Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples.
BACKGROUND
There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints.


METHODS
A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed.


RESULTS
The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR.


CONCLUSIONS
The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy.
Pirnia
Farzaneh
F
Institute of Medical Oncology, University of Bern, Inselspital, Bern, Switzerland.
Frese
Steffen
S
Gloor
Beat
B
Hotz
Michel A
MA
Luethi
Alexander
A
Gugger
Mathias
M
Betticher
Daniel C
DC
Borner
Markus M
MM
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D000077143:Docetaxel; D004354:Drug Screening Assays, Antitumor; D004789:Enzyme Activation; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000378:metabolism; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008297:Male; D016685:Mitomycin / Q000494:pharmacology; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D043823:Taxoids / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D019394:Ki-67 Antigen; D009687:Nuclear Proteins; D043823:Taxoids; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins; D000077143:Docetaxel; D016685:Mitomycin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin