Extended Data Fig. 5: Effect of IDO inhibition and chemotherapy on lymph-node metastasis.

a, Expression of Ido1 measured by qRT-PCR from tumour (T) and non-tumour (NT) regions of metastatic LNs and from primary tumour of mice bearing 4T1 cells (n = 6 biological replicates for tumour and non-tumour, 4 biological replicates for tumour). Relative gene expression normalized to Gapdh and calculated using the 2ΔCT method. b, Schematic of treatment regimen. c, Measurement of primary tumour volume at time of resection for each treatment group. d, Weight of tumour draining axillary lymph node after treatment. Untreated (n = 16); 1M-DL-Try (n = 16); CYC (cyclophosphamide) (n = 15); 1M-DL-Try + CYC (n = 15). e, Incidence of metastatic lymph nodes, as determined by cytokeratin staining of serial lymph node sections. f, Quantification of pulmonary macrometastatic nodules after treatment with indicated therapies. Untreated (n = 16); 1M-DL-Try (n = 16); CYC (n = 15); 1M-DL-Try + CYC (n = 16). g, Representative immunofluorescent staining of T cells (CD3, red) and cancer cells (cytokeratin, green) in lymph nodes from control (untreated), or treated (as indicated) animals. The inset shows single tumour cells within lymph nodes of cyclophosphamide-treated animals. DAPI (blue) stains all nucleated cells. Scale bars, 500 µm. Inset scale bar = 50 µm Four images were taken per group. Data plotted as the mean±s.e.m. Statistical significance was tested by one-way ANOVA with Tukey’s Honestly Significant Difference post-hoc test (c,d,f) and between categorical variables in e using a chi-square test.