht. Anschließend wurde mittels LIVE/DEAD® Cell Viability Assays die Viabilität des Zellmaterials zu unterschiedlichen Zeitpunkten der Kultivierung nachgewiesen. Eine Charakterisierung des Zellmaterials erfolgte durch immunzytochemische und immunhistochemische Verfahren. Dabei konzentrierten wir uns auf den Nachweis der Cytokeratine CK-5 und CK-8 sowie des Androgenrezeptors und des Proliferationsmarkers Ki67. Die genannten Oberflächenmerkmale konnten in unserer Kultur in unterschiedlich starker Exprimierung nachgewiesen werden. Schließlich wurde mittels CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) die Proliferationsfähigkeit des Zellmaterials über einen Beobachtungszeitraum von vier Tagen nachgewiesen. Unter Berücksichtigung der spezifischen Eigenschaften des Prostatagewebes ist es gelungen, ein primäres 3-dimensionales in vitro Sphäroid-Modell neu zu etablieren, welches auch über die vorliegende Arbeit hinaus weiterentwickelt wird. Es können dadurch neue experimentelle Forschungsansätze verfolgt werden. -------

Prostate cancer is the most frequent malignant tumor in men. It occurs mostly in older men, with a high morbidity and mortality. Against the background of an aging society the relevance of this disease will increase in the future. The currently available diagnostic and therapeutic options are limited because the biology of this disease is not fully understood. This is particularly related to a lack of suitable preclinical models for prostate cancer that can mimic primary prostate cancer. In vitro culturing of localized as well as metastasized prostate cancer cells has been particularly challenging. Many characteristics of prostate cancer impede in vitro culturing. There is on the one hand a very small amount of tumor tissue that can be asservated from each patient, on the other hand prostate cancer cells are growing very slowly. Fur-thermore, prostate cells require very specific environmental conditions that have not yet been entirely understood. The possibility of a reliable cultivation of prostate cancer would be an important step in a better understanding of prostate cancer biology. This could help to find better diagnostic and therapeutic options and also reduce costs in health economy. By considering new in vitro research of other tumor entities, such as findings in stem cell research, a novel in vitro model for LuCaP Xenografts has been successfully established by Peehl et al. They could demonstrate a cell viability of LuCaP Xenografts over several passages. Innovations that have been contributed to this success were a continual mainte-nance of cell-cell contacts as well as the adding of so called small molecules such as the Rock-Inhibitor Y-27632, which enhances cell-cell contacts. In the present work we attempted to transfer the successful in vitro model of LuCaP Xenografts to primary prostate cancer tissue. Cancer tissue was asservated by radical prostatectomy. We proceeded the tissue by limited digestion and cultivated it as three dimension-al spheroids. We could demonstrate viability of the spheroid cells over several months. Morphology was observed and documented by microscopy and histological examination. Cell viability was tested by LIVE/DEAD® Cell Viability Assays. To characterize the cell material we performed immunocyto- and immunohistochemistry. We used the markers CK-5, CK-8, Androgenreceptor and Ki67. They all could be found in different concentrations. Finally, we performed CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) to demonstrate cell proliferation. By an observation period of 4 days, we could demonstrate cell proliferation. By integrating the innovations mentioned above we established a novel three dimensional spheroid culture model that is applied and developed constantly. This enables new approaches in prostate cancer research.

Prostate cancer is the most frequent malignant tumor in men. It occurs mostly in older men, with a high morbidity and mortality. Against the background of an aging society the relevance of this disease will increase in the future. The currently available diagnostic and therapeutic options are limited because the biology of this disease is not fully understood. This is particularly related to a lack of suitable preclinical models for prostate cancer that can mimic primary prostate cancer. In vitro culturing of localized as well as metastasized prostate cancer cells has been particularly challenging. Many characteristics of prostate cancer impede in vitro culturing. There is on the one hand a very small amount of tumor tissue that can be asservated from each patient, on the other hand prostate cancer cells are growing very slowly. Furthermore, prostate cells require very specific environmental conditions that have not yet been entirely understood. The possibility of a reliable cultivation of prostate cancer would be an important step in a better understanding of prostate cancer biology. This could help to find better diagnostic and therapeutic options and also reduce costs in health economy. By considering new in vitro research of other tumor entities, such as findings in stem cell research, a novel in vitro model for LuCaP Xenografts has been successfully established by Peehl et al. They could demonstrate a cell viability of LuCaP Xenografts over several passages. Innovations that have been contributed to this success were a continual maintenance of cell-cell contacts as well as the adding of so called small molecules such as the Rock- Inhibitor Y-27632, which enhances cell-cell contacts. In the present work we attempted to transfer the successful in vitro model of LuCaP Xenografts to primary prostate cancer tissue. Cancer tissue was asservated by radical prostatectomy. We proceeded the tissue by limited digestion and cultivated it as three dimensional spheroids. We could demonstrate viability of the spheroid cells over several months. Morphology was observed and documented by microscopy and histological examination. Cell viability was tested by LIVE/DEAD® Cell Viability Assays. To characterize the cell material we performed immunocyto- and immunohistochemistry. We used the markers CK-5, CK-8, Androgenreceptor and Ki67. They all could be found in different concentrations. Finally, we performed CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) to demonstrate cell proliferation. By an observation period of 4 days, we could demonstrate cell proliferation. By integrating the innovations mentioned above we established a novel three dimensional spheroid culture model that is applied and developed constantly. This enables new approaches in prostate cancer research.