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Supplementary Table 1, Figures 1-3 from EphB4 Promotes Site-Specific Metastatic Tumor Cell Dissemination by Interacting with Endothelial Cell–Expressed EphrinB2

Prostate cancer is a major global health concern with limited treatment options for advanced disease. Its heterogeneity challenges the identification of crucial driver genes implicated in disease progression. Activating protein-1 (AP-1) transcription factor is associated with cancer since the first identification of its subunits, the proto-oncogenes JUN and FOS. Whereas both JUN and FOS have been implicated in prostate cancer, this study provides the first functional evidence that FOS acts as a tumor suppressor during prostate cancer progression and invasion. Data mining revealed decreased FOS expression in prostate cancer and a further downregulation in metastatic disease, consistent with FOS expression in cell lines derived from different prostate cancer stages. FOS deficiency in prostate cancer cell lines increases cell proliferation and induces oncogenic pathway alterations. Importantly, in vivo CRISPR/Cas9-mediated Fos and Pten double mutation in murine prostate epithelium results in increased proliferation and invasiveness compared to the abrogation of Pten alone. Interestingly, enhanced Jun expression is observed in the murine prostatic intraepithelial neoplasia lacking Fos. CRISPR/Cas9-mediated knockout of Jun combined with Fos and Pten deficiency diminishes the increased proliferation rate in vivo but not the ability to form invasive disease. Overall, we demonstrate that loss of Fos promotes disease progression from clinical latent prostate cancer to advanced disease through accelerated proliferation and invasiveness, partly through Jun.

Available tools for prostate cancer (PC) prognosis are suboptimal but may be improved by better knowledge about genes driving tumor aggressiveness. Here, we identified FRMD6 (FERM domain-containing protein 6) as an aberrantly hypermethylated and significantly downregulated gene in PC. Low FRMD6 expression was associated with postoperative biochemical recurrence in two large PC patient cohorts. In overexpression and CRISPR/Cas9 knockout experiments in PC cell lines, FRMD6 inhibited viability, proliferation, cell cycle progression, colony formation, 3D spheroid growth, and tumor xenograft growth in mice. Transcriptomic, proteomic, and phospho-proteomic profiling revealed enrichment of Hippo/YAP and c-MYC signaling upon FRMD6 knockout. Connectivity Map analysis and drug repurposing experiments identified pyroxamide as a new potential therapy for FRMD6 deficient PC cells. Finally, we established orthotropic Frmd6 and Pten, or Pten only (control) knockout in the ROSA26 mouse prostate. After 12 weeks, Frmd6/Pten double knockouts presented high-grade prostatic intraepithelial neoplasia (HG-PIN) and hyperproliferation, while Pten single-knockouts developed only regular PIN lesions and displayed lower proliferation. In conclusion, FRMD6 was identified as a novel tumor suppressor gene and prognostic biomarker candidate in PC.

Immunotherapy is a fundamental pillar in the treatment of lung cancer but remains inefficient in half of the treated patients. Recent clinical trials showed synergistic effects of combining chemotherapy and immunotherapy for lung cancer, suggesting that increased DNA damage imposed by chemotherapy modulates the tumor microenvironment to aid the efficacy of immunotherapy. But the immunological mechanisms related to this remains to be fully elucidated. The cGAS-STING pathway senses cytosolic DNA introduced by DNA-damaging agents such as chemotherapy. Activation of this pathway and induction of inflammatory cytokines may contribute to better immunotherapy efficacy. In particular, the release of type I IFN is a hallmark for STING pathway activation. However, we also know that IFNλ – a type III IFN – can be induced by STING-signaling. Despite the relevance of IFNλ in controlling inflammatory responses, its contribution in cancer remains unclear. Here, we investigated cGAS-STING expression and signaling in a panel of lung cancer cells. We identified IFNλ1 as an alternative hallmark for cGAS-STING DNA-sensing with higher expression after DNA-stimulation than IFNb in 6 out of 8 cell lines with one cell line having no IFNb expression at all. We then tested IFN induction after chemotherapeutic treatment in three different NSCLC cell lines and saw significant increase in both IFNb and IFNλ1 expression across four different chemotherapeutics, but with a higher fold change for IFNλ1. The induction of both IFNb and IFNλ1 was abolished when STING KO cell lines were treated with doxorubicin pointing to a STING-dependent response. To access the effect of IFNλ1 on macrophages, a prevalent cell type in the tumor microenvironment expressing IFNLR1, we performed RNA-sequencing on four primary human macrophage donors stimulated with IFNλ1. This stimulation led to a significant increase in well-known interferon-stimulated genes including the chemokines CXCL10 and CXCL11. We also explored whether IFNλ1 stimulation led to increased macrophage-dependent activation of autologous CD8+ T-cells. Interestingly, we found increased expression of both IFNg (p = 0.0215, n = 6) and Granzyme B (p = 0.0033, n = 6) measured by flow cytometry and ELISA (p > 0.0001, n = 6 for both). The addition of IFNλ1 during direct co-culture of macrophages and lung tumor organoids furthermore dampened the induction of the signal regulatory protein-a (SIRPa), a phagocytosis-inhibiting receptor, (p = 0.0041, n = 4) otherwise induced by the tumor organoids, but not matched healthy organoids. In support of this, there was an increased activation of CD8+ T-cells measured by IFNg production in an organoid-macrophage-CD8+ T-cell co-culture model. To summarize, we demonstrate that IFNλ1 may be a strong and broad marker of STING activation induced by chemotherapy in NSCLC and that IFNλ1 has the ability to prime a wider immune response by targeting macrophages. Hence, we propose that an IFNλ1 induced immune response has the potential to support and boost the response induced by existing immunotherapies. Citation Format: Kristine R Gammelgaard, Stine H Godsk, Albert G Olivier, Maria Riedel, Silke D Nielsen, Allard van Renterghem, Trine V Larsen, Anders Etzerodt, Emile Voest, Martin R Jakobsen. IFNλ1 is a marker of DNA damage-triggered STING-signaling in lung cancer that induces immune activation through macrophage stimulation [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A50.

Objective: Regarding the global pandemic scenario, the aim of this article is not only to describe the old and current outlines of the civil public managers’ responsibilities but also understanding at which level they are responsible by their choices and legal motivations, as well as when they can be considered responsible under contemporary circumstances. Even though the initial hypothesis is the most varied from common perception, the real answer to the problem comes from scientific research.Methodology: Considering the dogmatic methodology of deductive and exploratory research, in which the construction of concepts in legislation, doctrine, and jurisprudence. Result: It will be demonstrated – in a concise and structured way – that the motivation of public agents' decisions is very important to determine the degree and how they will take responsibilities. Contribution: The article represents an innovative and highly relevant theoretical tool in view of the COVID-19 pandemic context, explored in the national academic debate, to understand the process of interpretation and accountability of public agents. The present study, therefore, may be of great value to guide the application of the device in this specific context.Keywords: Responsibility; Motivation; Public funding policy; Pandemic; Public agent. RESUMOObjetivo: Diante do cenário da pandemia que se instaurou em âmbito global, o escopo precípuo deste artigo passa a ser, além de descrever os antigos e atuais contornos da responsabilidade civil do gestor público, de entender como os mesmos podem ser responsabilizados na motivação de suas escolhas, bem como em que momento isso pode/deve acontecer. As hipóteses iniciais podem ser as mais variadas oriundas de uma percepção comum.Metodologia: A real resposta ao problema vem a partir de uma pesquisa científica, considerando a metodologia dogmática de pesquisa dedutiva e exploratória, na qual se demonstrará a construção de conceitos na legislação, na doutrina e na jurisprudência de forma concisa e estruturadaResultado: O artigo evidenciará que a maneira pela qual se constrói a motivação das decisões dos agentes públicos é determinante no grau e na forma de sua responsabilização. Contribuição: O artigo representa uma ferramenta teórica inovadora e de grande relevância tendo em vista o contexto de pandemia da COVID-19, explorado no debate acadêmico nacional, para entender o processo de interpretação e responsabilização dos agentes públicos. O presente estudo, por este motivo, poderá ser de grande valia para orientar a aplicação do dispositivo nesta conjuntura específica.Palavras-chave: Responsabilização; Motivação; Política pública de fomento; Pandemia; Agente público.

Simple Summary Research in large animal models has been hampered by the complexity to introduce new gene alterations, but this has been simplified by the discovery of the CRISPR/Cas system. Here, we have cloned a Cas9 minipig to generate a porcine model for pre-clinical research. Six viable piglets were produced and backcrossed to Göttingen minipigs for two generations. Primary cells from different organs were isolated, and multiple gene alterations were performed by CRISPR in vitro. In vivo activation of the Cas9 expression was conducted by viral delivery of the FlpO expression to the skin. Overall, we successfully cloned a Cas9-expressing minipig and confirmed gene alterations introduced by the CRISPR/Cas system to porcine cells. Abstract The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

Simple Summary Lung cancer is by far the leading cause of cancer induced mortality worldwide with a median five-year survival rate of 19 percent. Genome sequencing of lung cancer samples has revealed several key mutated genes, which could be implicated in lung cancer formation. This study applied a mouse model of lung cancer based on CRISPR/Cas9 technology to functionally address key regulators of the mTor pathway, STK11 and PTEN. Our study revealed that loss of Stk11 drives lung adenocarcinoma progression, whereas Pten is dispensable. These functional mouse studies reveal that loss of Pten is non-essential for lung adenocarcinoma, which is in agreement with the low mutation rates of PTEN in human adenocarcinoma. In contrast, loss of Stk11 drive tumor progression and is often found mutated in human samples of lung adenocarcinoma. Abstract This study focused on STK11, PTEN, KRAS, and TP53, which are often found to be mutated in lung cancer. We compared Stk11 and Pten implication in lung cancer in combination with loss of Trp53 and gain of function of Kras in a CRISPR/Cas9 mouse model. Mice with loss of Stk11, Trp53, and KrasG12D mutation (SKT) reached human endpoint at around four months post-initiation. In comparison, mice with loss of Pten, Trp53, and KrasG12D mutation (PKT) survived six months or longer post-initiation. Pathological examination revealed an increase in proliferation in SKT deficient lung epithelia compared to PKT. This difference was independent of Pten loss, indicating that loss of Pten is dispensable for cell proliferation in lung adenocarcinoma. Furthermore, tumors with loss of Stk11, Trp53, and KrasG12D mutation had a significantly higher progression rate, monitored by PET/MRI scanning, compared to mice with loss of Pten, Trp53, and KrasG12D mutation, revealing that mutations in Stk11 are essential for adenocarcinoma progression. Overall, by using the CRISPR/Cas9 mouse model of lung adenocarcinoma, we showed that mutations in Stk11 are a key driver, whereas loss of Pten is dispensable for adenocarcinoma progression.

Summary Single-cell transcriptomics (scRNA-seq) has revolutionized the understanding of the spatial architecture of tissue structure and function. Advancing the “transcript-centric” view of scRNA-seq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyze post-translational modifications. Here, by combining spatial cell sorting with transcriptomics and quantitative proteomics/phosphoproteomics, we established the spatially resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signaling mechanisms. Phosphorylation of receptor tyrosine kinases was detected preferentially in the central vein area, resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation identified Tie receptor signaling as a selective and specific regulator of vascular Wnt activity orchestrating angiocrine signaling, thereby controlling hepatocyte function during liver regeneration. Taken together, the study has yielded fundamental insight into the spatial organization of liver endothelial cell signaling. Spatial sorting may be employed as a universally adaptable strategy for multiomic analyses of scRNA-seq-defined cellular (sub)-populations., Graphical abstract, Highlights • ScRNA-seq-guided spatial sort enables multiomic dissection of the liver vasculature • Liver sinusoidal endothelial cells have a hybrid vascular-lymphatic phenotype • Tyrosine phosphorylation of endothelial cell molecules is enriched on central vein • Endothelial Tie1 shapes hepatic Wnt signal zonation and promotes liver regeneration, Inverso, Shi et al. generate a multiomic encyclopedia of liver endothelial cells (L-ECs) with spatial resolution of transcriptome, proteome, and phosphoproteome. The study provides insight into liver vascular zonation and a template for scRNA-seq-data-guided spatial proteome and phosphoproteome analyses.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, and the incidence of HCC is increasing. Recently, cancer immunotherapy has emerged as an efficient treatment against some cancers. Here we have used a mouse model of mutagen-induced HCC to explore the therapeutic usefulness of targeting the DNA-activated STING pathway in HCC. STING-deficient mice exhibited unaltered initial development of HCC, but had higher number of large tumors at late stages of disease. In the liver of STING-deficient HCC mice, we observed reduced levels of phospho-STAT1, autophagy, and cleaved caspase3. These responses were activated in the liver by treatment with a cyclic dinucleotide (CDN) STING agonist. Importantly, CDN treatment of mice after HCC development efficiently reduced tumor size. Initiation of CDN treatment at an even later stage of disease to allow HCC detection by MR scanning revealed that the majority of tumors regressed in response to CDN, but new tumors were also detected, which were unresponsive to CDN treatment. Overall, the modulation of the STING pathway affects the development of HCC, and holds promise for a use as a treatment of this disease, most likely in combination with other immunomodulatory treatments such as PD1 inhibitors or with standard of care.

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease. Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane. This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

The aim of the project is to generate a toolbox pig model. This is being done because the pig probably is the best non-primate model in translational medicine. Unfortunately, pig models have proven difficult to produce. The two main complications have been the cloning of viable pigs and that just a few genes are altered, which often is not sufficient to induce the desired disease. Especially, models based on multiple genetic modifications of the pig genome, e.g. cancer, are challenging to generate. This was changed with the development of the CRISPR-Cas9 technology, which made it possible to perform multiple in vivo gene editing. We have cloned an inducible Cas9 expressing minipig and have obtained healthy F1 offspring. Activation of the Cas9 expression is attained by transduction with adeno-associated virus that delivers both the recombinase and the guide RNA to the designated tissue. This design provides spatial and temporal control of the activation. The Cas9 expression is mediated by a ubiquitous promotor, which also co-expresses fluorescent proteins for expression analysis. Transgenic Cas9 expression is observed in all major organs with strong expression in the epithelia, muscles and hepatocytes. Successful in vitro gene editing has been conducted in primary keratinocytes and fibroblast obtained from the transgenic pigs, which confirms the genetic design. By delivery of our construct by adeno-associated virus to the skin of the pigs, In vivo activation of the transgenic construct has been achieved. To validate the model, lung cancer has been induced in nine pigs by alteration a panel of cancer related genes including STK11, TP53, PTEN, and KRAS, all key drivers of human lung cancer. To obtain the constitutive active KRAS-G12D isoform of the KRAS oncogene a repair template was supplied. The pigs are being followed by PET/CT scanning in combination with 18F-FDG as a tracer. Preliminary data indicates abnormalities/cancer initiation in the pigs eight months after induction. In conclusion, an inducible Cas9 expressing minipig allows rapid gene editing to study different scenarios of human cancer in a pig model. The development of the Cas9 pig will extend the variety of pig disease models, both to generate a model of lung cancer as proposed but also models for other cancers. Citation Format: Martin F. Berthelsen, Maria Riedel, Mikkel H. Vendelbo, Aage K. Alstrup, Frederik Dagnæs-Hansen, Yonglun Luo, Simone S. Møller, Henrik Callesen, Jannik E. Jakobsen, Martin K. Thomsen. The CRISPR-Cas9 minipig: A transgenic toolbox pig to produce specific genome editing in designated tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3706.

Prostate Cancer is amongst the most frequently diagnosed malignancies and the second leading cause of cancer-related death in men worldwide accompanied by an increasing incidence. Until now its heterogeneity has been a major challenge in the establishment of good in vivo models for fast validation of potential driver genes. Classic in vivo models are costly, time-consuming and often target the majority of the prostate epithelium. We successfully introduced a novel prostate cancer mouse model based on CRISPR/Cas9 technology, ensuring multiplexed gene editing. In this model, specific in vivo gene editing was obtained in murine prostate epithelium cells of a transgenic mouse strain, harboring the CRISPR associated protein 9 (Cas9) endonuclease. Prostate epithelium cells were transduced by an Adeno-associated virus (AAV), carrying multiple single guide RNAs (sgRNA). Genetically different viral constructs were designed, each expressing different sgRNA combinations against Pten, representing the main driver in prostate cancer, tumor suppressor Trp53 and either one of the AP1 transcription factor subunits Junb or Fos, or tumor suppressor Smad4. Since viral transduction occurs in only a few cells, edited cell clones can clonally expand and undergo a natural selection process. Furthermore, the simultaneous gene knockouts reflect the human scenario of tumor heterogeneity. Mouse prostates were isolated and analyzed three to nine months post-injection to obtain insight on whether or not a gene appears crucial for tumor development in a specific tissue and wherein different gene combinations correlate with tumor severity. Histological analysis revealed increased proliferation, increased AKT activation, as well as invasiveness in samples with multiple gene knockouts compared to controls with single Pten knockout. Furthermore, loss of SMAD4 accelerated cancer progression when compared to the loss of the AP-1 transcription factor. Surprisingly, knockout of Trp53 was rarely observed in prostate samples while it occurred frequently in other tumor types in the same model system. This indicates that knockout of Trp53 may not be required for prostate cancer initiation. Overall, we established an in vivo model system of simultaneous, multiplexed gene editing in the prostate epithelium to address the biological cross-talk between various, altered pathways in prostate cancer initiation and progression. Citation Format: Maria Riedel, Latifa Bakiri, Martin F. Berthelsen, Michael Borre, Mikkel H. Vendelbo, Erwin F. Wagner, Martin K. Thomsen. A new mouse model for rapid identification of key factors driving prostate cancer progression and invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4632.

Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A165). NRP1 is also the receptor for the CendR peptides, a class of cell- and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A165, a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.

The combination of controlled radical polymerization (CRP) reactions and click chemistry offers high potential for the preparation of multifunctional polymers and significantly broadens the application scope of functional soft matter materials. In order to demonstrate the strategies as well as the potential of this methodology combination, examples for end-group and side-chain modification of polymers produced by CRP methods and the use of the resulting materials in functional polymer films are given.

Two phase separating block copolymers equipped with functional groups (acid and alkyne) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Thin films of these materials were prepared and examined with regard to surface morphology, surface composition, and film stability. Self-assembled structures with domain sizes of about 40 nm were detected through atomik force microscopy (AFM) analysis while X-ray photoelectron spectroscopy measurements revealed a balanced surface exposure of the two segregated phases. Thus, reactive groups being present in both phases are specifically provided within nanoscopic surface areas. The films showed good stability on exposure to various solvents but the self-organized surface patterns were only resistant toward ethanol. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011

Block copolymers consisting of a pentafluorostyrene (PFS) block and a hydrophilic block were synthesized by RAFT polymerisation. The hydrophilic blocks consist of methacrylate derivatives, 4-hydroxystyrene or 4-vinylpyridine monomers. The block copolymers were obtained with narrow molecular weight distributions and the molecular weights were in good agreement with the theoretical values. In addition, a model thiol was reacted with the PFS moieties of the block copolymers. This polymer–analogous reaction was performed under ambient conditions in high yields resulting quantitatively in para-substitution of the pentafluorophenyl rings. Finally, thin films consisting of block copolymers that showed strong phase-segregation behaviour and ordered nanostructured surfaces consisting of both blocks were obtained.

The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4–expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. Mol Cancer Res; 8(10); 1297–309. ©2010 AACR.

Objective— To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Methods and Results— Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain–containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. Conclusion— Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell–expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

The vascular endothelium is a crucial interface that controls the recruitment of circulating leukocytes. Based on the luminal expression of the ephrin-B2 ligand by endothelial cells (ECs) and the expression of EphB receptors (EphBRs) by circulating monocytes, we hypothesized that EphBR-ephrinB interactions are involved in monocyte adhesion. Adhesion experiments with monocytic cells were performed on ECs that overexpressed either full-length ephrin-B2 or cytoplasmically truncated ephrin-B2 (ΔC-ephrin-B2). Atomic force microscopy confirmed similar adhesive strengths of EphBR-expressing J774 cells to ECs that either overexpressed full-length ephrin-B2 or truncated ΔC-ephrin-B2 (1-minute interaction). Yet, adhesion experiments under static or flow conditions for 30 minutes demonstrated the preferential adhesion of monocytic cells to ECs that overexpressed full-length ephrin-B2 but not to ΔC-ephrin-B2 or to ECs that had been mock transduced. Adhesion was blocked by ephrin-B2-specific and EphBR-specific antibodies. Correspondingly, adhesion of EphB4-receptor-overexpressing monocytes to ephrin-B2-positive ECs was further augmented. Trafficking experiments of cell-surface molecules revealed that, prior to internalization, the resulting EphB4-receptor–ephrin-B2 complex translocated from the luminal surface to inter-endothelial junctions. Lastly, full-length ephrin-B2 in ECs was also involved in monocyte transmigration. Collectively, our study identifies a role of EphBR-ephrinB interactions as a new step in the cascade of events leading to monocyte adhesion and transmigration through the vascular endothelium.

The human prostate gland is a well known source of H1 and H2 relaxin but information is lacking on the expression and potential role of the INSL3 peptide hormone within the prostate gland. In the present study we have investigated the expression of human INSL3 in patients with benign prostate hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma tissues. Of the prostate epithelial cells, strongest INSL3 expression was detected in the basal epithelial cell compartment. Weaker INSL3 mRNA expression and immunoreactive INSL3 production were observed in secretory epithelial cells and in interstitial smooth muscle cells. Prostate epithelial cells were also a source for transcripts encoding the INSL3 receptor LGR8 suggesting the presence of an autocrine/paracrine INSL3-LGR8 ligand-receptor system within the human prostate. Three human prostate carcinoma cell lines displayed differential gene activity for INSL3 and LGR8. While LNCaP was devoid of INSL3, the androgen-insensitive PC-3 and the stromal prostate cell line hPCP co-expressed INSL3 and LGR8 transcripts. In addition to expressing INSL3 mRNA, the LGR8-negative DU-145 also expressed an INSL3 splice form formerly demonstrated in thyroid carcinoma cells. When incubated with recombinant INSL3, PC-3 cells showed significantly increased intracellular cAMP levels indicating functional LGR8 receptors. INSL3 did not alter the proliferative or metabolic activity of PC-3 carcinoma cells. Instead, PC-3 responded to INSL3 with significantly enhanced tumor cell motility and a transcriptional down-regulation of ErbB receptors and EGF. All-trans-retinoic acid was demonstrated in PC-3 to up-regulate LGR8 gene activity in a dose- and time-dependent manner while having no effect on INSL3 gene activity. In conclusion, we have identified a functional INSL3-LGR8 ligand-receptor system in human prostate carcinoma cells.

Prostate cancer is the second most diagnosed cancer in men. It is a slow progressing cancer, but when the disease reaches an advanced stage, treatment options are limited. Sequencing analyses of cancer samples have identified genes that can potentially drive disease progression. We implemented the CRISPR/Cas9 technology to simultaneously manipulate multiple genes in the murine prostate and thus to functionally test putative cancer driver genes in vivo. The activating protein-1 (AP-1) transcription factor is associated with many different cancer types, with the proto-oncogenes JUN and FOS being the two most intensely studied subunits. We analyzed expression of FOS and JUNB in human prostate cancer datasets and observed decreased expression in advanced stages. By applying CRISPR/Cas9 technology, the role of these two transcription factors in prostate cancer progression was functionally tested. Our data revealed that loss of either JunB or Fos in the context of Pten loss drives prostate cancer progression to invasive disease. Furthermore, loss of Fos increases Jun expression, and CRISPR inactivation of Jun in this context decreases cell proliferation. Overall, these in vivo studies reveal that JunB and Fos exhibit a tumor suppressor function by repressing invasive disease, whereas Jun is oncogenic and increases cell proliferation. This demonstrates that AP-1 factors are implicated in prostate cancer progression at different stages and display a dual function as tumor suppressor and as an oncogene in cancer progression.