4T1 cells were cultured in RPMI-1640 with 10% fetal calf serum medium. Six-week-old female Balb/c mice were acclimated and caged in groups of 6 or less. Celecoxib was manufactured by Pharmacia (Peapack, NJ, USA), rVEGF was purchased from Roche Applied Science (Basel, Switzerland) and a rat anti-mouse CD31 monoclonal antibody was from BD-Pharmingen. Celecoxib was fed on diet after 14 days of tumor cells implanted on the back of the mice, rVEGF was intraperitoneally injected daily at a dose of 5 mg/kg.

All animal studies were reviewed and approved by the Animal Care and Use Committee of the Shandong Provincial Animal Board (Jinan, China). Mice (6–8 weeks old) labeled with enhanced green fluorescent protein (EGFP) were used as donors of bone marrow (BM). BM cells (BMCs) were prepared according to procedures previously described (1). BMDCs were flushed out with RPMI-1640 medium from femurs and tibiae using 21-gauge needles. Then cells were harvested by centrifugation, resuspended with phosphate-buffered saline (PBS), and intravenously injected at a total number of 1×10⁶ cells per 100 μl PBS into recipient syngenic Balb/c mice that had received whole body irradiation of 900 rad immediately before the transplantation.

Total RNA was extracted from PBS perfused lungs with the RNeasy kit (Qiagen) according to the manufacturer’s protocol. First-strand cDNA was synthesized from 1 μg total tissue RNA using the RevertAid™ First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) with random primers. Then cDNA was amplified for quantitative real-time PCR. The specific primers used were: Cox-2, forward, 5′-ATCAGGTCATTGGTGGAG-3′ and reverse, 5′-ACACTCTGTTGTGCTCCC-3′; β-actin, forward, 5′-CTGTCCCTGTATGCCTCTG-3′ and reverse, 5′-ATGTCACGCACGATTTCC-3′. Real-time PCR reactions were performed at: 95°C, 10 sec (denaturation); 55°C, 30 sec (annealing); 72°C, 30 sec (extension) for 35 cycles. Real-time PCR reactions were performed on the ABI 7000 Fast real-time PCR system with SYBR-Premix Ex Taq™ according to the manufacturer’s instructions.

Balb/c mice were injected with 4T1 cells; control mice received PBS only. Twelve days after tumor-cell inoculation, mice were euthanized and lungs were perfused with PBS to completely remove blood from the lung vasculature. Cleaned lung lobes were immersed in RNAlater™ to stabilize RNA. Total RNA was isolated and used for subsequent microarray analysis. Affymetrix Gene Chip mouse genome 430 2.0 array was applied to analyze the expression profile of tissue samples. The microarray chip consisting of 27,326 different human cDNAs (Angilent, Wilmington, DE, USA) was employed to the analysis of the MPVECs treated with lipopolysacharide (LPS) and VEGF.

4T1 cells were labeled with DiI, then 2×10⁴ labeled cells were infused i.v. In mice with primary tumors, the labeled cells were infused when the tumor reached 6 mm in diameter. In mice without tumors, labeled cells were infused i.v. 6–15 min after VEGF treatment. Five and 24 h after cancer cell infusion, the lungs were perfused with PBS under physical pressure to remove any circulating tumor cells and were then excised. Three lung tissue fragments (3 mm in diameter) were randomly selected and two 50-μM sections per fragment were examined with a confocal microscope. The number of labeled cancer cells was normalized by total tissue surface area.

4T1 cells (1×10⁵) labeled with DiI were intravenously injected at 2 weeks after the subcutaneous implantation of tumor cells (2×10⁶) into the mice. Two weeks after injection, animals were sacrificed and lungs were resected. Surface metastasis was examined with light microscopy and further identification was performed with scanning for DiI-positive metastasis and processed for histological analysis. The metastasis was quantified as the total number of metastatic foci per reference area.

The method of MPVEC isolation was described previously (2). MPVECs were cultured in a 24-well plate with various concentrations of VEGF for 4 h and then fixed by 4% paraformaldehyde. After washing with PBS, cells were incubated with 3% bovine serum albumin at room temperature for 30 min, followed by incubation of celecoxib at a dilution of 1:100 overnight at 4°C. For the immunofluorescence of lung tissue, the procedures of the first day were described above. Similarly, an FITC-conjugated fluorescent secondary antibody was added on the next day, and cells (or tissue sections) were detected under an inverted fluorescent microscope.

MPVECs were cultured in 24-well plates with various concentrations of VEGF for 6 h. 4T1 cells were suspended at a concentration of 1×10⁶ cells/ml in DMEM and labeled with 2′,7′-bis-5-carboxyfluorescetin acetoxymethyl ester (BCECF, AM) at 37°C for 15 min. Then, the labeled 4T1 cells were added to the MPVEC 24-well plates, and incubated at 37°C for 30 min. After washing 3 times with PBS to remove unattached cells, the number of adhered AM-labeled 4T1 cells was counted using an inverted fluorescent microscope.

The RayBio Custom mouse cytokines antibody array kit was purchased from RayBiotech and used according to the manufacturer’s instructions. Briefly, after blocking with 1X blocking buffer, membranes were incubated for 1.5 h with the experimental serum. The membranes were washed and incubated with biotin-conjugated antibodies for 1.5 h. The membranes were washed again and incubated with streptavidin-conjugated horseradish peroxidase for 2 h, washed, and developed using an enhanced chemiluminescent substrate for horseradish peroxidase. Chemiluminescence was detected using EpiChemi3^® Darkroom imaging system and LabWorks^® densitometry software. Data was corrected for background signal and normalized to positive controls using RayBio^® Analysis Tool software (UVP Bioimaging, Upland, CA, USA).

The data were analyzed using JMP ver. 6. The groups were compared using Chi-square test and Fisher’s exact test. The analyses yielded 95% confidence intervals and P-values. Values of P<0.05 were considered to indicate a statistically significant difference.

We examined the effects of breast cancer-cell induction during the pre-metastatic phase on the morphology of lung tissues excised from 4T1 tumor-bearing mice. Edema and lung wet/dry-weight ratios were significantly increased by breast cancer cell induction, compared with controls (Fig. 1). Hematoxylin and eosin (H&E) staining also showed a higher level of inflammatory cell infiltration in the lungs of tumor-bearing mice, compared with non-tumor-bearing mice (Fig. 1A).

Immunohistochemical analysis with CD31 antibody showed that blood vessels in the lungs of 4T1-tumor-bearing mice appeared as primitive and dilated sinusoidal vascular structures, consisting of disorganized, tortuous, and interconnected vascular plexuses. Total blood vessel density was markedly increased in the lungs of tumor-bearing mice compared with non-tumor-bearing mice (Fig. 1A and B).

During tumor progression, BMDCs have been shown to promote inappropriate growth, invasion and ultimately metastasis. In the present study, BMDC mobilization and recruitment changed in response to breast cancer cell inoculation. Green fluorescent protein (GFP) was used to label BMDCs and pre-metastatic lung fluorescence was detected by confocal microscopy following 4T1-tumor-cell injection in mice. Mice inoculated with PBS were used as controls. No GFP⁺ BMDCs were observed in lungs without tumor cell injection (Fig. 2A), but GFP⁺ BMDCs were found scattered in lung tissue by day 7 after tumor implantation in tumor-bearing mice. No BMDCs were observed in control lungs at the same stage. The difference increased at day 14, when clusters of GFP⁺ BMDCs were detected in lung tissues in tumor-bearing mice and near terminal bronchioles, while minimal GFP⁺ BMDCs were observed in lung tissues in a small number of control mice (Fig. 2A and B).

The homing of tumor cells and their subsequent survival are critical for metastasis. To investigate the effects of BMDC recruitment on breast cancer cell homing in mouse lungs, DiI-labeled 4T1 cells were injected into mice 2 weeks after tumor inoculation. Clusters of GFP⁺ BMDCs were detected near terminal bronchioles in 4T1-tumor-bearing mice (Fig. 2C). A subsequent metastasis assay revealed that the number of metastatic foci in lung lesions in 4T1-tumor-bearing mice was significantly higher than that in the lungs of non-tumor-bearing mice (Fig. 2C and D).

We investigated the factor secreted by breast cancer cells responsible for inducing the pre-metastatic niche and BMDC recruitment. Mouse sera and 4T1 cell culture media were assayed using a RayBio Custom mouse cytokine antibody array kit. As shown in Fig. 3A, levels of VEGF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) were significantly increased in sera from 4T1-tumor-bearing mice. However, only VEGF was detected in culture media from 4T1 cells, at levels as high as 20 pg/ml (Fig. 3B); levels of GM-CSF, IL-6 and tumor necrosis factor-α (TNFα) were too low for detection, suggesting that 4T1 cell-derived VEGF may be the critical triggering molecule responsible for initiating the formation of the pre-metastatic niche.

To investigate the role of VEGF in the initiation of the pre-metastatic niche, mice were treated daily with recombinant VEGF for 7 consecutive days and BMDC recruitment in the lung was tested. As expected, the frequency of BMDCs within the lungs, as well as angiogenesis, increased significantly in response to VEGF (Fig. 3C). To determine the effect of VEGF on the metastatic potential of breast cancer cells, mice were treated with VEGF for 7 consecutive days, and 4T1 cells were then injected through the tail vein. The number of visible metastatic foci in lungs revealed increased metastases (Fig. 3C and D), and examination of the lungs showed enlarged lung masses in VEGF-treated mice, compared with controls. These results suggest that VEGF is a crucial triggering molecule initiating the formation of the pre-metastatic niche.

The important role of PGE2 in tumor angiogenesis and metastasis suggests that its expression may be correlated with VEGF. We therefore compared PGE2 levels, and levels of its synthesis regulator cyclooxygenase-2 (COX-2) gene, in pre-metastatic lungs in mice with and without 4T1 tumors. As shown in Fig. 4, COX-2 expression in lung tissues was upregulated at both mRNA and protein levels by inoculation of 4T1 cells, and the increase in PGE2 production confirmed the COX-2-expression results.

Notably, the expression profiles in MPVECs induced by VEGF and PGE2 were similar. Among the genes with >2-fold changes in expression in response to stimulation, 48 genes were upregulated and 35 were downregulated in response to VEGF, and 65 and 33, respectively, in response to PGE2. Notably, 33 upregulated genes and 14 downregulated genes were common to both responses, suggesting that they used the same downstream pathways.

PGE2 is a secreted protein previously characterized as a proinflammatory factor. We defined the role of PGE2 in VEGF-induced BMDC recruitment and lung metastasis in primary mouse lung endothelial cells exposed to VEGF. As expected, VEGF stimulation significantly increased COX-2 expression and PGE2 levels in endothelial cells (Fig. 5A and B). We also evaluated the role of PGE2 induction by VEGF in endothelial cell adhesion to metastatic cancer cells in an in vitro cancer cell adhesion assay. VEGF stimulation increased the number of fluorescently-labeled metastatic cancer cells attached to lung endothelial cells (Fig. 5C and D). The addition of celecoxib abolished tumor cell adhesion to the endothelial cells (Fig. 5C and D). PGE2 overexpression resulted in a significant increase in the homing of infused metastatic cancer cells to the lungs of mice treated with VEGF (Fig. 5C and D), measured 48 h after metastatic cancer cell infusion (Fig. 5C and D). Collectively, these data show that blocking lung endothelial PGE2 production reduced VEGF-induced breast cancer metastasis to the lungs.

The role of COX-2 in VEGF-induced BMDC recruitment and lung metastasis was defined by treating mice with VEGF and the COX-2 inhibitor, celecoxib. Celecoxib treatment reduced the recruitment of BMDCs in lungs (Fig. 6A and B), and lung inflammation was also reduced, as shown by H&E staining (Fig. 6A). 4T1 cells were then injected into the tail vein of mice pretreated with VEGF in the presence or absence of celecoxib. Celecoxib treatment significantly reduced VEGF-induced lung metastases of 4T1 cells (Fig. 6A and B). These results indicate that PGE2 is involved in VEGF-induced pre-metastatic microenvironmental changes.