Abstract P5-01-01: Lymphoid tissue inducer cells: Identification of a novel immune cell within the breast tumour microenvironment and its role in promoting tumour cell invasion
Authors: Irshad S , Lawler K , Evans R , Flores-Borja F , Monypenny J , Grigoriadis A , Fruhwith G , Poland S , Barber P , Vojnovic B , Ellis P , Tutt A , Ng T 
Published: August 2014

Abstract
Within breast cancers, trans-endothelial migration of tumour cells through lymphatic vessels is the first step to tumour dissemination and lympho-vascular invasion has been shown to stratify breast cancer phenotypes into distinct prognostic groups. The exact molecular mechanisms mediating tumor cell entry and persistence within the lymphatic system remain unclear. Lymphoid tissue inducer (LTi) cells are members of the emerging family of retinoic acid related orphan receptor (ROR)gt+ innate lymphoid cells (ILCs), and their interaction with stromal cells induces production by the stromal cells of VEGF-C and “lymphoid” chemokines, essential for lymphoid organogenesis. We hypothesized that tumour cells manipulate the normal processes that govern chemokine-dependent, trans-lymphatic migration of immune cells, including LTi cells; shaping its microenvironment. Results: We analyzed the expression of lymphoid chemokines genes (CXCL12, CXCL13, CCL19, CCL20 and CCL21) and their corresponding receptors (CXCR4, CXCR5, CCR6 and CCR7) within the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) Tissue Bank. An unsupervised hierarchical cluster analysis revealed co-expression of these genes, categorizing breast tumors as relatively high/low expressors. Tumors exhibiting relatively high expression of these genes were found to be enriched for “basal-like” breast cancers according to PAM50 intrinsic subtype assignments. Immunofluorescence of the primary tumour sections identified cells that were comparable in phenotype to LTi cells. In a blinded study, we observed that patients with high LTi counts within the tumour microenvironment were also likely to have a gene expression corresponding to high expression for the lymphoid chemokines. IHC for the lymphatic marker, podoplanin found that the LTi count correlated with both an increased lymphatic vessel density and tumor invasion into lymphatic vessels. Within the basal and HER2+ve subtypes, patients with more than 4 lymph nodes were found to exhibit higher numbers of intratumoural LTi cells. In vitro studies, alongside multi-photon in vivo imaging were performed to investigate the interaction between intra-tumoural LTi and mesenchymal stromal cells. CXCL13 was shown to be essential for LTi clustering around stromal cells in vitro, and, the administration of a blocking antibody in vivo delayed the onset of lymph node metastasis in a murine mammary tumour (4T1.2) model. CXCLl3 has been identified as having independent prognostic significance in breast cancer, but we and others report that breast cancer cell lines are not the source of CXCL13. We show that an increase in stromal CXCL13 concentration within the tumour microenvironment following LTi recruitment promotes an EMT phenotype in the 4T1.2 cancer cell line, possibly via activation of the RANKL/RANK axis promoting tumorigenesis. We report for the first time, the identification of LTi cells within the human breast cancer tumour microenvironment and propose a pivotal role for these cells, through stromal cell interactions in the tumour microenvironment, in facilitating lymphatic invasion of tumour cells by modulation of the local lymphoid chemokine profile.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-01.