All tissues and biologic samples were collected with the approval and in accordance with the requirements of the Institutional Review Board of the Massachusetts General Hospital, Boston, Massachusetts.
Paraffin-embedded tissue samples were obtained from the files of the Department of Pathology of the Massachusetts General Hospital, Boston, Massachusetts. All specimens had an established diagnosis at the time of assessment. A total of 4 normal pancreata, 15 chronic pancreatitis, 14 PanIN I, 26 PanIN II, 15 PanIN III and 41 PDAC, 8 liver metastasis, 11 lymph node metastasis, 10 with matching primary tumors, and 9 peritoneal metastasis were obtained. For the assessment of Plec1 expression in extra-pancreatic human cancer, a commercial tumor tissue microarray (MTU951, US Biomax, Rockville, MD) was used.
Mice & cell lines
All animal procedures were approved by the University of Virginia Animal Care and Use Committee and the Massachusetts General Hospital Subcommittee on Research Animal Care. Nude mice (nu/nu) were purchased from the National Cancer Institute (Fredericksburg, MD). FVB/NJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained in a germ-free environment and had access to food and water available ad libitum.
The L3.6pl pancreatic cancer cell line was originally derived from a repeated cycle of injecting COLO-357 cells into the pancreas of nude mice, selecting for liver metastases, and re-injecting into the pancreas. AK134 cells were derived from spontaneous PDAC arising in Pft1-Cre; LSL-K-RasG12D; p53 ± mice in the background of an inbred FVB/NJ strain. The Panc1 pancreatic cancer cell line was obtained from ATCC. All cell lines were routinely verified by morphology and growth curve analysis, and tested for Mycoplasma.
Three orthotopic mouse models and one mouse model of liver metastasis were employed to assess the suitability of Plec1 as an in vivo imaging biomarker. 1×106 L3.6pl (n=10) or 11×106 Panc1 cells (n=5) in 50 μl Hanks Buffered Sterile Saline (HBSS) were injected into the head of the pancreas of nu/nu mice. 2.5×105 AK134 were injected into the pancreas of FVB/NJ mice (n=8). To obtain liver metastasis, 2.5×105 AK134 cells (n=5) in 50 μL of HBSS were injected into the capsule of the spleen. Seven days (AK134), 10 days (L3.6pl), 4 weeks (Panc1) or 15 days (AK134 liver metastasis) after injection, animals were imaged, sacrificed, and tPTP biodistribution assessed. All animals underwent gross inspection of the abdominal cavity and liver for metastasis. Histology was used to confirm macroscopic findings. As control animals, FVB/NJ (n=2) or nu/nu (n=5) mice were injected with 50 μL of HBSS into the pancreas and spleen and then imaged 1–4 weeks after injection.
Western blot analysis
50 mg of pancreatic tissue obtained as snap-frozen surgical specimens was homogenized in RIPA buffer (50mM Trizma Base, pH 7.4, 1% Triton X-100, 0.25% sodium desoxycholate, 100mM EDTA 150mM NaCl) in combination with a protease inhibitor cocktail (0,001mg/ml aprotinin, bestatin, pepstatin, leupeptin and 0.005 mg/ml 20mM PMSF, Sigma-Aldrich, St. Louis, USA). The lysate was cleared by centrifugation. To ensure equal loading, a highly sensitive and precise copper-based assay was used to determine the protein concentration of each sample (2-D Quant Kit, Amersham Biosciences, NJ). 20 μg protein/lane were separated via SDS-PAGE and transferred onto a nitrocellulose membrane. Equal transfer was verified by Ponceau staining. Antigen detection was performed using a rabbit monoclonal antibody against human Plec1 (Abcam, Cambridge, MA). The secondary antibody was a HRP-coupled goat anti-rabbit polyclonal antibody (Sigma-Aldrich, St. Louis, USA). Bands were visualized with ECL. (control: rat brain lysate, Santa Cruz Biotechnology, La Jolla, CA).
Immunohistochemistry for Plectin-1
Paraffin-embedded sections were deparaffinized, hydrated with TBS and blocked with H2O2. Antigen retrieval was achieved by boiling tissue in Retrievit (BioGenex, San Ramon, CA). After blocking with avidin/biotin (Vector Laboratories, Burlingame, CA) and 5% goat serum in TBS, slides were incubated overnight at 4 C with 1:250 Plec1 antibody (Abcam). Sections were washed three times in TBST, followed by incubation with biotinylated anti-rabbit goat secondary antibody (Vector Laboratories, Burlingame, CA), than developed using DAB (Invitrogen, Carlsbad, CA) and counterstained with hematoxylin. Slides were evaluated using a Y-FL microscope (Nikon, Japan).
Expression of Plec1 in nerves within each slide was used as a staining control and reference for staining intensity. Nerves were noted to have a moderate staining intensity. Staining intensity was recorded by two independent observers, and in case of discrepant results, evaluated by a third observer. Plec1 staining was classified as negative if the staining intensity was weaker than nerves. It was classified as positive if the staining was as least as strong as nerves.
In-vitro competition assay
Tetramericplectin-1-targeted peptide (tPTP-4(βAKTLLPTPGGS(PEG5000))KKKDOTAβA-NH2)) was synthesized in a GMP grade facility (CS Bio Company, Menlo Park, CA). As a control, non-binding tetramer (ncPTP-4(βAKHVMSKQGGS(PEG5000))KKKDOTAβA-NH2)) was also synthesized. For Indium labeling, peptide (100 μg) was dissolved in 20 μl PBS, then diluted in 100 μl ammonium acetate buffer (0.1M, pH 4.5). Indium chloride (5mCi in water; Cardinal Health, VA) was mixed with the peptide and allowed to equilibrate with mixing at 40°C for 15 min. The reaction mixture was purified by size exclusion using a PD10 desalting column pre-equilibrated with DPBS. For in vitro peptide validation experiments, cells were incubated at room temperature for 1 hr with tPTP or ncPTP with concentrations ranging from 10−3 to 10−9 and 5 μCi tPTP-In111 in triplicate. After 1 hr the cells were washed and lysed with 100μL 1M NaOH for 5 min. The mixture was then transferred to tubes and activity analyzed on a gamma counter.
Mice were injected with 1 mCi of 111In labeled tPTP, then imaged 4 hours post injection with a microSPECT/CT scanner designed and built at UVa. CT acquisition used 200 evenly spaced projections spanning 200 degrees over approximately 5 minutes. Pinhole SPECT scanning was then performed using two opposing gamma cameras simultaneously. The two cameras were fitted with 0.5 mm diameter tungsten pinholes. 60 evenly spaced projection views per camera were obtained over 180 degrees, for a total of 120 views at 3 degree increments over 360 degrees. The SPECT acquisition time was approximately 45 minutes. The reconstructed CT voxel size was 0.082×0.082×0.082 mm on a 640×640×768 image matrix. The reconstructed SPECT voxel size was 0.65×0.65×0.65 mm on an 80×80×80 image matrix. All SPECT images were corrected for radioactivity decay but not for gamma ray attenuation.
Biodistribution and blood half-life
After mice were imaged via SPECT/CT, animals were sacrificed and their organs harvested and placed into pre-weighed Eppendorf tubes. Each tube was then re-weighed to determine the weight of the organ and the radiation measured. Tubes containing organs were analyzed on a gamma counter. To determine the plasma lifetime of the probe, a mouse injected with the tPTP-Peg-111In was bled 0, 15, 30, 45, 60, and 120 min post injection, and the sample analyzed on a gamma counter. Tissue samples were then placed in histology cassettes and fixed for paraffin embedding. After the radioactivity in the tissue samples decayed, the blocks were sectioned on a microtome and evaluated by H&E.