Reagents, cell lines and tumors
Mouse monoclonal 60.11 and 74.5.2 anti-p32 antibodies were purchased from Chemicon (Temecula, CA). Rat monoclonal anti-mouse CD-31, rat anti-MECA-32, rat anti mouse CD-11b and R-Phycoerythrin (R-PE)-conjugated rat anti-mouse Gr-1 were from BD-PharMingen (San Jose, CA), the anti-epithelial membrane antigen, EMA (clone E29), was from Chemicon, anti-CD68 from Oncogene Research Products (San Diego, CA) and anti β-actin from Sigma-Aldrich (St. Louis, MO). Rat anti-podoplanin antibody was kindly provided by Drs. T. Petrova and K. Alitalo (University of Helsinki, Finland). Polyclonal anti LYVE-1 antibody has been described (3
). ChromPure Rabbit IgG (whole molecule) was from Jackson ImmunoResearch Laboratories (West Grove, PA) and purified Mouse IgG1 (mIgG) from BD-Pharmingen. Purified polyclonal anti-full-length p32 was a gift from Dr. B. Ghebrehiwet (Stony Brook University, NY). Polyclonal anti-peptide antibody against p32 was generated in rabbits against a mixture of peptides corresponding to amino acids 76-93 of mouse (TEGDKAFVEFLTDEIKEE) and human (TDGDKAFVDFLSDEIKEE) p32 protein. The peptides were coupled to keyhole limpet hemocyanin (Pierce, Rockford, IL) via a cysteine residue added at their N-termini. The antibody was affinity purified on the peptides coupled to Sulfolink Gel (Pierce) via the cysteine residue. Dr. A. Strongin (Burnham Institute for Medical Research, La Jolla, CA) kindly provided human p32 cDNA in pcDNA3.1 Zeo and pET-15b vectors. Oligonucleotide duplexes for transient siRNA knockdown of p32 (C1QBP-HSS101146-47-48 Stealth RNAi) and negative control duplexes (Stealth RNAi control low GC and medium GC) were purchased from Invitrogen (Carlsbad, CA). Tissue Arrays (core diameter 0.6 mm) of paraformaldehyde fixed and paraffin-embedded tumor and normal tissue samples were from Applied Phenomics LLC (Tartu, Estonia).
MDA-MB-435, MDA-MB-231, MCF7, C8161, BT549, HL60, Raji and 4T1 cells were maintained in DMEM supplemented with 10% FBS and 1% Glutamine Pen-Strep (Omega scientific) at 37°C/5%CO2. The MCF10-A cell line was from ATCC (Manassas, VA), while the MCF10-AT and MCF10-CA1a cells were from The Barbara Ann Karmanos Cancer Institute (Detroit, MI). Cells were maintained in DMEM supplemented with 10% FBS, 1% Glutamine Pen-Strep and 100ng/ml human EGF (Sigma-Aldrich, Saint Louis, MI).
To produce MDA-MB-435 and 4T1 tumors, BALB/c athymic nude or normal BALB/c mice were orthotopically injected into the mammary fat pad with 2×106 MDA-MB-435 cells suspended in 100μl of PBS. C8161 tumors were grown from cells inoculated subcutaneously on the flank. All animal experimentation received approval from the Animal Research Committee of Burnham Institute for Medical Research.
Pull-down assays and mass spectrometry
Cells were lysed in cold PBS/200mM n-octyl-β-D glucopyranoside (Calbiochem; San Diego, CA) and supernatant aliquots containing 1mg protein were pre-cleared with 40 μl of streptavidin beads for 2 h at 4°C prior to incubation over night at 4 °C with streptavidin beads loaded with biotinylated peptides (3 μg/10 μl beads). The material eluted from the beads was separated on a 4-20% polyacrylamide gel and visualized by silver staining (Invitrogen). Bands that appeared in the LyP-1 but not control peptide pull down were cut out, digested with trypsin, and the resulting peptides were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Profound software was used to query the information against a protein sequence data.
In vitro phage binding assays
Microtiter wells (Costar, Corning, NY) were coated with 5 μg/ml of either purified p32 or BSA (Sigma-Aldrich), blocked with Pierce Superblock buffer, and incubated with 108 pfu of LyP-1 or control phage in 100μl of TBS/0.05% Tween-20 for 16 h at 37°C. After 6 washes in TBS/0.05% Tween-20, bound phage were eluted with 200 μl of Tris-HCl 1M pH 7.5/0.5% SDS for 30 min and quantified by plaque assay. To test antibody inhibition of phage binding, 1.5×107 pfu of LyP-1 or insertless phage were allowed to bind for 6 h at 37°C to p32-coated wells coated in the presence of 20 μg/ml of monoclonal anti-p32 or mouse IgG. When the assay was performed with cells, 2×106 Raji cells were resuspended in 500μl of PBS/1% BSA and pre-incubated for 1h at 4°C with 40μg/ml of anti-p32 or mIgG. LyP-1 or insertless phage (108 pfu) were subsequently added to the cells and incubated at 4°C for 3 h.
Saturation binding assay
LyP-1 binding affinity to p32 was quantified by an ELISA-based assay. Microtiter wells coated with 3μg/ml of purified p32 protein were incubated for 1 hour at room temperature with various concentrations of biotinylated LyP-1 peptide in PBS (100μl/well). After washing with Tris-buffered saline containing 1mM CaCl2 and 0.01% Tween-20, streptavidin-conjugated horseradish peroxidase (Zymed, Carlsbad, CA) was added to the wells and incubated for 1 hour at room temperature. Peptide binding to p32 was quantified with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) as a substrate. The background that was determined from wells without p32 coating was subtracted. Kd values were calculated using Prism software.
FACS analysis and immunohistology
FACS analysis of cell surface p32 was performed on live cells. Approximately 2.5×105
cells were stained with polyclonal anti-full-length/N-terminus p32 or rabbit IgG (20μg/ml) and secondary antibody, each for 30 min at 4°C, and analyzed without permeabilization and by gating for propidium iodide-negative (live) cells (30
Immunohistochemical staining of frozen tissue sections was carried out using acetone fixation and reagents from Molecular Probes (Invitrogen). Antibody binding was detected with secondary antibodies, which were: AlexaFluor-594 goat anti-rat or rabbit IgG, AlexaFluor-488 goat anti-rabbit IgG. Hypoxyprobe-1 (pimonidazole hydrochloride; Chemicon) was injected into tumor-bearing mice and hypoxic areas in tumors were detected with a FITC-conjugated anti hypoxyprobe-1 antibody according to the manufacture’s instructions.
Paraffin-embedded normal and malignant human tissue array sections were deparaffinized and treated with Target Retrieval Solution (DakoCytometion, Carpinteria, CA). For sequential staining the sections were stained as described above, except that the sections were treated with DAKO Biotin Blocking system, and p32, EMA, and CD68 were detected with biotinylated anti-mouse IgG and Vectastain ABC kit (Vector Laboratories Inc, Burlingame, CA). We used CD68 as a macrophage marker because an antibody that detects this antigen in paraffin-embedded sections was available. In the double immunohistochemistry labelling, the tumor array was first stained for CD68 as indicated above. A diaminobenzidine chromogen (DakoCytomation) (brown color) was used for antibody detection. Next, the slide was incubated with rabbit anti N-terminus p32 followed by the use of Envision Rabbit-HRP detection system (DakoCytomation) and Vector VIP substrate kit for peroxidase (Vector laboratories), which produces a purple reaction product. Nuclei were counterstained with Vector Methyl Green (Vector laboratories). The slides were scanned on a Scanscope CM-1 scanner and subsequently processed using ImageScope software (Aperio Technology) with color deconvolution and separation algorithms.
The clinical samples were blindly analyzed by a pathologist in the Institute’s core facility. The intensity of p32 staining was visually graded on a scale of 0 to 3. Parallel staining of an epithelial membrane antigen was used to identify tumor cells and an immuno-score (scale 0 to 300) was assigned to each malignant sample based on the percentage of tumor cells within the tissue and their intensity of p32 staining. The tissue arrays used contained a total of 41 normal tissues and 81 tumor samples, each one categorized for histology and malignancy grade. The type of tumors present in the array were: breast carcinomas (ductal, lobular and mucinous), endometroid adenocarcinomas, ovarial adenocarcinomas (serous and mucinous), colon adenocarcinomas (tubular, tubular-villous, and mucinous), adenocarcinomas of the stomach, pancreatic ductal carcinomas, kidney carcinomas, skin melanomas, hepatocellular carcinoma, carcinomas of the testis (seminoma and embryonal), prostate carcinomas, lung squamous cells carcinomas and glioblastomas.