Clinical tissue samples
In this study, clinical tissue samples were surgical resection specimens obtained from Huadong Hospital Affiliated to Fudan University. Written informed consent was obtained from all patients participating in this study. Clinical diagnosis of all patients were confirmed by pathological examination, laboratory tests and clinical manifestation according to the international association for the study of lung cancer (IASLC) diagnosis criteria.
Cell lines and culture
The human lung adenocarcinoma cells A549 and Large-cell lung cancer cell line H460 (ATCC; Manassas, VA, USA) were purchased from Shanghai cell bank of Chinese academy of sciences. These cell lines were cultured in Dulbecco's modified Eagle's medium (Thermo Scientific Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Hyclone). Human umbilical vein endothelial cells (HUVECs) and its completed medium were obtained from AllCells biological technology (Shanghai) co., LTD. And all these cell lines were maintained in a 37℃ incubator with 5% CO2.
Lentivirus-mediated overexpression of CXCR7 and transduction
psPAX2 (gag-pol expressor) and pMD2G (VSV-G expressor) were used as the lentiviral packing vector, and pRRL.EF1α plasmid with human CXCR7(NC_000002.12) were synthesized by Genscript biological technology (Nanjing, China) co., LTD. Lentiviruses were produced by co-transfection of HEK293T cells with psPAX2, pMD2G and pRRL.EF1α-hCXCR7 vectors. For overexpression, A549 cells were transduced with the collected lentiviruses at 1:5 and 1:20 two different concentrations in the presence of 8ug/ml Polybrene (Sigma-Aldrich, St Louis, MO, USA). In order to further enrich CXCR7 expressing A549 cells, lentivirus transducted cells were digested with 0.25% EDTA-Trypsin (Sigma-Aldrich) and washed with FACS buffer (PBS+2%FBS) for two times. Subsequently, cells were then stained with 2μl PE-conjugated antiCXCR7 antibodies (Clone#11G8, R&D Systems, Minneapolis, MN) on ice for 30 min and washed with FACS buffer for two times. Finally, cells were resuspended in serum-free DMEM medium and filtered through a filter of 40μm pore size (BD Biosciences). All procedures were underwent in a sterile environment. FACS sorting was performed (FACS Aria, BD Biosciences, San Jose, CA) and analyzed on a FACS machine (Calibur, BD, Franklin Lakes, NJ).
Wound healing assay
Cells were seeded in ibidi culture-insert (7000 cells per well) in 70μl DMEM (serum free) with CXCL12 (100 ng/ml, R&D Systems) in the presence or absence of CXCR7 inhibitor CCX771(0.5μM) (Chemo Centryx, Mountain View, CA), and pre-incubated overnight in 37℃ 5%CO2, and then gently removed the culture-insert by using sterile tweezers, filled the used wells with free medium, and photograph it in 0,12 as well as 24 hours. and compared the width in different group and timepoint.
Transwell assay inserts (24mm diameter, 8μm pores, Costar, Corning, NY, USA) were placed in 24-well plates. Cells (2 × 104) were resuspended in 1% FBS-DMEM medium and placed in the upper chamber, whereas 20% FBS-DMEM alone or containing 100ng/ml CXCL12 (R&D Systems) or 0.5μM CCX771 (Chemo Centryx) was added to the lower chamber. After incubation for 12h, the inserts were fixed by methanol and DAPI. Non-invading cells were mechanically wiped using cotton swabs. The invading cells were counted with Zeiss 710 confocal microscope (×100 magnification).
Polydimethylsiloxane (PDMS) was sealed on PDL-coated glass bottom dishes (35 mm dish with 20 mm bottom well), and microchannels were formed between the PDMS mold and well. The mixture of BSA and CXCL12 or BSA alone was added to one end of the microchannels, and vacuum was applied to the other end of the microchannels to ensure BSA/CXCL12 or BSA filled in all the microchannels. PDMS molds were removed after drying overnight. Cells were then fixed using 4% paraformaldehyde (PFA), and permeabilized with 0.4% Triton-X in PBS on dishes. Cells were incubated with primary antibodies overnight, and Cultures were washed and then incubated with phalloidin and corresponding secondary antibodies for 1h at room temperature. Nuclear DNA was labeled with DAPI for 2min after the secondary antibody at room temperature. Cultures were washed with PBS thrice then images were taken by a Zeiss confocal microscope.
Certain number of cells were collected and total cellular proteins were extracted in M-per(R) Mammalian Protein Extraction Reagent (Thermo Scientific). Cellular proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to poly-vinylidene fluoride membranes (Millipore; Bedford, MA) for immunoblotting. After blocking with 5% skim milk (BD Biosciences), the membrane was probed with the primary antibodies rabbit monoclonal to GPCR RDC1 (abcam, Cambridge, MA.ab138509,1:1000 diluted) overnight at 4℃. After washing, membranes were labeled with the horseradish peroxidase conjugated goat anti-rabbit secondary antibodies (R&D Systems,1:4000) for 1h at room temperature. The immunoblots were colored by using an ECL Kit (Millipore). Anti-actin was used for loading control (Sigma-Aldrich). Blots were detected and imaged by Image Lab (Bio-Rad).
For cell surface chemokine receptor detection, approximate 1×105 cells were harvested ,washed and suspended in FACS buffer (PBS+2%FBS) in 50μl volume. Cells were then stained with 2μl Phycoerythrin (PE)-conjugated anti-CXCR7 antibodies (R&D Systems) or APC-conjugated anti-CXCR4 antibody (Clone#12G5, BD Systems) for 30min in a dark place on ice, and mouse anti-human IgG1 served as isotype control. Stainned cells were washed twice with FACS buffer, resuspended in FACS buffer in a proper volume (250~350μl) for analysis. Ten thousands cells from each sample were evaluated for fluorescence detection using FACS machine (Calibur, BD, Franklin Lakes, NJ), and the data were analyzed with FlowJo 7.6 software.
Xenograft model and Bioluminescence imaging
The SCID/Beige (Severe Combined Immune-deficiency/Beige, 6-8 weeks of age,
female) mice were purchased from SLRC Laboratory Animal (Shanghai, China) co., LTD. A total of 1.8×106 A549-GFPLuc or A549-GFPLuc-CXCR7 lung adenocarcinoma cells were injected intravenously via a tail vein in 200μl of serum-free DMEM medium. Bioluminescence imaging was used to monitor cell proliferation and metastasis every week. For bioluminescence imaging, mice were injected intraperitoneally with D-luciferin at a dose of 150mg/kg. After injection of D-luciferin, the mice were anesthetized with isoflurane/oxygen and placed on the imaging platform. The bioluminescence signals were monitored using an IVIS-200 bio-photonic imager (LB983, Bertold Technologies) within 20 minutes. The data were analyzed using the “IndiGO” software and total photon flux emission in the regions of interest were quantified.
Immunohistochemistry and Immunofluorescence
Lung tumor samples were dissected from mice, fixed in 4% paraformaldehyde, and embedded in paraffin for immunohistochemistry, and sectioned at 4μm intervals. Standard IHC techniques were performed according to the manufacturer’s recommendations. The slides were deparaffinized in xylene and rehydrated through a series of dilutions of alcohol. The slides were then placed in EDTA epitope retrieval buffer (pH 8.0) in a microwave for two 8min sessions. After blocking with 3% H2O2 and 3% BSA, the treated slides were then incubated with the primary antibodies against CXCR7 (gift of ChemoCentryx,1:200) for overnight at 4℃ and horseradish peroxidase conjugated goat anti-mouse secondary antibodies (Dako North America Inc., Carpinteria, CA, USA) for 1h at room temperature. DAB Color Development Kit were used for detection of the bound antibodies. Furthermore, Nuclei were counterstained with hematoxylin, washed, dehydrated and mounted. The stained cells were observed by using a bright-field microscope in each experimental group, imaged and analyzed. Nuclei were stained in blue and the CXCR7 expression positive cells in brown yellow. For immunofluorescence analysis, lung samples were embedded in OCT reagent (Sakura) and sectioned at 10μm intervals. The frozen sections were fixed with cold acetone for 10min and rinsed with PBS（PH7.4）for three 5min sessions. Finally, nuclei were counterstained using DAPI. Slides were mounted using resistance of fluorescence quenching reagent and then observed by inverted fluorescence microscope (Nikon Eclipse Ti-SR).
Data were shown as mean values with SEM. Statistically significant differences were analyzed by Graphpad Prism software (Avenida de la Playa, La Jolla, CA, USA). Wound healing assay and Transwell assay experiments were performed 3 times each, and representative data were presented and determined by cell migration distance. Photon flux values of each group were compared by using the t-test. For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001.