Cell lines and agents
The human pancreatic adenocarcinoma (PDA) cell lines MIA PaCa-2, PANC-1, AsPC-1, Capan-1 and Capan-2 were obtained from the American Type Culture Collection (Manassas, VA, USA) [21]. All cell lines were cultured in Dulbecco’s modified Eagle medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) and 10 % heat-deactivated fetal bovine serum. Gemcitabine (GEM) was purchased from Eli Lilly Japan (Kobe, Japan), five-fluorouracil (5FU) was purchased from Kyowa Hakko Kirin Co. (Tokyo, Japan), and oxaliplatin (L-OHP) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trastuzumab was a gift from Chugai, Inc. (Tokyo, Japan), and trastuzumab emtansine (T-DM1) was provided by Genentech Inc. (South San Francisco, CA, USA).
Flow cytometric analysis
To assess HER2 expression levels, cells were incubated either with phycoerythrin (PE)-labeled anti-human HER2 (24D2) or with corresponding isotype-control antibodies (BioLegend, San Diego, CA, USA) in buffer (1 % FBS, 2 mM EDTA and 0.1 % NaN3 in PBS) for 30 min at 4 °C, after which they were washed and then analyzed using a MACSQuant Analyzer (Miltenyi Biotech K.K., Bergisch Gladbach, Germany). Before using the analyzer, 4 μg/ml propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, USA) was added to each sample to exclude dead cells.
To assess T-DM1 binding to PDA cells, 0.5x106 cells were incubated with 30 μg/ml T-DM1 at 37 °C for 1 h. The cells were washed, incubated with PE-labeled anti-human IgG Fc (HP6017) (Biolegend) or corresponding isotype-control antibodies (Affymetrix, Santa Clara, CA, USA) in buffer for 30 min at 4 °C, washed, re-suspended and analyzed using a MACSQuant Analyzer (Miltenyi Biotech K.K.). Before using the analyzer, 4 μg/ml PI (Sigma-Aldrich) was added to the sample to exclude dead cells. The mean fluorescence intensity (MFI) of HER2 was analyzed using MACSQuantify Software.
Cell cycle analysis
MIA PaCa-2 cells were suspended in Hoechst 33342 (5 μg/ml) (Life Technologies) and incubated at 37 °C for 90 min, then washed and incubated with phycoerythrin (PE)-labeled anti-human HER2 (24D2) or the corresponding isotype control antibodies (BioLegend) for 30 min at 4 °C. Before using the analyzer, 1 μg/ml PI (Sigma-Aldrich) was added to the sample to exclude dead cells. In cell cycle analysis, the mean fluorescence intensity (MFI) of HER2 in each phase of the cell cycle was examined using MACSQuant Analyzer (Miltenyi Biotech K.K.) and MACSQuantify Software.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
The cells were lysed in RLT Plus Buffer (Qiagen, Hilden, Germany) and homogenized. From 2 μg of total RNA, cDNA was synthesized using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) and a GeneAmp PCR System 9700 (Applied Biosystems). For qRT-PCR detection of HER2 and 18S rRNA, 5 ng of cDNA was amplified using SYBR Premix Ex Taq II (Takara Bio Inc., Otsu, Shiga, Japan) and a 7300 Real-Time PCR System (Applied Biosystems). The PCR conditions consisted of an initial denaturation step (95 °C for 30 s), followed by 40 cycles (95 °C for 5 s and 62 °C for 31 s) and a dissociation step (95 °C for 15 s, 60 °C for 60 s and 95 °C for 15 s, and 60 °C for 15 s). The sequences of the primers (Operon Biotechnologies K.K., Tokyo, Japan) for HER2 and the 18S ribosomal RNA (rRNA) that were used in the present study were as follows: 5’-TCCTGTGTGGACCTGGAT-3’ as a forward primer and 5’-TGCCGTCGCTTGATGAG-3’ as a reverse primer for human HER2; 5’-CGGCTACCACATCCAAGGAA-3’ as a forward primer and 5’-GCTGGAATTACCGCGGCT-3’ as a reverse primer for human 18S rRNA. The data were analyzed using the comparative ΔΔCT method by calculating the difference between the threshold cycle (CT) values of the target and reference genes of each sample and then comparing the ΔCT values of each drug treatment to the non-treated group.
Immunoblot analysis
Cells were homogenized in 20 mM HEPES buffer containing 1 % Triton X-100, 100 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin and 0.9 M Na3VO4, and the protein concentrations of the homogenates were analyzed using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Protein samples (10 μg) were separated by electrophoresis on a 7.5 % sodium dodecyl sulfate-polyacrylamide gel (ATTO, Tokyo, Japan) and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 3 nonfat milk and 3 % bovine serum albumin for 1 h, the membrane was treated with Abs against HER2 (1:1000) (e2-4001 + 3B5, Thermo Fisher Scientific Inc., Waltham, MA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:600,000) (2D4A7, Abcam Inc, Cambridge, UK) and then with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology Inc., Danvers, MA, USA). Chemi-Lumi One Super (Nakalai Tesque Inc, Kyoto, Japan) was used for chemiluminescent detection.
When HER2 was detected by immunoblot analysis, it produced two bands of approximately 185 and 155 kDa. The 155 kDa band has been reported to represent cytoplasmic HER2, whereas the 185 kDa represents membrane-bound HER2 [22–24].
Enzyme-linked immunosorbent assay (ELISA)
GEM- and T-DM1-treated MIA PaCa-2 cells (1x106 cells) were homogenized in 20 mM HEPES buffer containing 1 % Triton X-100, 100 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin and 0.9 M Na3VO4. To quantify human IgG (T-DM1) binding to 1x106 MIA PaCa-2 cells, a human IgG total Ready-Set-Go kit (Affymetrix, Santa Clara, CA, USA) was used according to the manufacturer’s procedures.
Estimation of the anti-proliferative effects of the agents by an in vitro cell growth assay
MIA PaCa-2 cells and Capan-1 cells were treated with GEM (0, 30, 100 or 300 ng/ml) for 2 h. After washing the cells with PBS, they were incubated in medium containing T-DM1 (0, 10 or 30 μg/ml) for 96 h. Then, the cells were detached by trypsin-EDTA treatment, and the number of cells that were not stained with trypan blue was determined using a hemocytometer. Identical numbers of viable cells were seeded into 96-well plates (103/100 μl/well). After a 96-h incubation, cell growth was examined by spectrophotometry using the counting reagent SF (Nakarai Tesque Inc, Catalog No. 07553). The cell count reagent SF is a sensitive colorimetric reagent that utilizes tetrazolium salt, which is highly water-soluble. Because the absorbance at 450 nm is proportional to the number of viable cells in the medium, the viable cell number can be determined using the absorbance value of a previously prepared calibration curve. Fluorescence was measured at 450 nm using an iMark microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA). The treatment design is shown in Fig. 1.
Statistical analysis
All data are presented as the mean ± standard deviation (SD). Comparisons between the untreated control and drug-treated groups were performed by non-paired Student’s t-tests for two independent groups and with Dunnett’s method for multiple-group comparisons. A p-value of <0.05 was considered statistically significant. Statistical analyses were performed using Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA, USA) with the add-on software Statcel3 (OMS publishing Inc., Saitama, Japan).