BMSC harvest and culture methods
BMSCs were obtained from femurs and tibias of mice using an established protocol [12
]. Briefly, primary bone marrow cells were cultured in Mesencult or Iscove's Modified Dulbecco's (IMDM) medium (Stem Cell Technologies, Canada) in plastic dishes to confluence. We used an established colony-forming unit fibroblast assay to characterize the surface phenotype and the differentiation of BMSCs [3
]. BMSCs seeded at 25-5000 cells per well in 24-well plates were cultured in Mesencult medium for 14 days, fixed in methanol, and stained with crystal violet. For differentiation assays, BMSCs expanded ex vivo
for 3-8 passages were cultured in differentiation media for 2-4 weeks and stained with Alizarin red (for osteoblasts), Oil Red-O (for adipocytes), Alcian blue (for chondrocytes) or anti-αSMA and NG2 (for fibroblasts), as previously described [3
]. For in vivo
experiments, we isolated and expanded GFP+ BMSCs from transgenic mice (n=10 per group) expressing GFP constitutively (EF1α-GFP/FVB
mice and Actb-GFP/C57BL/6
]. LA-P2097 mouse lung carcinoma cells (syngeneic to FVB mice, established in the Steele Laboratory [16
]) and Lewis lung carcinoma cells (LLC1), purchased from ATCC (Manassas, VA) were cultured in DMEM supplemented with 10% FCS.
Phenotypic analysis using flow cytometry
The surface phenotype of plastic-adherent BMSCs isolated from the bone marrow of C57BL/6, Actb-GFP/C57BL/6, FVB and EF1α-GFP/FVB mice was investigated by immunostainingand cytometric analyses usinga LSR-II flow cytometer. Briefly, cultured BMSCs (passage 1, 3 or 20) were separated into 100μl aliquots and labeled with 2% PerCP-CD45, PE-Sca1, APC-CD11b, APC-Cy7(AF750)-CD80, PE-F4/80, APC-Cy7(AF750)-PDGFRb, APC-CD31 and APC-Cy7(AF750) Gr-1 antibodies. There were no differences between the phenotypes of BMSCs from C57BL/6 and Actb-GFP/C57BL/6 or between the BMSCs from FVB and EF1α-GFP/FVB mice, respectively. Thus, BMSCs constitutively expressing GFP (from EF1α-GFP/FVB and Actb-GFP/C57BL/6 mice) were used for in vivo studies.
Evaluation of the role of BMSCs in tumors in vivo
We co-implanted BMSCs (passage 3) at a 1:1 ratio with metastatic lung carcinoma cells either subcutaneously (s.c., 3 × 105
cells of each type) or in dorsal skinfold chambers (DSC, 2 × 105
cells of each type), as previously described [12
]. FVB mouse-derived BMSCs were co-implanted with LA-P2097 and C57BL/6 mouse-derived BMSCs were co-implanted with LA-P2097 (n=6 in DSC and n=10 s.c.). Intravital multiphoton laser-scanning microscopy was used to study and quantify the engraftment of the GFP+
BMSCs in tumors, as previously described [18
]. S.c. tumors were measured twice weekly using a caliper, and were resected when they reached a diameter of 10mm to induce formation of metastases in the lungs [12
]. All tumor tissues were collected for analysis by immunohistochemistry at the end of the experiments.
RNA extraction and cDNA synthesis, oligonucleotide array, and qRT-PCR
RNA was isolated from adherent EF1α-GFP/FVB (passage 3), Actb-GFP/C57BL/6 (passage 3), or C57BL/6 (passage 20) bone marrow derived cells (n=4) using TRIzol reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer's specifications. Qiagen RNeasy® Mini Kit was used to extract RNA prior to cDNA synthesis. cDNA was prepared for qRT-PCR and oligonucleotide arrays using the Thermoscript RNA PCR kit from Invitrogen. Oligonucleotide arrays were performed on cDNA extracted from cells that were isolated with the bone marrow cells used in our animal studies. This cDNA, along with cDNA extracted from three additional bone marrow collections, was used for qRT-PCR. The expression of angiogenesis genes in BMSCs from EF1α-GFP/FVB (passage 3), Actb-GFP/ C57BL/6 (passage 3), or C57BL/6 (passage 20) was profiled using a mouse angiogenesis PCR oligonucleotide array (SA Biosciences, PAMM-024) according to manufacturer's protocol. For qRT-PCR, SYBR green and LUX primers were designed with Primer Express Software (Applied Biosystems). Primers used in our study included: mGAPDH, mMMP9, mMMP2, mCTGF, mTNFα, mCXCL2, mCXCL1, mCCL2, mTie2, mI L1β, mIL6, mThbs1, mThbs2, and mHIF1α. Samples were run on the Applied Biosystems 7000 Sequence Detection System. Mouse GADPH was used as a qRT-PCR control.
Myeloid cell isolation and co-implantation with tumor cells
CD11b+ cells were isolated from whole bone marrow harvested from EF1α-GFP/FVB mice or Actb-GFP/C57BL/6 mice (n=8) by negative selection using magnetic bead separation techniques. Mice were sacrificed by cervical dislocation and the tibiae and femurs were removed and flushed with cold PBS containing 5% fetal bovine serum. Bone marrow cells were centrifuged, filtered and re-suspended in 10 ml of sterile PBS. Two hundred microliters of biotinylated mouse lineage cocktail, containing equal volumes of TER-119 (Ly-76), Gr-1 (Ly-6G/ C), CD45R (B220), and CD3e, were added (BD Pharmingen, San Jose, CA, Product number 559971). The cells were incubated on ice for 30 minutes and 400 μl of Biotin-Binding Dyna-beads (Invitrogen, Carlsbad, CA, Product number 110-47) were added. The cells were placed on a rocker and incubated for an additional 45 minutes on a shaker plate at 4°C. Cells were separated into 5 ml aliquots and the MCP-1 magnet was used for magnetic bead depletion (depletion step was repeated 4 times). Cells were centrifuged and resuspended in 2 ml PBS. Red blood cells were lysed using 5 ml ACK buffer. Cells were centrifuged and resuspended in a small volume of PBS. This procedure yielded approximately 2 million cells. Flow cytometry was used to confirm that more than 98% of the cells were positive for CD11b and negative for TER-119, Gr-1, CD45R, and CD3e. CD11b+ (myeloid) cells from FVB or C57BL/6 mice were co-implanted with syngeneic LA-P0297 or LLC1 tumor cells in immunocompetent mice or with LLC1 cells in SCID mice at a 1:1 ratio (n=8 mice per group, 250,000 tumor cells per mouse). Tumor growth was monitored as described above.
Subcutaneous and dorsal skinfold tumor studies were performed with 6-8 mice per group. Pooled bone marrow from 8-10 mice per group was used for flow cytometry and oligonucleotide analysis. For qRT-PCR analysis, RNA was isolated from pooled or single-animal collections of bone marrow. Data reported is from single animal bone marrow collections (n=3); however, there were no statistical differences between qRT-PCR analysis for pooled bone marrow versus single animal isolated bone marrow. Flow cytometry experiments were repeated at least 3 times. Student's t-test was used to compare experimental groups and a p <0.05 designated a statistically significant difference. The rates of spontaneous metastasis formation were compared using the Mann-Whitney U test (Wilcoxon rank-sum test).