ISOLATION OF VIABLE EPITHELIAL CELLS FROM HUMAN COLON CARCINOMA TISSUE PROTOCOL FOR LIBERASE DH AND DL RESERACH GRADE

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1 ISOLATION OF VIABLE EPITHELIAL CELLS FROM HUMAN COLON CARCINOMA TISSUE PROTOCOL FOR LIBERASE DH AND DL RESERACH GRADE AUTHORS: Tamas Micsik, MD Rational Drug Design Laboratories CRC 1 st Department of Pathology and Experimental Cancer Research Semmelweis University, Budapest, Hungary micsik@kkk-org.hu Istvan Petak, PhD, MD I. Patologia es Kiserleti Rakkutato Intezet Semmelweis University, Budapest, Hungary petak@kps.hu MATERIALS GIBCO RPMI 1640 Cell culture media Cell Strainer BD Sigma-Aldrich Verapamil V4629 Molecular Probes Calcein-AM C3100 Sigma-Aldrich Fetal bovine serum F-2442 GIBCO Hank s balanced salt solution GIBCO Phosphate-buffered saline Dako Anti Ber-EP4 mouse immunoglobuline M Dako mouse IgG1 X Jackson Immunoresearch Cy5-conjugated goat anti-mouse IgG Beckton Dickinson FACSCalibur Roche Liberase DL Research Grade Roche Liberase DH Reseach Grade Sigma-Aldrich Propidium-iodide Four samples of fresh tumor and control normal bowel mucosa were investigated. 1

2 PREPARATION OF CELL SUSPENSIONS 1. Transport the tumor sample in 15 ml vials filled with RPMI 1640 cell culture media. 2. Remove the media and transfer the samples into Petri dishes. 3. Cut the samples into small pieces using a surgical blade. If necessary, pour enough HBSS on the cut pieces in order to avoid dehydration. 4. Transfer the tissue into 1.5 ml microfuge tubes, each containing 1,000 µl enzyme solution of 14 Wünsch Unit activity of Liberase Dl and DH Research Grade. 5. Vortex briefly and incubate in a moving/vibrating water bath at +37 C for 10 minutes, or gently vortex vials several times during incubation. 6. Block the reaction by adding 200 µl of 10% fetal bovine serum. To prepare the cell suspension, filter the mixture using a 70 µm mesh-cell filter. 7. Add 1 ml of HBSS, with the tip of the pipettor pointing at the remainder solid tissue on the filter. 8. Harvest the cells by centrifugation at 2,000 x g for 1 minute at +4 C. Decant the supernatant. 9. Resuspend the pellet in an adequate amount of HBSS for cell counting. 10. Add trypan blue solution to 10 µl of the suspension, mix thoroughly, and allow to stand for 5 minutes. 11. Transfer 10 µl of the trypan blue cell suspension to a hemocytometer and count the viable cells. 12. Add adequate amount of HBSS to achieve a final volume of 4.2 ml (7 x 600 µl) cell suspension containing 1.4 to 3.5 million (7 x 200,000 to 500,000) viable cells. 2

3 MULTIDRUG RESISTANCE (MDR) ASSAY 1. Pipet 600 µl of the 4.2 ml enzyme/sample cell suspension into 7 FACS vials. 2. Add 200 µl of 250 µm MDR-inhibitor Verapamil solution to three parallel vials. 3. Add 200 µl of HBSS to three additional parallel vials. 4. Add 200 µl of 50 nmol MDR-substrate Calcein-AM to each of the 7 vials. 5. Vortex thoroughly and incubate the vials for 10 minutes at +37 C in a moving/vibrating water bath, or gently vortex vials several times during incubation. 6. After the incubation, inmediately place the vials on ice for 1 minute. 7. Spin (2,000 x g, 1 minute, +4 C) and remove supernatant. ANTIBODY STAINING 1. Dilute antibodies using HBSS containing 2 µg/ml propidium iodide. Antibody Dilutions Antibody (µl) HBSS + PI (µl) Cy BerEP IgG Resuspend the pellet in the antibody solutions: a. To the verapamil and HBSS vials, add 100 µl of primary mouse anti- Ber-EP4 IgG antibody and 100 µl of Cy5-conjugated secondary goat anti-mouse IgG antibody solutions. b. To the IgG vial, add 100 µl of negative control mouse IgG1 antibody and 100 µl of Cy5-conjugated secondary antibody. 3. Incubate the vials at room temperature for 30 minutes in the dark. 4. Spin (2,000 x g, 1 minute, +4 C) and remove supernatant. 5. Resuspend the pellet in 300 µl of HBSS containing 2 µg/ml propidium iodide. 6. Perform FACS analysis. 3

4 FACS ANALYSIS The purpose of the gating strategy was to detect viable epithelial cells. First, signals due to debris on the FSC-SSC graph were removed.the resulting cell population without debris was visualized in Graph 1. Graph 1 For further analysis, only this population was used, and gated onto the FL2-FL3 graph on Graph 2. Signal intensity of calcein was detected on FL2, and signal intensity of propidium iodide was detected on FL3; thus, compensation between the two channels was possible during analysis. Accordingly, the population of viable cells could be identified as those propidium iodide-negative and calcein-positive within the R1 region on Graph 2. R1 Graph 2 Epithelial cells were assigned as the Ber-EP4-positive cells against the IgG isotype control on the R2 part of Graph 3. On the lower part, the percentage of Ber-EP4- positive cells was calculated. 4

5 R2 R2 Sample ID: igg zn Patient ID: zn Acquisition Date: 19-Jan-05 Gate: G1 Total Events: Region Events % Total R R R Sample ID: ver1 zn Patient ID: zn Acquisition Date: 19-Jan-05 Gate: G1 Total Events: Region Events % Total R R R M1 M1 Graph 3 Finally, the G4 group of viable epithelial cells could be defined as the intersection of sets representing cells without debris from Graph1 and the R1 region of Graph 2 (representing the viable cells), and the R2 region from Graph 3 (representing epithelial cells). The analysis of the mean fluorescent activity revealed a shift in FL2 calcein intensity in the presence of the MDR inhibitor verapamil, compared to the control samples containing only HBSS (see Graph 4). Mean F Ver : 416 Mean F Ver : 399 Mean F HBSS : 318 Graph 4 Then MAF values were calculated using the mean fluorescent activity in FL2, according to the following formula: 100 X (F Ver -F HBSS )/F Ver This equals 100x ( )/408= 23 in this example. Mean F HBSS : 328 5

6 DATA ANALYSIS/RESULTS EPITHELIAL CELLS / CELLS FREE OF DEBRIS % VIABLE CELLS / CELLS FREE OF DEBRIS % , ,12 26, , Figure 1: Ratio of viable cells and epithelial cells expressed as a percentage of all cell events during flow cytometric analysis after cell isolation with Liberase DH and DL Research Grade. The data show that about one-fourth of all cell events are either viable or epithelial cells. Not all detected epithelial cells are viable. VIABLE EPITHELIAL CELLS / VIABLE CELLS % 15 14, , Figure 2: Ratio of viable epithelial cells expressed as a percentage of all viable cells after cell isolation with Liberase DH and DL Research Grade. About oneeighth of all viable cells in the suspension were found to be viable epithelial tumor cells. 6

7 VIABLE EPITHELIAL CELLS / EPITHELIAL CELLS % ,91 27,31 Figure 3: Ratio of viable epithelial cells expressed as a percentage of all epithelial cells after cell isolation with Liberase DH and DL Research Grade. Almost 30 percent of all epithelial tumor cells survived the isolation protocol using Liberase DH and DL Research Grade. VIABLE EPITHELIAL CELLS / CELLS FREE OF DEBRIS % 5 3,21 3,83 0 Figure 4: Ratio of viable epithelial cells expressed as a percentage of events free of debris during flow cytometric analysis after cell isolation with Liberase DH and DL Research Grade. The Liberase protocol yields viable tumor cells in about 3 to 4 percent of all cell events. LIBERASE is a trademark of Roche. For life science research only. Not for use in diagnostic procedures 7