(P 85) TARGETING CD99 ELICITS EWING SARCOMA CELL PHAGOCYTOSIS BY UNDIFFERENTIATED AND M2-LIKE MACROPHAGES AND INDUCES MACROPHAGE POLARIZATION TOWARD A M1-LIKE PHENOTYPE

Objective: Tissue microenvironment affects the functional states of cancer cells and their response to treatments but exploiting it for therapy has remained a challenge. In the tumor microenvironment of Ewing sarcoma (EWS), the second most common pediatric bone tumor, macrophages are the most abundant immune cells and were found to be associated with worse patient’s prognosis. In this study, we identified CD99, a hallmark of EWS cells that is also expressed on macrophages, as a novel relevant target that may be beneficial to improve immunotherapy of EWS.

Methods: Human M0 macrophages were differentiated ex vivo from circulating monocytes by culturing for seven days, checked for the expression of macrophage markers, and then used in a co-culture model to investigate the elimination of EWS cells treated or not with different anti-CD99 ABsbs or exosomes from CD99-genetically modified EWS cells: phagocytosis; time lapse-confocal microscopy; vital cell sounting were analyzed together with markers of M1-likevs M2-like phenotype (i.e.CD80, CD86, CD163, CD206, IL1β, IL6, IL10 and TNFα) by PCR and/or ELISA. Expression of eat-me and/or dont-eat me signals on EWS cells after anti-CD99 approaches was measured by cytoflorometry.EWS xenografts were treated with anti-CD99 Abs to check tumor growth and macrophage infiltration.

Results: Targeting CD99 on EWS cells downregulates the expression of the “don’t eat-me” CD47 molecule while increasing the exposure of the “eat.me” phospahtidylserine and calreticulin molecules on the outer leaflet of tumor cell membrane and it triggers the engulfment and digestion of EWS cells by macrophages. In addition, CD99 ligation can result in the re-programming of undifferentiated M0 macrophages, or M2-like macrophage toward the inflammatory M1-like phenotype. This was confirmed also in vivo and the final result is a a significant inhibition of EWS tumor growth. In addition, experiments with exosomes modified for CD99 expression are ongoing.to verify the effects on tumor and macrophage recipient cells.

EWS xenografts treated with anti-CD99 Abs confirmed the recruiment of M1-like macrophages and inhibition of growth

Conclusion: This work reveals a previously unrecognized function for CD99,. which induces tumor cell phagocytosis and re-programs of macrophages, thus suggesting that approaches against CD99 may be exploited to improve immunotherapeutic strategies in EWS.