Transcriptomic profiling of CXCR3+/- SUM-159-LM1 breast cancer cells

Cancer-associated fibroblasts promote the development of many primary malignancies, but their function in metastatic progression is poorly understood. Here, we demonstrate that colonization of the lungs by metastatic breast cancer cells induces an inflammatory phenotype in lung fibroblasts. CXCL9 and CXCL10 are induced in an NFκB-dependent manner in metastasis-associated fibroblasts in response IL-1α and IL-1β secreted by disseminated breast cancer cells. We find that the chemokine receptor CXCR3, that binds CXCL9/10, is expressed in a small subset of breast cancer cells that exhibits greater tumor-initiating ability when co-transplanted with fibroblasts. CXCR3-expressing cancer cells maintain JNK signaling that drives IL-1A/B expression, and thus rendering this subpopulation efficient in both inducing CXCL9/10 in lung fibroblasts and responding to the chemokines. Importantly, disruption of the CXCL9/10-CXCR3 axis significantly reduces metastatic colonization in xenograft and syngeneic mouse models suggesting an essential role of this paracrine crosstalk in metastatic progression and a potential therapeutic vulnerability for the treatment of metastatic breast cancer. For profiling of CXCR3+ and CXCR3- breast cancer cells, SUM-159-LM1 (SUM-LM1) cells were seeded into ultra-low attachment cell culture flasks in Onco2 medium, consisting of HuMEC-medium (Invitrogen) supplemented 50 U/ml penicillin (Sigma-Aldrich), 50 μg/ml streptomycin (Sigma-Aldrich), 10 ng/ml basic fibroblast growth factor (bFGF, Invitrogen), 20 ng/ml EGF (Sigma-Aldrich), 5 μg/ml human insulin (Sigma-Aldrich) and 2 % vol/vol B27 (Life Technologies) at a density of 25,000 cells/ml (10 ml per flasks) and incubated at 37 °C for 1 week. Cells were pelleted and dissociated using StemPro Accutase (Life Technologies), counted and stained with 5 μg PE-conjugated anti-human CD183 (CXCR3) antibody (clone G025H7, BioLegend) or PE-conjugated mouse IgG1, κ isotype control (BioLegend) per 1 Mio cells in 100 μl FACS buffer for 30 min on ice in the dark. CXCR3+ and CXCR3- cells were sorted into Arcturus PicoPure Extraction Buffer (ThermoFisher Scientific) on a BD FACSAria1 machine. Three biological replicates were analyzed (3 independent sorts).