Tissue microarray analysis
Eighty seven grade 3 invasive breast ductal carcinomas and 6 non-tumoral mammary tissues were fixed in 10% paraformaldehide (PFA) and embedded in paraffin. Two representative tissue cores (1 mm of diameter) of each one were included in a tissue microarray. The main clinical pathology and molecular features of this series had been previously reported . Tissue sections were subjected to a heat-induced antigen retrieval step prior to exposure to the primary antibody. Anti-CB1 receptor antibody was generously donated by Dr. Ken Mackie, Indiana University, Indiana, and anti-CB2 receptor antibody was from Affinity Bioreagents/Thermo Fisher Scientific, Rockford, Illinois. ErbB2 expression was evaluated using a HercepTest (Dako, Carpenteria, CA) according to manufacturer's instructions. Immunodetection was performed using the LSAB method (DAKO) with DAB as the chromogen. In negative controls, the primary antibody was omitted or replaced with an irrelevant antibody. Cases were reviewed by two independent pathologists (J.P. and G.M-B) and were scored as positive for cannabinoid receptors when more than 25% of the neoplastic cell showed intense immunostaining for the corresponding antibody. ErbB2-staining was scored according to HercepTest manufacturer's guidelines: scores 0 and 1+ were considered as negative, and 2+ and 3+ as positive for ErbB2 overexpression.
Animals and treatments
All procedures involving animals were performed with the approval of the Complutense University Animal Experimentation Committee according to the European official regulations. FVB/N-Tg(MMTVneu)202 Mul/J mice (more commonly designated as MMTV-neu mice) were obtained from The Jackson Laboratory (Bar Harbor, Maine). Females were palpated twice weekly for mammary gland nodules and cannabinoid treatment was started when the first tumor in each animal was detected. Δ9-Tetrahydrocannabinol (THC, The Health Concept, Richelbach, Germany) and JWH-133 (kindly donated by John W. Huffman, Clemson University, South Carolina) were prepared in DMSO (0.2 mg/μL and 0.02 mg/μL, respectively) and diluted in PBS supplemented with 5% BSA (100 μL/dose for tumors ≤ 1000 mm3 and 200 μl/dose for tumors >1000 mm3). Cannabinoid peritumoral treatment (0.5 mg THC or 0.05 mg JWH-133/animal/day, twice a week) was maintained for 90 days, and only the first tumor in each animal was treated. Tumors were routinely measured during this period with external caliper, and volume was calculated as (4π/3) × (width/2)2 × (length/2). At the end of the treatment, animals were sacrificed and tumors and organs were collected. Tumors were divided in four portions for 1) preparation of tissue sections for immunofluorescent staining [frozen in Tissue-Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands)], 2) preparation of tissue sections for hematoxylin-eosin staining (fixed in buffered 4% PFA), 3) protein extraction (snap frozen) and 4) RNA isolation (snap frozen), and were stored at -80°C until analysis (except PFA-fixed tumor fractions, that were kept at room temperature). Brain, spleen, liver, kidneys and lungs were fixed in PFA. For xenograft experiments, subcutaneous tumors were induced in 6 week-old athymic female mice (Harlan Interfauna Iberica, Barcelona, Spain) by subcutaneous injection of 5 × 105 N202.1A cells. When tumors reached ca. 100 mm3, they were treated with THC (0.5 mg/animal/day), JWH-133 (50 μg/animal/day), SR144528 (50 μg/animal/day), a combination of cannabinoid and SR144528 or vehicle for 3 weeks, 3 times a week, measured, and processed as described above. For Akt-related experiments, half of the animals were injected with N202.1A cells stably expressing myristoylated Akt (N202.1A-pBABE-myr-Akt), and the other half with N202.1A cells stably expressing the corresponding empty vector (N202.1A-pBABE). Tumors were treated with THC, JWH-133 or vehicle and processed as described for N202.1A xenografts.
Collected organs were visually analyzed for macroscopic metastases. Microscopic metastases were determined by histological analysis of PFA-fixed paraffin-embedded hematoxylin-eosin stained sections. Radiographs were taken to evaluate the presence of bone metastases using a conventional X-ray equipment (Diagnost 93, Philips Medical Systems, Eindhoven, The Netherlands), and mammography cassette (Kodak MIN-R 2000 screen cassette) and film (Kodak MIN-R S film) (Eastman Kodak Company, Rochester, New York).
Cell culture and viability
N202.1A cells were kindly given by Dr. Vincenzo Bronte (Istituto Oncologico Veneto, Padova, Italy). This cell line was established from a MMTV-neu-derived tumor . BT474, MDA-MB-231, MCF-7 and SkBr3 human breast cancer cells, Jurkat human leukemic cells, and U373 human glioblastoma cells were from ATCC-LGC (Barcelona, Spain). All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were transferred to a low (0.5%)-FBS medium immediately before cannabinoid challenge. Cell viability was determined by the 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide thiazol blue test (Sigma, St. Louis, Missouri) according to manufacturer's instructions.
Plasmids, transfections and infections
Stable expression of myr-Akt was achieved by retroviral infection. N202.1A cells were transduced for 4 h with supernatants obtained from Phoenix ecotropic cells previously transfected with a retroviral vector carrying HA-tagged myr-Akt (kindly provided by Dr. Pier P. Pandolfi, Harvard University, Boston, Massachusetts) or the corresponding empty construction (pBABE). Infected cells were selected with puromycin.
Tissue-tek embedded tumor sections were fixed in PFA and incubated with anti-CB1 receptor, anti-CB2 receptor, anti-CD31 (Pharmingen/BD Biosciences, San Jose, California), anti-CD45 (Pharmingen/BD Biosciences), anti-Ki67 (Neomarkers/Lab Vision, Fremont, California) or anti-cleaved-caspase 3 (Cell Signaling Technology, Danvers, Massachusetts) antibodies. Secondary anti-rabbit antibodies AlexaFluor 594 and AlexaFluor 488 were from Invitrogen (Carlsbad, California). Cell nuclei were stained with Hoescht 33342 (Invitrogen). Fluorescence images were acquired using Metamorph Premier Offline software (Molecular Devices, Sunnyvale, California). Blood vessel size was calculated with ImageJ software.
Real-time quantitative PCR (RTQ-PCR) and reverse-transcriptase PCR (RT-PCR)
RNA was isolated with Trizol Reagent (Invitrogen), including a DNase digestion step, with the Real Star Kit (Durviz, Valencia, Spain), and cDNA was obtained with Transcriptor Reverse Transcriptase (Roche Applied Science, Penzberg, Germany). The primers used for RTQ-PCR amplification were: mouse CB1 receptor, sense 5'-GGGCAAATTTCCTTGTAGCA-3', antisense 5'-GGCTCAACGTGACTGAGAAA-3'; mouse CB2 receptor, sense 5'-ATTCAGGAGATCTGTTAAGACAAGG-3', antisense 5'-GACATCTATGAAGTTGAGGCAGTG-3'; mouse MMP2, sense 5'- GCGCTTTTCTCGAATCCAT-3', antisense 5'-GGGTATCCATCTCCATGCTC-3'; mouse MMP9, sense 5'-ACGACATAGACGGCATCCA-3', antisense 5'- GCTGTGGTTCAGTTGTGGTG-3'; rat ErbB2 (neu), sense 5'-GCTCAGAGACCTGCTTTGGA-3', antisense 5'-AGGAGGACGAGTCCTTGTAGTG-3'; mouse ErbB2, sense 5'-AACAGCTCGGAGACCTGCTA-3', antisense 5'-GTAGTGGGCACAAGCCTCA-3'. Probes were from the Universal Probe Library (Roche Applied Science). Multispecies 18S RNA was used as reference (sense 5'- GCTCTAGAATTACCACAGTTATCCAA-3', antisense 5'- AAATCAGTTATGGTTCCTTTGGTC-3'). The primers use for RT-PCR were: mouse MMP2, sense 5'- TCTGCGATGAGCTTAGGGAAAC-3', antisense 5'-GACATACATCTTTGCAGGAGACAAG-3'; mouse MMP9, sense 5'-GGACGACGTGGGCTACGT-3', antisense 5'- CACGGTTGAAGCAAAGAAGGA-3'. GAPDH was used as reference (sense 5'-GGGAAGCTCACTGGCATGGCCTTCC-3', antisense 5'-CATGTGGGCCATGAGGTCCACCAC-3').
Western blot analysis
Cell lysates from tumors and cell lines were subjected to SDS-PAGE, and proteins transferred onto polyvinylidene fluoride membranes. Blots were incubated with the following antibodiess: anti-CB1 receptor, anti-CB2 receptor (Affinity Bioreagents), anti-MMP9 (Chemicon International INC, Temecula, California), anti-ErbB2 (Santa Cruz Biotechnology, Santa Cruz, California), anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-S6 ribosomal protein (Cell Signaling) and anti-α-tubulin (Sigma). Luminograms were obtained with the Amersham Enhanced Chemiluminescence Detection Kit (GE Healthcare, Uppsala, Sweden) and densitometric analysis was performed with Quantity One software (Bio-Rad).
MMP activity assay
MMP2 (Gelatinase A) and MMP9 (Gelatinase B) activities were determined by gelatin zymography. Briefly, SDS-PAGE were run in the presence of 0.1% gelatin, washed with a 2.5% Triton X-100 containing buffer, and incubated overnight at 37°C in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.1% Triton X100. Gels were then stained with Coomasie Blue and digested bands quantified by densitometric analysis as described above.
ANOVA with a post hoc analysis by the Student-Newman-Keuls' test was routinely used. For the analysis of metastases and the number of tumors per animal, a Pearson Х2 test was used. To determine the correlation between immunohistochemical (CB1 and CB2 expression) and clinical pathology (ErbB2) data, the Х2 test with Yates correction, or Fisher's exact test, was used. The SPSS for Windows program (SPSS, Inc., Chicago, IL, version 17.0) was used for this analysis. All P-values were two-sided. Unless otherwise stated, data are expressed as mean ± s.e.m.