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Ex Vivo Assessment of Chemotherapy-Induced Apoptosis and Associated Molecular Changes in Patient Tumor Samples

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Ex Vivo Assessment of Chemotherapy-Induced Apoptosis and Associated Molecular Changes in Patient Tumor Samples ANTICANCER RESEARCH 26: 1765-1772 (2006) Ex Vivo Assessment of Chemotherapy-induced Apoptosis and Associated Molecular Changes in Patient Tumor Samples FARZANEH PIRNIA1, STEFFEN FRESE2, BEAT GLOOR3, MICHEL A. HOTZ4, ALEXANDER LUETHI1, MATHIAS GUGGER5, DANIEL C. BETTICHER1 and MARKUS M. BORNER1 1Institute of Medical Oncology, 2Clinic for Thoracic Surgery, 3Department of Visceral and Transplantation Surgery and 4Nose and Throat Surgery, Institute of Pathology University of Bern, Inselspital, Bern, Switzerland Abstract. Background: There are inherent conceptual problems It is notoriously difficult to study the molecular effects of in investigating the pharmacodynamics of cancer drugs in vivo. anticancer drug treatment in vivo. To date, the most common One of the few possible approaches is serial biopsies in patients. approach is to perform serial biopsies in tumor patients However, this type of research is severely limited by undergoing treatment. Such studies are associated with methodological and ethical constraints. Materials and Methods: discomfort, costs and significant morbidity, even mortality. A modified 3-dimensional tissue culture technique was used to Thus, patients are not motivated to participate and Ethics culture human tumor samples, which had been collected during Committees are very reluctant to support this kind of routine cancer operations. Twenty tumor samples of patients with research. An approach to circumvent these problems is ex vivo non-small cell lung cancer (NSCLC) were cultured ex vivo for tissue culture. Tumor tissue is obtained from patients during 120 h and treated with mitomycin C, taxotere and cisplatin. The routine operation and is then processed in vitro. Hoffman et cytotoxic activity of the anticancer agents was quantified by al. pioneered this approach by establishing a protocol for ex assessing the metabolic activity of treated tumor cultures and vivo cultivation of 3-dimensional tissue samples (1-3). Tumors various assays of apoptosis and gene expression were performed. are processed into small cubes, which can be grown on a Results: The proliferative activity of the tissue was maintained in collagen gel support. Three-dimensional culture methods have culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and been compared with 2-dimensional culture methods, which taxotere reduced the metabolic activity of the tumor tissue still are the most frequently used culture method in cancer cultures by 51%, 29% and 20%, respectively, at 120 h. The research. Two-dimensional cell culture techniques have the decrease in metabolic activity corresponded to the induction of advantage of simple handling and high reproducibility. apoptosis as demonstrated by the typical morphological changes, However, 2-dimensional and 3-dimensional culture techniques such as chromatin condensation and nuclear fragmentation. In have shown critical biological differences. The most obvious addition, activated caspase-3 could be verified in apoptotic cells are differences in the expression of genes, which are involved by immunohistochemistry. To verify functional aspects of in attachment, adhesion and the organization of the apoptosis, the induction of chemotherapy-induced cell death was 3-dimensional tissue architecture. Even more importantly for inhibited with the caspase inhibitor z-VAD.fmk. RNA was cancer research, the spatial organization of cells has a clear extracted from the tissue cultures after 120 h of ex vivo drug impact on their sensitivity to anticancer drugs and other treatment and was of sufficient quality to allow quantitative PCR. therapeutic manipulations (4-7). Thus, 3-dimensional tissue Conclusion: The 3-dimensional ex vivo culture technique is a cultures seem to reflect more appropriately the in vivo patient useful method to assess the molecular effects of pharmacological situation than the commonly used 2-dimensional monolayer interventions in human cancer samples in vitro. This culture cell cultures. technique could become an important tool for drug development Practicability is an important requirement before a new and for the prediction of in vivo drug efficacy. culture technique can be widely used in cancer research. A critical issue in this context is how to assess cytotoxicity as a major end-point for therapeutic drug action in culture. To date, viability in 3-dimensional tissue cultures has mainly been Correspondence to: Markus M. Borner, MD, Institute of Medical measured with the 3-(6)-2, 5-diphenyl tetrazolium bromide Oncology, Inselspital, 3010 Bern, Switzerland. Tel: +41 (31) 632 (MTT) dye assay or with the (8) thymidine incorporation 84 42, Fax: +41 (31) 382 41 19, e-mail: [email protected] assay. Both assays are very labor intensive and an additional Key Words: Apoptosis, ex vivo, non-small cell lung cancer, important disadvantage of the (8) thymidine incorporation quantitative PCR. assay is the requirement for radioactivity. Thus, the possibility 0250-7005/2006 $2.00+.40 1765 ANTICANCER RESEARCH 26: 1765-1772 (2006) of using Alamar Blueì to assess cytotoxic drug action in assay measurement and the tissue was fixed in 4% paraformaldehyde. 3-dimensional tissue cultures was explored. Alamar Blueì is The metabolic activity of the tissue was measured with Alamar Blueì a non-fluorescent compound, which yields a fluorescent in a Spectramax Gemini Fluorometer (Molecular Devices, Sunnyvale, CA, USA) at 560 nm excitation wavelength and 590 nm emission product after metabolic reduction by living cells. In addition to wavelength. Less than 5% reduction of metabolic activity was avoiding high costs and the use of radioactivity, this method considered as non-responsive. allows cytotoxicity to be measured without processing the tissue cultures, since the dye can be directly added to the Hoechst staining. Two-Ìm paraffin sections were placed on SuperFrost culture medium. Alamar Blueì is not cytotoxic and tissue Plus slides (Roth, Karlsruhe, Germany). The slides were dewaxed and cultures can be further incubated in its presence (8-11). stained with Hoechst 33342, as previously described (15). Continued growth leads to a reduced environment (pink Immunohistochemistry of activated caspase-3. The paraffin sections color), while inhibition of growth maintains an oxidized were dewaxed and dehydrated. For antigen unmasking, the tissue environment (blue color; see Figure 1B). sections were boiled in 10 mM sodium citrate buffer (pH 6.0) for The molecular program of apoptosis has been identified as 1 min at full power followed by 9 min at medium power in a a major determinant of chemotherapy responsiveness in microwave oven and were then cooled and washed with PBS. After cancer. In addition to a great diversity of individual drug- blocking endogenous peroxides with 1% H2O2 for 10 min and 5% target interactions, all anticancer agents achieve their horse serum for 1 h, the slides were either incubated with activated therapeutic goal of inducing cell death by exhibiting the caspase-3 antibody (Cell Signaling Technology, MA, USA) (1:50 dilution) or Ki-67 polyclonal antibody (Santa Cruz, CA, USA) for stereotypical morphological features of programmed cell 1 h. For detection of the immunosignal, the ABC Reagent death (apoptosis) (12-14). Different aspects of apoptosis (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA, induction by chemotherapy drugs were examined in USA) was used. The sections were counterstained with hematoxylin. 3-dimensional ex vivo cultures of non-small cell lung cancer The cells were examined on a Leica DMRB microscope using a 40x (NSCLC) patient samples. The goal of this work was to magnification lens. establish this culture technique for use in cancer research. Caspase inhibitor. The caspase inhibitory peptide benzyloxycarbonyl- Materials and Methods Val-Ala-Asp- fluoromethyl ketone (z-VAD.fmk) was obtained from Bachem (Bubendorf, Switzerland). The caspase inhibitors were used The histoculture drug response assay. Tumor tissue from 20 patients at a concentration of 100 ÌM 2 h prior to chemotherapy exposure. (6 female, 14 male) with NSCLC was collected upon resection of the primary tumor. All patients gave their written consent to be included RNA extraction and reverse transcription. Total RNA was extracted in this study. Gelfoam (Pharmacia & Upjohn, Kalamazoo, MI, USA) and cDNA synthesis was performed as previously described (16). pieces of 1 cm2 were placed into each well of 12-well plates. The gelfoam was allowed to soak for 2 h with culture medium (RPMI Real-time quantitative RT-PCR. Real-time PCR was performed as 1640 medium) containing 10% (v/v) heat-inactivated fetal bovine previously described (16). Briefly, real-time PCR was used for serum and 100 units/ml antibiotic antimycotic solution from Sigma, quantitation of p73 N-terminal variants, while 7S RNA was used for Switzerland. NSCLC tissue samples were taken from the operating the internal standard. Each PCR was carried out in a total volume of room immediately after resection and placed in transport culture 25 Ìl containing cDNA reverse-transcribed from 25 ng and 0.375 ng medium. Areas containing tumor were identified by routine total RNA for p73 and 7S, respectively. Dual-labelled (FAM/TAMRA) histological examinations. The specimens were dissected into 2 to gene-specific probes and TaqMan Universal PCR Master Mix 3 mm cubes. About 40-45 Ìg of tumor tissue was placed on top of the (Applied BioSystems, Rotkreuz, Switzerland) were used for the PCR. gelfoam in the 12-well plates, which were incubated at 37ÆC with 5% ¢N p73 was amplified using 5'GGAGATGGGA AAAGCG AAAAT-3' as the forward primer, 5'CTCTCCCGCTC GG-TCCAC- CO2 overnight. After 24 h, the chemotherapeutic drugs were added directly to the 3' as the reverse primer (both 300 nM) and a ¢N p73 probe respective well and the tissue cultures were incubated for the 5'CAAACGG-CCCGCATGTTCCC-3' (150 nM). For TA p73, the indicated time period (Figure 1A). Taxotere (1 Ìg/ml) was purchased primers were forward 5'-GC-ACCACGTTTGAGCACCTC-3', from Rhöne-Poulenc Rorer (Thalwil, Switzerland), cisplatin reverse 5'-TAATGAGGTGGTGGGCGGA-3' (both 300 nM), and (10 Ìg/ml) and mitomycin C (MMC) (5 Ìg/ml) were purchased from the probe was 5'-TTCGACCTTCCCCAGTCA AGCCG-3' (150 nM).
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