Endothelial deficiency of L1 reduces tumor

Downloaded September 1, 2014 from The Journal of Clinical Investigation. doi:10.1172/JCI70683.

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The Journal of Clinical Investigation

Endothelial deficiency of L1 reduces tumor angiogenesis and promotes vessel normalization Elena Magrini,1 Alessandra Villa,1,2 Francesca Angiolini,1,2 Andrea Doni,3 Giovanni Mazzarol,2 Noemi Rudini,4 Luigi Maddaluno,4 Mina Komuta,5 Baki Topal,6 Hans Prenen,7 Melitta Schachner,8 Stefano Confalonieri,2 Elisabetta Dejana,4 Fabrizio Bianchi,1,2 Massimiliano Mazzone,9,10 and Ugo Cavallaro1,2 Department of Experimental Oncology and 2Molecular Medicine Program, European Institute of Oncology, Milano, Italy. 3Department of Inflammation and Immunology, Humanitas Clinical and

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Research Center, Rozzano, Milano, Italy. 4IFOM, FIRC Institute of Molecular Oncology, Milano, Italy. 5Department of Morphology and Molecular Pathology, 6Department of Abdominal Surgery, and Department of Gastroenterology, University Hospitals Leuven, Leuven, Belgium. 8W.M. Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience,

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Rutgers University, Piscataway, New Jersey, USA. 9Laboratory of Molecular Oncology and Angiogenesis, Vesalius Research Center, Vlaams Instituut voor Biotechnologie (VIB), Leuven, Belgium. 10Department of Oncology, Katholieke Universiteit (KU) Leuven, Leuven, Belgium.

While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6–mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.

Introduction

Cancer growth strictly depends on the expansion of the host vasculature, which not only supplies oxygen and nutrients to the tumor tissue, but also provides cancer cells with the metastatic route to colonize distant organs. Therefore, angiogenesis represents a critical process during tumor initiation and malignant progression (1). Different strategies have been developed to reduce angiogenesis and thus control tumor progression, as exemplified by the inhibition of VEGF-dependent pathways. However, while some cancer types show a certain degree of therapeutic response, the benefits of such antiangiogenic agents are transient and the initial response is often followed by the establishment of resistance and escape mechanisms, leading to tumor relapse (2, 3). This highlights the need for a more comprehensive understanding of the biological processes that underlie tumor vascularization, which, in turn, would set the stage for additional angiogenesis-targeted therapies.

Authorship note: Alessandra Villa and Francesca Angiolini contributed equally to this work. Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: April 26, 2013; Accepted: July 17, 2014. Reference information: J Clin Invest. doi:10.1172/JCI70683.

Compared with their normal counterparts, tumor vessels are aberrant in almost all aspects of their structure and function. They are heterogeneous and tortuous, branch chaotically, and have an uneven vessel lumen. In addition, they frequently lack pericyte coverage and show an abnormal basement membrane, resulting in vascular instability and altered permeability. These vessel abnormalities generate a promalignant microenvironment, characterized by hypoxia, low pH, and high fluid pressure, which can select for more malignant cancer cells and facilitate their dissemination through leaky vessels, thus causing poor response to therapy (4). These findings raise the question of whether tumor vessel normalization provides an alternative therapeutic opportunity in order to reduce metastatic spread and enhance tumor responses to chemotherapy and radiotherapy (4). However, the characteristics that make tumor-associated endothelial cells (ECs) different from normal ECs are not yet fully identified, and their properties are usually extrapolated from the behavior of ECs during vascular development. Moreover, while it is well known that different and morphologically distinct EC types, such as tip, stalk, and phalanx cells, coexist during normal vascularization, the heterogeneity of cancer-associated ECs is still poorly understood. Hence, a deeper understanding of the phenotypical heterogeneity and specific molecular signature of tumor vasculature is essential to elucidat-

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Figure 1. Ablation of endothelial L1 in Tie2-Cre;L1floxed mice and related effects on tumor development and mouse survival. (A) Sections of normal pancreas and Panc02 tumors from L1floxed and Tie2-Cre;L1floxed mice were costained for L1 (green) and the vascular marker PECAM-1 (red), followed by confocal analysis. Arrows indicate vessels coexpressing PECAM-1 and L1; arrowheads indicate L1-positive nerves that served as internal control. Scale bars: 10 μm. (B and C) The volume (B) and weight (C) of pancreatic tumors from L1floxed and Tie2-Cre;L1floxed mice were recorded 14 days after Panc02 injection. Data represent mean ± SEM from 10 mice per group. (D) Images of explanted tumors. (E) Survival rates in Panc02 tumor–bearing L1floxed (n = 14) and Tie2-Cre;L1floxed mice (n = 13). **P < 0.01; ***P < 0.001.

ing the mechanisms of pathological angiogenesis and to identifying novel cancer vessel–specific markers. L1 (also known as L1CAM or CD171) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily and is composed of an extracellular portion, containing 6 Ig-like domains and 5 fibronectin type III repeats, followed by a transmembrane region, and a highly conserved cytoplasmic tail (5). L1 was discovered and characterized as a cell-adhesion molecule in the nervous system (6), where it is involved in neurite outgrowth and fasciculation as well as cell adhesion and migration. In addition to homophilic binding, L1 can establish cis- or trans-interactions with different binding partners, such as integrins, CD24, neurocan, neuropilin-1, and other members of the neural cell adhesion family (7). 2

Besides the nervous system, L1 is expressed in many human cancers, including ovarian and endometrial carcinoma, pancreatic ductal adenocarcinoma (PDAC), melanoma and glioblastoma. L1 expression confers motile and invasive properties to tumor cells, supporting cancer growth, metastasis, and chemoresistance and often acting as a marker of poor prognosis (8). L1 has also been detected in the hematopoietic system, in particular in immune cells of myelomonocytic and lymphoid origin (9), and we have previously reported L1-dependent transmigration of dendritic cells across the endothelium (10). An intriguing aspect of L1 biology is its expression in the vascular system: while no or very little L1 is detectable in the vasculature of most normal tissues, its level is markedly increased in the vas-

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Table 1. Number of mice with metastases and number of sitespecific metastases in Panc02 tumor–bearing L1floxed and Tie2Cre;L1floxed mice Mice with metastasis Site-specific metastases (number of nodules) Stomach Intestine Liver Kidney Urogenital tract Diaphragm Abdominal wall

L1floxed

Tie2-Cre;L1floxed

5/7 (71%)

2/7 (29%)

1 10 1 3 1 3 11

0 0 1 0 0 3 8

n = 7 mice per group.

cular endothelium associated with pathological conditions, most prominently cancer and inflammatory diseases. Furthermore, various cytokines and angiogenic growth factors abundantly released in the tumor microenvironment, such as VEGF-A, TNF-α, IFN-γ, and TGF-β1, upregulate L1 expression in ECs (10, 11). Taken together, these observations point to L1 as a tumor vessel–specific molecule. However, it remains elusive whether L1 is causally involved in the formation and/or function of cancer-associated vasculature. In this study, we investigated the role of vascular L1 in a mouse model of pancreatic tumor in which the endothelial-specific ablation of L1 was achieved by Cre-LoxP technology. This approach was complemented by in vitro studies on ECs in which the expression of L1 was manipulated in order to induce overexpression or silencing of the gene. Our data revealed that L1 plays a pivotal role in tumor-associated vessels, thus influencing cancer growth, metastasis, and mouse survival. In particular, L1 was found to modulate different EC functions and to promote endothelial-tomesenchymal transition (EndMT), a process causally linked to neovascularization (12). We also demonstrated that antibodymediated targeting of L1 in tumor-bearing mice leads to decreased neovascularization and to vessel normalization, resulting in reduced tumor growth. Finally, we obtained mechanistic insights into the function of L1 in ECs by showing that (a) L1 modulates several transcriptional programs in ECs, including pathways that govern vascular development and function; and (b) L1 promotes the activation of the IL-6/JAK/STAT signaling pathway, and the latter mediates L1-induced stimulation of ECs.

Results

Endothelial L1 regulates pancreatic tumor growth and metastasis in mice. To define the role of vascular L1 in tumor growth, we combined the genetic inactivation of endothelial L1 with an orthotopic mouse model of pancreatic carcinoma. This model is based on the injection of mouse Panc02 cells into the head of the pancreas of syngeneic C57BL/6 mice, resulting in the formation of highly vascularized and metastatic pancreatic tumors (13). Panc02 cells do not express L1, either in culture (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/ JCI70683DS1) or upon tumor formation in mice (not shown). The

endothelial-specific ablation of L1 in mice was achieved by crossing Tie2-Cre with L1floxed mice (10). Indeed, L1 was readily detectable in the tumor vessels of control, L1floxed mice, while no L1 immunoreactivity was observed in the vasculature of Tie2-Cre;L1floxed tumors (Figure 1A). Interestingly, the vessels of normal pancreatic tissue in either L1floxed or Tie2-Cre;L1floxed mice showed no L1 expression (Figure 1A). As expected, L1 expression in peripheral nerves was not affected by Tie2-Cre–mediated recombination, thus acting as an internal control (Figure 1A and Supplemental Figure 2). Given that the Tie2 gene promoter is also active in hematopoietic precursors and L1 is expressed in certain immune cell lineages (10), we first checked whether Tie2-Cre–mediated ablation of L1 affected immune cell infiltration into the tumor. No difference in the number of tumor-infiltrating immune cells, assessed either as total CD45+ leukocytes or as individual hematopoietic cell subpopulations identified by lineage-specific markers, was observed between control and Tie2-Cre;L1floxed mice (Supplemental Figure 3, A and B). In agreement with our previous results (10), the ablation of L1 in Tie2+ hematopoietic progenitors did not cause major defects in mouse hematopoiesis, as blood cell counts for the different leukocyte populations gave very similar values for L1floxed and Tie2-Cre;L1floxed littermates (Supplemental Figure 3C). Overall, these data indicate that in the Panc02 tumor model, L1 deficiency does not affect immune cell migration into cancer tissue and, therefore, any effect on tumor behavior upon Tie2 promoter–driven ablation of L1 in Tie2-Cre;L1floxed mice should be attributed to endothelial L1 rather than to L1 expressed in immune cells. We then analyzed Panc02 tumors 14 days after implantation. Pancreatic tumor burden was markedly reduced in Tie2-Cre;L1floxed mice (Figure 1, B and C), implicating vascular L1 in cancer growth. This effect was accompanied by an increase in TUNEL-positive apoptotic cells in Tie2-Cre;L1floxed tumors (Supplemental Figure 4A), while cancer cell proliferation, as assessed by staining for either Ki-67 or phospho-histone H3, was not affected (Supplemental Figure 4, B and C). These data indicated that vascular L1 deficiency is associated with reduced tumor growth and increased apoptosis. A macroscopical examination revealed that tumors of L1floxed mice exhibited intensely red areas, suggesting high tumor vascularization and/or hemorrhages. Notably, this feature was greatly reduced in tumors from Tie2-Cre;L1floxed mice (Figure 1D), possibly implicating endothelial L1 in tumor angiogenesis and/or vascular permeability. Vascular L1 also appeared to be involved in the dissemination of Panc02 tumors, since only 29% of Tie2-Cre;L1floxed mice (2/7) showed metastatic lesions in different abdominal organs, while metastases were detected in 71% of L1floxed mice (5/7) (Table 1). Consistent with the reduced cancer growth and dissemination, tumor-bearing Tie2-Cre;L1floxed mice also showed longer survival times than their control littermates (Figure 1E). Overall, these results indicated that the ablation of endothelial L1 results in decreased tumor malignancy. Endothelial L1 regulates tumor angiogenesis and vascular normalization. To determine whether endothelial L1 plays any role in cancer vascularization, we measured the microvessel density in tumor tissue. Panc02 tumors in Tie2-Cre;L1floxed mice exhibited a reduced number of vessels as compared with L1floxed mice, indicating that endothelial L1 is involved in tumor angiogenesis (Figure 2, A and B). These findings were further supported by in vivo

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The Journal of Clinical Investigation Figure 2. Endothelial L1 deficiency results in reduced tumor angiogenesis and in vessel normalization. (A) Representative images of Panc02 tumor sections stained for PECAM-1 (red) to visualize vessels. Scale bars: 50 μm. (B) Quantitation of vessel density in tumors from L1floxed (n = 9) and Tie2-Cre;L1floxed mice (n = 5). (C) Representative images of Panc02 tumor sections costained for PECAM-1 (red) and the pericyte marker NG-2 (green) to visualize pericyte coverage. Scale bars: 50 μm. (D) Quantitation of pericyte coverage in tumor vessels from L1floxed (n = 8) and Tie2-Cre;L1floxed mice (n = 5). (E) Representative images of Panc02 tumor sections costained for the endothelial apical marker podocalyxin (cyan), the junctional marker VE-cadherin (yellow), and the basement membrane marker collagen IV (red). Arrows indicate the localization of VE-cadherin at cell-cell contact in Tie2-Cre;L1floxed tumor vessels (right), which is lost or dramatically reduced in L1floxed vessels (left). Scale bars: 10 μm. (F) Quantitation of collagen IV deposition in tumor vessels from L1floxed (n = 3) and Tie2-Cre;L1floxed mice (n = 3). (G) Representative images of Panc02 tumor sections from mice injected with Texas red–dextran. Sections were costained for Texas red (red) and PECAM-1 (green). Scale bars: 50 μm. (H) Quantitation of vascular permeability, expressed as the percentage of vessels showing extravasated dextran in tumors from L1floxed (n = 3) and Tie2-Cre;L1floxed mice (n = 3). *P < 0.05; ***P < 0.0005.

Matrigel plug assays, where Tie2-Cre;L1floxed mice showed a marked decrease in the neovascularization induced by the angiogenic factor FGF-2 (Supplemental Figure 5). We also tested the hypothesis that L1 regulates vascular integrity in cancer tissue. To this goal, given that the interaction of pericytes with the endothelium is causally linked to vessel stabilization and integrity (14), we analyzed pericyte coverage in the vasculature of Panc02 tumors. The extent of pericyte coverage, as assessed by costaining for PECAM-1 and the pericyte marker NG-2 (15), was significantly increased in Tie2-Cre;L1floxed mice (Figure 2, C and D), thus implying that endothelial L1 negatively regulates the recruitment of pericytes to the vascular wall. Next, we asked whether endothelial polarity, another prerequisite for vessel integrity, was affected by L1. Panc02 tumors were stained for collagen IV, a major constituent of the vascular basement membrane that is essential for capillary stability and organization (16), for the apical marker podocalyxin and for VE-cadherin as a marker of endothelial junctions (17). In line with the notion that the architecture of cancer-associated vasculature is irregular and disorganized (4), the tumors of L1floxed mice showed a dramatic 4

disruption of endothelial polarity, with no or very low level of collagen IV, the irregular distribution of podocalyxin within the vessel wall, and a lack of VE-cadherin–marked junctions (Figure 2E). In contrast, the vasculature of Tie2-Cre;L1floxed tumors displayed abundant deposition of collagen IV in the basement membrane (Figure 2, E and F), localization of podocalyxin at the luminal surface, and a regular pattern of VE-cadherin localization, consistent with its accumulation at cell-cell contacts (Figure 2E). These findings implicated L1 in the maintenance of endothelial polarity. Vascular abnormalities such as defective pericyte coverage, altered polarity, and disorganized endothelial junctions, lead to disruption of endothelial barrier function and, hence, to vascular leakage, thus implying that L1 might regulate vascular permeability. To test this hypothesis, we assessed the extravasation of intravenously injected 40-kDa dextran in Panc02 tumors. Indeed, tumor vascular permeability to dextran was remarkably higher in control, L1floxed mice as compared with Tie2-Cre;L1floxed mice (Figure 2, G and H), thus implicating L1 in tumor vessel leakiness. Taken together, these observations point to endothelial L1 as a causal player in enhanced tumor angiogenesis as well as in the dis-

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Figure 3. Treatment with anti-L1 antibodies reduces tumor growth and angiogenesis while increasing pericyte coverage in tumor vessels. The volume (A) and weight (B) of pancreatic tumors from mice treated with anti-L1 antibodies (n = 6) or control (ctrl) IgG (n = 6) were recorded 14 days after Panc02 injection. Data represent means ± SEM. (C) Images of explanted tumors. (D) Quantitation of vessel density in tumors from mice treated with anti-L1 antibodies (n = 5) or control IgG (n = 5). (E) Quantitation of pericyte coverage in tumor vessels from mice treated with anti-L1 antibodies (n = 6) or control IgG (n = 5). *P < 0.05.

ruption of vascular integrity and stability that is commonly associated with cancer development. In vivo targeting of L1 reduces tumor growth and angiogenesis and promotes vessel normalization. The results obtained with the genetic ablation of L1 in Panc02 tumor–bearing mice implied that L1 might be a therapeutic target for preventing cancerassociated neovascularization and/or enhancing vessel normalization. To explore this possibility, we generated a polyclonal antibody against the ectodomain of mouse L1. First, we tested the function-blocking activity of the antibody in vitro. ECs were transfected with L1 and then subjected to functional assays in the presence of the antibody. As described in more detail in the next section, L1 induced EC proliferation, migration, and tube formation, and the anti-L1 antibody inhibited all these activities (Supplemental Figure 6), thus confirming its neutralizing activity. The antibody was then used to target host L1 in the Panc02 mouse tumor model. As shown in Figure 3, A and B, tumor growth was significantly reduced in mice treated with the anti-L1 antibody as compared with mice treated with control antibody. Furthermore, the intensely red areas present in control tumors were markedly reduced in anti-L1–treated tumors (Figure 3C), in agreement with our observations in Tie2-Cre;L1floxed mice (Figure 1D). The anti-L1 antibody caused a significant decrease of vessel density in Panc02 tumors (Figure 3D), thus supporting the

notion that targeting L1 might be a suitable anti-angiogenic strategy. Finally, tumor vessels from anti-L1–treated mice showed a higher rate of pericyte coverage than tumors treated with control antibody (Figure 3E). Thus, the treatment of tumor-bearing mice with the anti-L1 antibody, besides confirming the result of genetically ablating endothelial L1, revealed that the pharmacological inactivation of L1 might represent a novel strategy to interfere with tumor neovascularization and to enhance vessel stabilization. L1 orchestrates the angiogenic behavior of ECs. The reduced tumor microvessel density upon ablation of L1 in the endothelium prompted us to test whether L1 is involved in the critical EC processes that underlie angiogenesis, i.e., proliferation, migration, and tubulogenesis. Toward this goal, we employed immortalized mouse lung ECs (luECs) because, due to the expression of moderate levels of endogenous L1 (Supplemental Figure 7A), they were amenable to both gain- and loss-of-function studies. The luECs were stably transfected with the murine L1 cDNA (Supplemental Figure 7, A and B). As shown in Figure 4A, forced expression of L1 resulted in increased luEC proliferation. This result was confirmed in a classical in vivo assay for EC proliferation (18), where luECs were injected subcutaneously into immunodeficient mice and allowed to form hemangioma-like lesions. As shown in Figure 4B and C, hemangiomas formed by L1-transfected luECs exhibited a markedly higher growth rate than those formed by control luECs, consistent with our in vitro results on L1-induced proliferation of ECs. To further validate and extend these observations, we employed a loss-of-function approach. The knockdown of L1 with 2 different siRNAs (Supplemental Figure 8A) was accompanied by a decrease in luEC proliferation compared with control siRNAtransfected cells (Supplemental Figure 8B). In addition, L1 silencing also reduced the proliferative response of luECs to TNF-α stimulation (Supplemental Figure 8C). Thus, L1 plays a pivotal role in EC proliferation. Next, a scratch-wound assay was used to evaluate the possible effect of L1 on EC migration. L1-transfected luECs showed a higher migration rate than control cells (Figure 4D and Supplemental Figure 7C). Conversely, L1 gene silencing resulted in decreased luEC migration (Supplemental Figure 8, D and E). These data demonstrate that L1 is causally involved in EC migration. We also assessed the effect of L1 in in vitro angiogenesis assays, determining the ability of ECs to form tube-like structures in 3D reconstituted extracellular matrix. As shown in Figure 4E and Supplemental Figure 7D, forced expression of L1 significantly enhanced luEC tube formation as compared with control ECs. Thus, L1 regulates EC proliferation, migration, and tubulogenesis, considered to be key cellular processes during the angiogenic cascade.

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Figure 4. L1 confers an angiogenic phenotype to ECs and enhances endothelial permeability. (A) Proliferation curves of mock- and L1-transfected luECs. (B) Growth curves of hemangiomas formed by mock- or L1-transfected luECs injected subcutaneously into nude mice, as determined by volume measurement at the indicated time points. Representative images of hemangiomas explanted are shown (insets). (C) Weight of hemangiomas explanted 33 days after injection of mock- or L1-transfected luECs. (D) Migration assays of mock- and L1-transfected luECs were performed as described in Methods. (E) Matrigel-based tube formation assays of mock- and L1-transfected luECs were performed as described in Methods. (F) Mock- or L1-transfected luECs were stained for PECAM-1 (red) or VE-cadherin (green) prior to confocal analysis. Scale bars: 10 μm. (G) qRT-PCR analysis of claudin-5 mRNA in mock- and L1-transfected luECs. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mocktransfected cells (n = 3). (H) FITC-dextran permeability assays were performed on monolayers of mock- and L1-transfected luECs as described in Methods. Data in A, D, E, and H represent the mean ± SD from a representative experiment performed at least in triplicate. Data in B and C represent mean ± SEM from 10 to 12 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Our data on Tie2-Cre;L1floxed mice indicated that L1 modulates the permeability of tumor vessels. Since vascular permeability is strictly related to the organization and function of cell-cell junctions in the endothelium, we determined whether L1 affected the expression and localization of junctional proteins in luECs. Staining for 2 prototypical adhesion molecules of the endothelium, PECAM-1 and VE-cadherin, showed that the architecture of cell-cell boundaries was disorganized in L1-transfected luECs (Figure 4F), despite the fact that the total level of either adhesion molecule was unaffected (Supplemental Figure 9A). This observation was consistent with the in vivo data showing that the altered localization of VE-cadherin in the vasculature of Panc02 tumors was reverted upon endothelial ablation of L1 in Tie2-Cre;L1floxed mice (Figure 2E). We found no differences in the levels of other components of the interendothelial adhesion complexes, such as 4 catenin family members (α, β, γ, and p120-catenin) and junctional adhesion molecule-A (Supplemental Figure 9B). We then tested whether L1 affected the expression of claudin-5, an essential component of tight junctions widely implicated in the barrier function of endothelium (19). The level of claudin-5 mRNA was 6

significantly downregulated in L1-overexpressing luECs as compared with mock-transfected cells (Figure 4G), indicating that L1 negatively regulates its expression. The immunoblotting analysis also confirmed the decrease of claudin-5 in L1-transfected luECs (Supplemental Figure 10A). In line with these findings, the immunofluorescence (IF) staining of Panc02 tumors revealed that no or very little claudin-5 was present in L1floxed tumor vessels, while the protein was readily detectable in the tumor vasculature of Tie2Cre;L1floxed mice (Supplemental Figure 10B), implicating L1 in the regulation of claudin-5 expression in ECs. Interendothelial junctions play a key role in the integrity of vascular barrier (20). Therefore, given the junctional alterations induced by L1, we tested whether endothelial permeability was modified by L1 overexpression. Indeed, the permeability of confluent luEC monolayers to FITC-labeled dextran was dramatically increased upon forced expression of L1 (Figure 4H), in agreement with the reduced permeability observed in the vasculature of Tie2Cre;L1floxed mouse tumors (Figure 2, G and H). Taken together with our findings on L1-dependent alterations in tumor vessels (Figure 2), these data indicate that endothelial L1

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Figure 5. L1 promotes EndMT. (A) Immunoblotting analysis of mock- and L1-transfected luECs for S100A4/FSP1, Id1, KLF4, CD44, N-cadherin, fibronectin, and collagen IV. Actin, tubulin, and vinculin served as loading controls. (B) qRT-PCR analysis of the indicated genes in mock- and L1-transfected cells. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

destabilizes the vasculature not only by altering pericyte coverage and collagen IV deposition, but also via EC-autonomous effects on the localization and/or expression of certain junctional components, therefore orchestrating intercellular adhesion. L1 promotes EndMT. The morphological and functional changes that L1 imparted on ECs, such as increased permeability, loosening of cell-cell junctions and a migratory phenotype, represent typical aspects of the EndMT, a process that has been causally linked to cancer progression (12). In the course of EndMT, ECs gain the expression of mesenchymal markers. Therefore, to verify whether L1 induced bona fide EndMT, we tested whether it promoted the acquisition of mesenchymal markers. The overexpression of L1 in luECs, indeed, resulted in the upregulation of S100A4, N-cadherin, fibronectin, and Id1 (Figure 5A), all events associated with EndMT (21). Notably, we also observed markedly reduced levels of collagen IV in L1-overexpressing luECs (Figure 5A), which, besides being another hallmark of EndMT (21), was consistent with the increased collagen IV deposition in L1-deficient Panc02 tumor vessels (Figure 2, E and F). Furthermore, L1-overexpressing luECs exhibited enhanced expression of the stemness-associated factors KLF4 and CD44 (Figure 5A), in line with the notion that EndMT is accompanied by the acquisition of a stem-like phenotype (21). Most of the markers above were validated by quantitative real-time PCR (qRT-PCR) (Figure 5B), implying a regulation at the transcriptional level. In contrast, the mesenchymal marker vimentin was not affected by L1 overexpression (Figure

5B). Besides mesenchymal markers, L1 was able to enhance the expression of transcription factors (TFs) that are thought to drive EndMT, such as Zeb1 and Zeb2, as well as Tbx20 (Figure 5B), which has been implicated in EndMT-like processes during heart development (22). These data point to L1 as a regulator of EndMT, possibly implicating such an activity in L1-dependent alterations of EC behavior. L1 regulates EC transcriptome. In an attempt to elucidate the molecular mechanisms underlying the multiple roles of L1 in endothelium, we checked whether manipulating its expression affected the EC transcriptome. To this purpose, we compared the gene expression profiles of L1-overexpressing luECs with those of control luECs by Affymetrix microarray technology, and we used the Significance Analysis of Microarrays (SAM) (23) to identify genes whose expression was altered by L1. This analysis revealed a remarkable effect of L1 overexpression on luEC transcription (Figure 6A), with 361 genes that were upregulated and 580 that were downregulated (q value < 5%; 1.5-fold change difference; Supplemental Table 1). From the list of L1-regulated genes, we selected 16 candidates for qRT-PCR validation, based on their biological relevance. For all of them, including Cdk6, Adamts9, Hoxb9, Stmn2, Ebf1, Dll4, Vegfa, Vegfc, Ccnb1, Il13ra2 (Figure 6B), CD44, N-cadherin, S100A4, Tbx20, Klf4 (Figure 5B), and Cldn5 (Figure 4G), the regulation in L1-overexpressing luECs was confirmed by qRT-PCR. Many of these genes, including Stmn2, Ebf1, Dll4, Il13ra2, and Vegfc, exhibited a concordant L1 dependence upon siRNA-mediated silencing of L1 (Figure 6C), indicating that L1 is required and sufficient for their modulation. Next, we used ingenuity pathway analysis (IPA) to analyze the effect of L1 overexpression on genes involved in biologically relevant functions. The “bio-functions” analysis revealed that L1 affects the expression profiles of several genes involved in celcycle regulation, DNA replication, cellular assembly, and organization (top 5 biofunctions, P value < 10 –7, Benjamini-Hochberg correction; Figure 6D), which is consistent with the L1-induced proliferation of ECs (Figure 4A). Importantly, IPA also identified genes involved in cell migration and development (Figure 6D). In particular, the gene expression profile of L1-transfected luECs was consistent with the activation of pathways involved in EC movement (Supplemental Figure 11), thus supporting our observations on L1-dependent functional changes (Figure 4, D and E). L1 regulates EC function via the IL-6/JAK/STAT3 pathway. To get further insights about possible effectors of L1 biological function, we performed an unsupervised ingenuity upstream regulator analysis, which predicts the activation of specific gene expression modulators (i.e., TFs, microRNA, etc.). This analysis predicted the L1-induced activation of the TFs STAT1, STAT2, STAT3, IRF7, and ATF4. In particular, we identified a network of 105 L1-regulated genes downstream of the above-mentioned 5 TFs (Figure 7A).

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Figure 6. L1 regulates EC transcriptome. (A) Hierarchical clustering of genes differentially expressed in L1-transfected versus control luECs. Three independent experimental replicates of L1-overexpressing and control cells were screened by gene expression microarray. Data were log2 transformed before clustering analysis. Red, upregulated genes; blue, downregulated genes. A total of 361 upregulated and 580 downregulated genes (i.e., 496 and 743 probe sets, respectively) were identified in L1-overexpressing cells. (B) qRT-PCR analysis of the indicated genes in mock- and L1-transfected luECs. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). (C) qRT-PCR analysis of the indicated genes in luECs transfected either with 2 different L1 siRNAs (A and B) or with a control siRNA. Transcript levels are shown as fold changes in L1 siRNA–transfected cells relative to control cells (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (D) IPA analysis of L1-regulated genes. The enriched biofunctions were selected based on their significance (P < 0.05; Benjamini-Hochberg correction). Blue bars indicate the –log(P value) of enrichment.

To investigate in more detail the molecular mechanisms underlying the effect of L1 on endothelium, we focused on STAT3, which has been implicated in tumor angiogenesis and EC activation (24). STAT3 activation is classically induced by IL-6 (25); therefore, we checked whether L1 affected the expression of this cytokine in luECs. Indeed, not only IL-6, but also its receptor IL-6Rα, was significantly upregulated in L1-overxpressing ECs, while the expression of the IL-6 coreceptor gp130 was not affected (Figure 7B). These observations were further confirmed by the increased levels of IL-6 in the conditioned medium (Figure 7C) and of IL-6Rα in the lysate of L1-overexpressing luECs (Figure 7D). The forced expression of L1 also resulted in the strong induction of STAT3 phosphorylation (Figure 7D), which was abolished by antibody-mediated neutralization of IL-6Rα (Figure 7E). These results indicated that L1 promoted STAT3 activation via the IL-6/IL-6Rα axis. 8

Next, we checked whether STAT signaling is involved in L1-dependent regulation of EC function. Since STAT activation occurs through JAK-mediated phosphorylation, we treated luECs with the JAK inhibitor I (JAKi) which, indeed, repressed L1-induced phosphorylation of STAT3 (Figure 7F). Importantly, JAK blockade repressed L1-dependent EC proliferation and migration (Figure 7, G and H). Together, our transcriptomic, biochemical, and cell biological data support the notion that L1 regulates EC function via the JAK/STAT pathway. Endothelial L1 is upregulated in human pancreatic carcinoma and in other tumor types. Based on our findings on the Panc02 orthotopic mouse model, we asked whether the expression of L1 in tumor vessels also occurs in human pancreatic carcinoma. Immunohistochemistry for L1 was performed on 18 tissue samples of PDAC and 11 samples of noncancerous pancreatic tissue, using

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Figure 7. L1 regulates EC function via the IL-6/JAK/STAT3 pathway. (A) Gene network of L1-regulated genes. In bold, IPA-predicted upstream modulators. Lines connect modulators to direct targets, and colors indicate the consistency with the predicted activity with the expression change observed in L1-overexpressing luECs (i.e., target expression). Orange, consistent predicted activation of TFs; blue, consistent predicted inhibition of TFs; yellow, inconsistent predicted activation of TFs; grey, not defined activity. (B) qRT-PCR analysis of the indicated genes in mock- and L1-transfected luECs. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). (C) The amount of IL-6 released in the culture medium by mock- and L1-transfected luECs was quantified by ELISA. (D) Immunoblotting analysis of mock- and L1-transfected luECs for IL-6Rα, phosphorylated STAT3, and total STAT3. (E) Immunoblotting analysis for phosphorylated and total STAT3 in mock- and L1-transfected luECs, treated either with anti–IL-6Rα antibody or with control IgG. (F) Immunoblotting analysis for phosphorylated and total STAT3 in mock- and L1-transfected luECs, treated either with vehicle (DMSO) or with 20 μM JAKi. Actin in D–F served as loading control. (G) Proliferation curves of mock- and L1-transfected luECs treated either with vehicle (DMSO) or with the indicated concentration of JAKi. (H) Mock- and L1-transfected luECs treated either with vehicle (DMSO) or with 20 μM JAKi were subjected to 24-hour migration assays. Data in G and H represent the mean ± SD from a representative experiment performed in triplicate. **P < 0.01; ***P < 0.001. jci.org

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tumor vascularization and enhanced vessel normalization, thus delaying tumor growth. These findings were supported by in vitro data that revealed that L1 induces EC proliferation, migration, and tubulogenesis, and confers a mesenchymal phenotype to ECs. Previous studies have shown that a soluble form of the L1’s ectodomain enhances EC proliferation and migration (31–33), implying a model whereby exogenous (e.g., tumor cell derived) L1 stimulates ECs. While this remains a possibility, particularly in the case of L1-expressing tumors, our findings on the upregulation of L1 in cancer-associated vessels and on the proliferative and migratory response of L1-expressing ECs, point to a cell-autonomous effect of L1 in pathological vasculature. It is noteworthy that L1 promotes the expression of various genes causally linked to tumor vascularFigure 8. L1 is upregulated in human PDAC-associated vessels. (A) Consecutive sections from human noncancerous or PDAC tissue were stained for PECAM-1 (left panels) and ization, such as VEGF-A, VEGF-C, Dll4, and HOXB9 L1 (right panels) in order to visualize L1-positive vessels (arrows). Scale bars: 100 μm. (B) (34–36). On the other hand, the endothelial expresQuantitation of L1-positive vessels in noncancerous (n = 11) and tumor tissue (n = 18), sion of L1 itself is induced by classical angiogenic expressed as percentage of L1-positive over PECAM-1–positive vessels. **P < 0.01. factors, including VEGF-A, ANGPTL4, TNF-α, and IFN-γ (10, 11). Taken together, these observations an antibody against PECAM-1 to identify the vessels on consecuimplicate L1 as a central hub in the transmission and amplification tive sections. L1 expression was markedly enhanced in PDAC of angiogenic stimuli within the tumor microenvironment. vasculature as compared with noncancerous tissue (Figure 8, A Our data also indicate that L1 promotes vascular permeability. and B), confirming and extending previous observations (11). The Since VEGF-A, ANGPTL4, TNF-α and IFN-γ (i.e., the same cytokines presence of L1 in vascular endothelium was further validated by that induce endothelial L1 expression) are among the most potent confocal microscopy on PDAC tissue costained for L1 and for the inducers of vascular permeability (37, 38), it is conceivable that L1 endothelial marker VE-cadherin (Supplemental Figure 12). Taken acts as a general effector of cytokine-induced vascular permeability. together with our data on the Panc02 tumor model and on cultured Notably, we discovered that L1 overexpression induces EndECs, the expression pattern of L1 in clinical samples supports the MT, a process that may underlie, or at least contribute to, many hypothesis that vascular L1 contributes to pancreatic malignancy. aspects of the EC response to L1, such as migration and tubuloTo test whether the vascular expression of L1 also occurs in genesis as well as increased tumor angiogenesis and vascular perother cancer types, we performed the immunohistochemical meability (12). Various lines of evidence support the notion that staining for L1 on tissue microarrays (TMAs) containing various EndMT recapitulates most of the cellular and molecular events tumors and their noncancerous tissue counterparts. As shown in occurring during epithelial-to-mesenchymal transition (EMT) Figure 9 and Supplemental Figure 13, in several tumor types, the (12). The role of L1 in EMT has been clearly established in various percentage of L1-positive vessels was markedly higher as comexperimental models (39–41), including tumor cells in which the pared with their corresponding nonneoplastic tissues. Thus, the expression of L1 per se promoted EMT (42, 43). We now provide induction of L1 expression in the vasculature is common to a broad evidence that L1 expression is sufficient to confer a mesenchymal spectrum of human tumors. phenotype to ECs, which entails not only an increased migratory activity but also the upregulation of several mesenchymal markers, such as N-cadherin, CD44, S100A4/FSP1, and fibronectin, Discussion and even of TFs that are considered to be EMT/EndMT drivers, The expression of L1 in tumor vasculature has been reported in namely KLF4, Zeb1, Zeb2, and Tbx20 (22, 44, 45). These findings several cancer types, including breast, ovarian, colon, and pancresuggest that L1 acts as a key mediator of the multiple EndMTatic carcinoma (10, 11), neural tumors (26), smooth muscle tumors inducing factors that can occur in tumor microenvironment and (27), and melanoma (28). While vascular L1 has been implicated as a master orchestrator of cancer-associated EndMT (12). in the adhesion and transendothelial migration of L1-expressing One of the most intriguing and unexpected results of our study cancer cells via homophilic binding (11, 29, 30), it remains elusive is the global effect of L1 on EC transcriptome, with the modulation whether L1 induces cell-autonomous effects in tumor endotheliof approximately 1,000 genes. While these include individual facum and whether this has an effect on cancer development. tors that per se might account for several aspects of the endotheHere, we report for what we believe is the first time that L1 lial response evoked by L1, such as VEGF-A, VEGF-C, and Dll4 for orchestrates the EC behavior in tumor vasculature. In particular, tumor angiogenesis (see above), or occludin and claudin-5 for vasthe endothelial deficiency of L1 in a mouse model of cancer led to cular permeability (19), our data rather implicate L1 in the control enhanced vessel stability and decreased tumor angiogenesis, resultof whole gene networks, resulting in the modulation of signaling ing in reduced tumor growth and metastasis and prolonged mouse pathways that mediate the EC response. survival. Consistently, antibody-mediated targeting of L1 reduced 10

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Figure 9. Vascular L1 is upregulated in various human tumor types. TMAs containing different tumor types and their normal tissue counterparts were stained for PECAM-1 (A and C) and L1 (B and D). Examples of thyroid carcinoma (A and B) and gastric carcinoma (C and D) are shown. Scale bars: 200 μm. (E) Quantitation of L1-positive vessels in noncancerous and tumor tissue expressed as percentage of L1-positive over PECAM-1–positive vessels. **P < 0.002 (n = 5 for each tissue type).

In this context, it is noteworthy that L1 induces the expression of IL-6 and IL-6Rα. IL-6 is a potent angiogenic cytokine that promotes neovascularization in various solid tumors, and it has been proposed not only as a therapeutic target for antiangiogenic therapies but also as a biomarker to predict the response to such treatments (46). Thus, it is reasonable to speculate that the IL-6/ IL6–Rα axis acts as a key mediator of the angiogenic response elicited by vascular L1, an intriguing hypothesis that warrants further investigation. In addition, L1-mediated regulation of IL-6 expression might also occur in different biological contexts where IL-6 signalling has been implicated. For example, the IL-6/IL-6Rα system plays a major role in inflammation and in several cancer cell functions (47, 48), and our findings imply that the established function of L1 in both pathological conditions (8, 10) might be accounted for, at least to some extent, by the induction of IL-6 expression and activity. Among the signalling cascades that are elicited by IL-6, the JAK/STAT pathway appears particularly important in the context of L1-dependent regulation of EC function. Indeed, L1 induces high phosphorylation of STAT3, a TF that plays a key role in EC activation and pathological angiogenesis (49, 50), consistent with the hypothesis that L1-dependent regulation of tumor vasculature is mediated by the IL-6/JAK/STAT pathway. Our data on the inhibition of L1-induced EC proliferation and migration upon blockade of JAK/STAT signaling not only support this hypothesis, but provide mechanistic insights into the role of L1 in pathological vessels and shed light on a signaling axis that links L1 to the JAK/ STAT pathway. Given the broad spectrum of functions that have been ascribed to L1 in different cellular contexts, including cellcell adhesion, axon guidance, tumor cell invasion, and stem cell self renewal (7, 8, 51), we propose that at least some of these activities are mediated by JAK/STAT signaling. The expression and function of L1 in cancer vessels has relevant translational implications. First, it provides the rationale to test L1 as an imaging biomarker suitable for visualizing pathological angiogenesis. In this application, due to its absence or

low levels in normal vasculature, L1 might show higher specificity than other molecular biomarkers that are currently in use or under clinical testing, such as integrins and VEGFR2 (52). The feasibility of this approach is supported both by the preclinical imaging of cancer vessels by targeting other immunoglobulinlike adhesion molecules such as NCAM (53) and by the suitability of L1 antibodies for tumor-imaging purposes as shown in mouse models (54). Second, based on the positive role of L1 in tumor neovascularization, in EC proliferation and migration, and in EndMT, it is reasonable to speculate that neutralizing L1 could represent a novel antiangiogenic strategy. Indeed, we showed that treating tumor-bearing mice with an L1-neutralizing polyclonal antibody delays tumor growth and reduces tumor vascularization, strengthening the rationale for developing L1-targeting agents as therapeutic tools. In this context, promising results have been obtained with anti-L1 monoclonal antibodies in preclinical models of solid tumors and of endometriosis (55, 56). Third, the observation that L1 destabilizes cancer-associated vessels implies that targeting endothelial L1 and interfering with its function might result in vessel normalization, a process that has been proposed to improve the delivery into the tumor of systemically administered chemotherapeutics (4). Thus, our results set the stage for exploring the clinical relevance of L1 expression and function in cancer vessels, possibly opening new avenues for targeted treatments of malignancies.

Methods Mice Tie2-Cre;L1floxed mice were generated in the C57BL/6 genetic background as previously described (10). Since the L1 gene is located on chromosome X (and hence, only 1 copy is present in the male genome), Cre-mediated ablation of L1 was expected to be more efficient in males. Therefore, only Tie2-Cre–positive males carrying the floxed L1 allele were used throughout the study, with L1floxed males serving as controls.

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In vivo models Pancreatic carcinoma model. The syngeneic mouse model of pancreatic cancer has been described previously (13). Briefly, 10- to 12-week-old C57BL/6 mouse males were anesthetized by intraperitoneal injection of 500 mg/kg avertin (Sigma-Aldrich), the stomach was exteriorized via abdominal midline incision, and Panc02 tumor cells (1 × 106 cells in 30 μl PBS) were injected into the head of the pancreas using a 29-gauge needle. The intrapancreatic injection was considered successful with the appearance of a fluid bleb without intraperitoneal leakage. Peritoneum and abdominal wall were closed with individual surgical sutures. Where indicated, mice were treated every 48 hours with 7 mg/kg of affinity-purified anti-L1 polyclonal antibodies (obtained from rabbits immunized with mouse L1-Fc) or control, nonimmune rabbit IgG (Sigma-Aldrich), starting from the day after Panc02 injection. At day 14, primary tumors were removed, and tumor weight and volume were recorded. Tumor volume was calculated using the formula V = π × (d1 × d2 × d3)/6, where d1, d2, and d3 are the 3 tumor axes. To assess tumor invasion to adjacent organs and metastasis, mice were sacrificed at day 26 and subjected to whole-mount body fixation with 4% paraformaldehyde. Necropsy was performed by the Mouse and Animal Pathology Laboratory, Fondazione Filarete (Milan, Italy). Hemangioma model. For EC transplantation, 1 × 105 mock- or L1transfected mouse luECs in 200 μl of PBS was injected subcutaneously into the right flank of CD-1 nude (nu/nu) mice (9 weeks old, female; Charles River Laboratories) as previously described (18). Hemangioma volumes were recorded at the indicated time points and calculated using the formula V = π × (d2 × D)/6, where d and D are the minor and the major hemangioma axes, respectively. Chemicals and antibodies The JAK inhibitor 1 was provided by Calbiochem. The following monoclonal antibodies were used as indicated: anti-mouse L1 (clone S10.33 [ref. 10, 5 μg/ml in IF]; clone 555 [1 μg/ml in IF]; clone 324 [ref. 6] hybridoma supernatant [1:2 in IF]; and clone I4.2 [ref. 10] hybridoma supernatant [1:2 in Western blotting (WB)]); anti-human L1 (clone UJ127; 1:30 in IHC on human tissues; Thermo Scientific); anti–PECAM-1 (clone 2H8; 1:500 in IF on cells and mouse tissues, Millipore; clone JC70A; 1:30 in IHC on human tissues, Dako); anti–claudin-5 (clone 4C3C2; 1:200 in IF, 1:250 in WB, Life Technologies); anti-actin (clone AC-40; 1:1000 in WB; Sigma-Aldrich); anti–α-catenin (clone 5/α-catenin; 1:1000 in WB; BD Biosciences); anti–β-catenin (clone 14/β-catenin; 1:200 in IF and 1:500 in WB; BD Biosciences); anti–γ-catenin (clone 15/γ-catenin; 1:1000 in WB; BD Biosciences); anti-CD44 (clone IM7; 1:100 in WB; BD Biosciences); anti–JAM-A (clone BV19/BV20) (57); anti–N-cadherin (clone 32/N-cadherin; 1:2000 in WB; BD Biosciences), anti–p120-catenin (clone 98/pp120; 1:1000 in WB; BD Biosciences); anti–phosphoStat3 (Cell Signaling, clone D3A7; 1:1000 in WB); anti-Stat3 (clone 124H6; 1:1000 in WB; Cell Signaling); anti–VE-cadherin (clone 11D1.4; 1:200 in IF on cells and mouse tissues; BD Biosciences); antivinculin (clone hVIN-1; 1:2000 in WB; Sigma-Aldrich); anti-tubulin (clone DM1A; 1:2000 in WB; Sigma-Aldrich). The following polyclonal antibodies were used as indicated: anti-collagen IV (1:900 in IF and 1:400 in WB; Serotec); anti-fibrin/fibrinogen (1:1000 in IF; Dako); anti-fibronectin (1:4000 in WB; Abcam); anti-S100A4/FSP1 (1:1000 in WB; Millipore); anti-Id1 (C-20; 1:400 in WB; Santa Cruz Biotechnology Inc.); anti–IL-6Rα (1:1000 in WB; R&D Systems); 12

anti–Ki-67 (1:100 in IF; Abcam); anti-KLF4 (1:1000 in WB; R&D Systems); anti–mouse L1 (obtained by rabbit immunization with mouse L1-Fc; 1 μg/ml in IF and WB); anti–PECAM-1 (M-20; 1:500 in WB; Santa Cruz Biotechnology Inc.); anti–NG-2 (1:200 in IF; Millipore); anti–phospho-histone H3 (1:250 in IF; Millipore); anti-podocalixin (1:400 in IF; R&D Systems); anti–VE-cadherin (C-19; 1:500 in WB and 1:200 in IF on human tissues; Santa Cruz Biotechnology Inc.); anti–β-tubulin III (1:500 in IF; Covance). Antibodies used for FACS analysis are listed in the corresponding section.

Cells Mouse luECs were immortalized with polyoma middle T antigen as previously described (18) and cultured in MCDB131 medium (Gibco; Life Technologies) supplemented with 20% FBS (Invitrogen), 2 mM l-glutamine (Lonza), 1 mM Na-pyruvate (Gibco; Life Technologies), 100 μg/ml heparin (Sigma-Aldrich), and 50 μg/ml EC growth supplement (ECGS) obtained from calf brain. ECs were seeded on 0.1% gelatin (Sigma-Aldrich), except as otherwise indicated. To enhance EC adhesion, plates were coated with glutaraldehyde–crosslinked gelatin as follows. Plates were incubated overnight with 1% gelatin at 37°C followed by a crosslinking with 2% glutaraldehyde for 15 minutes at room temperature (RT). Glutaraldehyde was replaced with 70% ethanol for 1 hour at RT. After 5 washes with PBS, plates were incubated for 2 hours at 37°C with 2 mM glycine in PBS. Prior to cell seeding, plates were washed 5 times with PBS. Murine Panc02 (pancreatic carcinoma) and MOVCAR7 (ovarian carcinoma) cell lines were provided by S. Sebens (Institute for Experimental Medicine, Kiel, Germany) and D. Connolly (Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA), respectively, and cultured in RPMI 1640 medium (Lonza) supplemented with 10% FBS, 2 mM l-Glutamine, and 1 mM Na-pyruvate. All cells were cultured in a humidified incubator with 5% CO2. Cell transfection and RNA interference Mouse cDNA encoding full-length L1 was cloned into the pcDNA3.1/ Hygro(-) expression vector (Invitrogen) using standard DNA cloning procedures. The parental pcDNA3.1/Hygro vector and pcDNA3.1/ Hygro-L1 were amplified in competent bacteria cells (TOP10) and purified using Maxi-Prep kits (QIAGEN) according to the manufacturer’s instructions. Transfection with either pcDNA3.1/Hygro or pcDNA3.1/Hygro-L1 was performed with LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. Transfected cells were selected with 300 μg/ml of Hygromycin-B (Roche) and maintained under selection conditions as a bulk cell population. Stealth RNAi Duplexes and the corresponding Medium GC Stealth RNAi Control Duplexes (Invitrogen) were used to knock down L1 in murine ECs. The following L1 target sequences were used: 5′-CCUGGUACCCGGACCAUCAUUCAAA-3′ (siRNA A) and 5′-UGCACCUUUCUUCUCAAUUGCGCUC-3′ (siRNA B). ECs were subjected to 2 rounds of transfection with 40 nM siRNA the 2 days prior to the experiment. Transfection was performed with LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. Cell proliferation assay ECs were seeded at a density of 2 × 103 cells/well in 96-well plates coated with glutaraldehyde–crosslinked gelatin. After overnight incubation in medium containing 5% FBS, cells were stimulated with

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The Journal of Clinical Investigation medium containing 20% FBS, 100 μg/ml heparin, and 50 μg/ml ECGS. Where indicated, cells were treated with JAK inhibitor 1 or vehicle (DMSO) or with 3 μg/ml of polyclonal anti-L1 antibodies or control, nonimmune rabbit IgG throughout the assay. Cells were fixed at 0, 24, 48, and 72 hours, followed by staining with 0.1% crystal violet in 20% methanol. Bound dye was solubilized with 10% acetic acid, and the absorbance at 590 nm was measured. Cell growth was normalized on absorbance measured at 0 hours. The experiments were performed in quintuplicate and repeated at least 3 times.

Cell migration assay To assess cell migration, we employed the wound-healing assay. Briefly, confluent monolayers of luECs seeded on fibronectin-coated (1 μg/cm2) 24-well plates were starved for 24 hours in medium containing 0.5% FBS medium. Monolayers were wounded with a plastic pipette tip to induce EC migration into the wound, and images were acquired at 0 and 24 hours. Where indicated, cells were pretreated for 1 hour with 20 μM JAK inhibitor 1 or vehicle (DMSO), or with 3 μg/ml anti-L1 polyclonal antibodies or control, nonimmune rabbit IgG; such treatments were maintained throughout the assay. Optical images of the wounds were acquired at 0 and 24 hours. The width of the wounds was measured with ImageJ (http://imagej.nih.gov/ij/) software, and the distance covered luECs, expressed as μm in 24 hours, was calculated according to this formula: (wound width at 0 – wound width at 24 hours)/2. The experiments were repeated 3 times, each time with triplicate wells. Tube formation assay A Matrigel-based tubulogenesis assay was performed to assess the ability of ECs to form an organized capillary-like network. Confluent ECs were starved overnight with medium containing 1% FBS. Growth factor–reduced Matrigel (BD Biosciences) was thawed overnight at 4°C on ice and, the day of the assay, plated on the bottom of a 96-well plate and left at 37°C for 1 hour for gelification. Thereafter, 1 × 104 cells/well were seeded on Matrigel and incubated at 37°C. Where indicated, cells were treated with 3 μg/ml anti-L1 polyclonal antibodies or control, nonimmune rabbit IgG. Optical images of the wells were acquired after 8 hours at ×4 magnification. The tubes in each well were manually counted. The experiments were repeated 3 times, each time with triplicate wells. Vascular permeability assays In vitro. The luECs were cultured on Transwell inserts (clear, 24-well plate, 0.4-μm pores; 6.5 mm diameter; tissue culture–treated; Costar), coated with glutaraldehyde–crosslinked gelatin, and allowed to form confluent monolayers (typically after 4 days). The day of the experiment, the medium in the upper chamber was replaced with complete medium containing 1 mg/ml FITC-labeled 40-kDa dextran (Sigma-Aldrich). At each time point, 50 μl of medium in the bottom chamber were taken, and fluorescence was measured at 488 nm. The medium taken from the bottom chamber was replaced each time with fresh complete medium in order to maintain a constant volume in the bottom chamber. The experiments were performed in quintuplicate and repeated 3 times. In vivo. Texas red–labeled 40-kDa dextran (Life Technologies) was administered intravenously by retroorbital injection into anesthetized Panc02 tumor–bearing mice (0.25 mg/mouse). Thirty minutes after injection, mice were sacrificed and their tumors were fixed in 2% para-

formaldehyde and embedded in paraffin. Sections were then subjected to IF costaining with rat anti-CD34 (BD; clone RAM34; 1:100) and rabbit anti–Texas red antibodies (1:100; Life Technologies). Vascular permeability was assessed as the ratio between the number of leaky vessels (i.e., vessels showing perivascular, extravasated Texas red staining) and the total number of CD34-positive vessels in 5 randomly selected fields (13).

Gene expression profiling ECs were seeded on plates coated with glutaraldehyde–crosslinked gelatin and cultured in complete medium for 4 days to reach confluence. Total RNA was extracted with RNeasy Mini Kit (QIAGEN). Quality control of the RNA samples was performed using Agilent Bioanalyzer 2100 (Agilent Technologies). Three different RNA extractions were processed for each of the cell lines under analysis. Each sample was labeled and hybridized to a Mouse Gene 1.0 ST Genechip array according to the manufacturer’s instructions (Affymetrix). Data were normalized using the Robust Multi-array Average (RMA) (ref. 58; raw and normalized data were deposited in the NCBI’s Gene Expression Omnibus [GEO GSE45859]). All analyses were performed on log2 data using parametric tests. BRB ArrayTools (http://linus.nci.nih.gov/BRB-ArrayTools.html) was used to run SAM analysis (http://www-stat.stanford.edu/~tibs/SAM/). A false discovery rate of less then 5% (i.e., the q value) was used to select statistically significant differentially expressed genes. Cluster 3.0 for Mac OS X (http://bonsai.hgc.jp/~mdehoon/software/cluster/) and Java Treview (http://jtreeview.sourceforge.net) were used for hierarchical clustering analysis using the uncentered correlation metric and centroid clustering method. Ingenuity Pathway Analysis and Upstream Regulator Analysis were performed using the online available web tool (http://www.ingenuity.com/). For gene network analysis, only direct relationships in mammals (i.e., human, mouse, and rat) were considered. P values for biofunction enrichment were corrected for multiple testing using the Benjamini-Hochberg correction. Immunoblotting ECs were cultured on petri dishes coated with glutaraldehyde–crosslinked gelatin for 4 days. Where indicated, cells were incubated for 2 or 24 hours with 2 μg/ml goat polyclonal anti–IL-6 Rα (R&D Systems) or control goat IgG. Total proteins were extracted by solubilizing cells in boiling Laemmli buffer (4% SDS, 16% glycerol, 40 mM Tris-HCl [pH 6.8]). Lysates were incubated for 15 minutes at 90°C to allow protein denaturation and then centrifuged for 5 minutes at 14,000 g to discard cell debris. The supernatants were collected, and the concentration of protein was determined using a BCA Protein Assay Kit (Pierce) according to the manufacturer’s instructions. Equal amounts of protein were separated on SDS polyacrylamide gel (SDS-PAGE), transferred to a Protran Nitrocellulose Transfer Membrane (Whatman), and blocked for 1 hour at RT in TBS, 0.1% Tween 20 containing 5% nonfat milk or BSA (blocking solution). The membranes were incubated overnight at 4°C or for 1 hour at RT with primary antibodies diluted in blocking solution. Membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies (Bio-Rad). The signal was detected by the ECL system (GE Healthcare) using Hyperfilm (Amersham Biosciences). The molecular masses of proteins were estimated relative to the electrophoretic mobility of contransferred prestained protein marker Precision Plus Protein Standards (Bio-Rad).

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qRT-PCR analysis ECs were cultured on petri dishes coated with glutaraldehyde–crosslinked gelatin for 4 days. Total RNA was isolated by extraction with RNeasy Mini Kit (QIAGEN), and 1 μg was reverse-transcribed with random hexamers (SuperScript Vilo cDNA Synthesis Kit; Invitrogen) according to the manufacturer’s instructions. cDNA (5 ng) was amplified in triplicate in a reaction volume of 15 μl with the TaqMan Gene Expression Assay (Applied Biosystems) and an ABI/Prism 7900 HT thermocycler (Applied Biosystems) using a pre-PCR step of 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 5°C and 60 seconds at 60°C. Preparations of RNA template without reverse transcriptase were used as negative controls. For each sample, the expression level was normalized against the geometric mean of the housekeeping genes encoding GAPDH and 18S. Normalized expression changes were determined with the comparative threshold cycle (Ct) method (59). IF ECs were cultured on 35-mm petri dishes coated with glutaraldehyde– crosslinked gelatin for 4 days and then fixed with 4% PFA for 10 minutes at room temperature. Panc02 tumors were fixed with 2% PFA overnight at +4°C and embedded in OCT after a graded sucrose series. Fixed cells and tumor sections (5 μm) were permeabilized with PBS and 0.5% Triton X-100 and incubated for 1 hour at RT with a blocking solution of PBS, 2% BSA, 5% donkey serum, and 0.05% Triton X-100. Samples were then incubated overnight at +4°C with primary antibodies diluted in blocking buffer, followed by secondary antibodies (2 hours at room temperature). Samples were then washed and mounted with Vectashield mounting medium with DAPI (Vector Laboratories). For staining of L1 on cells or on tumor sections, the permeabilization step was omitted and Tween 20 was used instead of Triton X-100 in all the buffers. TUNEL staining (Roche) was performed according to the manufacturer’s instructions. Confocal microscopy was performed with a Leica SP2 confocal microscope equipped with a motorized stage and violet (405 nm laser diode), blue (488 nm Argon), yellow (561 nm laser diode), and red (633 nm HeNe laser) excitation laser lines. In some cases, tile scans of the tumor sections were acquired using ×20 magnification. Mosaic images were created using the Leica LCS software. The number of Ki-67+, PHH3+, or TUNEL+ cells/mm2 were calculated by counting the number of positive cells in 10 different fields at ×63 magnification for each section. Density of PECAM-1–positive blood vessels was measured on images of tumor sections acquired as reported above and quantified with a custom-written plug-in of ImageJ software. Vessel pericyte coverage was calculated on the images of tumor sections and quantified with a custom-written plug-in, as previously described (60). Collagen IV and fibrin(ogen) deposition was measured on images of tumor sections and quantified with ImageJ as the mean of the fluorescence intensity of the pixels in the tumor area analyzed. Fluorescence microscopy was performed with a Olympus BX61 automated upright wide-field microscope equipped with a Photometrics Coolsnap Camera. IL-6 detection ECs were seeded on plates coated with 0.1% gelatin and cultured in complete medium for 4 days to reach confluence. At day 4, medium was replaced by fresh complete medium, and supernatants were collected after 24 hours. Mouse IL-6 was quantitated using the Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions. 14

Immunohistochemistry The clinicopathological data of PDAC patients are described in Supplemental Table 2. Formalin-fixed, paraffin-embedded specimens were prepared from both cancerous (n = 18) and noncancerous pancreatic tissue samples (n = 11). Sections were rehydrated through xylene and graded alcohols. Antigen retrieval was accomplished using 1 mM EDTA and 0.05% Tween. Samples were incubated with 3% H2O2 for 5 minutes,followed by blocking in 2% goat serum in PBS for 1 hour. Primary antibodies were incubated for 2 hours at RT in 2% goat serum. Horseradish peroxidase–conjugated secondary antibodies were used. Samples were developed with DAB and counterstained with hematoxylin. The vascular expression of L1 on tissue sections was measured as the ratio between L1-positive vessels and the total number of vessels identified by PECAM-1 staining on consecutive sections. The analysis was performed on 10 fields per sample at ×40 magnification. TMAs were prepared with formalin-fixed, paraffin-embedded samples from different tumor types (thyroid, gastric, colon, lung, and prostate carcinoma) and from their normal tissue counterparts. Representative areas were first selected on H&E-stained sections by a trained pathologist; 2 representative core biopsies for every tissue block were included in the TMAs. TMAs were assembled on a custombuilt tissue arrayer (Beecher Instruments) as previously described (61). TMAs were stained for PECAM-1 and L1, and the vascular expression of L1 was measured on serial sections as described above. FACS analysis Tumors were collected 14 days after Panc02 injection, washed in PBS, and digested in RPMI medium containing 0.1% collagenase type I (Gibco; Life Technologies) and 10 μg/ml DNase (Roche) for 45 minutes at 37°C. Digested tissue was passed 10 times through a 20-gauge needle and filtered through a 40-μm pore–sized mesh. After red blood cell lysis, cells were resuspended in FACS buffer (PBS, 1% FBS, 0.05% NaN3) and subjected to FACS staining. After a blocking incubation in FACS buffer containing 1% normal mouse serum (Sigma-Aldrich) for 45 minutes on ice, cells were stained for 45 minutes on ice with the indicated antibodies against different immune cell markers. Finally, cells were washed 3 times and fixed with 1% PFA. Percentage of positive cells was measured by FACSCalibur flow cytometer and analyzed with CellQuest software. The following monoclonal antibodies, all purchased by BD Biosciences except otherwise indicated, were used for FACS staining: PE anti-CD4 (clone RM4-5; 1:200), PE anti-CD8 (clone 53-6.7; 1:50), PE anti-CD11b (clone M1/70; 1:100), PE anti-CD11c (clone HL3; 1:50), FITC anti-CD14 (clone rmC5-3; 1:50), FITC anti-CD19 (clone 1D3; 1:200), FITC anti-CD45 (clone 104; 1:100), PerCP anti-CD45 (clone 30-F11; 1:100), FITC anti-F4/80 (Caltag Laboratories, clone CI:A31; 1:100); PE anti–Gr-1 (clone RB6-8C5; 1:50); and PE anti–I-A/I-E (clone M5/114.15.2; 1:200). Statistics Data are expressed as mean ± SEM, except as otherwise indicated. Student’s 2-tailed nonpaired t test or ANOVA and Bonferroni’s multiple comparison tests were used to determine statistical significance (GraphPad Prism 4). Tumor-free survival was drawn using the KaplanMeier method and compared by the log-rank test. Differences were considered significant at P < 0.05.

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Research article

The Journal of Clinical Investigation Study approval Mouse housing and all experimental animal procedures were performed in accordance with the guidelines established in the Principles of Laboratory Animal Care (directive 86/609/EEC) and approved by the Italian Ministry of Health. Specimens of PDAC and noncancerous pancreatic tissue were obtained upon informed consent from patients operated at University Hospitals Leuven. All tissue samples used for the TMAs were collected via standardized operative procedures approved by the Institutional Ethical Board of the European Institute of Oncology, and informed consent was obtained for all samples linked with clinical data.

Acknowledgments

We are grateful to Chiara Luise and Valentina Mattei for technical assistance; Monica Corada and Alicja Gruszka for technical advices; 1. Ziyad S, Iruela-Arispe ML. Molecular mechanisms of tumor angiogenesis. Genes Cancer. 2011;2(12):1085–1096. 2. Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell. 2009;15(3):232–239. 3. Paez-Ribes M, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 2009;15(3):220–231. 4. Goel S, Wong AH, Jain RK. Vascular normalization as a therapeutic strategy for malignant and nonmalignant disease. Cold Spring Harb Perspect Med. 2012;2(3):a006486. 5. Moos M, Tacke R, Scherer H, Teplow D, Fruh K, Schachner M. Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature. 1988;334(6184):701–703. 6. Rathjen FG, Schachner M. Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion. EMBO J. 1984;3(1):1–10. 7. Maness PF, Schachner M. Neural recognition molecules of the immunoglobulin superfamily: signaling transducers of axon guidance and neuronal migration. Nat Neurosci. 2007;10(1):19–26. 8. Kiefel H, et al. L1CAM: a major driver for tumor cell invasion and motility. Cell Adh Migr. 2012;6(4):374–384. 9. Pancook JD, Reisfeld RA, Varki N, Vitiello A, Fox RI, Montgomery AM. Expression and regulation of the neural cell adhesion molecule L1 on human cells of myelomonocytic and lymphoid origin. J Immunol. 1997;158(9):4413–4421. 10. Maddaluno L, et al. The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells. J Exp Med. 2009;206(3):623–635. 11. Issa Y, et al. Enhanced L1CAM expression on pancreatic tumor endothelium mediates selective tumor cell transmigration. J Mol Med. 2009;87(1):99–112. 12. Potenta S, Zeisberg E, Kalluri R. The role of endothelial-to-mesenchymal transition in cancer progression. Br J Cancer. 2008;99(9):1375–1379. 13. Mazzone M, et al. Heterozygous deficiency of

Tania Roskams for providing tissue samples; Giovanni Bertalot for helping with vessel counting; and Pier Paolo Di Fiore for his support. This work was supported by research grants from the Association for International Cancer Research (AICR grant 10-0091), the Associazione Italiana Ricerca sul Cancro (AIRC grants 2214, 1378, and 5815), the Fondazione Cariplo (grant 2004-1587 and 20082375, the Fondazione Telethon (grant GGP04078), and the Italian Ministry of Health (Ricerca Finalizzata), and by a travel grant to E. Magrini from the European Society of Cardiology. Address correspondence to: Ugo Cavallaro, Molecular Medicine Program, European Institute of Oncology, Via G. Ripamonti 435, I-20141 Milano, Italy. Phone: 39.02.9437.5165; E-mail: ugo. cavallaro@ieo.eu.

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Supplemental Table 1. Genes regulated by L1 in lECs. ProbeSet 10605113 10502791 10360391 10524621 10462621 10591853 10505717 10490818 10492815 10488608 10371332 10430174 10375360 10462623 10608638 10425049 10451287 10427131 10549222 10541307 10483110 10600044 10384539 10505734 10474972 10571984 10360382 10569102 10393887 10507500 10395039 10416505 10496580 10462618 10562905 10531415 10461423 10462613 10349138 10496592 10437224 10582275 10496569 10385526 10566583 10385518 10385533

Symbol Fold-‐change (SAM L1 vs. dc(i) ontrol) (t-‐statistic) control (Geometric L1 ((Geometric mean -‐ chip mean intensity) -‐ chip intensity) L1cam 13.33 -‐12.667 116.11 1556.84 Ifi44 11.63 -‐8.38 194.08 2258.22 Ifi203 9.09 -‐8.11 73.65 697.69 Oasl2 8.33 -‐8.742 308.45 2580.88 I830012O16Rik 6.67 -‐7.266 157.08 1073.99 Tbx20 6.67 -‐9.407 56.61 377.89 Adamtsl1 6.25 -‐10.088 132.31 810.96 Stmn2 6.25 -‐10.299 44.55 282.96 Tmem154 6.25 -‐8.511 59.14 363.65 Trib3 6.25 -‐6.622 228.7 1396.81 Aldh1l2 5.88 -‐4.193 73.09 438.21 Apol9a 5.88 -‐8.023 215.55 1305.02 Ebf1 5.88 -‐7.166 98.28 573.45 Ifit1 5.56 -‐8.832 676.44 3687.47 Klra33 5.56 -‐6.272 28.64 159.58 Apol9b 5.26 -‐10.016 216.09 1134.87 Isg15 5.26 -‐8.347 90.58 484.56 Soat2 5.26 -‐5.096 81.94 431.71 Bcat1 4.76 -‐4.912 266.16 1273.24 Usp18 4.76 -‐6.924 557.93 2667.21 Ifih1 4.55 -‐8.909 180.76 835.71 Prrg3 4.55 -‐12.812 58.73 262.64 Slc1a4 4.55 -‐6.589 418.04 1860.51 Adamtsl1 4.35 -‐6.647 103.24 447.92 Chac1 4.35 -‐5.646 982.36 4218.55 Ddx60 4.35 -‐5.118 52.53 232.72 Ifi204 4.35 -‐4.554 186.9 804.23 Irf7 4.35 -‐6.344 243.46 1066.67 Pycr1 4.35 -‐7.238 379.7 1618.44 Slc6a9 4.35 -‐4.68 407.7 1753.79 Cmpk2 4.17 -‐5.614 112.36 463.95 Kctd4 4.17 -‐7.698 120.16 494.84 Gbp3 4.00 -‐8.638 101.93 413.01 Ifit3 4.00 -‐7 1194.35 4834.4 Atf5 3.85 -‐7.657 258.28 989.6 Cxcl10 3.85 -‐4.404 165.84 636.08 Fads3 3.85 -‐7.811 480.82 1828.13 Ifit2 3.85 -‐5.232 213.07 823.35 Serpinb11 3.85 -‐6.276 16.2 63.38 Gbp2 3.70 -‐4.576 73.31 273.92 Mx2 3.70 -‐5.277 57.23 208.3 Slc7a5 3.70 -‐5.847 1430.62 5389.8 Gbp6 3.57 -‐6.818 124.78 450.71 9930111J21Rik2 3.45 -‐9.58 274.85 952.46 Gm8995 3.45 -‐8.251 620.96 2130.31 Tgtp1 3.45 -‐4.904 77.21 266.35 Tgtp1 3.45 -‐4.512 81.92 285.21

10385513 10607738 10420488 10599348 10366881 10399710 10473356 10573626 10402347 10554240 10434778 10382321 10545672 10550332 10546432 10360406 10499899 10360377 10585214 10391207 10534075 10498024 10546450 10462390 10545130 10525158 10519693 10433101 10566574 10563338 10544320 10409278 10439249 10493860 10546454 10546434 10480139 10393573 10415248 10378068 10537561 10381122 10376326 10569458 10438769 10522895 10496727 10420483 10607484 10350516

9930111J21Rik2 Car5b D14Ertd668e Gria3 Ddit3 Rsad2 Ube2l6 Gpt2 Ifi27l2a Isg20 Rtp4 Kcnj2 Mthfd2 Slc1a5 Adamts9 Ifi205 Sprr1a AI607873 Cryab Dhx58 Psph Slc7a11 Adamts9 Cd274 Gadd45a Oas1b Sema3d Gpr84 Gvin1 Ppp1r15a 1810009J06Rik Nfil3 Parp14 Sprr2b Adamts9 Adamts9 C1ql3 Lgals3bp Pck2 Xaf1 1810009J06Rik Fkbp10 Irgm2 Cars Cldn1 Csn3 Ddah1 Phf11 Ptchd1 Ptgs2

3.33 3.33 3.33 3.33 3.23 3.23 3.23 3.13 3.13 3.13 3.13 2.94 2.94 2.94 2.86 2.86 2.86 2.78 2.78 2.78 2.78 2.78 2.70 2.70 2.70 2.70 2.70 2.63 2.63 2.63 2.56 2.56 2.56 2.56 2.50 2.50 2.50 2.50 2.50 2.50 2.44 2.44 2.44 2.38 2.38 2.38 2.38 2.38 2.38 2.38

-‐9.346 -‐7.961 -‐8.837 -‐4.498 -‐5.087 -‐4.655 -‐6.096 -‐8.077 -‐5.913 -‐5.831 -‐5.193 -‐8.778 -‐7.676 -‐4.05 -‐12.775 -‐2.932 -‐9.522 -‐3.878 -‐5.819 -‐5.996 -‐6.688 -‐3.907 -‐11.539 -‐2.895 -‐3.351 -‐5.384 -‐5.955 -‐3.318 -‐5.843 -‐2.812 -‐5.378 -‐4.086 -‐5.576 -‐4.941 -‐11.065 -‐10.741 -‐5.413 -‐7.297 -‐6.948 -‐5.578 -‐4.81 -‐7.596 -‐5.173 -‐5.855 -‐3.689 -‐3.877 -‐15.218 -‐2.763 -‐6.816 -‐3.419

286.47 38.08 409.92 48.99 273.94 590.48 303.9 226.45 123.5 277.89 973.64 48.57 957.74 623.51 302.52 137.55 263.77 542.28 198.03 358.4 254.26 1938.95 375.48 149.77 182.72 135.45 36.52 295.24 1280.38 491.03 74.21 145.32 221.51 31.86 310.74 333.93 161.1 933.78 892.89 482.41 70.68 89.73 268.87 735.05 27.48 47.95 168.92 40.79 78.78 1385.76

968.49 126.17 1384.85 162.33 873.53 1894.21 968.24 697.61 389.19 868.38 3019.32 142.02 2827.82 1844.39 867.75 391.33 743.52 1514.59 542.69 1000.15 712.23 5357.6 1028.5 407.17 494.53 370.25 97.82 773.86 3332.83 1293.88 188.31 371.34 575.06 82.57 769.44 837.68 402.39 2354.31 2236.69 1206.1 170.72 218.9 662.74 1733.02 66 114.14 403.14 97.95 186.62 3298.27

10354741 10455970 10389207 10363173 10566571 10566578 10588691 10569618 10345445 10561104 10359929 10390850 10358434 10347948 10427772 10571788 10361091 10424559 10471129 10448004 10432236 10383233 10535852 10367224 10435497 10546430 10467470 10431935 10485405 10568369 10403727 10373157 10367641 10544133 10383214 10485674 10443885 10455961 10458607 10603087 10466410 10352936 10408613 10453057 10452118 10606689 10541071 10472538 10405094 10414360

Rftn2 BC023105 Ccl5 Gja1 Gm8979 Gm8979 Hyal1 Ano1 Arid5a Axl Ddr2 Krt20 Pla2g4a Sp100 Tars Vegfc Atf3 Khdrbs3 Ncs1 Phf10 Rnd1 Rnf213 Slc7a1 Stat2 Stfa2l1 Adamts9 Aldh18a1 Amigo2 Cd44 Cox6a2 Gli3 Mars Mthfd1l Parp12 Rnf213 2700007P21Rik Cyp4f40 Iigp1 Lars Pir Psat1 Sox17 Tubb2b Cyp1b1 Lonp1 Timm8a1 8430408G22Rik Dhrs9 Iars Lgals3

2.38 2.33 2.33 2.33 2.33 2.33 2.33 2.27 2.27 2.27 2.27 2.27 2.27 2.27 2.27 2.27 2.22 2.22 2.22 2.22 2.22 2.22 2.22 2.22 2.22 2.17 2.17 2.17 2.17 2.17 2.17 2.17 2.17 2.17 2.17 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.08 2.08 2.08 2.04 2.04 2.04 2.04

-‐4.422 -‐3.847 -‐3.114 -‐4.571 -‐5.885 -‐5.768 -‐5.398 -‐6.006 -‐3.235 -‐4.079 -‐2.574 -‐5.794 -‐3.835 -‐6.604 -‐6.313 -‐4.732 -‐4.265 -‐3.877 -‐10.089 -‐5.689 -‐4.348 -‐5.016 -‐5.389 -‐5.03 -‐8.304 -‐7.241 -‐5.737 -‐6.895 -‐5.755 -‐3.465 -‐4.482 -‐6.854 -‐5.965 -‐6.023 -‐5.574 -‐5.828 -‐2.806 -‐3.631 -‐5.434 -‐9.105 -‐6.583 -‐3.512 -‐5.004 -‐5.638 -‐3.423 -‐4.317 -‐4.852 -‐3.057 -‐7.677 -‐5.048

123.21 10.54 321.03 898.54 490.07 613.92 42.09 157.42 285.73 153.02 50.88 302.32 851.07 399.99 504.37 244.96 1055.96 67.13 178.28 1398.33 166.8 828.02 980.63 605.94 48.54 746.33 1811.36 536.43 175.71 148.91 70.03 1236.45 419.94 841.4 1210.24 1174.84 119.64 20.07 1062.51 277.47 1800.42 158.84 134.72 53.62 692.81 130.76 181.33 53.95 1968.82 271.9

292.63 24.55 754.09 2097.72 1131.17 1416.56 97.01 356.4 648.52 347.4 114.43 680.07 1949.74 917.88 1139.44 556.63 2335.28 149.98 393.05 3086.97 369.22 1830.99 2186.65 1352.25 107.4 1630.69 3922.6 1170.41 379.38 321.28 150.95 2661.61 920.71 1845.3 2655.88 2486.95 255.9 42.59 2262.24 590.05 3868.8 336.5 289.3 110.88 1441.71 270.79 372.68 109.48 4055.84 559.47

10475532 10456756 10566346 10584124 10564507 10425364 10580829 10571162 10500272 10384223 10567995 10533246 10607486 10393341 10361760 10376021 10562416 10411229 10397351 10484488 10373202 10379630 10467149 10546452 10362968 10399882 10406205 10373452 10574087 10380622 10375515 10424119 10502240 10566366 10518358 10579331 10450075 10491970 10429560 10459405 10346191 10359181 10575550 10353775 10350003 10432372 10415319 10385500 10533256 10566326

Sqrdl Zbtb7c 9230105E10Rik Arhgap32 Arrdc4 Atf4 Cngb1 Eif4ebp1 Gm129 Igfbp3 Nupr1 Oas1g Ptchd1 Rhbdf2 Timm8a1 41160 Cebpg F2r Jdp2 P2rx3 Shmt2 Slfn2 2010002M12Rik Adamts9 Bves Dus4l Erap1 Gm129 Herpud1 Hoxb9 Ifi47 Nov Npnt Trim30d 2610305D13Rik Gdf15 H2-‐K1 Lhfp Ly6i Nars Stat1 Tor3a Aars Bend6 Cyb5r1 Dhh Irf9 Irgm1 Oas1a Trim12a

2.04 2.04 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 1.96 1.96 1.96 1.96 1.96 1.96 1.96 1.92 1.92 1.92 1.92 1.92 1.92 1.92 1.92 1.92 1.92 1.92 1.92 1.89 1.89 1.89 1.89 1.89 1.89 1.89 1.89 1.85 1.85 1.85 1.85 1.85 1.85 1.85 1.85

-‐6.707 -‐12.218 -‐3.623 -‐3.685 -‐2.79 -‐5.419 -‐7.792 -‐5.733 -‐4.522 -‐12.32 -‐5.83 -‐3.993 -‐7.785 -‐6.677 -‐4.319 -‐5.91 -‐4.868 -‐3.863 -‐3.889 -‐5.199 -‐5.88 -‐5.911 -‐7.049 -‐8.148 -‐3.386 -‐3.817 -‐6.694 -‐5.258 -‐4.582 -‐4.841 -‐4.953 -‐6.165 -‐5.948 -‐3.129 -‐4.111 -‐4.321 -‐3.455 -‐7.973 -‐4.349 -‐5.973 -‐4.693 -‐4.804 -‐6.296 -‐9.483 -‐2.978 -‐6.543 -‐4.706 -‐5.386 -‐3.777 -‐4.211

1147.79 386.34 131.87 79.17 230.57 673.13 136.58 1978.58 501.57 2976.44 2191.16 311.34 643.5 300.4 166.29 1144.79 559.38 96.42 493.57 109.23 2539.52 1600.41 25.49 355.97 64.96 279.91 168.44 707.11 1365.45 137.19 27.65 2320.47 3262.59 309.17 89.9 244.89 240.76 293.95 56.91 2948.86 1715.49 778.82 953.43 130.12 911.59 436.82 1247.95 1847.45 215.51 312.03

2325.22 784.43 264.4 158.89 461.14 1335.95 273.22 3921.71 1009.35 6007.21 4418.46 616.53 1293.4 604.9 335.52 2260.5 1104.2 188.36 960.06 216.16 4943.74 3107.84 49.2 682.37 125.14 538.08 322.08 1354.14 2602.13 262.76 53.5 4423.09 6281.06 599.37 169.65 464.01 453.3 556.23 108.3 5525.64 3243.67 1464.53 1765.4 242.13 1676.32 814.15 2328.78 3412.08 395.48 577.78

10451198 10372877 10543067 10428310 10445347 10356262 10362201 10586248 10352514 10504450 10487506 10478938 10499705 10400095 10365559 10424188 10462683 10560709 10440099 10474048 10508420 10523547 10515500 10418927 10407955 10345025 10444890 10381408 10429160 10393106 10550786 10542842 10527965 10574102 10469571 10401607 10518726 10456405 10394674 10508115 10376899 10560767 10550778 10560754 10590433 10599680 10601581 10463911 10538420 10513008

Vegfa Xpot Asns Azin1 Clic5 Csprs Ctgf Dennd4a Eprs Glipr2 Gm14005 Hax1 Hax1 Ifrd1 Igf1 Mtbp Pcgf5 Pvr St3gal6 Syt13 Yars Agpat9 B4galt2 Bmpr1a Epdr1 Iars Ier3 Ifi35 St3gal1 Trim47 Vmn1r132 Ccdc91 Cldn12 Nlrc5 Otud1 Pgf Slc25a33 Slmo1 Socs2 Stk40 Trim16 Vmn1r132 Vmn1r132 Vmn1r132 1700048O20Rik 3830403N18Rik 9230105E10Rik Add3 Gars Klf4

1.85 1.85 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.82 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.79 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.72 1.72 1.72 1.72 1.72 1.72

-‐4.406 -‐4.158 -‐5.485 -‐6.404 -‐9.993 -‐5.883 -‐4.739 -‐2.566 -‐3.685 -‐3.167 -‐3.991 -‐5.073 -‐4.918 -‐3.981 -‐2.58 -‐5.815 -‐3.566 -‐3.549 -‐3.63 -‐5.112 -‐6.553 -‐3.832 -‐7.646 -‐5.128 -‐5.541 -‐4.451 -‐3.44 -‐5.009 -‐10.584 -‐3.582 -‐3.331 -‐3.357 -‐4.415 -‐3.832 -‐4.388 -‐4.84 -‐3.036 -‐4.858 -‐3.922 -‐4.117 -‐3.853 -‐3.426 -‐3.426 -‐3.314 -‐5.289 -‐8.34 -‐3.607 -‐7.031 -‐4.778 -‐4.875

533.52 1278.34 4882.4 1261.07 599.39 182.85 212.55 239.95 1332.46 342.93 81.24 1519.07 1405.96 925.98 537.25 328.84 204.72 1558.62 77.52 195.24 1656.39 264.53 127.09 25.34 393.34 2831.77 116.66 712.17 1105.07 288.37 29.76 67.21 326.95 92.89 71.29 2661.82 107.91 89.85 847.7 865.25 659.92 26.18 26.18 31.04 36.01 360.24 205.71 864.57 2516.6 947.61

981.78 2383.56 8815.15 2307.39 1080.62 333.85 388.95 436.22 2406.77 623.36 147.23 2737.13 2553.23 1692.39 982.97 601.31 375.56 2844.82 139.8 355.98 2997.07 475.04 226.17 45.05 701.24 5101.96 207.82 1260.72 1970.18 514.64 53.02 117.39 574.65 161.62 125.9 4666.4 189.75 157.6 1500.03 1510.77 1149.32 45.59 45.59 54.83 61.91 624.91 356.52 1491.78 4309.29 1630.88

10608659 10366796 10429140 10350733 10347796 10431874 10566333 10397145 10361828 10578222 10488806 10458340 10436428 10578045 10441003 10456140 10528015 10598626 10463121 10542875 10438468 10584259 10580382 10482517 10576610 10521972 10602840 10604656 10423902 10549276 10479852 10561008 10360764 10595145 10458226 10438753 10385159 10360934 10380506 10566144 10577226 10369276 10519324 10440427 10400941 10488108 10460108 10394914 10413609 10404996

Klra22 Mettl1 Ndrg1 Rgs16 Rhbdd1 Slc38a1 9230105E10Rik Acot2 Cited2 Dlc1 Eif2s2 Hbegf Mina Nrg1 Runx1 Sh3tc2 Steap1 Tspan7 Zfp518a 3010003L21Rik Cyp2ab1 Fez1 Neto2 Nmi Pard3 Pcdh7 Sh3kbp1 Xlr Zfpm2 Bhlhe41 Camk1d Ceacam1 Enah Gm8074 Hspa9 Leprel1 Rars Rrp15 Tac4 Trim21 2610019F03Rik Ccdc109a Cdk6 D16Ertd472e Dhrs7 Esf1 Gnpnat1 Iah1 Mustn1 Ninj1

1.72 1.72 1.72 1.72 1.72 1.72 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.69 1.67 1.67 1.67 1.67 1.67 1.67 1.67 1.67 1.67 1.67 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.61 1.61 1.61 1.61 1.61 1.61 1.61 1.61 1.61 1.61

-‐3.639 -‐2.791 -‐12.278 -‐3.871 -‐3.932 -‐3.509 -‐3.673 -‐2.622 -‐5.684 -‐6.008 -‐3.899 -‐2.573 -‐3.461 -‐4.38 -‐2.827 -‐3.815 -‐3.519 -‐6.257 -‐3.031 -‐5.239 -‐4.446 -‐4.614 -‐2.717 -‐4.491 -‐5.522 -‐3.854 -‐3.273 -‐4.138 -‐7.972 -‐3.768 -‐4.896 -‐6.135 -‐8.243 -‐3.595 -‐4.931 -‐3.666 -‐4.48 -‐2.615 -‐2.764 -‐3.384 -‐4.562 -‐3.507 -‐5.776 -‐5.469 -‐4.849 -‐3.021 -‐3.084 -‐3.821 -‐5.145 -‐5.103

9.39 568.01 1061.42 215.8 1356.78 624.2 258.33 2100.36 567.13 157.46 823.2 303.61 927.79 119.4 166.52 291.71 25.24 2000.03 243.16 110.51 65.92 822.51 44.25 210.06 256.48 35.82 1506.8 34.35 204.81 162.38 237.86 2416.7 241.56 17.48 2453.77 501.32 3834.82 311.04 48.2 298.36 97.14 1213.41 618.81 506.1 312.66 1463.84 326.31 411.66 186.36 1907.59

16.06 983.46 1836.05 372.18 2357.25 1082.62 439.88 3561.16 966.61 267.77 1403.33 514.51 1572.48 200.95 280.26 490.64 42.83 3409.26 410.25 183.15 109.54 1371.07 74.23 350.44 429.44 59.9 2492.73 57.32 339.56 265.32 392.65 3938.23 398.27 28.83 4037.49 818.9 6327.47 507.08 79.15 490.16 155.67 1947.39 995.54 813.34 505.26 2364.65 523.81 660.52 302.65 3066.75

10529425 10435457 10366346 10489569 10455873 10370510 10411459 10585834 10423109 10457644 10478890 10578810 10476383 10502655 10454254 10347915 10419216 10467842 10397966 10359816 10461869 10447056 10471550 10567043 10501358 10495830 10529515 10493850 10435075 10433433 10548030 10538338 10464391 10548905 10402808 10598638 10580191 10429843 10407481 10385511 10363181 10445241 10371676 10489237 10582445 10366848 10483178 10474958 10385504 10606016

Nop14 Parp9 Phlda1 Pltp Slc12a2 Syde1 Tmem171 6030419C18Rik Adamts12 Cdh2 Cebpb Clcn3 Crls1 Cyr61 Dtna Gm7609 Gnpnat1 Got1 Otub2 Pogk Prune2 Qpct Rpl12 Rras2 Sars Sec24d Sorcs2 Sprr2a1 Tfrc BC024814 Cd9 Creb5 Emx2 Eps8 Jag2 Mid1ip1 Nfix Parp10 Pfkp Psme2 Stard6 Tnfrsf21 Utp20 9430008C03Rik Ankrd11 B4galnt1 Cobll1 Dll4 Gm5431 Il2rg

1.61 1.61 1.61 1.61 1.61 1.61 1.61 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.59 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.54 1.54 1.54 1.54 1.54 1.54 1.54

-‐3.647 -‐3.682 -‐2.973 -‐5.07 -‐3.105 -‐3.749 -‐6.249 -‐3.27 -‐3.499 -‐3.129 -‐2.761 -‐4.14 -‐3.987 -‐4.039 -‐3.188 -‐4.806 -‐3.267 -‐3.166 -‐3.802 -‐3.761 -‐4.373 -‐2.944 -‐2.923 -‐4 -‐5.522 -‐3.169 -‐4.999 -‐3.983 -‐2.797 -‐3.7 -‐4.169 -‐6.765 -‐3.696 -‐2.669 -‐6.96 -‐4.181 -‐3.584 -‐4.457 -‐8.308 -‐4.815 -‐6.784 -‐4.999 -‐2.625 -‐3.265 -‐5.56 -‐3.303 -‐3.701 -‐3.868 -‐4.012 -‐5.496

864.78 422.62 366.27 135.04 767.17 492.79 1122 160.31 139.4 407.2 139.9 645.42 288.11 198.23 32.48 20.37 321.27 2410.23 432.01 239.56 538.82 39.39 348.81 1202.47 2954.16 637.44 112.1 181.4 158.6 390.3 462.44 121.5 47.94 389.05 157.64 834.84 1074.29 297.08 298.65 653.75 111.4 284.46 640.42 591.07 932.35 190.38 250.83 1330.37 19.25 2272.02

1401.99 679.37 588.23 219.55 1245.86 789.02 1800.03 254.03 220.17 641.62 223.36 1029.39 458.91 314.86 51.73 32.14 509.34 3841.98 691.09 378.8 849.02 62.64 556.11 1907.94 4709.56 1007.83 178.45 289.93 251.63 607.58 726.71 190.69 75.45 611.89 246.03 1306.84 1679.12 461.92 466.22 1014.8 175.03 447.44 997.46 908.24 1423.47 291.53 387.71 2052.62 29.75 3509.24

10555197 10410264 10450611 10476401 10408097 10410709 10420216 10379646 10456556 10488378 10438555 10566358 10499514 10402585 10489850 10389005 10501971 10582458 10363007 10359982 10490632 10423274 10581737 10462231 10515095 10424853 10405211 10538590 10580183 10499655 10376033 10365817 10554521 10515399 10431792 10457959 10498332 10516956 10447891 10390032 10461930 10517967 10587194 10540118 10574976 10464754 10383731 10465089 10479765 10443535

Mtap6 Mterfd1 Nrm Plcb1 Prss16 Rfesd Sdr39u1 Slfn3 Stard6 Thbd Thpo Trim30a Trim46 Wars Znfx1 1110002N22Rik Ank2 Ankrd11 Ascc3 Atf6 BC006779 Cdh18 Ddx19b Dmrt2 Elavl4 Fam203a Gadd45g Herc6 Ier2 Il6ra Kif3a Ntn4 Pde8a Plk3 Pus7l Sft2d3 Siah2 Smpdl3b Acat2 Acsf2 D030056L22Rik Fblim1 Gnb5 Lsm3 Pla2g15 Rhod Smtn Snx32 Suv39h2 Tbc1d22b

1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.54 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 1.52 -‐1.50 -‐1.50 -‐1.50 -‐1.50 -‐1.50 -‐1.50 -‐1.50 -‐1.50 -‐1.50 -‐1.50 -‐1.50 -‐1.50

-‐5.433 -‐4.601 -‐4.62 -‐5.277 -‐3.709 -‐5.072 -‐5.074 -‐4.051 -‐6.464 -‐4.588 -‐5.098 -‐3.602 -‐3.354 -‐3.834 -‐4.89 -‐4.277 -‐3.055 -‐4.932 -‐3.25 -‐2.635 -‐5.89 -‐4.069 -‐5.235 -‐3.551 -‐3.472 -‐3.997 -‐2.822 -‐3.161 -‐3.516 -‐4.271 -‐3.229 -‐4.303 -‐4.822 -‐2.594 -‐2.615 -‐3.693 -‐3.423 -‐4.531 2.61 2.501 2.814 3.869 2.801 3.33 4.011 4.161 3.739 2.368 2.462 7.112

384.93 649.49 132.6 150.76 149.45 276.03 530.79 2230.02 112.88 2797.28 86.64 1893.96 237.39 2536.11 951.4 286.37 54.55 275.13 808.72 1510.5 662.01 95.76 121.1 65.75 26.89 460.97 349.24 329.79 225.68 296.12 272.59 171.47 573.96 1360.32 371.57 79.72 423.13 1183.97 572.29 820.62 313.1 1280.46 557.02 1561.51 312.43 954.17 2360.39 593.56 95.32 1743.1

591.04 1004.99 202.75 233.47 229.06 426.43 811.05 3412.2 172.98 4301.63 134.09 2919.99 366.37 3914.43 1465.33 434.25 82.09 418.83 1226.86 2282.05 1003.29 145.14 183.28 98.99 40.77 702.35 525.81 497.09 340.59 448.39 412.61 261.18 867.5 2062.4 564.84 121.54 639.64 1801.88 380.96 546.69 208.63 852.64 370.95 1039.62 208.66 634.56 1577.89 394.94 63.73 1158.81

10495416 10494655 10526754 10546031 10492341 10554129 10583847 10537179 10511429 10531987 10539119 10545308 10446928 10409424 10585395 10350188 10471474 10345046 10543118 10565958 10560644 10458843 10434758 10446063 10535231 10381260 10403081 10572398 10544501 10394593 10376208 10472958 10545921 10419744 10390103 10476021 10449242 10582778 10445702 10519612 10561927 10523297 10474223 10590060 10591660 10563085 10408321 10400971 10489759 10372831

Vav3 Zfp697 Ap4m1 Aplf Arhgef26 B130024G19Rik Bbs9 Bpgm Car8 Gbp4 Ggcx Kdm3a Ltbp1 Mxd3 Siva1 Tmem9 Ak1 Efhc1 Glcci1 P2ry6 Pvrl2 Sema6a St6gal1 Tnfaip8l1 Ttyh3 Tubg2 Wdr60 Crlf1 Ezh2 Fam49a Gm2a Hoxd10 Mxd1 Oxa1l Pdk2 Sirpa Snrpg Tarbp1 Usp49 9330182L06Rik Aplp1 Ccng2 Cd59b Ctdspl Epor Fcgrt Gmnn Six4 Sulf2 Tbc1d30

-‐1.50 -‐1.50 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.51 -‐1.52 -‐1.52 -‐1.52 -‐1.52 -‐1.52 -‐1.52 -‐1.52 -‐1.52 -‐1.52 -‐1.52 -‐1.52 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.53 -‐1.54 -‐1.54 -‐1.54 -‐1.54 -‐1.54 -‐1.54 -‐1.54 -‐1.54 -‐1.54 -‐1.54 -‐1.54

4.237 4.337 4.444 2.821 2.974 3.671 4.042 5.256 3.107 3.796 6.336 7.687 3.792 2.668 2.504 3.169 3.54 6.1 2.589 2.439 4.569 3.058 5.438 2.89 3.887 3.441 3.163 4.634 2.531 3.177 4.689 4.226 3.232 3.59 2.84 3.912 6.043 2.475 3.673 6.037 3.299 2.859 3.332 3.063 3.12 4.16 2.906 3.822 2.564 5.658

842.54 1808.53 689.25 168.22 61.59 319.88 333.37 269.54 504.77 4232.3 806.98 927.93 211.15 263.51 172.65 599.49 1485.91 229.92 283.84 275.55 915.61 1506.31 676.44 239.66 1678.05 209.09 182.86 195.15 1539.6 901.62 1177.05 157.68 428 1876.36 648.06 2482.25 1017.56 150.64 146.12 393.65 367.96 403.28 28.65 380.41 162.33 273.73 54.58 219.45 881.32 64

561.12 1205.79 457.87 111.25 40.84 211.5 221.09 178.18 335.07 2796.36 533.36 613.96 140.11 174.88 114.68 396.93 974.44 150.97 187.23 181.44 602.58 989.53 443.96 157.67 1102.57 137.47 120.03 127.91 1008.28 589.75 767.39 102.73 279.05 1223.27 422.53 1623.46 666.99 98.4 95.57 254.95 238.94 262.61 18.58 246.79 105.44 177.63 35.43 142.15 573.05 41.64

10363445 10521667 10562651 10582427 10589420 10409866 10446441 10357164 10444895 10439114 10508465 10531370 10460626 10380489 10476301 10443108 10528790 10354677 10601846 10500434 10490923 10496023 10497265 10583992 10421581 10460666 10537062 10606554 10437432 10574676 10508099 10536483 10377380 10549171 10414102 10598369 10603431 10547274 10484425 10435704 10587733 10498998 10488147 10373583 10350753 10473281 10546510 10489246 10572928 10471464

4632428N05Rik Bst1 C330019L16Rik Cbfa2t3 Cdc25a Ctla2b Ddx11 Epb4.1l5 Flot1 Iqcg Marcksl1 Naaa Rnaseh2c Samd14 Smox Syngap1 Xrcc2 Ankrd44 Arxes2 Bcl9 Car2 Casp6 Fabp4 Igsf9b Lrch1 Ltbp3 Mest Nap1l3 Nmral1 Nol3 Oscp1 Tes 1500010J02Rik 5730419I09Rik Mmrn2 Prickle3 Suv39h1 Zfp248 2700094K13Rik Cd80 Ctsh D930015E06Rik Flrt3 Gdf11 Glul Itgav Lrig1 Mafb Rasd2 St6galnac6

-‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.55 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.56 -‐1.57 -‐1.57 -‐1.57 -‐1.57 -‐1.57 -‐1.57 -‐1.58 -‐1.58 -‐1.58 -‐1.58 -‐1.58 -‐1.58 -‐1.58 -‐1.58 -‐1.58 -‐1.58 -‐1.58 -‐1.58

7.62 4.535 4.482 3.177 3.634 3.274 4.105 3.133 3.879 2.88 5.327 3.654 2.562 3.424 5.121 2.417 2.481 2.734 3.028 3.52 2.917 2.845 3.078 2.684 4.047 5.11 5.48 4.455 3.133 2.894 3.241 4.823 3.046 10.159 3.408 3.266 2.537 4.538 2.759 2.884 3.728 5.596 6.793 2.986 2.691 6.965 4.007 2.442 3.361 2.748

659.18 1629.72 207.21 584.72 507.88 728.73 320.22 236.61 446.49 325.74 2101.7 423.55 639.62 227.33 751.49 30.99 154.56 160.53 32.47 801.69 124.74 945.69 5684.95 469.14 1764.44 955.49 4984.16 130.64 388.91 145.71 335.64 1863.26 1154.98 708.02 6326.54 327.21 603.14 336.38 519.47 118.56 4268.22 227.61 944.18 448.02 1630.69 1625.65 516.81 1336.73 93.27 375.31

424.08 1054.32 133.42 376.53 327.64 471.43 206.46 152.41 287.69 210.7 1352.66 273.06 412.41 147.1 484.63 20.02 99.42 103.11 20.84 514.46 80.03 604.75 3643.64 299.94 1128.19 610.67 3199.36 83.92 248.92 93.54 214.64 1192.7 736.55 449.66 4025 209.03 383.74 213.87 329.72 75.14 2697.69 143.83 598.17 283.77 1032.47 1027.33 328.03 845.71 59.01 237.21

10552245 10447275 10519607 10472128 10406407 10384811 10444858 10417167 10493494 10475625 10605392 10528794 10606436 10359754 10469559 10460400 10407097 10485624 10467493 10489391 10567412 10499168 10447417 10546834 10396849 10455967 10355325 10402063 10564539 10529375 10588482 10597945 10542340 10346374 10551469 10446777 10582501 10491732 10451547 10579659 10395163 10455813 10595981 10451372 10402117 10492679 10587085 10471424 10346607 10600017

Tshz3 1700106N22Rik 4930420K17Rik Arl6ip6 Arrdc3 Ccdc104 Cchcr1 Clybl Efna3 Eid1 F8 Gm10471 Hmgn5 Mpzl1 Msrb2 Pcx Pde4d Prrg4 Tctn3 Ada Eri2 Kirrel Msh6 Rad18 Rad51l1 2610318N02Rik Bard1 Foxn3 Mctp2 Mxd4 Poc1a Tmem158 8430419L09Rik Aox1 Dyrk1b Ehd3 Fanca Fat4 Gm16494 Hmgn2 Lamb1 Lmnb1 Mras Ptk7 Rps6ka5 4930579G24Rik BC031353 Fam102a Fzd7 Hmgb3

-‐1.58 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.59 -‐1.60 -‐1.60 -‐1.60 -‐1.60 -‐1.60 -‐1.60 -‐1.61 -‐1.61 -‐1.61 -‐1.61 -‐1.61 -‐1.61 -‐1.61 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.62 -‐1.63 -‐1.63 -‐1.63 -‐1.63 -‐1.63

3.092 2.905 5.078 3.431 7.989 3.414 3.184 6.251 2.476 4.527 10.09 2.764 2.867 6.505 3.971 3.074 3.438 2.494 4.421 4.044 3.064 4.646 3.982 3.053 2.955 4.698 2.85 4.475 4.417 2.982 3.243 2.952 6.83 3.115 3.409 5.346 2.601 3.338 3.081 2.937 4.23 2.509 5.288 7.243 3.434 2.683 2.825 3.478 7.206 4.38

1154.5 441.67 606.4 799.99 1583.08 423.47 362.24 819.73 127.49 815.87 1785.81 37.79 216.57 1597.18 196.38 417.55 477.61 242.72 812.34 164.53 765.42 684.22 369.29 1221.36 75.79 144.99 151.89 783.5 99.89 1546.5 161.91 414.86 608.3 1218 979.27 1134.92 230.21 606.55 630.4 1839.83 897.32 418 1534.62 274.15 269.88 362.66 251.28 597.83 836.56 532.36

731.97 277.3 380.66 504.39 996.83 267.08 227.34 516.83 79.99 514.4 1122.71 23.76 136.44 1006.4 123.86 262.87 300.55 152.22 510.84 102.59 478.27 426.76 230.44 764.91 47.25 90.15 94.17 487.82 61.89 961.93 100.87 257.07 376.47 752.63 603.84 699.08 142.4 375.13 388.74 1132.43 553.67 257.34 946.55 169.61 166.55 222.9 154.01 367.19 513.71 327.13

10403108 10580061 10552516 10462035 10595979 10494817 10472400 10526120 10541877 10549842 10379953 10465844 10424779 10528077 10542677 10473312 10517141 10591773 10400649 10350864 10368720 10539933 10457587 10512949 10420320 10440953 10529613 10557326 10591270 10445293 10399011 10416736 10409990 10453049 10546685 10554463 10433887 10521626 10447023 10512236 10517116 10348739 10458033 10424543 10554045 10594631 10540935 10544906 10388234 10602068

Hmgn2 Il27ra Klk6 Ldhb Mras Ngf Scn2a1 Tpst1 Vamp1 Zfp667 4632419I22Rik Asrgl1 Cks2 Dbf4 Etnk1 Fam171b Hmgn2 Hmgn2 Pole2 Sec16b Slc16a10 Txnrd3 Zfp521 Abca1 Cenpj Donson Evc Il4ra Olfm2 Pla2g7 4930427A07Rik 6720463M24Rik 6720489N17Rik Cdc42ep3 Eif4e3 Hddc3 Pkp2 Cc2d2a Heatr5b Kif24 Rps6ka1 Sned1 Stard4 Wisp1 Adamts17 Aph1b Cand2 Ggct Gsg2 Mid2

-‐1.63 -‐1.63 -‐1.63 -‐1.63 -‐1.63 -‐1.63 -‐1.63 -‐1.63 -‐1.63 -‐1.63 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.64 -‐1.65 -‐1.65 -‐1.65 -‐1.65 -‐1.65 -‐1.65 -‐1.65 -‐1.66 -‐1.66 -‐1.66 -‐1.66 -‐1.66 -‐1.66 -‐1.66 -‐1.67 -‐1.67 -‐1.67 -‐1.67 -‐1.67 -‐1.67 -‐1.67 -‐1.68 -‐1.68 -‐1.68 -‐1.68 -‐1.68 -‐1.68

2.959 4.217 5.321 5.03 4.112 4.193 4.298 4.682 4.153 4.811 2.929 3.505 2.528 3.146 6.957 2.969 2.964 2.945 2.555 4.618 5.957 3.916 2.753 3.996 2.667 2.415 4.998 5.957 2.48 3.279 3.505 3.087 4.255 3.282 6.086 3.627 5.101 2.562 2.859 3.546 4.323 3.943 3.041 5.5 3.037 4.485 5.08 5.347 2.647 6.382

2226.78 200.15 150.7 91.52 832.97 610.63 16.63 657.42 252.83 226.32 691.47 112.8 1507.32 777.27 1576.42 571.79 1878.87 1809.15 556.88 83.64 455.68 870.04 1115.16 2391.77 230.74 236.23 276.16 3984.49 689.53 68 212.11 323.85 365.75 520.43 126.77 408.62 430.87 208.22 132.16 128.54 287.37 737.11 846.71 374.56 754.63 428.95 330.66 371.94 230.15 1613.14

1362.85 122.99 92.41 56.09 512.3 375.45 10.21 403.47 155.27 139.27 422.47 68.73 918.59 472.74 958.54 347.63 1144.66 1100.21 339.84 51.1 277.48 530.86 679.57 1448.87 139.79 143.56 167.44 2418.49 418.72 41.2 128.16 194.58 220.86 313.28 76.18 245.56 259.92 124.49 79.08 77.11 171.79 442.7 508.15 223.84 450.52 255.08 196.62 220.78 136.99 957.51

10574384 10349834 10485378 10383395 10433578 10379034 10355329 10396030 10427898 10472782 10356461 10462442 10357436 10449999 10537157 10353004 10604175 10465861 10358982 10545910 10393628 10572815 10410560 10477052 10582008 10405185 10494122 10555460 10409876 10352954 10498599 10350742 10528915 10435948 10434516 10404885 10382425 10346000 10587799 10607877 10586184 10442194 10481272 10453233 10522090 10400321 10524790 10451604 10380289 10412559

Ndrg4 Pik3c2b Prr5l Slc25a10 Snn Tlcd1 Bard1 Fancm Fbxl7 Hat1 Hjurp Il33 Mcm6 Zfp101 Akr1b10 Cks2 Fam70a Incenp Mr1 Pcyox1 Tbc1d16 Tmem38a Trip13 2310046K01Rik 2310061C15Rik Cks2 Rfx5 Stard10 Ctla2a Hmgb3 Ift80 Rnasel Tyms Ccdc80 Fam131a Gmpr Gprc5c Gulp1 Plscr2 Prps2 Tipin Zfp677 1700007K13Rik Slc8a1 Wdr19 1110002B05Rik Cit Mdfi Mmd Slbp

-‐1.68 -‐1.68 -‐1.68 -‐1.68 -‐1.68 -‐1.68 -‐1.69 -‐1.69 -‐1.69 -‐1.69 -‐1.69 -‐1.69 -‐1.69 -‐1.69 -‐1.70 -‐1.70 -‐1.70 -‐1.70 -‐1.70 -‐1.70 -‐1.70 -‐1.70 -‐1.70 -‐1.71 -‐1.71 -‐1.71 -‐1.71 -‐1.71 -‐1.72 -‐1.72 -‐1.72 -‐1.72 -‐1.72 -‐1.73 -‐1.73 -‐1.73 -‐1.73 -‐1.73 -‐1.73 -‐1.73 -‐1.73 -‐1.73 -‐1.74 -‐1.74 -‐1.74 -‐1.75 -‐1.75 -‐1.75 -‐1.75 -‐1.75

4.732 2.355 5.759 2.46 3.793 4.53 2.503 2.426 4.756 2.979 3.093 7.512 2.504 2.822 6.539 2.454 2.947 2.438 3.929 4.71 5.739 3.107 2.767 4.787 2.356 2.454 4.3 4.738 4.762 5.322 4.291 4.434 2.633 3.059 4.184 4.83 4.098 7.572 3.368 3.727 2.525 5.849 3.29 4.606 4.343 3.212 2.611 8.641 3.125 3.109

390.98 1259.71 644.57 511.77 456.52 220.9 278.79 152.36 169.08 1680.16 1195.57 65.8 980.76 169.32 605.92 1482.97 351.94 728.88 2045.68 1893.89 382.27 358.89 177.76 317.66 1019.63 1221.23 323.6 569.33 1992.63 508.79 402.21 148.97 738.55 93.43 479.28 244.57 243.12 66.92 691.64 1609.82 636.83 130.62 95.27 197.57 337.31 307.31 304.57 1526.88 267.74 643.99

232.66 749.6 384.39 304.02 271.56 131.74 165.11 90.11 100.34 995.26 708.74 38.92 581.3 100.09 356.13 873.32 206.81 428.66 1202.79 1116 224.67 211.07 104.64 185.65 595.76 714.12 189.74 332.15 1159.22 295.46 233.95 86.84 428.2 53.92 276.98 141.34 140.75 38.73 400.69 928.09 369.05 75.7 54.64 113.41 193.82 176.07 174.34 873.15 152.98 367.67

10400006 10535807 10395612 10474381 10406877 10584334 10371506 10545644 10440794 10443730 10454851 10399584 10369993 10499854 10576586 10462005 10360187 10556266 10488459 10606058 10373542 10525365 10539017 10385466 10596931 10522060 10478160 10446001 10544891 10515257 10396840 10458285 10498620 10484261 10601303 10425066 10460468 10594251 10351491 10526514 10420372 10456492 10523923 10586491 10394990 10491805 10358057 10430032 10421697 10601312

Ahr Flt1 G2e3 Kif18a Serf1 Siae Stab2 Wbp1 2610039C10Rik Abcg1 Cxxc5 G730007D18Rik Gstt3 S100a1 Slc35f3 Tmem2 Vangl2 Wee1 Zfp442 Cxcr3 Dgka Hvcn1 Reep1 Sgcd Wdr6 Fam114a1 Fam83d Fsd1 Nod1 Rad54l Rdh12 Spata24 Trim59 Cerkl Chic1 Csf2rb Ctsf Kif23 Olfml2b Cldn15 Cryl1 D18Ertd653e Ccdc18 Dapk2 Mboat2 Plk4 Shisa4 Tonsl 9030625A04Rik Chic1

-‐1.76 -‐1.76 -‐1.76 -‐1.76 -‐1.76 -‐1.76 -‐1.76 -‐1.76 -‐1.77 -‐1.77 -‐1.77 -‐1.77 -‐1.77 -‐1.77 -‐1.77 -‐1.77 -‐1.77 -‐1.77 -‐1.77 -‐1.78 -‐1.78 -‐1.78 -‐1.78 -‐1.78 -‐1.78 -‐1.79 -‐1.79 -‐1.79 -‐1.79 -‐1.79 -‐1.79 -‐1.79 -‐1.79 -‐1.80 -‐1.80 -‐1.80 -‐1.80 -‐1.80 -‐1.80 -‐1.81 -‐1.81 -‐1.81 -‐1.82 -‐1.82 -‐1.82 -‐1.82 -‐1.82 -‐1.82 -‐1.83 -‐1.83

3.488 2.985 4.146 2.655 4.73 4.304 7.82 4.242 3.433 7.678 7.571 4.49 4.043 3.655 3.702 6.812 4.491 2.382 6.261 6.721 4.272 3.762 4.554 6.289 2.451 4.674 2.629 3.549 3.479 2.881 5.566 3.609 3.724 3.662 3.923 2.523 4.41 2.853 4.531 3.853 3.754 6.237 3.05 4.758 5.302 2.58 5.554 2.694 4.344 3.759

3286.87 1082.5 838.86 196.69 288.01 876.81 138.58 1922.25 268.04 489.98 710.53 212.25 778.98 289.26 103.28 1630.56 445.63 918.48 186.16 191.69 1283.4 320.18 527.49 394.92 617.65 516.06 526.34 205.08 769.06 183.14 319.05 332.02 481.04 121.34 759.91 564.28 338.13 1534.93 158.14 624.94 815.23 644.12 108.44 346.77 345.87 635.26 1018.67 172.37 202.5 1319.78

1869.18 615.08 477.88 111.81 163.88 499.3 78.67 1089.74 151.43 277.36 400.56 120.22 440.04 163.65 58.39 919.31 251.71 517.82 104.92 107.68 719.33 180.3 296.95 221.59 347.56 288.08 293.35 114.58 429.72 102.55 178.06 185.95 269.09 67.55 422.99 313.96 187.75 852.92 88.03 345.92 450.93 355.32 59.57 190.44 189.55 348.62 558.89 94.84 110.5 720.87

10420957 10503259 10395457 10574378 10510215 10419779 10540059 10495945 10604620 10465912 10548761 10606163 10559568 10446027 10393926 10577164 10601844 10390050 10416406 10353733 10366983 10478572 10565712 10379989 10541034 10362294 10571696 10544462 10452709 10349051 10355806 10506571 10516723 10544751 10352798 10547088 10366277 10517655 10585286 10503617 10380566 10359648 10442932 10467637 10406934 10436666 10466800 10485963 10357875 10554281

Ptk2b Trp53inp1 Etv1 Gins3 Gm13139 Haus4 Slc41a3 4930422G04Rik Fam122b Fen1 Hebp1 4930519F16Rik 6030429G01Rik Chaf1a Dcxr Gas6 Bhlhb9 Eme1 Htr2a Prim2 Tmem194 Ube2c Acer3 Fam33a Anubl1 Arhgap18 Casp3 Fam115a Ndc80 Tnfrsf11a Tuba4a Dhcr24 Hcrtr1 Hoxa2 Kcnh1 Mbd4 E2f7 Pla2g5 Arhgap20 Mms22l Phospho1 Scyl3 Tmem8 Arhgap19 Etv1 Jam2 Pgm5 Arhgap11a Btg2 Fanci

-‐1.83 -‐1.83 -‐1.84 -‐1.84 -‐1.84 -‐1.84 -‐1.84 -‐1.85 -‐1.85 -‐1.85 -‐1.85 -‐1.86 -‐1.86 -‐1.86 -‐1.86 -‐1.86 -‐1.87 -‐1.87 -‐1.87 -‐1.87 -‐1.87 -‐1.87 -‐1.88 -‐1.88 -‐1.89 -‐1.89 -‐1.89 -‐1.89 -‐1.89 -‐1.89 -‐1.89 -‐1.90 -‐1.90 -‐1.90 -‐1.90 -‐1.90 -‐1.91 -‐1.91 -‐1.93 -‐1.93 -‐1.93 -‐1.93 -‐1.93 -‐1.94 -‐1.94 -‐1.94 -‐1.94 -‐1.95 -‐1.95 -‐1.95

5.273 7.852 5.884 2.492 4.105 2.699 5.604 2.61 3.343 2.551 6.957 4.186 5.885 2.523 3.882 2.387 5.994 2.521 4.734 2.691 3.036 2.538 7.849 2.707 4.376 7.286 6.578 6.592 2.574 5.012 4.813 3.748 4.292 6.808 6.307 4.115 2.731 6.696 4.342 2.38 5.063 3.015 8.786 2.786 6.625 6.232 2.841 2.768 4.586 2.718

976.66 1720.64 148.57 371.18 276.2 368.99 708.15 181.64 397.31 495.37 370.38 43.3 242.61 377.04 753.55 1095.39 125.01 323.52 137.05 1313.28 812.21 1265.61 1142.78 1167.22 348.43 265.39 900.82 721.11 344.07 300.37 777.84 1170.27 290.89 623.79 220.7 347.07 816.26 442.52 74.91 161.29 285.36 301.53 298.52 499.41 194.5 5544.93 252.12 579.71 2703.66 191.88

534.51 938.06 80.68 201.35 150.46 200.02 383.86 98.37 214.63 268.26 200.01 23.34 130.48 203.07 404.19 588.3 66.93 172.74 73.3 702.7 433.42 677.6 607.45 620.42 184.54 140.1 477.56 380.59 181.97 159.03 411.47 616.9 153.05 329.13 116.26 182.85 427.3 231.57 38.89 83.77 147.67 156.37 154.54 256.87 100.02 2852.25 129.66 296.85 1385.95 98.61

10538658 10401852 10421361 10545835 10379363 10523012 10539818 10603135 10569181 10511333 10453857 10415411 10607225 10403229 10586194 10592084 10366293 10546163 10565570 10356780 10481931 10495574 10375065 10475362 10413482 10597470 10443007 10583242 10426016 10583952 10351047 10399308 10421877 10504957 10515744 10369815 10502638 10555695 10591369 10423855 10499545 10569335 10377560 10521090 10426669 10350341 10377372 10538299 10550102 10415408

Herc3 4930534B04Rik Bmp1 1700040I03Rik Atad5 Dck Gp9 Fancb Lrdd Plag1 Gata6 Nynrin Lrch2 Itgb8 Megf11 St3gal4 Csrp2 Mcm2 4632434I11Rik Pask Phf19 Sass6 Sh3pxd2b Wdr76 Wnt5a Cmtm8 Neurl1b Sesn3 Gtse1 Ncapd3 Cenpl Fkbp1b Diap3 Smc2 Cdc20 Cdk1 Clca5 Rrm1 Dnmt1 Rims2 Efna3 H19 Sat2 Tacc3 Troap Mir181b-‐1 Slc25a35 Hoxa3 Lig1 Nynrin

-‐1.95 -‐1.96 -‐1.96 -‐1.97 -‐1.97 -‐1.97 -‐1.97 -‐1.98 -‐1.98 -‐1.98 -‐1.99 -‐1.99 -‐2.00 -‐2.02 -‐2.02 -‐2.02 -‐2.03 -‐2.03 -‐2.04 -‐2.04 -‐2.04 -‐2.04 -‐2.04 -‐2.04 -‐2.04 -‐2.05 -‐2.05 -‐2.05 -‐2.06 -‐2.06 -‐2.07 -‐2.07 -‐2.08 -‐2.08 -‐2.09 -‐2.09 -‐2.09 -‐2.09 -‐2.10 -‐2.10 -‐2.11 -‐2.11 -‐2.11 -‐2.11 -‐2.11 -‐2.12 -‐2.12 -‐2.13 -‐2.13 -‐2.13

8.128 3.747 3.886 2.855 2.359 2.422 2.435 2.465 3.547 5 4.714 4.628 6.289 5.023 4.384 7.656 6.522 2.948 2.641 3.245 3.218 2.724 5.253 2.599 5.062 3.743 4.633 5.572 3.595 2.933 3.196 10.254 2.776 2.721 2.576 2.644 5.908 2.438 2.606 4.845 3.617 7.021 5.117 2.688 2.966 3.788 3.733 4.612 2.622 4.173

232.88 218.84 873.76 486.16 158.37 486.57 720.04 225.2 382.75 124.72 310.72 1007 279.38 129.5 307.14 1055.65 557.62 1247.76 1372.26 317.17 307.35 879.18 678.67 368.47 147.98 413.54 249.52 503.36 885.56 657.28 174.13 238.28 338.08 740.36 737.06 2735.73 376.03 3159.3 980.02 90.26 394.07 302.03 162.68 375.26 512.06 150.59 302.71 351.74 560.79 975.15

119.3 111.45 445.35 246.54 80.41 247.3 364.73 113.84 193.62 63.09 155.77 506.13 139.8 64.05 151.77 522.81 274.92 614.81 671.51 155.28 150.31 431.86 332.31 180.88 72.37 201.98 121.44 246.06 429.09 319.19 83.95 115.12 162.8 356.59 353.04 1307.04 179.71 1508.54 467.41 43 186.48 142.96 77.21 177.89 242.54 71.17 142.65 165.48 262.93 457.17

10427369 10490104 10462140 10477187 10367973 10557156 10594774 10371770 10379127 10539617 10417004 10569393 10437945 10474825 10555205 10355967 10516246 10538832 10418506 10415413 10497253 10382435 10399478 10458195 10534974 10385542 10537712 10350297 10594426 10453231 10479811 10430825 10525591 10346843 10515836 10466304 10554445 10346365 10513818 10354168 10446074 10562637 10411739 10385583 10607366 10508986 10401673 10389606 10497451 10487577

Pde1b Aurka Dock8 Tpx2 Aig1 Plk1 Ccnb2 Gas2l3 Spag5 Alms1 Dzip1 R74862 Mcm4 D2Ertd750e Gdpd5 Ap1s3 Cdca8 Mad2l1 Stab1 Nynrin Pmp2 Gprc5c Lpin1 Cdc25c Mcm7 Btnl9 Gstk1 Kif14 Zwilch Slc8a1 Mcm10 Cenpm Kntc1 Nrp2 Ccnb1 Dtx4 Prc1 Sgol2 Stmn1 Tbc1d8 Uhrf1 Ccnb1 Ccnb1 Ltc4s Shroom2 Stmn1 Tgfb3 Prr11 Cpa3 Ckap2l

-‐2.13 -‐2.14 -‐2.14 -‐2.14 -‐2.15 -‐2.15 -‐2.16 -‐2.16 -‐2.16 -‐2.17 -‐2.17 -‐2.17 -‐2.18 -‐2.19 -‐2.19 -‐2.20 -‐2.21 -‐2.21 -‐2.23 -‐2.24 -‐2.24 -‐2.25 -‐2.26 -‐2.27 -‐2.27 -‐2.28 -‐2.28 -‐2.28 -‐2.28 -‐2.29 -‐2.30 -‐2.31 -‐2.31 -‐2.31 -‐2.33 -‐2.33 -‐2.33 -‐2.33 -‐2.33 -‐2.33 -‐2.33 -‐2.34 -‐2.34 -‐2.34 -‐2.34 -‐2.34 -‐2.34 -‐2.35 -‐2.36 -‐2.37

6.376 2.569 6.303 2.503 5.188 2.386 2.663 4.222 2.755 7.022 10.434 6.516 2.593 2.763 4.108 7.319 2.523 3.019 9.227 4.477 2.59 3.483 6.433 3.044 2.482 7.727 6.201 3.367 3.077 5.29 2.354 2.499 2.369 10.146 2.688 6.434 2.483 2.829 2.538 5.777 2.668 2.699 2.609 3.196 5.954 2.513 7.868 2.84 4.735 2.518

349.85 853.19 426.53 948.95 139.32 2175.39 2870.65 1131.88 396.58 318.31 398.76 588.94 1015.03 988.06 792.87 538.92 322.85 2611.46 5392.63 1312.17 97.77 342.83 312.5 186.64 1319.08 407.85 1181.87 242.47 469.35 97.71 371.24 830.52 328.34 2705.56 1225.02 390.05 1016.65 2106.16 2282.24 306.98 693.4 1540.82 1286.06 2609.82 411.29 2384 3509.91 1198.36 69.92 421.83

163.9 399.62 199.4 443.6 64.75 1010.54 1331.28 523.51 183.67 146.45 183.35 271.15 466.23 451.53 362.02 245.25 145.81 1180.6 2419.05 585.4 43.73 152.24 138.13 82.17 580.44 179.15 518.58 106.13 206.02 42.59 161.71 359.34 142.18 1171.73 524.93 167.08 436.62 902.3 980.06 131.95 297.21 657.8 548.77 1116.5 175.57 1018.32 1501.67 510.12 29.61 177.88

10542079 10372139 10572282 10540408 10568150 10382998 10485213 10497520 10571715 10462973 10495316 10568714 10390707 10538640 10350392 10591781 10422321 10361995 10414315 10497831 10517287 10471721 10451805 10605711 10436106 10396068 10546421 10495083 10514177 10421029 10585699 10475437 10450374 10406581 10416437 10371591 10540738 10370587 10541729 10601705 10513061 10564165 10391811 10587508 10590494 10413874 10358894 10564169 10361110 10539342

Foxm1 Nts Hapln4 Itpr1 Kif22 Birc5 Cd82 Ect2 Enpp6 Hells Psrc1 Mki67 Top2a Abcg2 Aspm Anln Dzip1 Fam54a Cdkn3 Ccna2 Man1c1 Ptgs1 Sgol1 Pdk3 C330027C09Rik Lrr1 Prickle2 Kcnd3 Bnc2 Cdca2 Fabp5 Sord D17H6S56E-‐5 Dhfr Lcp1 4930547N16Rik Fancd2 Shc2 Cdca3 Cenpi Ctnnal1 Snord116 Kif18b Ttk Kif15 Ogdhl Sord Snord116 Dtl Rtkn

-‐2.37 -‐2.37 -‐2.38 -‐2.38 -‐2.38 -‐2.39 -‐2.39 -‐2.39 -‐2.40 -‐2.40 -‐2.40 -‐2.41 -‐2.41 -‐2.42 -‐2.42 -‐2.43 -‐2.43 -‐2.43 -‐2.45 -‐2.46 -‐2.46 -‐2.47 -‐2.47 -‐2.48 -‐2.49 -‐2.49 -‐2.49 -‐2.50 -‐2.51 -‐2.51 -‐2.52 -‐2.52 -‐2.53 -‐2.55 -‐2.55 -‐2.56 -‐2.56 -‐2.56 -‐2.57 -‐2.57 -‐2.57 -‐2.57 -‐2.58 -‐2.58 -‐2.59 -‐2.60 -‐2.60 -‐2.61 -‐2.62 -‐2.63

3.172 4.303 8.762 16.211 2.433 2.931 8.633 2.764 3.035 2.588 4.791 2.581 2.451 3.544 2.749 2.693 8.949 2.703 3.192 2.515 7.356 4.502 2.603 7.856 3.101 2.483 2.992 7.001 10.464 2.91 3.739 5.935 2.923 2.424 9.989 2.601 3.113 10.637 2.67 2.471 11.736 7.112 2.731 2.665 2.93 6.414 6.223 6.206 2.803 6.191

661.21 348.67 350.49 503.74 478.7 296.93 616.48 1066.9 265.49 699.87 1298.99 1915.81 1440.75 886.62 451.68 1160.67 914.91 405.78 540.13 2887.22 1292.27 1436.53 564.01 578.1 576.62 349.32 662.57 413.38 193.57 511.65 6298.79 1611.12 2278.7 423.89 1263.44 352.14 226.09 207.77 653.91 207.19 1294.61 140.99 1096.78 230.89 305.33 251.13 1777.2 109.52 1365.74 355.36

278.5 146.9 147.47 211.29 200.74 124.46 257.58 446.77 110.7 292.05 541.01 794.2 597.9 366.05 186.74 477.98 377.05 166.76 220.14 1174.49 525.19 580.48 228.08 233.55 232 140.54 266.54 165.62 77.16 203.86 2500.75 639.78 900.19 166.55 496.33 137.49 88.24 81.32 254.47 80.49 503.72 54.97 425.41 89.57 117.72 96.64 682.36 42.04 521.48 134.91

10554325 10492689 10419296 10377405 10474769 10389395 10562989 10352767 10564177 10474875 10469167 10490838 10400589 10391461 10547943 10573261 10540085 10602009 10462132 10562563 10352048 10462632 10515431 10419062 10564183 10419323 10474902 10571680 10585381 10507112 10485718 10406968 10503264 10474984 10564159 10400510 10548086 10462866 10454709 10486255 10544596 10564161 10420426 10483401 10399391 10394978 10438378 10563883 10563780 10564163

5730590G19Rik Pdgfc Wdhd1 Aurkb Bub1b Brip1 Cpt1c Nek2 Snord116 Casc5 Sfmbt2 Fabp5 Mis18bp1 Brca1 Ncapd2 Asf1b Fbln2 Rnf128 E030010A14Rik Ccne1 Exo1 Kif20b Kif2c Sh2d4b Snord116 Dlgap5 Rad51 Mlf1ip Slc35f2 Stil Ano3 Cenpk Ccne2 Nusap1 Snord116 Clec14a Rad51ap1 Cep55 Kif20a Oip5 Tmem176b Snord116 F630043A04Rik Spc25 Gen1 Rrm2 Cdc45 Depdc1a E2f8 Snord116

-‐2.64 -‐2.64 -‐2.64 -‐2.65 -‐2.65 -‐2.66 -‐2.66 -‐2.66 -‐2.66 -‐2.67 -‐2.67 -‐2.68 -‐2.69 -‐2.70 -‐2.71 -‐2.73 -‐2.74 -‐2.74 -‐2.76 -‐2.77 -‐2.77 -‐2.77 -‐2.77 -‐2.77 -‐2.77 -‐2.78 -‐2.79 -‐2.80 -‐2.80 -‐2.80 -‐2.81 -‐2.81 -‐2.82 -‐2.82 -‐2.83 -‐2.84 -‐2.86 -‐2.87 -‐2.87 -‐2.88 -‐2.88 -‐2.89 -‐2.90 -‐2.90 -‐2.91 -‐2.92 -‐2.93 -‐2.93 -‐2.93 -‐2.93

2.714 6.912 2.685 2.475 3.089 2.516 12.728 3.51 6.459 2.678 6.155 3.825 2.955 2.888 3.033 2.581 9.829 7.936 7.78 2.566 2.575 3.202 2.964 5.944 6.551 2.834 2.64 2.402 9.641 2.66 5.847 2.866 2.546 2.533 7.663 3.7 2.841 3.265 3.056 2.62 20.094 6.72 2.72 2.6 2.746 2.818 3.126 2.476 3.123 6.902

362.48 676.06 489.85 2005.92 671.86 286.69 252.84 564.54 144.32 721.85 109.37 5690.66 623.38 317.53 1016.75 1466.9 363.9 329.97 1059.77 1044.51 339.59 396.91 440.78 629.98 152.85 695.4 421.89 289.61 1373.33 447.59 426.46 161.2 473.76 1665.56 151.17 1071.57 234.06 798.49 1142.86 231.65 3841.83 139.35 512.91 666.51 605.28 1818.08 1112.12 324 720.53 139.77

137.3 256.03 185.71 755.6 253.81 107.63 95.08 212.44 54.3 270.86 40.93 2125.24 231.99 117.74 375.39 537.09 132.87 120.38 383.89 376.84 122.76 143.43 158.85 227.82 55.19 250.31 151.24 103.42 489.74 159.75 151.66 57.44 167.85 590.95 53.51 376.81 81.92 278.21 398.12 80.44 1333.08 48.22 177.01 230.2 207.85 622.44 379.65 110.72 245.64 47.67

10564167 10564171 10564173 10564175 10564179 10564181 10564185 10564187 10564189 10564191 10564193 10564195 10564197 10564199 10564201 10564205 10564207 10361375 10487480 10577508 10530615 10462796 10359890 10496204 10497122 10385248 10399087 10427336 10360985 10521498 10487340 10450519 10350838 10521731 10460738 10438358 10466888 10548729 10502845 10436967 10462702 10368918 10571252 10450957 10485745 10448506 10586448 10559509 10534168 10429515

Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Snord116 Fbxo5 Bub1 Ckap2 Ociad2 Kif11 Nuf2 Cenpe Depdc1a Hmmr Ncapg2 Nckap1l Cenpf Crmp1 Ncaph Tcf19 2810417H13Rik Ncapg Cdca5 41157 Glis3 Mansc1 Fam73a Cbr1 Hectd2 Sobp Tex15 Cenpq Ano3 Ccnf 2810417H13Rik Cdc42ep5 Auts2 Lynx1

-‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.93 -‐2.98 -‐3.00 -‐3.01 -‐3.02 -‐3.03 -‐3.03 -‐3.04 -‐3.06 -‐3.06 -‐3.09 -‐3.09 -‐3.10 -‐3.10 -‐3.11 -‐3.11 -‐3.12 -‐3.12 -‐3.15 -‐3.17 -‐3.18 -‐3.18 -‐3.20 -‐3.22 -‐3.23 -‐3.24 -‐3.27 -‐3.29 -‐3.32 -‐3.33 -‐3.41 -‐3.48 -‐3.51 -‐3.52

6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 6.902 2.419 2.872 2.828 4.962 2.854 2.814 3.361 2.446 3.214 2.685 7.879 3.525 10.292 2.914 2.371 2.563 2.751 3.132 6.209 8.541 13.713 4.956 13.243 6.828 8.06 7.569 3.05 6.713 3.464 2.569 7.643 5.271 10.695

139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 139.77 759.11 920.94 751.29 599.76 1731.25 698.62 635.37 346.44 395.19 511.9 314.39 546.25 1335.29 1563.19 2445.33 1654.55 489.83 611.29 1436.45 1540.44 182.46 288.22 319.21 255.85 983.46 60.54 297.22 501.56 714.59 1191.33 667.83 534.44 1128.79

47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 47.67 254.61 306.72 249.51 198.64 571.75 230.74 208.77 113.2 129.14 165.77 101.71 176.46 430.59 502.16 787.39 530.37 156.93 193.93 452.43 484.9 57.41 89.97 98.99 79.19 303.11 18.52 90.31 151.09 214.66 349.01 191.7 152.13 321.11

10420877 10604694 10381096 10536667 10357488 10538150 10429520 10467206 10607206 10473444 10604528 10553833 10599174 10400609 10400607 10492964

Esco2 Mtap7d3 Igfbp4 Ptprz1 Cd55 Tmem176a Ly6d Ppp1r3c Il13ra2 Aplnr Mbnl3 Ndn Il13ra1 Mdga2 Mdga2 Cd5l

-‐3.55 -‐3.56 -‐3.64 -‐3.70 -‐3.72 -‐4.03 -‐4.13 -‐4.60 -‐6.07 -‐6.18 -‐7.18 -‐7.67 -‐9.95 -‐9.98 -‐10.57 -‐15.86

2.699 7.118 7.545 4.145 8.062 16.334 5.748 5.25 5.254 14.766 10.086 9.394 25.008 15.251 11.749 6.37

789.27 82.4 2809.59 580.5 277.97 1267.92 412.74 578.01 946 1060.33 552.59 416.64 363.69 953.7 1043.61 1823.6

222.6 23.15 772.44 156.78 74.64 314.69 99.89 125.58 155.9 171.6 77 54.32 36.54 95.52 98.76 114.98

Supplementary Table S2. Clinico-pathological data of pancreatic carcinoma patients. Patient Gender Age Tumor number (years) diameter 85 >4 cm F 61 221 >4 cm F 53 76 M 60