Title: <scp>VEGF</scp> blockade enhances the antitumor effect of <scp> BRAF <sup>V</sup> </scp> <sup>600E</sup> inhibition
Abstract: Research Article14 December 2016Open Access Source DataTransparent process VEGF blockade enhances the antitumor effect of BRAFV600E inhibition Valentina Comunanza Valentina Comunanza Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Davide Corà Davide Corà Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Center for Molecular Systems Biology, University of Torino, Orbassano, Italy Search for more papers by this author Francesca Orso Francesca Orso Center for Molecular Systems Biology, University of Torino, Orbassano, Italy Molecular Biotechnology Center (MBC), Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy Search for more papers by this author Francesca Maria Consonni Francesca Maria Consonni Humanitas Clinical and Research Center, Rozzano, Italy Search for more papers by this author Emanuele Middonti Emanuele Middonti Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Federica Di Nicolantonio Federica Di Nicolantonio orcid.org/0000-0001-9618-2010 Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Anton Buzdin Anton Buzdin Laboratory of Bioinformatics, D. Rogachyov Federal Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia National Research Centre "Kurchatov Institute", Centre for Convergence of Nano-, Bio-, Information and Cognitive Sciences and Technologies, Moscow, Russia Search for more papers by this author Antonio Sica Antonio Sica Humanitas Clinical and Research Center, Rozzano, Italy Department of Pharmaceutical Sciences, Università del Piemonte Orientale "Amedeo Avogadro", Novara, Italy Search for more papers by this author Enzo Medico Enzo Medico orcid.org/0000-0002-3917-2438 Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Dario Sangiolo Dario Sangiolo Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Daniela Taverna Daniela Taverna Center for Molecular Systems Biology, University of Torino, Orbassano, Italy Molecular Biotechnology Center (MBC), Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy Search for more papers by this author Federico Bussolino Corresponding Author Federico Bussolino [email protected] Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Center for Molecular Systems Biology, University of Torino, Orbassano, Italy Search for more papers by this author Valentina Comunanza Valentina Comunanza Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Davide Corà Davide Corà Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Center for Molecular Systems Biology, University of Torino, Orbassano, Italy Search for more papers by this author Francesca Orso Francesca Orso Center for Molecular Systems Biology, University of Torino, Orbassano, Italy Molecular Biotechnology Center (MBC), Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy Search for more papers by this author Francesca Maria Consonni Francesca Maria Consonni Humanitas Clinical and Research Center, Rozzano, Italy Search for more papers by this author Emanuele Middonti Emanuele Middonti Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Federica Di Nicolantonio Federica Di Nicolantonio orcid.org/0000-0001-9618-2010 Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Anton Buzdin Anton Buzdin Laboratory of Bioinformatics, D. Rogachyov Federal Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia National Research Centre "Kurchatov Institute", Centre for Convergence of Nano-, Bio-, Information and Cognitive Sciences and Technologies, Moscow, Russia Search for more papers by this author Antonio Sica Antonio Sica Humanitas Clinical and Research Center, Rozzano, Italy Department of Pharmaceutical Sciences, Università del Piemonte Orientale "Amedeo Avogadro", Novara, Italy Search for more papers by this author Enzo Medico Enzo Medico orcid.org/0000-0002-3917-2438 Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Dario Sangiolo Dario Sangiolo Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Search for more papers by this author Daniela Taverna Daniela Taverna Center for Molecular Systems Biology, University of Torino, Orbassano, Italy Molecular Biotechnology Center (MBC), Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy Search for more papers by this author Federico Bussolino Corresponding Author Federico Bussolino [email protected] Department of Oncology, University of Torino, Candiolo, Italy Candiolo Cancer Institute IRCCS, Candiolo, Italy Center for Molecular Systems Biology, University of Torino, Orbassano, Italy Search for more papers by this author Author Information Valentina Comunanza1,2, Davide Corà1,2,3, Francesca Orso3,4, Francesca Maria Consonni5, Emanuele Middonti1,2, Federica Di Nicolantonio1,2, Anton Buzdin6,7, Antonio Sica5,8, Enzo Medico1,2, Dario Sangiolo1,2, Daniela Taverna3,4 and Federico Bussolino *,1,2,3 1Department of Oncology, University of Torino, Candiolo, Italy 2Candiolo Cancer Institute IRCCS, Candiolo, Italy 3Center for Molecular Systems Biology, University of Torino, Orbassano, Italy 4Molecular Biotechnology Center (MBC), Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 5Humanitas Clinical and Research Center, Rozzano, Italy 6Laboratory of Bioinformatics, D. Rogachyov Federal Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia 7National Research Centre "Kurchatov Institute", Centre for Convergence of Nano-, Bio-, Information and Cognitive Sciences and Technologies, Moscow, Russia 8Department of Pharmaceutical Sciences, Università del Piemonte Orientale "Amedeo Avogadro", Novara, Italy *Corresponding author. Tel: +39 011 9933347; E-mail: [email protected] EMBO Mol Med (2017)9:219-237https://doi.org/10.15252/emmm.201505774 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The development of resistance remains a major obstacle to long-term disease control in cancer patients treated with targeted therapies. In BRAF-mutant mouse models, we demonstrate that although targeted inhibition of either BRAF or VEGF initially suppresses the growth of BRAF-mutant tumors, combined inhibition of both pathways results in apoptosis, long-lasting tumor responses, reduction in lung colonization, and delayed onset of acquired resistance to the BRAF inhibitor PLX4720. As well as inducing tumor vascular normalization and ameliorating hypoxia, this approach induces remodeling of the extracellular matrix, infiltration of macrophages with an M1-like phenotype, and reduction in cancer-associated fibroblasts. At the molecular level, this therapeutic regimen results in a de novo transcriptional signature, which sustains and explains the observed efficacy with regard to cancer progression. Collectively, our findings offer new biological rationales for the management of clinical resistance to BRAF inhibitors based on the combination between BRAFV600E inhibitors with anti-angiogenic regimens. Synopsis A combination of bevacizumab and vemurafenib analog PLX4720, which targets BRAF(V600E), sustains the decrease of tumor hypoxia, accumulation of antitumor macrophages, and reduction in cancer-associated fibroblasts, delaying the onset of PLX4720 resistance. The association between a BRAF inhibitor with an antibody neutralizing VEGF induces a de novo genetic program, which is not observed in the presence of either agent alone. Provides a combinatorial benefit inducing an apoptotic program in cancer cells. Promotes vascular normalization improving tissue perfusion and oxygenation. Reprograms the tumor microenvironment promoting ECM remodeling and favoring M1-like macrophage infiltration. Delays the onset of acquired resistance to the BRAF inhibitor. Introduction Activating mutations in the BRAF oncogene occur in approximately 7% of human malignancies, including 50–60% of melanomas and 5–8% of colorectal cancers (CRCs) (Davies et al, 2002). BRAF mutations are associated with adverse clinical outcomes in melanoma, thyroid carcinoma, non-small-cell lung cancer, and CRC (Cantwell-Dorris et al, 2011). The most frequent BRAF mutation (V600E) (Halilovic & Solit, 2008) affects the kinase domain, leading to constitutive activation of the protein. Oncogenic BRAF induces downstream phosphorylation of MEK and ERK, which in turn triggers cell autonomous proliferation even in the absence of extracellular growth factors (Davies et al, 2002). Specific inhibitors of BRAFV600E, including vemurafenib and dabrafenib, have been approved for treating BRAF-mutant metastatic melanomas (Bollag et al, 2010; Flaherty et al, 2012; Hauschild et al, 2012). Although these drugs show remarkable clinical efficacy and improve overall survival, almost all patients develop resistance and subsequently relapse (Bollag et al, 2010; Flaherty et al, 2012). A combination of BRAF and MEK inhibition further improves time to progression and overall survival in patients with metastatic melanomas when compared with single-agent BRAF inhibition (Paraiso et al, 2010). It is likely that the limited efficacy of combinations targeting a single oncogenic pathway is due to the plasticity and ability of cancer cells to circumvent such a blockade. Additional strategies must be exploited to increase the efficacy of BRAFV600E inhibitors and circumvent or delay the onset of resistance. Several studies on the effects of BRAFV600E inhibitors on the cellular transcriptional landscape have envisaged new, attractive preclinical combinations with molecules affecting specific processes, including glucose metabolism (Parmenter et al, 2014), the immune response (Hu-Lieskovan et al, 2014), and autophagy (Goodall et al, 2014), to improve the antitumor effects of BRAFV600E inhibitors. In physiological angiogenesis, the effects of pro-angiogenic molecules are counterbalanced by those of endogenous inhibitors. During tumor angiogenesis, this balance is tipped in favor of new vessel formation. However, the resulting vessels are highly abnormal both structurally and functionally. This balance could be restored by removing the excess of VEGF-A (VEGF) or by blocking VEGF signaling, which would induce pruning of abnormal vessels, resulting in vascular normalization characterized by improved perfusion and alleviation of hypoxia. Hence, strategies that favor vascular normalization could improve the efficacy of cancer therapies (Jain, 2014). We and others have previously demonstrated that BRAFV600E triggers an angiogenic response by modifying the expression profile of angiogenic inducers (Durante et al, 2011; Bottos et al, 2012; Sadow et al, 2014). Thus, enhanced angiogenesis and tumor–stroma cross-talk may represent an additional therapeutic target in the context of BRAFV600E-driven tumors. We reported that BRAFV600E inhibition by the vemurafenib analog PLX4720 in xenografts did not reduce the number of tumor capillaries but instead favored vascular normalization (Bottos et al, 2012). Based on this premise, we hypothesized that BRAF-targeted inhibitors could cooperate with anti-angiogenic regimens in the treatment of BRAF-mutant tumors. Specifically, in this work, we investigated the combined effects of PLX4720 and bevacizumab, an anti-VEGF humanized monoclonal antibody, in xenograft models of melanoma and CRC. We report that this dual treatment induces a new genetic program that regulates myeloid cell recruitment and extracellular matrix remodeling and is more efficient than either single agent for controlling tumor growth and the onset of resistance. Results Dual BRAFV600E and VEGF targeting provides a combinatorial benefit against BRAFV600E mutants tumor growth in vivo Cohorts of CD1-immunocompromised mice bearing A375 BRAFV600E-mutant melanoma xenografts were treated with PLX4720, bevacizumab, or a combination of both (COMBO). PLX4720 or bevacizumab alone caused a clear delay in tumor growth, resulting in a 45% or 56% reduction in tumor volume, respectively, compared with vehicle alone. However, neither single treatment induced a regression of initial tumor size. Concurrent administration of the two drugs at the same doses increased antitumor activity, with 88% reduction in tumor volume compared to vehicle and shrinking by 58% the initial tumor size (Fig 1A). Similar results were obtained by treating CRC COLO205 xenografts. PLX4720, bevacizumab, and COMBO, respectively, inhibited of 61, 57, and 80% tumor growth (Fig 1B). These data indicate that bevacizumab improves the efficacy of the therapeutic inhibition of the oncogenic driver BRAFV600E. Figure 1. Effect of the combination of PLX4720 and bevacizumab on the growth of A375, COLO205, and MC-1 cells harboring BRAFV600E transplanted in athymic nude mice Mice bearing established A375 were treated with vehicle (n = 7), PLX4720 (n = 5); bevacizumab (n = 7) or COMBO (n = 5). Tumor growth is expressed as % change of the initial tumor. **P < 0.01, ***P < 0.001 versus vehicle (PLX4720 P = 0.0049; bevacizumab P = 0.0003; COMBO P = 4.86E-06), ŦP = 0.015 compared to PLX4720. Mice bearing established COLO205 were treated with vehicle (n = 7), PLX4720 (n = 8); bevacizumab (n = 7) or COMBO (n = 8). Tumor growth is expressed as % change of the initial tumor. ***P < 0.001 versus vehicle (PLX4720 P = 2.75E-05; bevacizumab P = 2.96E-05; COMBO P = 2.13E-07), ŦP = 0.039 compared to PLX4720. Melanoma MC-1 cells (5 × 105) were injected into the tail vein of CD1 mice. Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs. The mice analyzed were as follows: start point, n = 6; vehicle, n = 7; PLX4720, n = 6; bevacizumab, n = 6; COMBO, n = 7. *P = 0.031 versus vehicle. Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated. Bar graphs indicate the Ki67+ area/tumor area (n = 4 tumors). ***P < 0.001 versus vehicle (PLX4720 P = 2.83E-08; bevacizumab P = 4.25E-13; COMBO P = 6.16E-08). Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated. Bar graphs indicate the TUNEL+ area/tumor area (n = 3 tumors). ***P = 2.07E-10 versus vehicle. Data information: The scale bars represent 1 cm (A, B), 100 μm (D), and 50 μm (E). The results are given as the mean ± SEM. Significance was assessed by one-way ANOVA test followed by post hoc pairwise analysis test (A–E). Download figure Download PowerPoint Then, we evaluated the effect of PLX4720, bevacizumab, and COMBO on lung colonization of the MC-1 cell line, which is an highly metastatic variant of A375 cells (Orso et al, 2016) (Fig 1C). Treatments were started 12 weeks after i.v. cell injection and maintained up to week 14. Lung staining by hematoxylin–eosin (HE) revealed that only COMBO treatment significantly reduced lung nodules area compared to controls (vehicle, 5.1 ± 1.4%; COMBO, 0.6 ± 0.1%). To investigate the mechanism sustaining the observed tumor shrinkage, we measured cellular proliferation and apoptosis in A375 tumors. The number of proliferating cells did not differ significantly among the three treatment groups. Bevacizumab, PLX4720, and COMBO decreased Ki67 staining by 69, 55, and 58%, respectively, compared to controls (n = 4 tumors; Fig 1D). By contrast, only COMBO treatment induced a significant increase in apoptosis (22.4 ± 3.3%, n = 3 tumors) compared with vehicle (5.7 ± 1.2%, n = 3 tumors) as revealed by TUNEL staining; Fig 1E) and by the accumulation of caspase-cleaved cytokeratin 18 fragment (Appendix Fig S1). These findings support that the antitumor activity observed with COMBO treatment depends on an apoptotic program rather than modification of tumor cell cycle. Dual BRAFV600E and VEGF targeting promotes vascular normalization and improves tissue perfusion in BRAFV600E xenografts We analyzed the vascular effects of bevacizumab and PLX4720 on A375 and COLO205 xenografts, by evaluating microvessel density (MVD) and microvascular area (MVA). In A375 tumors, MVD was reduced by 51% after bevacizumab treatment compared with the control (capillaries/mm2: control, 87.9 ± 3.5; bevacizumab, 42.7 ± 2.1). PLX4720 alone or associated with bevacizumab slightly reduced MVD (n = 5 tumors; Fig 2A). The treatment with PLX4720, bevacizumab, or COMBO resulted in a similar reduction in MVA compared with the control group (vehicle, 11398.3 ± 497.0 μm2; PLX4720, 7186.7 ± 522.3 μm2; bevacizumab, 5472.4 ± 423.5 μm2; COMBO 6881.1 ± 615.8 μm2) (n = 6 tumors; Fig 2A). Figure 2. Vascular response of A375 and COLO205 xenograft tumors to PLX4720 and bevacizumab Representative images of vasculature stained by an anti-CD31 antibody in A375 xenografts treated as indicated. Bar graphs indicate quantitative microvessel density (MVD) and microvessel area (MVA) analysis (n = 5 tumors). ***P < 0.001 versus vehicle (MVD: bevacizumab P = 5.94E-25; COMBO P = 1.84E-04) (MVA: PLX4720 P = 4.98E-09; bevacizumab P = 1.16E-19; COMBO P = 3.00E-08). Representative images of vessel lumen in A375 xenografts treated as indicated. Bar graphs indicate the quantitative analysis of lumen diameters in A375 and COLO205 xenografts (n = 3 tumors). *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 0.0078; COMBO P = 0.011) (COLO205: PLX4720 P = 2.01E-06; COMBO P = 1.33E-07). Representative images of perfusing fluorescent beads and their relationship with microvessels in A375 xenografts treated as indicated. Bar graphs indicate the % of CD31+ vessels co-stained with fluorescent beads (n = 4 tumors). **P < 0.01 versus vehicle (PLX4720 P = 0.0031; COMBO P = 0.0033). Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated. Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts (n = 5 tumors). **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 6.94E-12; COMBO P = 3.34E-11) (COLO205: PLX4720 P = 0.0015; bevacizumab P = 0.0004; COMBO P = 0.0003). Data information: The scale bars represent 100 μm in (A, D) and 50 μm in (B, C). The results are given as the mean ± SEM. Significance was assessed by one-way ANOVA test followed by post hoc pairwise analysis test. Download figure Download PowerPoint The analysis of MVD and MVA in COLO205 xenografts (Appendix Fig S2A) showed a different effect, characterized by increased values of MVD and MVA following PLX4720 treatment, confirming previous observations (Bottos et al, 2012). On the contrary, bevacizumab and COMBO reduced both parameters in this model. Then, we analyzed the vessel lumen diameter in both models. In A375 xenografts, the average vessel lumen diameter decreased from 52.7 ± 3.6 μm in vehicle-treated tumors to 35.3 ± 3.7 μm and 33.6 ± 4.8 μm upon treatment with PLX4720 and COMBO, respectively (n = 3 tumors; Fig 2B). Similar results were observed in COLO205 (vehicle, 50.3 ± 3.2 μm; PLX4720, 35.5 ± 2.3 μm; COMBO, 33.6 ± 1.8 μm). Bevacizumab did not modify the lumen area compared with vehicle in both tumor types. In spite of the cell line-specific differences (Bottos et al, 2012), these data indicate that COMBO and PLX4720 influence vascular morphology by reducing vessel lumen areas as compared to bevacizumab and vehicle. This hypothesis was further investigated in the A375 xenografts. The vessel lumen was resolved and categorized into different groups (from < 100 μm2 to > 600 μm2). PLX4720 alone or in combination with bevacizumab increased the number of the smallest capillaries (surface < 100 μm2) and reduced the number of larger vessels (surface > 500 μm2; Appendix Fig S2B). None of the different therapeutic treatments modified the number of vessels with surface areas from 100 to 500 μm2. These data indicate that BRAF inhibition and VEGF removal have distinct effects on vascular size and that the reduction in number of tumor capillaries induced by VEGF withdrawal is counteracted by BRAFV600E inhibition, which influences capillary shape. Modification of the vascular area is a component of vascular normalization upon treatment with anti-angiogenic compounds (Jain, 2014). A specific hallmark of this process is the improvement of vascular perfusion, which was studied by the i.v. delivery of orange fluorescent microspheres. PLX4720 or COMBO increased vascular perfusion in A375 xenografts compared with treatment with vehicle or bevacizumab (Fig 2C). As expected, PLX4720 treatment restored tissue oxygenation in this model (Fig 2D) as well in COLO205 xenografts in agreement with our earlier report (Bottos et al, 2012). Interestingly, this positive effect was maintained when bevacizumab was combined with PLX4720. Since normalization process may include improvement of pericyte coverage, we assessed the presence of pericytes surrounding capillaries using the specific NG2 marker. We observed that none of the treatments modified pericyte coverage compared with untreated tumors in A375 xenografts (Appendix Fig S2C). Consistent with our previous study (Bottos et al, 2012), we confirmed that PLX4720-mediated BRAFV600E inhibition changed the architecture and functionality of vessels and abrogated tumor hypoxia. Although bevacizumab reduced blood perfusion, its association with PLX4720 did not. Furthermore, the lack of any evident combinatorial effects between bevacizumab and PLX4720 on tumor vessels, and on the amount of murine VEGF detected in the xenografts (not shown), suggests that the enhanced antitumor activity observed with COMBO is likely independent of angiogenesis. Gene expression profiling highlights extracellular matrix remodeling and immunomodulation after dual BRAFV600E and VEGF inhibition In A375 xenografts, the stromal microenvironment is contributed by the mouse. Therefore, species-specific analysis of gene expression can discriminate the effects of different therapeutic regimens on tumor (human) and stroma (mouse) compartment. Human and mouse microarrays were analyzed separately to determine the dynamic changes within each compartment of the xenograft upon treatment with PLX4720, bevacizumab, or COMBO. Of the 23,025 human genes that were above background, a supervised comparison (one-way ANOVA with Benjamini–Hochberg (BJ) false discovery rate (FDR) correction) among the four treatment arms revealed a set of 68 differentially modulated genes (Appendix Fig S3A). Sample clustering analysis based on these 68 genes highlighted minor variations in bevacizumab-treated samples with respect to vehicle and a strong and concordant transcriptional shift in PLX4720- and COMBO-treated samples. A comparison between each treatment arm and the vehicle group by LIMMA (Smyth, 2005) defined a subset of 340 genes modulated by PLX4720 alone (145 up-regulated and 195 down-regulated), 201 genes modulated by COMBO (152 up-regulated and 49 down-regulated), and only four genes modulated by bevacizumab alone (two up-regulated and two down-regulated). The Venn diagrams in Fig 3A illustrate the overlapping subsets of modulated genes, and the volcano plots (Appendix Fig S3B) show the changes in the log2-fold change and P-values for all genes in the three separate comparisons with respect to vehicle. DAVID was used to identify the biological functions enriched in the different treatments in human A375 melanoma cells, considering up- and down-regulated genes separately (significant P-value < 0.05 after BJ FDR method; Appendix Fig S3C). Genes that were down-regulated in PLX4720 or COMBO groups were highly enriched in gene sets involved in "oxygen levels" and "response to hypoxia". This result is consistent with the observation that tumors are less hypoxic after treatment with PLX4720 or COMBO. Genes that were up-regulated in the PLX4720 and COMBO groups showed enrichment in the Gene Ontology (GO) categories of "immune response", "defense responses", and "inflammatory response". Interestingly, in the GO categories related to immune and inflammatory responses, we identified a cluster of cytokine and chemokine genes. Genes involved in extracellular matrix (ECM) organization, extracellular structure organization, biological adhesion, and cell adhesion were only modulated by treatment with PLX4720 alone (Appendix Tables S1 and S2). Taken together, these analyses indicate highly but not completely overlapping functional processes triggered by PLX4720 and COMBO in the human cell compartment of the tumor. This model does not indicate a significant direct functional signature for bevacizumab treatment on the human cell compartment. Figure 3. Transcriptional profiling in A375 xenografts in response to PLX4720, bevacizumab, and COMBO Venn diagram showing the overlapping subsets of modulated human genes (fold change |log2| ≥ 1, P-value < 0.01) in the PLX4720, bevacizumab, and COMBO treatments versus vehicle. Venn diagram showing the overlapping subsets of modulated murine genes (fold change |log2| ≥ 1, P-value < 0.01) in the PLX4720, bevacizumab, and COMBO treatments versus vehicle. Summary of the functional categories of mouse genes significantly enriched in response to PLX4720 and COMBO. GO analyses were performed individually on down- or up-regulated genes using DAVID tool (biological process). GO terms are ranked by P-value corrected by BH method, and the number of genes is indicated. For a complete list of significantly enriched GO groups see Appendix Tables S3 and S4. GSEA enrichment plots for "Reactome Cytokine Signaling in Immune System" and "Reactome Innate Immune System" (upper panels) and "Reactome Extracellular Matrix Organization" and "Reactome Collagen Formation" (lower panels) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO-treated tumors as compared to the other treatments (vehicle, PLX4720, bevacizumab). Heatmap representation of gene expression changes within the "Reactome Innate Immune System" (left panel) and "Reactome Extracellular Matrix Organization" (right panel) gene set. Genes in heatmaps are shown in rows, and samples are shown in columns. Expression level is represented as a gradient from high (red) to low (blue). V, P, B and C, respectively, indicate vehicle, PLX4720, bevacizumab, and COMBO. Download figure Download PowerPoint To further characterize the effects of COMBO on the microenvironment of A375 xenografts, we performed mouse-specific gene expression profiling. Of the 14,533 mouse genes that were above background, ANOVA highlighted a larger number of stromal mouse genes that were significantly altered in the four treatment arms compared to the human arrays (n = 806, one-way ANOVA after BJ FDR correction). In this case, sample cluster analysis highlighted expression similarities between the vehicle and bevacizumab groups and between the PLX4720 and COMBO groups, with PLX4720 and COMBO inducing a substantially wider gene modulation (Appendix Fig S4A). A subset of bevacizumab-modulated genes related to angiogenesis (compared with vehicle, fold change threshold | log2 FC | of 0.5) is reported and further validated by quantitative PCR analysis (Appendix Fig S4B and C). Volcano plots show the changes in the log2-fold change and P-values for all genes for the different treatments (Appendix Fig S4D), and Venn diagrams illustrate the overlapping subsets of modulated genes (Fig 3B). Among the 805 genes modulated by PLX4720 identified by LIMMA analysis, 517 were down-regulated, and 288 were up-regulated. COMBO had a reduced modulatory effect (414 genes; 214 suppressed and 200 induced). GO analysis was performed on the three treatment arms compared with vehicle using DAVID (P < 0.05 with BH FDR correction), considering up- and down-regulated genes separately. Among the down-regulated genes, PLX4720 treatment mainly targeted genes belonging to GO terms characterizing functions and processes of the vasculature ("blood vessel development", "vasculature development", "blood vessel morphogenesis", "angiogenesis"; Fig 3C and Appendix Tables S3 and S4) in accordance with the observed effect on tumor vessel shape and functions (Fig 2). DAVID analysis of the COMBO response revealed that the top enriched functions belonged to the immune response (up-modulated genes), blood vessel, and cell adhesion (down-modulated genes; Fig 3C). A comparison betwe