5T4 Regulates CXCR4 Chemotaxis CXCL12). (B), MAb dose response of inhibition of chemotaxis towards CXCL12 in differentiating WT-ES cells. (C), The chemotactic migration exhibited by primary WT MEF was abolished in the presence of the m5T4 specific mAb B1C3 (10 mg) but not in presence of mAb P1C9 (10 mg) or an irrelevant control antibody (10 mg). MAbs B3F1 and B5C9 (10 mg) reduced the chemotactic response. doi:10.1371/journal.pone.0009982.g009 It is clear that not all CXCL12/CXCR4 responsive cells express 5T4 molecules. 5T4 cannot be an absolute requirement for CXCR4 activation because 5T4 knockout mice are viable, whereas both CXCL12 and CXCR4 KOs are lethal [49,50]. Clearly, there must be some redundancy and other molecules must be able to regulate CXCR4 trafficking to and/or retention at the cell surface. Additionally, there is mounting evidence that chemokine receptors are able to form discrete functional units via heterodimerisation with other G-protein coupled receptors. In the case of CXCR4, heterodimerisation with the chemokine receptor CXCR7, which binds the same ligand CXCL12, can alter both the kinetics and the dynamics of CXCR4 responsiveness to CXCL12 [51]. CXCL12 is a homeostatic chemokine that, unlike other ELR- CXC chemokines, is angiogenic. CXCL12 binds to the widely expressed CXCR4 (exclusively) and the more restricted CXCR7 (which also binds CXCL11) [26]. CXCL12 through CXCR4 regulates cardiac and neuronal development, stem cell motility, neovascularisation and tumorigenesis [23]. Besides acting as a cofactor for HIV, CXCR4 mediates the CXCL12-directed migration of cancer cells to metastatic sites through the promotion of angiogenesis and migration of tumor cells in breast, lung, ovarian, renal, prostate and neuroblastoma [22,23,24]. It is significant that all these tumor types are known to express the 5T4 glycoprotein [2,9,52]. Importantly, these CXCR4-positive tumors preferentially spread to tissues with high levels of CXCL12 such as lung, liver, lymph nodes, brain and bone marrow which are key metastatic sites [22,23,24]. In addition, the stromal environment (often 5T4 positive [2,7,8]) can have a tumor- imprinted promotional influence [53]; and chemokines can sometimes induce proliferation rather than chemotaxis enhancing tumorigenesis [26,54]. We have investigated the relationship between expression of 5T4, CXCR4 and chemotaxis in several human tumor cell lines including choriocarcinoma, breast and ovarian and all exhibited CXCL12-mediated chemotaxis and showed evidence of a 5T4/CXCR4 complex in the cell membranes (Southgate et al., unpublished). The regulation of CXCR4 surface expression by 5T4 molecules may provide a new way to control response to the chemokine CXCL12 in normal circumstances but could be selected to advantage the spread of a tumor from its primary site. If the latter events are preferentially and constitutively expressed properties of tumors then targeting the CXCR4/5T4 complex might offer new opportunities for therapeutic intervention. Materials and Methods Ethics Statement All animal work was performed in accordance with the UK Animal Scientific Procedures Act 1986 and was covered by both Project and Personal licences that were issued by the Home Office and reviewed by the Paterson Institute for Cancer Research ethical committee. Professor Peter L Stern Home Office Project Licence numbers 40-2666 (years 2003–2008) and 40/3200 (years 2008–2013) and Named Animal Care & Welfare Officer Licence number 40/3085 covered all procedures and breeding. Local Ethics Committee approval was provided prior to submission of all subsequently approved project licence applications. Ethical Review Process advises the Certificate Holder regarding Project licence applications and standards of animal care and welfare; they also develop initiatives leading to the widest possible application of the 3Rs so that procedures are refined to minimise suffering, numbers of animals used are reduced and animal use is replaced wherever possible. Mice are housed in individually ventilated cages. These cages prevent the spread of potential disease from one cage to another and each cage has an individual Hepa filtered air supply that gives approximately 72 air changes per hour and a fixed exhaust system. All the cages are provided with environ- mental enrichment, in the form of nesting material, a variety of mouse houses, wooden chew blocks or play tunnels. The addition of these items increases socialisation and environmental stimula- tion for the mice and reduces aggression amongst some strains of males. Routine health screening from our colonies is performed to ensure that the mice are free from a list of specific pathogens (SPF) and any new strains brought into the unit are health screened before introduction into the facility. 5T4 Knockout (KO) mice We have constructed a 5T4KO mouse by replacing the second exon of 5T4, which encodes the entire protein, with an IRES- LacZneo reporter gene in ES cells. These cells were used to produce chimeric mice and germline progeny; 5T4 KO heterozygote mice were backcrossed to the C57BL/6 background. The 5T4KO C57BL/6 animals are viable but adult animals show some structural disorganization within the brain and exhibit a high frequency of hydrocephalus. The frequency of hydrocephalus is approximately 13%, with the median age of death, (animals requiring termination) at 49 days, (range 38–83). We have made and characterized 5T4KO ES cells in order to study aspects of the role of 5T4 in EMT [16]. The 5T4KO mice were used to generate monoclonal antibodies specific for m5T4 (B3F1 (IgG2a); B5C9 (IgG1); B1C3 (IgG2a); P1C9 (IgG2b) and P1H10 (IgG2b). Primary murine embryonic fibroblasts (MEF) of all three genotypes were prepared from day 13 embryos following mating of male and female 5T4 heterozygote C57BL/6 transgenic mice by methods previously described [55]. Cell lines E14TG2a [56], (here referred to as WT-ES) and 5T4KO-ES cells [16] were cultured on pre-prepared 0.1% gelatine (Sigma) coated tissue culture flasks. ES cells were grown in Knockout DMEM, (Invitrogen) supplemented with 10% Hyclone fetal calf serum, (Perbio), 2 mM L-glutamine, 1% non-essential amino acids, (Sigma), nucleosides [6 ml of the following solution/500 ml DMEM: adenosine, (80 mg), guanosine, (85 mg), cytidine, (73 mg), uridine, (73 mg) and thymidine, (24 mg) dissolved in 100 ml double distilled water; Sigma], 2-mercaptoethanol, (50 mM; Invitrogen), leukemia inhibitory factor, (LIF; 1000 units/ml of ESGRO; Chemicon Int.), 100 units/ml penicillin and 100 mg/ml streptomycin (Gibco). For differentiation cells were grown in media that was not supplemented with LIF. Media was changed daily. MEF, A9 fibroblast cell lines, B16neo and B16m5T4 melanoma cell lines [57,58] were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin. The hybridoma cell lines producing the anti-m5T4 monoclonal antibodies B5C9, P1H10, P1C9, B1C3 and B3F1 were cultured in DMEM supplemented with heat inactivated 10% fetal calf serum PLoS ONE | www.plosone.org 14 April 2010 | Volume 5 | Issue 4 | e9982