The floxed Fbw7 (Fbxw7tm1Kei
) strain was obtained from A. Balmain (27
). The floxed p53 (Trp53tm1Brn/J
) and Villin-Cre [Tg(Vil-cre)997Gum
] strains were purchased from Jackson Laboratory (13
). All strains were backcrossed onto C57BL/6 at least 5 generations prior to transfer to our laboratory, and experimental mice were segregated for the C57BL/6J and S129 genomes at an approximate 90:10 ratio. Animal studies and all animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were carried out at the Fred Hutchinson Cancer Research Center (FHCRC). To analyze growth of FPV cell lines in vivo
/SzJ (NSG) mice (originally obtained from Jackson Laboratory and bred at FHCRC) were treated with low-dose radiation (275 rads) prior to injection of 5 million tumor cells subcutaneously (n
≥ 2 per cell line) (FPV1, FPV2, and FPV3) according to standard protocols. Animals with subcutaneous tumors were sacrificed when masses were palpable, generally by 4 weeks postinjection.
Mice were sacrificed via carbon dioxide inhalation, and full necropsies were performed according to standard techniques. In all animals, the gastrointestinal tract (from stomach to anus) and the cecum were harvested, flushed with ice-cold phosphate-buffered saline (PBS), opened longitudinally, divided into 4 sections (proximal small intestine, mid-small intestine, distal small intestine, and colon) and fixed flat on bibulous paper in 10% formalin for at least 24 h at room temperature. After fixation, intestines were rolled and stored in 70% ethanol until submitted for pathological evaluation. Individual tumors were sometimes dissected and fixed separately. A full gross inspection of all animals, including lymph nodes, breast tissue, thymus, lungs, heart, stomach, liver, spleen, pancreas, kidneys, and reproductive organs, was performed. In a subset of representative animals (>10 per genotype), all tissues were fixed in 10% formalin for 7 days and then submitted for pathological evaluation. In subsequent necropsies, all tissues were examined grossly and any abnormal tissues were submitted for pathology. After fixation, samples were submitted to the Experimental Histopathology Core Facility at the FHCRC, where tissues were processed, embedded in paraffin, cut onto slides, and stained according to standard techniques. The stains used included hematoxylin/eosin (H&E), periodic acid-Schiff (PAS), and Alcian blue (AB). In some cases, tumors were harvested and embedded in OCT compound (Tissue-Tek) and frozen at −80°C. Slides were cut from these blocks both for hematoxylin/eosin staining and for laser capture microdissection with an Arcturus Veritas laser capture microdissection system to enrich for tumor tissue. All tissue slides were reviewed by a board-certified veterinary pathologist (S. E. Knoblaugh).
Immunohistochemistry was performed by the Experimental Histopathology Core Facility at the FHCRC. Four-micrometer sections were cut, deparaffinized, and rehydrated in Dako wash buffer (Carpinteria, CA). Steam antigen retrieval was performed in a Black and Decker steamer. Ki-67 slides were steamed for 40 min and β-catenin slides were steamed for 20 min in preheated target retrieval solution (pH 6; Dako) and cooled for 20 min. Lysozyme slides were treated with proteinase K (Dako) for 5 min. Slides were rinsed 3 times in wash buffer, and all subsequent staining steps were performed at room temperature using the Dako Autostainer. Endogenous peroxide activity was blocked using 3% H2O2 for 8 min, followed by protein blocking with 0.25% casein and 0.1% Tween 20 in Tris-buffered saline (TBS) for 10 min. In addition to protein blocking, the Ki-67 slides were blocked with biotin block (Dako) for 10 min. Ki-67 was detected using a rat monoclonal antibody from Dako (Tec-3 M7249) at a dilution of 1:25 for 30 min, followed by biotinylated goat anti-rat antibody at 1:200 for 30 min (112-065-167; Jackson ImmunoResearch) and horseradish peroxidase (HRP)-labeled streptavidin (016-030-084; Jackson ImmunoResearch) at 1:2,000 for 30 min. Lysozyme was detected using rabbit polyclonal antibody from Dako (A0099) at 1:200 for 30 min, followed by Mach 2 anti-rabbit HRP-labeled polymer (Biocare Medical, Walnut Creek, CA) for 30 min. β-Catenin staining was detected using a mouse monoclonal antibody from BD Biosciences (610154; San Jose, CA) at a dilution of 1:100, conjugated to a rabbit anti-mouse Fab fragment (Jackson ImmunoResearch; 315-007-003), followed by Mach 2 anti-rabbit HRP-labeled polymer for 30 min. The staining for all slides was visualized with 3,3′-diaminobenzidine (DAB) (Dako) for 8 min, and the sections were counterstained with hematoxylin (Dako) for 2 min. Concentration-matched isotype control slides were run for each tissue sample (Jackson ImmunoResearch Laboratories).
All images were obtained using a Nikon Eclipse 50i microscope and a Digital Sight DS-L1 camera. Images were imported into Photoshop (Adobe Systems), and the white balance was automatically adjusted using the levels tool.
Quantitation of proliferation and differentiation in normal intestines.
To evaluate proliferation and differentiation in normal tissues, intestinal rolls from mice of each genotype were stained for lysozyme (to stain Paneth cells) (n = 2 per genotype), PAS/AB (to stain goblet cells) (n = 3 per genotype), or Ki-67 (to stain proliferating cells) (n = 3 per genotype) as outlined above. To quantitate Paneth cells, stained sections were microscopically examined using the 10× objective. The positive crypts per field were counted for at least 5 fields. Goblet cells were quantitated by counting 30 villi per mouse, and Ki-67-positive cells were counted in 25 crypts per mouse, both using the 20× objective.
Small intestines were harvested from mice, flushed extensively with PBS, and opened longitudinally. To enrich for crypt cells, we modified a published protocol (22
). Briefly, intestines were gently scraped with a microscope slide. They were then cut into ~1-cm pieces, incubated in Hanks' balanced salt solution (HBSS) and 8 mM EDTA for 5 min at room temperature, and then shaken vigorously. The intact tissue pieces were allowed to settle, and the supernatant was discarded. These initial steps serve to remove the majority of the villi from the intestine pieces. Next, the tissue pieces were resuspended in 10 ml of ice-cold HBSS and 8 mM EDTA in 15-ml conical tubes and then incubated at 4 degrees with constant rotation for a total of 45 min. Every 15 min, the tubes were shaken vigorously for 2 min. At the end of this incubation, the intact tissue fragments were removed with forceps and the remaining material was pelleted in a refrigerated centrifuge for 5 min at 1,000 × g
. The resulting pellet (~500 μl of volume) was highly enriched for crypt cells. It was resuspended in PBS and equally aliquoted to 4 microcentrifuge tubes, followed by a second spin at 1,000 × g
at 4 degrees. The supernatant was removed, and pellets were stored at −80°C.
For immunoblotting, cell pellets were lysed in TENT buffer (50 mM Tris-Cl [pH 8.0], 2 mM EDTA, 150 mM NaCl, 1% Triton X-100) and analyzed using standard techniques. The following primary antibodies were used: c-Myc (a mixture of monoclonal antibodies 143 and 274, both provided by N. Ikegaki, University of Illinois, Chicago, IL) (6
), Myc-pT58 (ab28842; Abcam), cyclin E (26
), c-Jun (H-79; Santa Cruz Biotechnology), transforming growth factor interacting factor (TGIF) (H-172; Santa Cruz Biotechnology), p53 (1C12; Cell Signaling Technology), bax (1063-1; Epitomics), and gamma tubulin (C-20; Santa Cruz Biotechnology). Cyclin E-associated kinase assays were performed as previously described using histone H1 as the substrate (5
Crypt fractions were isolated as described above, and total RNA was purified from cell pellets using TRIzol (Invitrogen). cDNA was synthesized with a high-capacity cDNA reverse transcription kit (Applied Biosystems), and quantitative reverse transcription-PCR (qRT-PCR) was performed using Platinum SYBR green qPCR SuperMix UDG (Invitrogen) with an ABI 7900HT real-time PCR system. Data were analyzed using SDS 2.3 software (Applied Biosystems). Results were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression and compared across genotypes. The results are expressed as fold change relative to the control genotype (FP). The following primers were used (forward primer listed first in each case): Atoh1 (TATCCCGTCCTTCAACAACGA and TGGTCATTTTTGCAGGAAGCT), Myc (TCCCTGAATTGGAAAACAACG and TGCTCGTCTGCTTGAATGGA), Fbxw7 (GAGACTTCATCTCCTTGCTTCCTAAA and CGCTTGCAGCAGGTCTTTG), Gapdh (GCAAAGTGGAGATTGTTGCCA and ATTTGCCGTGAGTGGAGTCAT), Hes1 (TCAACACGACACCGGACAAA and TTATTCTTGCCCTTCGCCTC), Hes5 (GGTACAGTTCCTGACCCTGCA and CCGCTGGAAGTGGTAAAGCA), Insm1 (CAGGTGATCCTCCTTCAGGT and CTCTTTGTGGGTCTCCGAGT), Lgr5 (CCAATGGAATAAAGACGACGGCAACA and GGGCCTTCAGGTCTTCCTCAAAGTCA), Neurod1 (CCTGCGCTCAGGCAAAA and GCTGGGACAAACCTTTGCA), ngn3 (CCGGATGACGCCAAACTTA and GAGTCAGTGCCCAGATGTAGTTGT), and Trp53 (GCAACTATGGCTTCCACCTG and CTCCGTCATGTGCTGTGACT).
All analyses were performed in the flow cytometry resource at the University of Washington. For formalin-fixed paraffin-embedded (FFPE) tumors, four 60-μm sections were cut, dewaxed, rehydrated, and digested with 1% pepsin for 1 h at 37°C. Samples were washed in bovine serum albumin saline-Tris buffer (ph 7.8), filtered though nylon mesh, and resuspended in isotonic (pH 7.4) buffered solution with 0.1% Nonidet P-40 detergent (NP-40) and 10 μg/ml diamidino-2-phenylindole (DAPI). Flow cytometric analysis was performed on a Cytopeia InFlux cytometer using UV excitation. A known diploid cell line and normal cells contained within each tumor were used to determine the position of the diploid peak. A total of 50,000 cells were analyzed, if available, and in all cases, acceptable histograms contained at least 10,000 cells and a coefficient of variation (CV) below 6.0%. DNA content and cell cycles were analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, CA). For FPV cell lines, cell pellets of approximately 500,000 cells were resuspended in 1,000 μl of an isotonic (pH 7.4) buffered solution with 0.1% NP-40 detergent and 10 μg/ml DAPI and analyzed as described above. Normal mouse kidney cells were used to determine the position of the diploid peak.
Derivation of FPV cell lines and tumor allografting.
Tumors were rinsed 5 times with Dulbecco's modified Eagle medium (DMEM) containing 10% serum as well as penicillin-streptomycin (pen-strep) and gentamicin. After washing, tumors were minced with a razor blade, scraped into 10 ml of 0.5% trypsin-EDTA, and incubated at 37 degrees for 1 h. At the end of this incubation, tissue fragments were put through a 25-gauge needle several times to further break up cell clumps. Cells were pelleted for 5 min at 800 × g, resuspended in DMEM with 20% serum and the antibiotics listed above, and plated into tissue culture dishes. These cells were considered passage 0 (p0). Cells were allowed to grow to confluence and then were harvested via trypsinization and replated into new dishes. After initial passage, cells were grown in DMEM with 10% serum and pen-strep. Cells were frozen back at various passages (p5, p10, and p15). After p20, cells were considered to be stable cell lines.
A total of 50 ng of DNA was whole-genome amplified (WGA) using the GenomePlex WGA kit (Sigma). A total of 500 ng each of the amplified control and the tumor DNA was fluorescently labeled with either Cy3 or Cy5 using the genomic DNA ULS labeling kit (Agilent Technologies). The labeled control and tumor DNAs were combined, and mouse Cot-1, hybridization buffer, and blocking agent were added. The hybridization mixture was heated to 95°C for 3 min, followed by incubation at 37°C for 30 min. An Agilent comparative genomic hybridization (CGH) block was then added, and the entire mixture was hybridized to Agilent SurePrint G3 mouse CGH 4×180K microarrays at 65°C for 24 h with rotation. Hybridization was followed by washes using Agilent array CGH (aCGH) commercial wash solutions according to Agilent's aCGH procedures. Arrays were subsequently scanned using the Agilent microarray scanner system, and feature intensity data were extracted from the images using the Agilent feature extraction software.