Supplementary Methods
EGFR immunohistochemistry of PDAC tissue microarrays
Tissue microarrays were constructed from formalin-fixed, paraffin-embedded material from the Australian Pancreatic Cancer Genome Initiative (APGI), with each specimen represented by a minimum of 3 x 1 mm tissue cores. Immunohistochemistry was performed on 4-µm serial sections mounted on SuperFrost slides (Menzel-Glaser, Braunschweig, Germany). Immunohistochemistry for EGFR was performed with the mouse anti-human EGFR antibody clone H11 at a dilution 1:50 (Dako, USA) as previously described (1). Immunostaining was scored semiquantitatively as follows: 0 (absent staining), 1 (cytoplasmic or incomplete membranous staining), 2 (weak to moderate complete membranous staining) and 3 (strong, complete membranous staining). Standardization of scoring was achieved by comparison of scores between at least two specialist pancreatic histopathologists and/or translational researchers experienced in peripancreatic pathology (A.C. and M.P.). Any discrepancies were resolved by consensus after conferencing. Positive EGFR expression was defined as any positive staining. Preclinical models used for efficacy screened were highly positive for EGFR protein (staining intensity 2). Median survival was estimated using the Kaplan-Meier method, and the difference was tested using the log-rank test. The 5-year survival rate was estimated using the life-table method. P values<.05 were considered statistically significant. Statistical analysis was performed using StatView 5.0 software (Abacus Systems, Berkeley, CA). Overall and disease-specific survival were used as the primary end points in this study.
Patient derived xenografts (PDXs) and cell lines (PDCLs) generation
Six-eight week-old female NOD/SCID/interleukin 2 receptor [IL2R] gamma (null) (NSG) mice and athymic balb/c-nude mice were used for the establishment of the patient derived xenograft (PDX) models. All mice were bred at the Australian Bioresources (ABR) under research protocols approved by the Garvan Institute Animal Ethics Committee (ARA 09/19, 11/23, 11/09 and 12/21). The PDXs were generated according to methodology published elsewhere with modifications (2). Briefly, surgical non-diagnostic specimens of patients operated at the various APGI clinical sites were implanted subcutaneously (s.c.) into three NSG and three balb/c nude mice for each patient, with two small pieces per mouse (left and right flank; engraftment stage). Once established, tumors were grown to a size of 1500 mm3, at which point they were harvested, divided, and re-transplanted into further mice to bank sufficient tissues for experimentation (1st passage and 2nd passage). Utilization of the NOG mouse model, which is characterized by high immune deficiency in this study has enabled establishment of a significant cohort of PDXs (70) xenografts, with a high rate of successful engraftment and propagation (76%, data not shown).
The selected patient-derived cell lines used in this study, named The Kinghorn Cancer Centre (TKCC) lines, were established in the laboratory by plating and growing cells from an enzymatically digested xenograft on a collagen matrix for approximately 1 week prior to removal of fibroblasts from the mixture using flow cytometry with anti-mouse CD140a-PE (BD Biosciences, USA) and anti-mouse MHCI-H2KD antibody, eBiosciences, USA). All cell lines were profiled by short tandem repeat (STR) DNA profiling as unique (CellBankaustralia.com).
Cell culture
PANC-1 and BxPC-3 were cultured in DMEM and RPMI-1640, respectively, and supplemented with 10% fetal calf serum (FCS). The EGFR mAb hybridoma (mAb clone 225, ATCC®) was cultured in RPMI-1640+10% FCS. The PDCLs TKCC-07 and TKCC-10 were cultured in M199/Ham’s F12 media mixture (50%:50% v:v) supplemented with HEPES (15 mM), glutamine (2 mM), EGF (20 ng/mL), hydrocortisone (40 ng/mL), transferrin (5 mg/mL), insulin (8 mg/mL), triiodothyronine (0.5 ng/mL), MEM vitamin solution (1X), O-phosphorylethanolamine (2 mg/mL), glucose (0.6%) and fetal calf serum (FCS 7.5%). All media and supplements were purchased from Sigma-Aldrich®. All cell lines were profiled by short tandem repeat (STR) DNA profiling as unique (CellBankaustralia.com).
Treatments in vitro
For in vitro treatments, sub-confluent cultures of PANC-1 cells were treated with escalating doses of gemcitabine (Selleck Chemicals LLC, TX, USA) in the absence or presence of the Chk1 inhibitor PF-477736 (provided by Pfizer, Compound transfer program grant WS835429). The Chk1 inhibitor (Chk1i), at 180 nM final concentration, was added concurrently or 16 after gemcitabine. Cells were collected at several time points (24-96 hours) for standard DNA content analysis by flow cytometry using BD FACSCanto II™ flow cytometer (BD Biosciences, CA, USA). Standard clonogenic survival assay were performed after the treatment of sub-confluent cultures of PANC-1 cell in the absence or presence of anti-EGFR mAb unlabeled or labeled with 177Lu, absence or presence of gemcitabine (40 ng/mL) and the absence or presence of Chk1i (180 nM).
Histological and immunoblotting analyses of PDXs post-treatment
The TKCC-PDX-07 model was used to perform histological and immunoblotting analyses after treatments. Briefly, tumors from control and treated mice were collected at 7 days after treatment start and were bisected at a symmetrical plane where one half was fixed in 10% saline-buffered formalin solution and the second half was used to prepare protein lysates. Formalin fixed, paraffin embedded tumors were used to prepare 7 mm tissue sections for standard H&E, Masson's trichrome or Sirius red staining. Immunohistochemical staining (IHC) was performed to detect double stand DNA breaks (DSBs) using the anti-phospho-histone H2AX (Ser139, gH2AX) mAb (clone JBW301, Merk Millipore. MA, USA) or apoptosis using ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Merk Millipore) as per manufacturer instructions. For IHC quantification, images of ten random regions from each tumor section were analyzed using ImageJ (3) (V1.46d) with ImmunoRatio plugin (4). Masson’s trichrome and Sirius red staining were analyzed using ImageJ as previously described (5) using images of ten random regions from each tumor section. Standard immunoblotting was performed using lysates prepared from freshly isolated tumors. Membranes were probed with antibodies against CDC25A and RAD51 (Santa Cruz Biotech, CA, USA), total Erk1/2 and phosphorylated Erk1/2 (Cell Signaling Technology Inc., MA, USA) as per manufacturer instructions. Antibody against COX IV (Cell Signaling Technology) was used as a loading control. Membranes developed using chemiluminescence reagent plus (Merk Millipore).
References for Supplementary Methods
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4. Tuominen VJ, Ruotoistenmaki S, Viitanen A, Jumppanen M, Isola J. ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67. Breast Cancer Res. 2010;12:R56-R67.
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