An integer linear programming approach for finding deregulated subgraphs in regulatory networks

Deregulation of cell signaling pathways plays a crucial role in the development of tumors. The identification of such pathways requires effective analysis tools that facilitate the interpretation of expression differences. Here, we present a novel and highly efficient method for identifying deregulated subnetworks in a regulatory network. Given a score for each node that measures the degree of deregulation of the corresponding gene or protein, the algorithm computes the heaviest connected subnetwork of a specified size reachable from a designated root node. This root node can be interpreted as a molecular key player responsible for the observed deregulation. To demonstrate the potential of our approach, we analyzed three gene expression data sets. In one scenario, we compared expression profiles of non-malignant primary mammary epithelial cells derived from BRCA1 mutation carriers and of epithelial cells without BRCA1 mutation. Our results suggest that oxidative stress plays an important role in epithelial cells of BRCA1 mutation carriers and that the activation of stress proteins may result in avoidance of apoptosis leading to an increased overall survival of cells with genetic alterations. In summary, our approach opens new avenues for the elucidation of pathogenic mechanisms and for the detection of molecular key players

Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model

<div><p>CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.</p></div

Tumor and host samples show distinct cytokine secretion profiles upon RG7356 treatment.

<p>(A) Cluster analysis of Luminex cytokine panel. Samples were analyzed at 8 and 168 hours after treatment in both human- and mouse-specific assays. Rows represent analytes, columns represent samples. Only analytes covered by both panels were used for clustering. Circles represent human samples, triangles represent mouse samples; open and solid symbols represent 8 and 168 hours of treatment, respectively, while color codes depict the different treatment groups. (B) and (C) Side-by-side comparison of human (left) and mouse (right) analyte concentrations (pg/ml) for the three different treatments. Shown are concentrations for GM-CSF (B) and MIP-1a (C).</p

The increased amounts of MIP-1a and MCP-1 decline to baseline levels after one week.

<p>Time-course study of MDA-MB231 xenografts over one week after treatment with RG7356. MIP-1a (A) and MCP-1 (B) concentrations were analyzed in obtained tumor lysates by ELISA.</p

RG7356 treatment leads to phagocytosis of tumor cells.

<p>(A) <i>Ex vivo</i> analysis of MDA-MB-231 xenograft immune infiltrates. Left panel: percentage of CD45⁺CD11b⁺ myeloid cells in total infiltrate. Right panel: tumor-associated macrophages (F4/80^high) and myeloid-derived suppressor cells (F4/80^low) as percentage of the myeloid compartment. (B) RG7356-opsonized MDA-MB-231 and HCC-1937 cells were pre-incubated with macrophages and phagocytosis was assessed by flow cytometry after 30 min as described in Material and Methods. Results represent the outcome of 3 independent experiments. (C) MDA-MB-231 (upper panels) and HCC-1937 (lower panels) cells were pre-incubated with RG7356 (left panels) or isotype control (right panels) and cultured together with macrophages, as described in Materials and Methods. Pictures were taken every 30 min for 10 hours (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159716#pone.0159716.s018" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159716#pone.0159716.s019" target="_blank">S2</a> Videos). Pictures shown here correspond to the 0.5h time point and are representative of at least 3 independent experiments. Arrowheads indicate engulfed tumor cells. Macrophages are shown in yellow, tumor cells in green. (D) Time-course quantification of total tumor cells (CFMDA signal) in MDA-MB-231/macrophages co-cultures in the presence of RG7356 (open circles) or isotype control (filled circles).</p

Tumor responses to RG7356 involve activation, migration and differentiation of immune cells.

<p>Tumor responses to RG7356 involve activation, migration and differentiation of immune cells.</p

Host responses to RG7356 involve recruitment of immune cells and cell survival.

<p>Host responses to RG7356 involve recruitment of immune cells and cell survival.</p

RG7356 treatment leads to a deregulation of genes in both tumor and host at early time points.

<p>(A) Number of differentially expressed human and mouse transcripts at 3 different time points of anti-CD44 treatment bearing the IgG1 backbone compared to vehicle control. (B) Number of differentially expressed human and mouse transcripts at 3 different time points of anti-CD44 treatment bearing the IgG4 backbone compared to vehicle control. (C) Heatmap view of predicted activity of biological processes obtained from IPA® downstream processes analysis. Activated processes are shown in different shades of orange, inhibited processes are shown in different shades of blue. If no activity assessment was possible, the biological process is shown in grey.</p