Animals
Two-month-old virgin female BALB/c mice (Animal Facility, Instituto de Biología y Medicina Experimental) were housed in groups of four per cage. Transgenic mice expressing enhanced GFP under the direction of the human ubiquitin C promoter were purchased from the Jackson Laboratories, Bar Harbor, Maine. These mice express Green Fluorescent Protein (GFP) in all tissues examined. The animals were in an air-conditioned room at 20 ± 2°C with a 12-hour light/dark cycle and had access to food and water ad libitum. Animal care and manipulation were in agreement with institutional guidelines and the Guide for the Care and Use of Laboratory Animals [14]. Protocols are approved by the Institutional Bioethical Committee.
Tumors
C4-HD, a transplantable ductal mammary tumor, was originally induced by the continuous administration of MPA to a BALB/c female mouse [9, 15] and was subsequently maintained by serial subcutaneous (sc) transplantations into syngeneic MPA-treated female mice. This HD tumor expresses high levels of estrogen (ER) and progesterone receptors (PR) [16] and shows a nearly diploid karyotype [17]. The C4-HI variant arose from the C4-HD tumors when it started to grow in the absence of MPA. C4-HI cells also exhibit high ER and PR levels but have acquired an aneuploid karyotype [17, 18].
Primary cultures
Primary cultures were prepared from C4-HI and C4-HD tumors as described previously [16, 19]. Briefly, tumors were aseptically removed, minced, washed with DMEM/F12 medium (Dulbecco's modified Eagle's medium: Ham's F12, 1:1, without phenol red, Sigma Chem. Co.), suspended in 5 ml of enzymatic solution [2.5 mg/ml trypsin (Life Technologies Inc.), 5 mg/ml albumin (Life Technologies Inc.), and 850 U/ml collagenase type II (Life Technologies Inc.) in phosphate buffered saline], and incubated at 37°C for 20 minutes with continuous stirring. The liquid phase of the suspension was then removed, and the undigested tissue was incubated for an additional 20 minutes with fresh enzymatic solution. Enzyme action was interrupted by adding a washing medium with 10% fetal calf serum (FCS, BioSer, Buenos Aires, Argentina). Epithelial (EPI) and fibroblastic cells (CAF) were isolated by resuspending the digested material in 20 ml of medium plus 10% FCS and allowing it to settle for 20 minutes. The upper 7 ml, containing single cells, were seeded into plates and the medium was changed after two hours. This time period is enough for the CAF to settle and not enough for the EPI to attach to the plastic plates. The fraction corresponding to the lower 5 ml, containing the sedimented cells, was resuspended in another 20 ml of washing medium (plus 5% FCS) and allowed to settle for 20 minutes. The upper 15 ml were discarded and the procedure was repeated 10 times, or until no single cells were observed in the upper fraction. Instead of plating the purified EPI, they were directly used for in vivo experiments. CAF, on the other hand, were obtained from primary cultures that were grown for one week prior to inoculation.
Immunofluorescence
Purified EPI-HI, CAF-HI or EPI-HI+CAF-HI were seeded onto chamber slides. Slides were fixed with 100% ethanol for 30 minutes at -20°C, washed and treated with 0.25% Triton X-100 for 20 minutes. Slides were rinsed and blocked with 5% FCS for one hour at room temperature. Incubation with cytokeratin (CK; polyclonal rabbit antibody Z0622, 1:250 dilution, DakoCytomation, Carpinteria, CA) or smooth muscle actin (SMA; monoclonal mouse antibody Sigma Aldrich) was performed overnight at 4°C. After rinsing in PBS, slides were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit (for CK) or anti-mouse (for SMA) IgG at 1:100 dilution (Vector Laboratories, Burlingame, CA). Nuclei were counterstained with propidium iodide (PI). The signal was viewed in a Nikon Eclipse E800 confocal microscope.
In vivo experiments
Tumor growth
In the first set of experiments, untreated female BALB/c mice were sc inoculated with 1.5 × 104 EPI-HD or EPI-HI alone or together with the same number of CAF-HI (n = 6 mice/group). Isolated CAF-HI were also inoculated to rule out the possibility that contaminating EPI-HI cells could account for the observed effects. In the second set of experiments, 1.5 × 104 EPI-HI or EPI-HI+CAF-HI were sc inoculated in untreated female BALB/c mice and the animals were euthanized at 2, 7, 12 and 17 days post-inocula (n = 3/group). These cell injections were mixed with trypan blue prior to inoculation to localize the area of inoculation. After removal, isografts were immediately fixed in 10% formalin, embedded in paraffin and prepared for histological evaluation.
Tracking inoculated CAF-HI
Female CAF-HI in male BALB/c mice
EPI-HI (1.5 × 104) alone or together with an equal number of CAF-HI were sc inoculated into female (n = 4-6/group) or male untreated BALB/c mice (n = 4/group). Animals were followed closely, and tumor width and length were measured 3 times a week using a Vernier Caliper. Animals were euthanized after one month. Some of the tumors were frozen for in situ hybridization studies.
GFP-labeled CAF-HI in BALB/c mice
EPI-HI (1.5 × 104) alone or together with an equal amount of CAF-HI-GFP were sc inoculated in BALB/c mice. Trypan blue was used to visualize the site of injection.
Unlabeled CAF-HI in BALB/c-GFP mice
EPI-HI (1.5 × 104) alone or together with an equal amount of unlabeled CAF-HI were sc inoculated in BALB/c-GFP. Trypan blue was used to visualize the site of injection.
Tumor transplant growing in BALB/c-GFP mice transplanted in BALB/c mice
EPI-HI were sc inoculated into BALB/c-GFP mice. Once the tumor grew a small piece was transplanted into BALB/c mice.
For all in vivo experiments performed with BALB/c-GFP mice or inoculated CAF-HI-GFP, before the tumors were excised, the animals were perfused with a cold saline solution (0.9% NaCl) followed by 4% paraformaldehyde (PFA). The inoculated area was excised at different time points (4, 7, 13 and 28 days), kept in cold 4% PFA overnight and then transferred to 20% sucrose for another 24 hours. Tissues were embedded in OCT (Tissue-Tek O.C.T Compound), frozen sections were cut and the endogenous fluorescence was analyzed by confocal microscopy. Nuclei were counterstained with PI. Primary cultures followed by SMA immunostaining were performed with some of these tumors.
Mast and PMN cell quantification
BALB/c mice (n = 3-10/group) were sc inoculated with saline, CAF-HI (1.5 × 104), EPI-HI (1.5 × 104), or EPI-HI+CAF-HI (1.5 × 104 + 1.5 × 104). Trypan blue was used to visualize the site of injection. The animals were euthanized at 7 and 12 days post inocula; tumors were then fixed with 10% formalin and embedded in paraffin.
Mast cells-toluidine blue method
Tumor sections were deparaffinized and rehydrated, stained for 30 minutes in aqueous toluidine blue (0.1% pH 1), washed and nuclei were counterstained with methyl green. Tissues were dehydrated and mounted on Permount and observed under a microscope. Mast cells were identified by their characteristic metachromasia. Total mast cells within the inoculated area were counted using a Nikon Eclipse E200 microscope, magnification 400×. The tumor area was measured using "Image J" Software and the numbers of mast cells were calculated per area.
Polymorphonuclear cells quantification
Hematoxylin and eosin (H&E) staining was observed under a Nikon Eclipse E200 microscope. PMN cells were identified by standard morphological criteria (Magnification: 1000×). All fields within the inoculated area were counted and PMN numbers of cells were referred per field.
Fluorescence in situ hybridization (FISH) and immunofluorescence
Frozen tissue sections of the tumors were fixed in a cold solution of methanol:acetic acid (3:1) for one hour. The tissue sections were denatured in 70% formamide/2× standard saline-citrate. Mouse paint biotinylated DNA probes (Cambio, Cambridge, UK) for chromosomes X and Y were denatured and preannealed. The hybridization was performed overnight at 37°C. After hybridization, the slides were washed and the probes were detected with Avidin-TexasRed (Vector Laboratories, Burlingame, CA). Immunofluorescence using a CK antibody (polyclonal rabbit antibody Z0622, 1:250 dilution, DakoCytomation, Carpinteria, CA) was performed on the same slides. A secondary anti-rabbit-FITC coupled antibody was used. Nuclei were counterstained with DAPI. The signal was viewed in a Nikon Eclipse E800 confocal microscope.
Angiogenesis assay in vivo
Each BALB/c mouse was implanted intradermally on the ventral surface with the cells. Saline, CAF-HI (1.5 × 104), EPI-HI (1.5 × 104) or EPI-HI+CAF-HI (1.5 × 104 + 1.5 × 104) were injected in a volume of 50 μl together with one drop of 0.4% trypan blue to visualize the sites of injection. After 5 days, the mice were sacrificed, the skin carefully separated, and photographs captured under a Trinocular Zoom Stereomicroscope (Leica). The number of vessels was scored using a grid, subdivided into squares of 0.16 mm2, and superimposed onto the photograph of the injection site.
Statistical Analysis
Tumor growth curves were studied using regression analysis and the slopes were compared using ANOVA followed by parallelism analysis. Unpaired t-tests were used as needed. Values are considered significant if p < 0.05.