Anti ccl18

The OSCC cell line, CAL27, was purchased from American Type Culture Collection (Manassas, VA, USA). The OSCC cell lines (HSC-6 and CAL33) and normal oral keratinocytes (NOK) were kindly provided by J. Silvio Gutkind (NIH, Besthesda, MD). HSC-6, CAL33 and CAL27 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Inc., Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). NOK cells were maintained in KSFM (Gibco, USA). Cells were incubated in a 5% CO2 atmosphere at 37°C. A neutralizing antibody against CCL18 (anti-CCL18) was purchased from R & D (R & D Systems, Minneapolis, MN, USA). Recombinant human CCL18 (rCCL18) was purchased from Peprotech (Peprotech, Inc., Princeton, NJ, USA).

Ex vivo tumor slice migration assay was performed as previously described^{23 (link)}. Briefly, freshly resected samples were embedded in 5% agarose (type VII-A; Sigma-Aldrich), 400 μm slices were cut with a vibratome (VT 1000S; Leica), overlaid with 2 × 10⁵ autologous naive CD4⁺ T cells isolated from PB and labeled with CFSE, and incubated in 24-well plates containing RPMI 1640 plus 10% autologous serum for 3 h. The slices were then rinsed, fixed and stained with anti-CCL18 (Cat# MAB394, R&D). Cells were counted in at least 10 fields of view per section at 400× magnification.