Nerve Growth Factor in Breast Cancer Cells Promotes Axonal Growth and Expression of Calcitonin Gene-related Peptide in a Rat Model of Spinal Metastasis

Materials and Methods

ELISA of NGF. RL-1666 were cultured in McCoy’s 5A medium in a 10-cm dish. The medium was changed every two days. When CRL-1666 grew to 100% confluency, the protein in the cultured cells was extracted using a 3-min Total Protein Extraction Kit (101Bio, San Francisco, CA, USA) according to the manufacturer’s instructions.

The supernatant of the cultured CRL-1666 and the protein extracted from the CRL-1666 cells were assayed separately using a ChemiKine NGF Sandwich ELISA Kit (cat. No. CYT304; Chemicon, Temecula, CA, USA) according to the manufacturer’s instructions. In brief, an ELISA plate was coated with a sheep anti-mouse NGF polyclonal antibody and then nonspecific binding sites were blocked. The plates were washed four times and samples were added. The protein extract from the CRL-1666 cells and their supernatants were used as samples, each tested in duplicate. After the samples were added, a specific anti-NGF mouse monoclonal antibody was added to bind the captured NGF. After washing four times, the amount of specifically bound monoclonal antibody was detected using diluted donkey anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase. The plates were washed and incubated with chromogen before determining the absorbance of the well contents at 450 nm using a microplate reading spectrophotometer.

Cell culture. Cultures of the rat breast adenocarcinoma cell line CRL-1666 were maintained in McCoy’s 5A medium with 1% penicillin and streptomycin and 10% fetal bovine serum (FBS). The cells were incubated in a humidified incubator at 37°C under an atmosphere of 5% CO₂ 95% air.

Lumbar DRG were harvested from neonatal Wistar rats (P7-P9). DRG cells were collected by centrifugation (1,500 rpm, 5 min) twice. We added 1 ml 0.25% trypsin and 1 μg of DNase I to the DRG cells in Eppendorf tubes and incubated them for 15 min at 37°C. After incubation, the cell mixture was centrifuged (1,500 rpm, 5 min) once. The mixture was triturated, and 10% FBS was added to the mixture in the Eppendorf tube, which was then processed by centrifugation (3,000 rpm, 2 min). The procedure was repeated twice after which the medium was replaced with Dulbecco’s Modified Eagle’s Medium (DMEM). This mixture was centrifuged at 3,000 rpm for 2 min, and the procedure was repeated twice (11).

DRG cells were cultured in 2 groups in a chamber slide: (i) 2×10³ rat DRG cells were cultured in DMEM medium, (ii) 1×10² CRL-1666 cells and 2×10³ rat DRG cells were cocultured in DMEM. We added 10 μl of B-27 Supplement 50× (Gibco Invitrogen, Grand Island, NY, USA) in both groups. The cells were then cultured for 24 h in a humidified incubator at 37°C under an atmosphere of 5% CO₂ 95% air. We cultured DRG cells without CRL-1666 as a control.

Tuj-1 and CGRP immunocytochemistry. Following the culture procedure, the DRG cells were fixed in 4% paraformaldehyde for 15 min at room temperature and washed with phosphate buffered saline (PBS) three times for 5 min. DRG cells were immersed in 0.3% Triton X-100 (5% BSA + 10% Triton X-100) for 10 min and in 5% BSA for 30 min. For immunocytochemistry, the cells were labeled with an antibody to the neuronal marker β-tubulin (Tuj-1; mouse monoclonal antibody against neuronal class III β-tubulin; 1:800; Covance, Emeryville, CA, USA) and with rabbit antibodies to CGRP (1:1,000; Immunostar, Hudson, WI, USA) that were diluted with a blocking solution. After incubation with the Tuj-1 and CGRP specific antibodies for 20 h at 4°C, cells were double stained with species-specific antibodies conjugated to fluorescent dyes: Alexa 488-conjugated goat anti-mouse IgG (as a marker of Tuj-1 immunoreactivity, 1:1,000; Invitrogen, Eugene, OR, USA) and Alexa 594-conjugated goat anti-rabbit IgG (1:1,000; Invitrogen, Eugene, OR, USA). After each step, the fixed cells were rinsed three times in PBS. The cells were then observed using a fluorescence microscope.

Proportion of CGRP-ir cells, number of growing axons, and average axonal length. The proportion of the CGRP-ir DRG neurons to Tuj-1-ir neurons, and the number of axons growing from the CGRP and Tuj-1 double-stained DRG neurons were determined for both DRG cells cultured alone and those cocultured with CRL-1666. We determined the number of CGRP-ir DRG neurons and average length of axon growth from 100 CGRP-ir and Tuj-1-ir DRG neurons. All measurements were made using ImageJ (NIH, Bethesda, MD, USA).

A rat model of spinal metastasis and retrograde Fluoro-Gold labeling. All animal procedures and protocols were approved by the IRB of the authors’ affiliated Institution and were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (1996 revision).

We used a rat model of spinal metastasis reported by Mantha et al. (12). We used 36 female, 8-week-old Sprague Dawley (SD) rats weighing 200-250 g. They were anesthetized with a mixture of 0.15 mg/kg body weight (b.w.) medetomidine, 2.0 mg/kg b.w. midazolam, and 2.5 mg/kg b.w. butorphanol and treated aseptically throughout the surgery. We made a dorsal midline longitudinal incision to expose the L6 vertebra under a microscope, and then drilled a hole 1 mm left from the midline with an 18-gauge needle to a depth of 1.5 mm. Subsequently, in 18 rats (control group), FG was placed in the hole using a 26-gauge needle whose tip was filled with Fluoro-Gold crystals (FG; Fluorochrome, Denver, CO, USA). In 18 other rats, FG was placed in the hole and a tumor block (1 mm × 1 mm × 1 mm) implanted with microsurgical instruments (metastasis group). The tumor block had been excised from a donor rat with a subcutaneous tumor of CRL-1666 (11). After applying the FG or implanting the tumor and applying the FG, the hole was sealed with Bone Wax (Lukens Corp., Richmond, VA, USA) and both the fascia and skin were closed in rats of both groups.

On the 7^th day after applying the FG alone or together with the implanted tumor block, the rats were anesthetized with the mixture of 0.15 mg/kg body weight (b.w.) medetomidine, 2.0 mg/kg b.w. midazolam, and 2.5 mg/kg b.w. butorphanol, and perfused transcardially with 0.9% saline, followed by 500 ml of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). Bilateral DRG from L1 to L6 were resected and fixed by soaking in 4% paraformaldehyde in phosphate buffer overnight at 4°C. The fixed DRG were immersed in 20% sucrose in 0.01 M phosphate-buffered saline (PBS) for 20 h at 4°C for cryoprotection. Subsequently, each DRG was sliced at 20 μm thickness on a cryostat and sections mounted on poly-L-lysine-coated slides.

Immunohistochemistry for CGRP. The mounted sections were incubated in a solution (PBS containing 0.3% Triton X-100 and 3% skim milk) to block nonspecific binding sites for 90 min at room temperature. Next, the sections were processed for CGRP immunohistochemistry by incubating them with rabbit antibodies to CGRP (1:1,000; Immunostar) for 20 h at 4°C, followed by incubation with goat anti-rabbit Alexa 488-fluorescein-conjugated antibody (1:400; Molecular Probes). The fluorescent marker was observed using a fluorescence microscope (Olympus, Sinjuku, Tokyo, Japan). We counted the numbers of FG-labeled and CGRP-ir DRG cells in control and metastasis groups.

Statistical analyses. All results are expressed as the mean±standard error unless otherwise indicated. Differences in the number of CGRP-ir neurons, and that of axons growing from DRG neurons were determined using the Chi-squared test. Differences in axonal length were determined using Student t tests. Differences in the proportions of FG-labeled and CGRP-ir DRG neurons were determined using Mann–Whitney U-tests. p<0.05 was considered significant.