Sphingosine kinase 2 deficient tumor xenografts show impaired growth and fail to polarize macrophages towards an anti-inflammatory phenotype Andreas Weigert 1 , Susanne Schiffmann 2 , Divya Sekar 1 , Stephanie Ley 1 , Heidi Menrad 1 , Christian Werno 1 , Sabine Grosch 2 , Gerd Geisslinger 2 and Bernhard Br€ une 1 * 1 Institute of Biochemistry I/ZAFES, Goethe-University, Theodor-Stern-Kai 7, Frankfurt, Germany 2 Institute of Clinical Pharmacology/ZAFES, Goethe-University, Theodor-Stern-Kai 7, Frankfurt, Germany A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor-associ- ated macrophages (TAMs), which are polarized towards an anti- inflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage pheno- type are urgently needed to address potential anti-cancer strat- egies along this line. One potential immune modulating com- pound, sphingosine-1-phosphate (S1P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of sphingo- sine kinase 2 (SphK2), one of the S1P-producing enzymes for tu- mor progression. The growth of SphK2-deficient MCF-7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2-deficient and con- trol tumors was comparable. However, TAMs from SphK2-defi- cient tumors displayed a pronounced anti-tumor phenotype, show- ing an increased expression of pro-inflammatory markers/media- tors such as NO, TNF-a, IL-12 and MHCII and a low expression of anti-inflammatory IL-10 and CD206. These data suggest a role for S1P, generated by SphK2, in early tumor development by affecting macrophage polarization. ' 2009 UICC Key words: cancer; angiogenesis; macrophages; sphingolipids; inflammation Macrophages exhibit a high plasticity in their functional responses and thus acquire different phenotypes in response to incoming signals generated by their microenvironment. 1,2 A major task of macrophages is to fight pathogens closely associated with initiation as well as resolution of inflammation. 3 During resolution of inflammation, macrophages contribute to several aspects of wound healing such as tissue remodeling. 4 Because tumors can be viewed as ‘‘wounds that do not heal,’’ 5,6 it is not surprising that macrophages actively participate in tumor progression. Tumor-associated macrophages (TAM) are a major cellular constituent of the leukocyte infiltrate in tumors, 7 where they become polarized towards an anti-inflammatory M2 phenotype. 8 In contrast to M1 macrophages, they show a reduced capacity in producing anti-tumor molecules such as TNF-a or NO, but sup- port tumor survival, growth, metastasis and play a major role in tu- mor angiogenesis as well as immune evasion. 8–10 With regard to the question how TAM polarization is achieved during cancer de- velopment, the production of immunomodulatory mediators such as IL-4, IL-10 or transforming growth factor (TGF)-b as well as tumor hypoxia were suggested. 8,9 This is comprehensible for well- established tumors that have acquired a series of suitable muta- tions. However, early in tumor formation, especially during the equilibrium phase of cancer immunoediting, 11 when shaping of tu- mor immune escape mechanisms takes place and small tumor nod- ules persist without outgrowth due to constant selective pressure built up by the immune system, more complex signaling circuits are involved. As an evolutionary process, early stages of tumor progression entail the continuous interaction of immune cells with dying tumor cells. In this regard, at least apoptotic cells (AC) are powerful immune regulators by delivering anti-inflammatory signals. 12 Ap- optotic tumor cells were reported to suppress macrophage cytotox- icity directed against vital tumor cells 13 and, most interestingly, preventing their interaction with the phagocyte in vivo induced tu- mor regression. 14 These immune modulating effects of AC may be attributed to their specific recognition by phagocytes or by secretion of immunomodulatory mediators. Among the factors released from AC is sphingosine-1-phosphate (S1P), 15,16 a lipid mediator that fosters M2 polarization of macro- phages in vitro either when added directly 17 or when being present in the supernatant of dying tumor cells. 18 Production of S1P by dying tumor cells demands the catalytic activity of SphK2. Along this line, a monoclonal S1P antibody reduced tumor growth by attenuating tumor angiogenesis and survival. 19 These observations support a direct link between S1P production and tumorigenesis. To verify SphK2 as a potential source for S1P in tumor devel- opment, we xenografted SphK2-deficient MCF-7 cells when com- pared with control MCF-7 cells into nude mice. We noticed a reduced growth in SphK2-deficient xenografts, concomitant with largely impaired macrophage polarization towards a M2 pheno- type. The growth inhibitory effect of the SphK2 knockdown resulted neither from reduced cell cycle progression nor an increased apoptotic capacity. Although SphK2-deficient MCF-7 cells grew faster and were less susceptible to apoptosis in vitro when compared with corresponding controls, their growth as xen- ografts in nude mice was markedly delayed. These results imply that the interaction of S1P-producing tumor cells with the host microenvironment such as infiltrating macrophages may be crucial to polarize these cells and thus to drive tumor progression. Material and methods Cell culture MCF-7 breast carcinoma cells 20 (originally obtained from DMSZ 21 ) were maintained in RPMI 1640, supplemented with 100 U/ml penicillin, 100 lg/ml streptomycin and 10% heat-inacti- vated FCS. LPS and neomycin were from Sigma-Aldrich and human recombinant IFN-g was obtained from Roche. Generation of sphingosine kinase 2 knockdown cells For SphK2 knockdown, pSilencer-siSphK2 was transfected into MCF-7 cells (MCF-7-siSphK2) using Nucleofector technology (Amaxa) as described. 18 SphK2-deficent MCF-7 cells used in this study were a mixed population and not derived from a single clone. MCF-7 cells carrying a nontargeting shRNA vector (MCF- 7-siNT) cells were generated by transfecting MCF-7 cells with the pSilencer 4.1-CMV neo control vector (Ambion). Also these cells were a mixed population. Grant sponsor: Deutsche Forschungsgemeinschaft; Grant numbers: Br 999, FOG 784, ECCPS; Grant sponsor: European Community (PRO- LIGEN); Grant sponsor: Deutsche Krebshilfe; Grant number: 106996; Grant sponsor: Sander Foundation; Grant number: 2007.070.1; Grant spon- sor: The LOEWE program/LiFF. *Correspondence to: Institute of Biochemistry I-Pathobiochemistry, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Ger- many. Fax: 149-69-6301-4203. E-mail: bruene@pathobiochemie1.de Received 1 April 2009; Accepted after revision 14 May 2009 DOI 10.1002/ijc.24594 Published online 28 May 2009 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 125, 2114–2121 (2009) ' 2009 UICC Publication of the International Union Against Cancer