E. Konze

Ex vivo analysis of antineoplastic agents in precision-cut tissue slices of human origin: effects of cyclooxygenase-2 inhibition in hepatocellular carcinoma.

Abstract: Cultures of precision‐cut tissue slices allow the investigation of substance effects on human tissues under in vivo‐like conditions over a limited time span. We have adapted the model for direct analyses of antineoplastic substances on tumor tissues. We have recently demonstrated that selective cyclooxygenase‐2 (COX‐2) inhibitors strongly suppress growth of human hepatocellular carcinoma (HCC) cells in vitro and nude mouse HCC implants by inducing apoptosis and reducing proliferation. We have now analyzed the effects of COX‐2 inhibition on human tumor tissue. Three hundred micrometer slices of tumorous and non‐tumorous liver tissue from three surgically resected HCCs were cultured with increasing concentrations of the selective COX‐2 inhibitor Meloxicam (20–200u2003μM) for 6, 12, 24, and 48u2003h. The cultured tissue slices were analysed morphologically and by immunohistology for proliferation (Ki‐67), apoptosis (M30), and COX‐2 expression. COX‐2 was expressed in all HCCs and in the non‐tumorous liver tissue. Cytoplasmic COX‐2 immunoreactivity in HCCs increased during culturing time. In two of three cases, COX‐2 inhibition significantly increased tumor cell apoptosis in HCCs, whereas the low basal apoptosis rate in the non‐tumorous liver parenchyma did not change. Tumor cell proliferation was mildly reduced, but the changes did not reach statistical significance. These results demonstrate that the precision‐cut tissue slice culture model is a useful tool to analyze directly drug‐dependent antitumorous or unwanted organ‐specific effects. The analysis of COX‐2 inhibition lends further support to the antineoplastic effects previously demonstrated in vitro and in animal models.

Cryopreservation of precision cut tissue slices (PCTS): Investigation of morphology and reactivity

Precision cut tissue slices (PCTS) represent a suitable and convenient tool for pharmacological, toxicological and morphological studies. Cryopreservation would enable to overcome the shortage of liver tissue, in particular in settings using human liver tissue. We investigated the potential of cryopreservation of porcine PCTS as a morphological tool by rapid freezing with 10% and 30% dimethyl sulfoxide as cryopreservation agents and with or without medium using a Brendel/Vitron tissue slicer. Incubation after thawing was done in a static incubation system. Slices were cultured for 3 h, 6 h, 24 h and 48 h and assessed histologically and immunohistologically for proliferation (Ki67) and spontaneous as well as induced apoptotic activity (M30Cytodeath). Vitality was tested using the Tox-8 test. After cryopreservation, morphology of PCTS was well preserved up to 24 h. A reduction of vitality rate took place. Compared to non-frozen PCTS, the rate of spontaneous proliferation of Kupffer cells and apoptosis of hepatocytes were significantly reduced independent of the freezing conditions. The reactivity of PCTS to apoptotic stimuli was significantly reduced in tissue slices after cryopreservation. Apoptotic stimuli could not induce the same amount of cell deaths compared to non-frozen sections. Thus, cryopreservation of PCTS does interfere with pathomechanisms of apoptosis in PCTS.

Epression therapieprädiktiver Faktoren in syn- und metachronen Lebermetastasen kolorektaler Karzinome im Vergleich zum Primärtumor

Bei fortgeschrittenen Tumorleiden nimmt die auf den individuellen Patienten und das biologische Potenzial des Tumors zugeschnittene Therapie durch die Entwicklung target-gerichteter Medikamente eine immer grosere Rolle ein. Die Expression pradiktiver Zielfaktoren im Tumor ist eine Voraussetzung fur eine solche Therapie. Es wurden Gewebearrays der Primartumoren und der Metastasen von 57 Patienten mit kolorektalen Adenokarzinomen mit Lebermetastasen (34 Manner und 23 Frauen; mittleres Alter 61,3 Jahre; mittlere Primartumorgrose 4,5 cm; 1 x T1, 2 x T2, 42 x T3 und 10 x T4; 42 und 15 metachrone Lebermetastasen mit durchschnittlichem Intervall von 14,6 Monaten; Metastasengrose 2,4 cm) angefertigt. Immunhistochemisch wurde die Expression von VEGF, EGFR, PDGF alpha, PDGF beta und CD117 (c-kit) bestimmt. VEGF ist in 97% der Lebemetastasen und 94% der Primartumoren exprimiert. Eine Metastase zeigte bei negativem Primartumor eine Expression. EGFR ist in 42,3% der Primartumoren und in 46% der Metastasen unterschiedlich stark nachweisbar. 6 Falle zeigten Unterschiede zwischen Primar- und Sekundartumor. PDGF alpha und beta sind in 91,2% der Primartumoren und in 81,3% (PDGFalpha) bzw. 60,4% (PDGFbeta) in Metastasen detektierbar. Abweichungen zwischen Primar- und Sekundartumor fanden sich in 2 (PDGFalpha) bzw. 13 (PDGFbeta) Fallen. C-kit ist in 17% der Primartumoren und in 21% der Metastasen teils jedoch auch nur schwach nachweisbar. 4 Falle zeigten Abweichungen. Eine therapeutische Option kann bei VEGF- oder PDGF- Antagonisten auch ohne histologische Prufung als wahrscheinlich betrachtet werden. EGFR und c-kit sollten bei einem palliativen Therapieansatz immunhistochemisch nachgewiesen werden. Bei der Variabilitat zwischen Primar- und Sekundartumor sollte die Bestimmung am Gewebe der Metastase selbst erfolgen.