Blocking the angiopoietin-2-dependent integrin ß-1 signaling axis abrogates small cell lung cancer invasion and metastasis.

Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin ß-1-dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin ß-1 or blocking Integrin ß-1 signaling by an anti-ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin ß-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1-targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin ß-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.

Tripartite antigen-agnostic combination immunotherapy cures established poorly immunogenic tumors.

BACKGROUND: Single-agent immunotherapy has shown remarkable efficacy in selected cancer entities and individual patients. However, most patients fail to respond. This is likely due to diverse immunosuppressive mechanisms acting in a concerted way to suppress the host anti-tumor immune response. Combination immunotherapy approaches that are effective in such poorly immunogenic tumors mostly rely on precise knowledge of antigenic determinants on tumor cells. Creating an antigen-agnostic combination immunotherapy that is effective in poorly immunogenic tumors for which an antigenic determinant is not known is a major challenge. METHODS: We use multiple cell line and poorly immunogenic syngeneic, autochthonous, and autologous mouse models to evaluate the efficacy of a novel combination immunotherapy named tripartite immunotherapy (TRI-IT). To elucidate TRI-ITs mechanism of action we use immune cell depletions and comprehensive tumor and immune infiltrate characterization by flow cytometry, RNA sequencing and diverse functional assays. RESULTS: We show that combined adoptive cellular therapy (ACT) with lymphokine-activated killer cells, cytokine-induced killer cells, Vγ9Vδ2-T-cells (γδ-T-cells) and T-cells enriched for tumor recognition (CTLs) display synergistic antitumor effects, which are further enhanced by cotreatment with anti-PD1 antibodies. Most strikingly, the full TRI-IT protocol, a combination of this ACT with anti-PD1 antibodies, local immunotherapy of agonists against toll-like receptor 3, 7 and 9 and pre-ACT lymphodepletion, eradicates and induces durable anti-tumor immunity in a variety of poorly immunogenic syngeneic, autochthonous, as well as autologous humanized patient-derived models. Mechanistically, we show that TRI-IT coactivates adaptive cellular and humoral, as well as innate antitumor immune responses to mediate its antitumor effect without inducing off-target toxicity. CONCLUSIONS: Overall, TRI-IT is a novel, highly effective, antigen-agnostic, non-toxic combination immunotherapy. In this study, comprehensive insights into its preclinical efficacy, even in poorly immunogenic tumors, and mode of action are given, so that translation into clinical trials is the next step.

EGFR Inhibition Strongly Modulates the Tumour Immune Microenvironment in EGFR-Driven Non-Small-Cell Lung Cancer.

EGFR-driven non-small-cell lung cancer (NSCLC) patients are currently treated with TKIs targeting EGFR, such as erlotinib or osimertinib. Despite a promising initial response to TKI treatment, most patients gain resistance to oncogene-targeted therapy, and tumours progress. With the development of inhibitors against immune checkpoints, such as PD-1, that mediate an immunosuppressive microenvironment, immunotherapy approaches attempt to restore a proinflammatory immune response in tumours. However, this strategy has shown only limited benefits in EGFR-driven NSCLC. Approaches combining EGFR inhibition with immunotherapy to stimulate the immune response and overcome resistance to therapy have been limited due to insufficient understanding about the effect of EGFR-targeting treatment on the immune cells in the TME. Here, we investigate the impact of EGFR inhibition by erlotinib on the TME and its effect on the antitumour response of the immune cell infiltrate. For this purpose, we used a transgenic conditional mouse model to study the immunological profile in EGFR-driven NSCLC tumours. We found that EGFR inhibition mediated a higher infiltration of immune cells and increased local proliferation of T-cells in the tumours. Moreover, inhibiting EGFR signalling led to increased activation of immune cells in the TME. Most strikingly, combined simultaneous blockade of EGFR and anti-PD-1 (aPD-1) enhanced tumour treatment response in a transgenic mouse model of EGFR-driven NSCLC. Thus, our findings show that EGFR inhibition promotes an active and proinflammatory immune cell infiltrate in the TME while improving response to immune checkpoint inhibitors in EGFR-driven NSCLC.

Notch1 Deficiency Induces Tumor Cell Accumulation Inside the Bronchiolar Lumen and Increases TAZ Expression in an Autochthonous Kras LSL-G12V Driven Lung Cancer Mouse Model.

Purpose: Abrogation of Notch signaling, which is pivotal for lung development and pulmonary epithelial cell fate decisions was shown to be involved in the aggressiveness and the differentiation of lung carcinomas. Additionally, the transcription factors YAP and TAZ which are involved in the Hippo pathway, were recently shown to be tightly linked with Notch signaling and to regulate the cell fate in epidermal stem cells. Thus, we aim to elucidate the effects of conditional Notch1 deficiency on carcinogenesis and TAZ expression in lung cancer. Methods: We investigated the effect of conditional Cre-recombinase mediated Notch1 knock-out on lung cancer cells in vivo using an autochthonous mouse model of lung adenocarcinomas driven by Kras LSL-G12V and comprehensive immunohistochemical analysis. In addition, we analyzed clinical samples and human lung cancer cell lines for TAZ expression and supported our findings by publicly available data from The Cancer Genome Atlas (TCGA). Results: In mice, we found induction of papillary adenocarcinomas and protrusions of tumor cells from the bronchiolar lining upon Notch1 deficiency. Moreover, the mutated Kras driven lung tumors with deleted Notch1 showed increased TAZ expression and focal nuclear translocation which was frequently observed in human pulmonary adenocarcinomas and squamous cell carcinomas of the lung, but not in small cell lung carcinomas. In addition, we used data from TCGA to show that putative inactivating NOTCH1 mutations co-occur with KRAS mutations and genomic amplifications in lung adenocarcinomas. Conclusion: Our in vivo study provides evidence that Notch1 deficiency in mutated Kras driven lung carcinomas contributes to lung carcinogenesis in a subgroup of patients by increasing TAZ expression who might benefit from TAZ signaling blockade.

Graph theoretical quantification of white matter reorganization after cortical stroke in mice.

Stroke is a devastating disease leading to cell death and disconnection between neurons both locally and remote, often resulting in severe long-term disability. Spontaneous reorganization of areas and pathways not primarily affected by ischemia is, however, associated with albeit limited recovery of function. Quantitative mapping of whole-brain changes of structural connectivity concerning the ischemia-induced sensorimotor deficit and recovery thereof would help to target structural plasticity in order to improve rehabilitation. Currently, only in vivo diffusion MRI can extract the structural whole-brain connectome noninvasively. This approach is, however, used primarily in human studies. Here, we applied atlas-based MRI analysis and graph theory to DTI in wild-type mice with cortical stroke lesions. Using a DTI network approach and graph theory, we aimed at gaining insights into the dynamics of the spontaneous reorganization after stroke related to the recovery of function. We found evidence for altered structural integrity of connections of specific brain regions, including the breakdown of connections between brain regions directly affected by stroke as well as long-range rerouting of intra- and transhemispheric connections related to improved sensorimotor behavior.

Inhibition of Tumor VEGFR2 Induces Serine 897 EphA2-Dependent Tumor Cell Invasion and Metastasis in NSCLC.

Anti-angiogenic treatment targeting vascular endothelial growth factor (VEGF)-VEGFR2 signaling has shown limited efficacy in lung cancer patients. Here, we demonstrate that inhibition of VEGFR2 in tumor cells, expressed in ∼20% of non-squamous non-small cell lung cancer (NSCLC) patients, leads to a pro-invasive phenotype. Drug-induced inhibition of tumor VEGFR2 interferes with the formation of the EphA2/VEGFR2 heterocomplex, thereby allowing RSK to interact with Serine 897 of EphA2. Inhibition of RSK decreases phosphorylation of Serine 897 EphA2. Selective genetic modeling of Serine 897 of EphA2 or inhibition of EphA2 abrogates the formation of metastases in vivo upon VEGFR2 inhibition. In summary, these findings demonstrate that VEGFR2-targeted therapy conditions VEGFR2-positive NSCLC to Serine 897 EphA2-dependent aggressive tumor growth and metastasis. These data shed light on the molecular mechanisms explaining the limited efficacy of VEGFR2-targeted anti-angiogenic treatment in lung cancer patients.

Cloud-based relational database for multimodal animal data.

Pre-clinical research builds on a large variety of in vivo and ex vivo tools such as non-invasive imaging, microscopy, and analysis of gene expression. To work efficiently with multimodal data and correlate results across scales, it is of particular importance to have easy access to all data points from different specimen, e.g. the magnetic resonance imaging (MRI) data from different time points, and the post-mortem histology. That requires an efficient data management, which is customizable and designed to relate all applied methods, raw data and analyses to one specific animal. Despite increasing demands to handle such complex data, most pre-clinical labs have not yet established such an electronic database. Here, we present a novel cloud-based relational database for multimodal animal data, which operates on commercial software. We have implemented data fields for various pre-clinical features such as MRI, histology and behaviour. Automated procedures replace manual and recurrent calculations. Pre-set plotting and printing features provide efficient analysis and documentation. The database template is useful for all labs working with laboratory animals and the adaption to specific research projects requires no prior scripting expertise. The database works operating-system independent through the web browser and allows multiple users to work simultaneously. The data entry is monitored and restricted for particular tests according to the user management in order to keep for example users during the experiment blinded for the experimental group. The database improves data accessibility, standardization of data recording and data handling efficiency in pre-clinical research.