Reagents
HGF was from R & D (Minneapolis, MN). 5'-Iodoresiniferatoxin (I-RTX), doxorubicin, crizotinib and SB366791 were from Tocris (R & D, Minneapolis, MN); rabbit monoclonal antibodies to, ERK1/2, CREB, pERK1/2, pCREB, p-Met, CAMKIIαand mouse monoclonal antibodies to c-Met, HRP-conjugated horse anti-mouse IgG and goat anti-rabbit IgG antibody were from Cell Signaling Technology (Danvers, MA); bafilomycin A1, rabbit polyclonal antibodies to TRPV1 and HGF, the goat polyclonal antibody against CGRP, and HRP-conjugated mouse monoclonal antibody against β-actin were from Abcam (Cambridge, UK); Alexa fluor 488-conjugated donkey anti-rabbit IgG and Alexa fluor 680-conjugated donkey anti-goat IgG and rhodamine red-X (RRX)-conjugated donkey anti-goat IgG antibody were from Jackson Laboratory (Bar Harbor, ME); Rhodamine phalloidin was from Life Technologies (Carlsbad, CA); and HRP-conjugated goat anti-chicken antibody was from Santa Cruz Biotechnology (Dallas, TX). Anti-a3V-ATPase antibody was a kind gift from Dr. Sun-Wada, Doshisha Women's College, Japan.
BC Cells
Mouse BC cells 4T1 have been maintained in our laboratory and mouse BC cells E0771 were purchased from CH3 Biosystems (#94A001, Amherst, NY). The 4T1 and E0771 cells and human BC cells MCF-7 and MDA-MB-231 were cultured in DMEM (Gibco, Carlsbad, CA) with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cell lines were analyzed and went through authentication by targeted genomic and RNA sequencing.
DRG SNs
The lumbar (L3-L5) DRG SNs were isolated as described 15. Primary rat DRG neuronal cells (Lonza, R-DRG-505, Alpharetta, GA) were cultured in primary neuron growth medium (Lonza, #CC-4461) with 2% FBS, L-glutamine, gentamycin sulfate-amphotericin (GA-1000, Lonza, #CC-4083) and neural survival factor-1 (NSF-1, Lonza, #CC-4323) as instructed. Immortalized rat DRG neuronal cells, 50B11, were a generous gift from Dr. Hoke, Johns Hopkins University and cultured in Neurobasal medium (Gibco) supplemented with 10% FBS, 1 x B27 supplement (Gibco), 0.2% glucose and 0.5 mM glutamine (neuron growth medium) 30. The rat DRG/mouse neuroblastoma hybrid cells, F11, were cultured as described 53.
Neurite outgrowth
Primary DRG neuronal cells (1 x 103, lower chamber) were co-cultured with 4T1 BC cells (5 x 103, upper chamber) in transwells (Corning, Corning, NY) for 72 h. Neurite outgrowth in lower chambers was visualized with calcein AM (1 µM, Life Technologies) and the length of outgrowing neurites was quantified under a fluorescent microscope using Neuron J.
Microfluidic culture platforms
Neurite outgrowth from F11 cells in response to extracellular microenvironment of 4T1 BC cells was determined using microfluidic culture platforms 54. Suspension of F11 cells (105 cells/10 µl) was introduced into the left channels of the microfluidic chambers (AXIS™, Millipore, Billerica, MA), and the right channels were filled with the neuron growth medium. After 12 h, 4T1 BC cells (106 cells/ml) were plated into the right channels and cultured for further 48 h. Non-adhered cells and debris were then washed out, and the chambers were labeled with calcein AM (1µM) for 10min. The number of F11 cells extending neurites longer than 50 µm toward 4T1 BC cells were counted under a fluorescent microscope using Neuron J.
Wound healing assay
Assay was performed as we described 55. The 4T1 BC cells were grown to confluence in 6-well tissue culture dishes, and a single scratch was made in the confluent monolayer using a sterile 200-µl pipette tip. The monolayer was washed with PBS, and then complete medium containing indicated inhibitors or vehicle was added. The number of cells that had migrated over the margins of the wound was counted after 12 h of treatment under a phase contrast microscope.
c-Met Knockdown
4T1 cells were infected with 20 µl of control (sc-108080) or c-Met (sc-35924-V) shRNA lentiviral particles (Santa Cruz) in the presence of 5 µg/ml polybrene for 24 h, and cultured in DMEM with 5% FBS for 7 days in the presence of 4 µg/ml puromycin (Gibco) to select cells stably expressing the shRNAs.
Microarray of DRGs
4-week-old female Spraque-Dawley (SD) rats weighting 250 to 300 g were used, respectively. The rats were maintained in plastic cages in a 12-hour light/12-hour dark cycle at a temperature of 21℃.
Rat pulmonary carcinoma cells (IP-B12) which show osteolytic tumor growth in bone marrow established by Dr Nakanishi40. IP-B12 (1x105/10µl) were inoculated into the bone marrow cavity of the right tibiae in rat under general anesthesia. DRGs were collected from rat followed by the isolation of RNA using NucleoSpin RNA Plus after 14days from inoculation. DRGs of left tibia were used as control. Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 100 ng total RNA. Following fragmentation, 12.5 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Rat Genome 230 2.0 Array. GeneChips were scanned using the GeneChip Scanner 3000 7G. The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings.
Intratibial injection
Mouse 4T1 or E0771 BC cells (2x105/10µl) or PBS (sham) were inoculated into the bone marrow cavity of the right tibiae in 4- to 6-week-old female BALB/c (Harlan Laboratories, Indianapolis, IN), or C57BL/6J (E0771 BC) (Jackson Laboratory, Bar Harbor, ME), respectively, under general anesthesia with ketamine (Ketaset; 90–150 mg/kg, ip) and xylazine (AnaSed; 5–10 mg/kg, ip). In some experiments, mice were treated with or without antagonists, inhibitors and anti-cancer drugs as indicated in the figure legends.
Evaluation of BCIBP
BCIBP was evaluated by hind-paw mechanical allodynia of 4T1 BC mice by von Frey test using the Dynamic Plantar Aesthesiometer (Ugo Basile, Gemonio, VA, Italy), as we described 11,15. The test determines the force by which rodents withdraw their hind-paw following poking by a single von Frey filament in response to automated increasing force. Rodents with pain exhibit paw withdrawal with lighter force than those without pain.
Immunoblotting
Cells or tissues were lysed in lysis buffer (Invitrogen, Carlsbad, CA), electrophoresed on a 10% SDS-PAGE, and blotted onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA). After blocking, the membranes were incubated with primary antibodies to HGF (1:1,000, Abcam, #ab83760), pc-Met (1:1,000, Cell Signaling, #3077), pERK1/2 (1:1,000, Cell Signaling, #4370), or pCREB (1:1,000, Cell Signaling, #9198) overnight at 4°C, and then with horse radish peroxidase (HRP)-conjugated secondary antibodies for 1h. Protein bands were visualized with a SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific, Carlsbad, CA) 15.
Histology and immunohistochemistry (IHC)
Bones were fixed in 10% neutral buffered formalin for 48 h, decalcified in 10% EDTA for two weeks and stained with hematoxylin and eosin (HE). Lungs were fixed with Bouin’s solution and the number of metastatic foci was macroscopically counted.
For IHC, bones were fixed, decalcified, embedded in OCT compound and sectioned at 10µm thickness using a cryostat. After blocking, sections were incubated with the primary antibodies overnight at 4°C, and secondary antibodies for 60 min, mounted with coverslips in VECTASHIELD anti-fade mounting medium (Vector Laboraories Inc, Burlingame, CA) and observed under TCS SP8 confocal laser scanning microscope (Leica Microsystems, Nussloch, Germany)
Tumors and DRGs were sectioned at 10µm thickness, and incubated with primary antibodies to CD-31 (1:50, Abcam, #ab28364), DeadEnd™ Colorimetric TUNEL System (Promega, #G7360, Madison, WI), Ki67 (1:400, Cell Signaling, #9129), PGP9.5 (1:200, Abcam, #ab8189), CGRP (1:200 Abcam, #ab36001), TRPV1 (1:1,000 Abcam, #ab31895), peripherin (1:1,000 Abcam, #ab4666), or HGF (1:200, Abcam, #ab83760) overnight at 4 ̊C, and a secondly fluorescent-labeled antibody (1:100) for 60 min or a streptavidin-biotin complex, EnVision HRP (Dako, Carpinteria, CA), for 60 min and visualized using a 3,3-diaminobenzidine (DAB) substrate-chromogen solution (Dako Cytomation Liquid DAB Substrate Chromogen System).
Statistics
Data were analyzed using a Mann-Whitney or an unpaired Student’s t test for comparisons of two groups. A one- or two-way analysis of variance (ANOVA), respectively, and a Dunn’s, Dunnet’s and Tukey’s test for the analysis of multiple group comparison, using PRISM statistical software (ver. 7.0). Results are expressed as the mean ± standard deviation (SD). P < 0.05 was considered significant.
Study Approval
All animal studies were approved by the Institutional Animal Care and Use Committee at Indiana University School of Medicine (Protocol #: 10553) and conducted according to. All study methods were performed in accordance with the ARRIVE guidelines, American Veterinary Medical Association Guidelines and regulations of this organization as well as the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan.