ORIGINAL PAPER Journal of Pathology J Pathol 2011; 223: 347–357 Published online 10 December 2010 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.2819 Pro-inflammatory chemokine – chemokine receptor interactions within the Ewing sarcoma microenvironment determine CD8 + T-lymphocyte infiltration and affect tumour progression Dagmar Berghuis, 1 Susy J Santos, 2 Hans J Baelde, 1 Antonie HM Taminiau, 3 R Maarten Egeler, 2 Marco W Schilham, 2 Pancras CW Hogendoorn 1 * and Arjan C Lankester 2 1 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands 3 Department of Orthopedics, Leiden University Medical Center, Leiden, The Netherlands *Correspondence to: Pancras CW Hogendoorn, Professor of Pathology, Department of Pathology, Leiden University Medical Center, L1-Q, PO Box 9600, 2300 RC Leiden, The Netherlands. e-mail: p.c.w.hogendoorn@lumc.nl Abstract Ewing sarcoma is an aggressive round cell sarcoma with poor patient prognosis, particularly in cases of advanced- stage disease. Dynamic tumor-host immune interations within the tumor microenvironment may polarize in situ immune responses and shape tumor development and/or progression. To gain insight into the nature of tumour–host immune interactions within the Ewing sarcoma microenvironment, the presence and spatial dis- tribution of infiltrating CD8 + /CD4 + T-lymphocytes were evaluated in therapy-naive Ewing sarcoma. Expression profiling of 40 different chemokines and several chemokine receptors was performed in therapy-naive tumours and cell lines by qPCR, immunohistochemistry, and flow cytometry. Considerable inter-tumour variation was observed regarding density, type, and distribution of infiltrating T-lymphocytes. Tumour-infiltrating T-cells contained significantly higher percentages of CD8 + T-lymphocytes as compared to stroma-infiltrating cells, suggesting preferential migration of this T-cell type into tumour areas. Gene expression levels of several type 1-associated, pro-inflammatory chemokines (CXCR3- and CCR5-ligands CXCL9, CXCL10, and CCL5 ) correlated positively with infiltrating (CD8 + ) T-lymphocyte numbers expressing corresponding chemokine receptors. Survival analyses demonstrated an impact of tumour-infiltrating, and not stroma-infiltrating, CD8 + T-lymphocytes on tumour progression. At protein level, both tumour and stromal cells expressed the IFNγ-inducible chemokines CXCL9 and CXCL10. CCR5-ligand CCL5 was exclusively expressed by non-tumoural stromal/infiltrating cells. Together, our results indicate that an inflammatory immune microenvironment with high expression of type 1-associated chemokines may be critical for the recruitment of (CD8 + ) T-lymphocytes expressing corresponding chemokine receptors. The observed impact of tumour-infiltrating (CD8 + ) T-lymphocytes is consistent with a role for adaptive anti-tumour immunity in the prevention of Ewing sarcoma progression. Recognition of the merits and exploitation/induction of an inflammatory microenvironment may improve the efficacy of natural immune responses against, and (adoptive) immunotherapeutic approaches for, Ewing sarcoma. Copyright  2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: Ewing sarcoma; bone/soft tissue tumour; chemokines; infiltrating T-lymphocytes; tumour microenvironment; immunotherapy; quantitative PCR; multicolour immunohistochemistry; flow cytometry; patient prognosis Received 16 August 2010; Revised 29 October 2010; Accepted 29 October 2010 No conflicts of interest were declared. Introduction Ewing sarcoma (EWS) is an aggressive round cell sar- coma characterized by specific gene fusions most com- monly containing TET gene family products, though rarely other activating transcription factors [1–3]. It arises most commonly in children, adolescents, and young adults. Despite multimodal therapy, no signifi- cant improvements have been achieved during the past decade with regard to patient survival [4]. Patients with refractory or (primary) metastatic EWS have the most unfavourable prognosis, with a 5-year overall survival of less than 30%, which has recently been demonstrated to be independent of EWS–ETS fusion type [5,6]. Dynamic and reciprocal interactions between tumour cells and components of the tumour microenviron- ment coordinate events critical to tumour progression [7,8]. Tumour–host immune interactions within the tumour microenvironment may polarize in situ immune responses and shape tumour development and evolu- tion [9,10]. Recruitment of tumour-reactive immune cell subsets into the tumour microenvironment may be of vital importance for initiation or execution of the effector phase of anti-tumour immunity. Indeed, studies on several human solid tumours have demonstrated that type and density as well as spatial distribution and functional orientation of infiltrating lymphocytes correlate with tumour progression and patient progno- sis [11 – 13]. Chemokines produced by both tumour and Copyright  2010 Pathological Society of Great Britain and Ireland. J Pathol 2011; 223: 347–357 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com