Volume 138, Issue 4, Pages 1429-1440 (April 2010) Biomolecular Network Reconstruction Identifies T-Cell Homing Factors Associated With Survival in Colorectal Cancer  Bernhard Mlecnik, Marie Tosolini, Pornpimol Charoentong, Amos Kirilovsky, Gabriela Bindea, Anne Berger, Matthieu Camus, Mélanie Gillard, Patrick Bruneval, Wolf–Herman Fridman, Franck Pagès, Zlatko Trajanoski, Jérôme Galon  Gastroenterology  Volume 138, Issue 4, Pages 1429-1440 (April 2010) DOI: 10.1053/j.gastro.2009.10.057 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Biomolecular network using gene expression data in a cohort of patients with CRC and predicted gene–gene interactions based on available knowledge. The network illustrated experimental data (colored nodes) and in silico prediction (white nodes surrounded by a red border). The gene expression data were acquired by a reverse-transcription PCR study for 47 genes in a cohort of 108 CRC patients (Supplementary Table 1). The network was reconstructed based on a subset of 12 genes, which reached a significant log-rank level for DFS. The network shows the top genes predicted in silico plus the genes analyzed by reverse-transcription PCR. CX3CL1 was the top predicted gene. All nodes surrounded by a red border were predicted by STRING. The node sizes of the network are based on the HR for DFS (Supplementary Table 1). Nodes surrounded by a black border had significant log-rank P values (P < .05). The edge weights of the network are based on the integrated score of the pairwise uncentered Pearson correlation value between the 47 reverse-transcription PCR genes and the combined edge scores for all genes predicted in silico provided by STRING (see Supplementary Materials and Methods section for details). The network node layout was based on Gene Ontology (go), gene expression correlations (blue lines), STRING scores (gray lines), and the integrated association strength between genes (edge thickness). Edge thickness levels show the relation strength based on the integrated score value between the nodes. Nodes are colored based on multiple occurrences in different GO categories (Supplementary Table 2). Gastroenterology 2010 138, 1429-1440DOI: (10.1053/j.gastro.2009.10.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Gene expression levels from 108 colorectal tumors (cohort 1) were analyzed by real-time quantitative PCR. (A) Kaplan–Meier curves for the duration of DFS, according to the expression of the predicted genes (CX3CL1, CXCL9, CXCL10, CCL2, CCL5, CCL11, and MADCAM1) were performed. Patients with high (Hi) expression for both genes (red line) or low (Lo) expression for both gene densities (black line), and heterogeneous expression (HiLo or LoHi, green line) are represented. (B) HRs were calculated for high and low gene expression compared with the whole cohort (108 patients). A HR-matrix (heatmap) followed by unsupervised hierarchical clustering was represented from favorable prognosis: HR, 0.4 (red) to poor prognosis HR, 2.5 (blue). All HR with HR less than 0.55 or HR greater than 1.66, were significant. Gastroenterology 2010 138, 1429-1440DOI: (10.1053/j.gastro.2009.10.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 (A) Tissue-microarray spots of representative tumors with low (top) or high (bottom) CX3CL1, CXCL9, and CXCL10 gene expression are illustrated. Stainings for tumor cells (cytokeratin-8+, blue staining) and cytotoxic T cells (CD8+, brown staining) were performed. (B) Comparison of immune cell densities as measured by flow cytometry from 39 freshly resected tumors. Cytotoxic (CD3+CD8+), memory (CD3+CD45RO+), and TCRαβ (CD3+TCRαβ+) T cells were analyzed in the tumors from patients with high - (blue histogram) or low- (white histogram) CX3CL1, CXCL9, or CXCL10 gene expression. (C) Hierarchical clustering of correlation matrix of the flow cytometry data from 39 freshly resected tumors. Pearson correlation coefficients (R) were calculated between the combination of 149 markers for major immune cell populations (“total” prefix) and specific T-cell subpopulations and CX3CL1, CXCL9, and CXCL10. Correlation coefficients were plotted with negative correlation (green), positive correlation (red), and R = 0 (yellow), in matrix representation followed by unsupervised Spearman hierarchical clustering. Six major clusters (A–F) are illustrated. (D) Comparison of the mean of immune cell densities (cell/mm2) as measured by tissue-microarrays from 108 paraffin-embedded tumors. Cytotoxic (CD8+, GZMB+), Th1 (T-Bet+), and memory (CD45RO+) T cells were analyzed in the tumors from patients with high- (blue histogram) or low- (white histogram) CX3CL1, CXCL9, or CXCL10 gene expression. *P < .05. Gastroenterology 2010 138, 1429-1440DOI: (10.1053/j.gastro.2009.10.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 T-cell repertoire analyses were performed on tumors from 10 randomly selected CRC patients using the quantitative expression of the 26 TCR Vβ chain families. Results were expressed in a Vβ/hypoxanthine phosphoribosyltransferase (HPRT) ratio. These quantitative data were represented by the height of the peak in the (A and C) TcLandscapes and on the (B and D) histograms. T-cell repertoire analysis was performed by combining qualitative alterations of Vβ use at the CDR3 length level (13 different CDR3 lengths) with the magnitude of expression of each Vβ mRNA species. (A and C) The CDR3 lengths were represented in the TcLandscapes. (E) Pearson correlation coefficients (R) were calculated between the quantity of all Vβ for each of the 13 different CDR3 lengths and CX3CL1, CXCL9, and CXCL10 gene expression. Correlation coefficients were plotted with negative correlation (green), positive correlation (red), and R = 0 (yellow), in matrix representation followed by unsupervised Spearman hierarchical clustering. Six major clusters (A–F) were represented on the matrix. Gastroenterology 2010 138, 1429-1440DOI: (10.1053/j.gastro.2009.10.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 (A and B) The CDR3 lengths of the TCRs from 10 randomly selected CRC patients (A–J) are represented in the histograms. (C and D) Kaplan–Meier curves illustrate the overall survival of patients according to particular TCR expression at the median of the dataset. (C) Three TCRs (Vβ2L04, Vβ5.2L05, or Vβ5.2L04) not associated with CX3CL1 gene expression, and (D) 3 TCRs (Vβ5.2L08, Vβ2L03, and Vβ2L07) significantly increased in patients with high expression of CX3CL1. Gastroenterology 2010 138, 1429-1440DOI: (10.1053/j.gastro.2009.10.057) Copyright © 2010 AGA Institute Terms and Conditions