P04.02 Membrane-bound CX3CL1 enhances NK cell–mediated cytotoxicity in ovarian cancer

Background The chemokine CX3CL1 (fractalkine) is a unique member of the chemokine family that exists as both a soluble and a membrane-bound molecule and exhibits distinct functions in cancer. Proteolytic shedding of membrane-bound CX3CL1 (mCX3CL1) by the metalloproteases ADAM10 and ADAM17 generates soluble CX3CL1 (sCX3CL1), which primarily mediates chemotactic immune cell recruitment, whereas the membrane-bound form is implicated in cell–cell adhesion, migration, and direct activation of cytotoxic lymphocytes. CX3CL1 has been described to display both tumor-suppressive and tumor-promoting properties depending on tumor entity and microenvironment and is a known modulator of natural killer (NK) cell recruitment, activation, and effector function via its receptor CX3CR1. Despite increasing evidence for a context-dependent role of the CX3CL1/CX3CR1 axis in cancer, the individual contributions of soluble versus membrane-bound CX3CL1 have not been systematically addressed in ovarian cancer. In this study, we specifically examine the aspect of NK cell cytotoxicity and whether increasing tumor cell–associated mCX3CL1, either through inhibition of ADAM10/17-mediated shedding or enforced CX3CL1 expression, can enhance NK cell–mediated cytotoxicity.Methods Human serous ovarian carcinoma cell lines OV-MZ-6 and OVCAR-3 were stimulated with the inflammatory cytokine TNF-α and/or treated with selective ADAM10 (GI-254023X) or ADAM17 (TAPI-2) inhibitors. CX3CL1 expression was quantified by ELISA for soluble CX3CL1 and by flow cytometry for membrane-bound CX3CL1. Transient overexpression of CX3CL1 was achieved using a pCMV6-CX3CL1 expression vector. NK cell–mediated cytotoxicity was assessed using a europium-based DELFIA cytotoxicity assay at multiple effector-to-target ratios. CX3CL1 dependency was further evaluated by antibody-mediated blockade during cytotoxicity assays.Results In OV-MZ-6 cells, TNF-α stimulation markedly increased CX3CL1 expression and was associated with enhanced NK cell–mediated cytotoxicity. Antibody-mediated blockade of CX3CL1 partially abrogated the enhanced cytotoxicity, indicating a functional contribution of tumor cell–associated mCX3CL1. Inhibition of ADAM17 significantly reduced the release of soluble CX3CL1 while concomitantly increasing membrane-bound CX3CL1, as demonstrated by flow cytometry. This resulted in a significant enhancement of NK cell–mediated lysis. In contrast, ADAM10 inhibition led to a lesser reduction of soluble CX3CL1 and a less pronounced increase in mCX3CL1 and showed no significant effect on NK cell–mediated lysis. Transient overexpression of CX3CL1 significantly increased NK cell–mediated lysis in OV-MZ-6 cells, whereas additional ADAM17 inhibition did not further augment cytotoxicity. In OVCAR-3 cells, TNF-α induced only minimal CX3CL1 expression and did not enhance NK cell–mediated lysis; however, CX3CL1 overexpression resulted in a non-significant trend toward increased cytotoxicity.Conclusion Membrane-bound CX3CL1 enhances NK cell–mediated cytotoxicity against ovarian cancer cells in a cell line–dependent manner. ADAM17 inhibition may represent a strategy to increase mCX3CL1 and improve NK cell–mediated tumor cell lysis in vitro. Further studies are required to understand the distinct functional roles of membrane-bound and soluble CX3CL1 and to explore their potential for future pharmacological exploitation.