Materials and reagents
AMD3100, a specific antagonist of Stromal cell-derived factor-1α (SDF-1α)’s receptor CXCR4, was purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine® 2000 was from Invitrogen (Carlsbad, CA, USA). Lipophilic fluorescence dye-CM DiI was from Molecular Probes (Carlsbad, CA, USA).
The bone marrow human MSCs (hMSCs) were isolated from healthy bone marrow transplantation donors by density-gradient centrifugation with Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK). Written informed consent was obtained under the approval of the Combined Internal Review Board (Ethical Committee) of the University of Hong Kong and The Hong Kong West Cluster of Hospital Authority. The immunophenotype and differentiation characteristics of MSCs were clarified by surface marker definition and differentiation assays. The homogenous hTertMSCs, an immortalized hMSCs cell line with human telomerase reverse transcriptase gene inserted, was a gift from Prof. D. Campana (St Jude Children’s Research Hospital, Memphis, TN, USA) . All hMSCs were cultured in vitro with Dulbecco’s Modified Eagles Medium-low glucose (DMEM-LG; GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin, 100mg/mL streptomycin and 2mM L-Glutamine.
Human neuroblastoma cell line SK-N-LP (a gift from Prof. NK Cheung, Memorial Sloan-Kettering Cancer Centre, NY, USA) was cultured with Dulbecco’s Modified Eagles Medium-high glucose (DMEM-HG, Invitrogen, Carlsbad, CA, USA) at 37oC supplemented with 10% FBS, 100U/mL penicillin, 100mg/mL streptomycin and 2mM L-Glutamine.
hMSCs were pre-labeled with the lipophilic fluorescence dye-CM DiI (Molecular Probes, Carlsbad, CA, USA) before in vivo transplantation. In brief, cells were washed and incubated with CM-DiI at concentration of 5µl/mL for 20 minutes at 37oC and then washed three times with normal growth medium according to the instructions of the manufacturer. The concentration and incubation period were optimized by series of tests. The labeling efficiency was detected to be more than 99% without cytotoxicity and the strong fluorescence signal has been proven to be persistent for more than one month.
Cell transfection and culture of bioluminescent human neuroblastoma cell line
Human neuroblastoma cell line SK-N-LP were transfected with plasmid expressing luciferase (a kind gift from Prof. Kwan Man) using Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. In brief, cells were cultured in 6-well plate and allowed to grow until they were 70-80% confluent. Plasmid DNA was diluted with DMEM without FBS and mixed with prepared Lipofectamine® 2000 solution. The mixture was added into cells and cultured at 37oC overnight. Stable cell line was obtained by neomycin (G418) selection. The bioluminescence of luciferase gene-transfected cells was confirmed under Xenogen IVIS 100 imaging system. Luciferase gene-transfected SK-N-LP cells were culture with DMEM-HG supplemented with 10% FBS at 37oC and sub-cultured when growing to 70-80% confluence.
Orthotopic neuroblastoma modeland xenografts of human luciferase-SK-N-LP cells
This in vivo project obtained the approval of Hong Kong Department of Health and Committee on the Use of Live Animals in Teaching and Research (CULATR), The University of Hong Kong. All procedures and animal care were under the surveillance of the committee. 6-week severe combined immunodeficiency (SCID)-beige mice (18.73±0.76g) were obtained from the Laboratory Animal Unit, The University of Hong Kong and were housed at specific pathogen-free facility with temperature of 22±1ºC, humidity of 55±5% and bred with autoclaved food and water ad libitum. Animals were monitored twice daily.
Luciferase-SK-N-LP cells were trypsinized from the culture flasks and prepared into single cell suspension. Cell viability was analyzed to be more than 99% using trypan blue exclusion assay. The single cell suspension at a concentration of 2×107/mL and 0.2×106 cells diluted with 10µl 50% matrigel (BD Bioscience, Bedford, MA, USA) were prepared in equal volume and maintained on ice. The mixture of SK-N-LP and hMSCs was prepared by mixing SK-N-LP and hMSCs in ratio of 2:1. The final injected number of SK-N-LP cells was 0.2×106.
Animals were anesthetized by intra-peritoneal injection of 100mg/kg pentobarbital. After the disinfection with alcohol and betadine, an incision was made vertically in the abdomen of anesthetized SCID-beige mice. Left kidney was exposed gently and 20µl of cell mixture was slowly injected into the fat pad of the adrenal gland adjacent to the upper pole of left kidney. The organs were carefully rearranged back and the incision was closed. The whole surgery was ensured to be aseptic avoiding infection. Mice were monitored until regaining consciousness. During the first 3 days post-surgery, the mice were given meloxicam in drinking water to minimize the pain at the dose of 0.3mg/kg.
Following the intraperitoneal injection with D-luciferin (Gold Biotechnology, St Louis, MO, USA), the bioluminescence of transplanted cells of SK-N-LP group (n=4) and hMSCs co-transplantation group (n=4) was compared by Xenogen IVIS 100 in vivo imaging which was used to evaluate the in vivo initiation and progression of neuroblastoma. Signal intensity of regions of interest was analyzed by Living Image® Software (Xenogen, corporation Alameda, CA). Study designed for exploring the role of hMSCs in the growth and metastasis of neuroblastoma was shown in Supplementary Figure 1.
Treatment and transplantation of hMSCs
CM DiI pre-labeled hMSCs (1×106) were cultured with PBS (Control group, n=4) or specific CXCR4 antagonist AMD3100 (10µg/mL) in suspension for 1 hour, respectively. Then they were intravenously injected into mice with implanted neuroblastoma 7 weeks post-surgery via tail vein (n=4). Before injection, the staining efficiency and cell viability were evaluated. Experimental design for exploring the tumor tropism property of hMSCs towards primary tumor and metastatic loci was illustrated in Supplementary Figure 1.
Tumor volume evaluation
Three dimensions of isolated tumors were measured using a digital caliper and the volume was calculated according to the following formula. Tumor volume=Length×Wideth×Height×1/2.
Tumor metastasis loci detection
Mice were sacrificed by an overdose of pentobarbital. Organs including brain, lung, heart, liver, spleen, gut and bone were harvested immediately and washed twice with PBS. The metastatic loci of neuroblastoma were detected using Xenogen IVIS 100 imaging.
Evaluation of hMSCs trafficking in vivo
The trafficking of hMSCs in vivo was traced using CRI MaestroTM imaging system by detecting the fluorescence signal of CM-DiI pre-labeled hMSCs in freshly harvested tumors and organs.
Comparison between means from different groups was analyzed using unpaired 2-tailed Student t test for tumor volume. The difference was considered as statistically significant only when P < 0.05. The statistical analysis and data graphs were conducted by GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA, USA).