M2-polarized macrophages in keratocystic odontogenic tumor: relation to tumor angiogenesis

The purpose of this study was to evaluate the presence of M2-polarized macrophages and their relationships to angiogenesis in keratocystic odontogenic tumor (KCOT). M2-polarized macrophages were detected in KCOT samples by immunohistochemistry and immunofluorescence. Meanwhile, microvessel density measured with antibody against CD31 was closely correlated with the presence of M2-polarized macrophages. In addition, macrophage colony-stimulating factor (M-CSF) significantly contributed to the activation of M2-polarized macrophages. Moreover, the results of in vitro wound healing, cell migration and tube formation assays further revealed the pro-angiogenic function of M2-polarized macrophage-like cells. This function might be associated with secretion of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and matrix metalloprotein-9 (MMP-9). This study demonstrates for the first time that M2-polarized macrophages are prevalent in KCOT, and their presence is dependent on M-CSF expression. More importantly, these tumor-supportive cells can also promote tumor angiogenesis by secreting angiogenic cytokines.


Cell culture.
According to previous description 6 , THP-1 cells, obtained from ATCC, were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. To generate polarized THP-1 macrophages, one million THP-1 cells were seeded into six-well culture plates and treated with 25 ng/mL phorbol myristate acetate (PMA) for 48 hours. To obtain M2-polarized cells, THP-1 cells were treated with 25 ng/mL PMA for 48 hours and further polarized them with 20ng/ml IL-4 and 20ng/ml IL-13 for 36 hours, added 12 hours after PMA. M2-polarized macrophages-like (M2L-macrophages) were generated from THP-1 cells culture with 25 ng/mL PMA for 48 hours and eKCOT tumor homogenate for final 36 hours (30%, added 30 hours after PMA). To inhibit the influence of M-CSF on the differentiation of M2-polarized macrophages, GW2580, a specific M-CSFR inhibitor, was added to the medium as previous description 7,8 . HUVECs, isolated from human umbilical cord veins by as our previously described 9 , were cultured in endothelial basal medium (EBM) supplemented with 20% fetal bovine serum, SingleQuot (Bio Science), and 100 U/mL penicillin, and 100 ng/mL streptomycin. Passages 2-7 of these cells were used in this study. Cell densities were counted by the Vi-CELL cell viability analyser (Beckman Coulter) as our previous studies 9 . All experiments were repeated more than three times.

Cell viability assay.
One million 25ng/mL PMA-treated THP-1 cells were seeded in 6-well plates. After THP-1 cells were cultured in 25 ng/mL PMA for 12 h, they were treated with serum-free RPMI 1640 medium containing GW2580 (a specific antagonist for CSF-1R, Germany) at various concentrations for 24, 36, and 48 h. Then, total cells were analyzed by using a Vi-CELL cell viability analyzer, based on trypan blue exclusion (Beckman Coulter, USA) as our previous description 9, 10 . All experiments were repeated three times at least.

MTT assay.
According to our previous steps 10 , ten thousand 25ng/mL PMA-treated THP-1 cells plated onto 96-well plates for 12 h then, incubated for 24, 36, and 48 h with 100 ml of various concentrations of GW2580 in serum-free RPMI 1640 medium. Then, 10 μL of MTT solution (5 mg/ml) was added to each well and incubated for another 4 h at 37 o C. After that, the medium was changed, 150 μL DMSO was added to the wells, and the absorbance was quantified at 490 nm by using a 96-well microplate reader (Bio-Tek). This experiment was repeated three times or more.

Real-time quantitative PCR.
As our previous description 11 , total RNA was isolated, the cDNA was synthesized and real-time qPCR was performed. GAPDH was selected as an internal control in our experiment. The primer nucleotide sequences for PCR were listed as follows: TNF-α:

Endothelial cell wound healing assay.
HUVECs were seeded in six-well culture plates as our previous description 9 . When these cells grew to 90% confluence, a gap of constant width scraped with a micropipette tip was made in the center of the cell monolayers. The medium was changed and replaced by EBM with 20% FBS, 50% PMA only-CM, 50% PMA + eKCOT-CM or 50% PMA + dKCOT-CM. Twelve hours later, the cells were fixed and The distance of cell migration was calculated by Image J. Data were expressed as the ratios of migration compared to control groups. This experiment was repeated at least three times.
Endothelial cell migration assay.
Endothelial cell migration was measured using a transwell Boyden chamber system (Becton-Dickinson). Briefly, HUVEC (5×10 5 ) cells were seeded into the upper chamber in 100μL of serum-deprived EBM, whereas EBM with 5 ng/ml VEGF, 50% PMA only-CM, 50% PMA + eKCOT-CM or 50%PMA + dKCOT-CM were added to the lower chambers as chemoattractants. After incubation at 37°C for 12 hours, the cells in the upper surface of the chamber were thoroughly removed with a cotton swab, and the migrated cells were fixed with buffered 4% paraformaldehyde, followed by staining with crystal violet, and then photographed and quantified. The experiment was repeated at least three times.

Tube formation assay.
As our preceding steps 9 , HUVECs (2×10 5 cells) in 500μL EBM with 10% FBS, 50% PMA only-CM, 50% PMA + eKCOT-CM or 50% PMA + dKCOT-CM were seeded onto forty-eight-well culture plates coated with BD Matrigel™ Matrix (Becton-Dickinson). After incubation for 24h at 37°C, cells were fixed, photographed under phase-contrast microscopy, and capillary-like structures were photographed at low-power magnification. Five random fields in each well were chosen to count and analyses. All experiments were repeated at least three times.