CCL20 and CXCR4 Interaction Figure 3. Regulation of CCL20 expression and function in various tumor cells. (A) CCL20 mRNA and protein expression in CCL20- transfected PC3 single-cell clones tested by semi-quantitative RT-PCR and ELISA. (B) PC3-CCL20 clones were seeded at 26104 cells/1 ml per well into a 24-well plate and incubated for 6 days. On day 6, the cells were harvested and viable cells were counted using PI staining and FACS analysis. Data is presented as mean6SD from triplicates (** P,0.05). (C) PC3-CCL20 single-cell clones were grown to confluence, harvested, resuspended in their conditioned culture medium and allowed to adhere to collagen I- and fibronectin-coated plates for 30 minutes. Non-adherent cells were washed, and adherent cells were collected in 300 ml FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean6SD from triplicates (** P,0.05). (D) Leukemic cell lines NB4 and HL60, primary human leukemic blasts, HT-29 cells, and normal human keratinocytes were incubated with various concentrations of CXCL12 for 48 hours. CCL20 secretion to culture medium was assessed using ELISA method. (E) CCR6 mRNA expression in leukemic cell lines NB4 and HL60 and colon cancer HT-29 cells assessed by semi-quantitative RT-PCR. b-actin confirmed comparable loading of RT- PCR products in each lane. (F) HL60 and HT-29 cells either untreated or treated with various concentrations of CCL20 were placed on collagen I coated plates (10 mg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 ml FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean6SD from triplicates (** P,0.05). Data is representative of three separate experiments. doi:10.1371/journal.pone.0005125.g003 cells developed larger tumors as measured by an increase in size compared to mice injected with mock-transfected PC3 cells (Fig. 4A). Moreover, tumors produced by PC3-CCL20.30 cells were more vascularized and invasive to the neighboring tissues (muscle and dermis) (Fig. 4B). These findings were confirmed by H&E-stained tissue sections of xenograft tumors. Histological analysis of PC-CCL20 tumors demonstrated an invasion of tumors to adjacent muscle tissue, development of necrosis that can be associated with rapid tumor growth, and massive aberrant vascularization of tumors. In contrast, as was previously described, PC3-mock tumors were encapsulated, non-invasive and no aberrant blood vessels were present (Fig. 4B). Mice injected with PC3- CCL20.10 cells also developed larger tumors as measured by an increase in size and weight compared to mice injected with mock- transfected PC3 cells. However, the difference between PC3- CCL20.10 and the parental cells were smaller (Fig. 4A). Both PC3-CCL20.30 as well as the parental PC3 cells developed a necrotic core, while tumors that overexpressed CCL20 continued to grow beyond this point, suggesting that CCL20 may facilitate angiogenesis in tumors. We studied this hypothesis by comparing the number of blood vessels in histological sections as well as by using an in vivo intra-tumoral vessel functionality MRI based assay in the CCL20 tumors (PC3- CCL20.30) versus that in control tumors (PC3-mock). Macro- scopic assessment of tumors overexpressing CCL20 revealed increased vascularization as compared to control tumors (Fig. 4B). To complement the data obtained from histology on blood vessel density, vessel functionality (DSo2) was measured by fMRI to study the actual in vivo perfusion of the tumor and the oxygen delivery efficiency into the tumor mass. Functionality of the vasculature was derived from GE images acquired during inhalation of air-CO2 and carbogen (95% oxygen+5% CO2) [26] PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5125